Content uploaded by Verónica Guarner
Author content
All content in this area was uploaded by Verónica Guarner on Nov 22, 2023
Content may be subject to copyright.
Citation: Pérez-Torres, I.;
Aisa-Álvarez, A.; Casarez-Alvarado,
S.; Borrayo, G.; Márquez-Velasco, R.;
Guarner-Lans, V.; Manzano-Pech, L.;
Cruz-Soto, R.; Gonzalez-Marcos, O.;
Fuentevilla-Álvarez, G.; et al. Impact
of Treatment with Antioxidants as an
Adjuvant to Standard Therapy in
Patients with Septic Shock: Analysis
of the Correlation between Cytokine
Storm and Oxidative Stress and
Therapeutic Effects. Int. J. Mol. Sci.
2023,24, 16610. https://doi.org/
10.3390/ijms242316610
Academic Editor: Dumitru
Constantin-Teodosiu
Received: 10 October 2023
Revised: 14 November 2023
Accepted: 18 November 2023
Published: 22 November 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
International Journal of
Molecular Sciences
Article
Impact of Treatment with Antioxidants as an Adjuvant to
Standard Therapy in Patients with Septic Shock: Analysis of
the Correlation between Cytokine Storm and Oxidative Stress
and Therapeutic Effects
Israel Pérez-Torres 1,† , Alfredo Aisa-Álvarez 2, † , Sergio Casarez-Alvarado 3, Gabriela Borrayo 4,
Ricardo Márquez-Velasco 3, Verónica Guarner-Lans 5, Linaloe Manzano-Pech 1, Randall Cruz-Soto 3,
Omar Gonzalez-Marcos 2, Giovanny Fuentevilla-Álvarez 5, Ricardo Gamboa 5,
Huitizilihuitl Saucedo-Orozco 6, Juvenal Franco-Granillo 2and María Elena Soto 3,7,8,*
1Cardiovascular Biomedicine Department, Instituto Nacional de Cardiología Ignacio Chávez,
Juan Badiano No. 1, Col. Sección XVI, Mexico City 14380, Mexico; pertorisr@yahoo.com.mx (I.P.-T.);
loe_mana@hotmail.com (L.M.-P.)
2Critical Care Department, American British Cowdray (ABC) Medical Center, PAI ABC Sur 136 No. 116,
Col. las Américas, Mexico City 01120, Mexico; alfredoaisaa@gmail.com (A.A.-Á.);
ogmarcos35@gmail.com (O.G.-M.); jfranco@abchospital.com (J.F.-G.)
3Immunology Department, Instituto Nacional de Cardiología Ignacio Chávez, Juan Badiano No. 1,
Col. Sección XVI, Mexico City 14380, Mexico; secazalv@gmail.com (S.C.-A.);
marquezric@hotmail.com (R.M.-V.); randallcruz44@hotmail.com (R.C.-S.)
4Instituto Mexicano del Seguro Social, Dirección de Prestaciones Médicas Coordinación de Innovación en
Salud, Ciudad de México 06700, Mexico; gabriela.borrayo@imss.gob.mx
5Physiology Department, Instituto Nacional de Cardiología Ignacio Chávez, Juan Badiano No. 1,
Col. Sección XVI, Mexico City 14380, Mexico; gualanv@yahoo.com (V.G.-L.);
fuentevilla_alvarez@hotmail.com (G.F.-Á.); rgamboaa_2000@yahoo.com (R.G.)
6Cardioneumology Department, Specialty Hospital, National Medical Center “La Raza”,
Mexico City 02990, Mexico; huitzilihuitls@outlook.com
7Research Direction Instituto Nacional de Cardiología Ignacio Chávez, Juan Badiano No. 1, Col. Sección XVI,
Mexico City 14380, Mexico
8Cardiovascular Line in American British Cowdray (ABC) Medical Center, PAI ABC Sur 136 No. 116,
Col. Las Américas, Mexico City 01120, Mexico
*Correspondence: elena.soto@cardiologia.org.mx; Tel.: +52-553888-0897
†These authors contributed equally to this work.
Abstract:
Cellular homeostasis is lost or becomes dysfunctional during septic shock due to the
activation of the inflammatory response and the deregulation of oxidative stress. Antioxidant
therapy administered alongside standard treatment could restore this lost homeostasis. We included
131 patients
with septic shock who were treated with standard treatment and vitamin C (Vit C),
vitamin E (Vit E), N-acetylcysteine (NAC), or melatonin (MT), in a randomized trial. Organ damage
quantified by Sequential Organ Failure Assessment (SOFA) score, and we determined levels of
Interleukins (IL) IL1
β
, Tumor necrosis factor alpha (TNF
α
), IL-6, monocyte chemoattractant protein-1
(MCP-1), Transforming growth factor B (TGF
β
), IL-4, IL-10, IL-12, and Interferon-
γ
(IFN
γ
). The SOFA
score decreased in patients treated with Vit C, NAC, and MT. Patients treated with MT had statistically
significantly reduced of IL-6, IL-8, MCP-1, and IL-10 levels. Lipid peroxidation, Nitrates and nitrites
(NO
3−
and NO
2−
), glutathione reductase, and superoxide dismutase decreased after treatment with
Vit C, Vit E, NAC, and MT. The levels of thiols recovered with the use of Vit E, and all patients
treated with antioxidants maintained their selenium levels, in contrast with controls (
p= 0.04
). The
findings regarding oxidative stress markers and cytokines after treatment with antioxidants allow us
to consider to future the combined use of antioxidants in a randomized clinical trial with a larger
sample to demonstrate the reproducibility of these beneficial effects.
Keywords: antioxidants; septic shock; melatonin; cytokines
Int. J. Mol. Sci. 2023,24, 16610. https://doi.org/10.3390/ijms242316610 https://www.mdpi.com/journal/ijms
Int. J. Mol. Sci. 2023,24, 16610 2 of 26
1. Introduction
Sepsis and septic shock are global health problems and their prevalence generates a
high economic cost; 51% of patients are admitted to intensive care units (ICUs), and 17.3%
are treated in intermediate care (IC) or coronary units (UCCs) [
1
]. Approximately 80% of
patients have a risk of developing multiple organ failure (MOF) [2].
In patients with sepsis and septic shock, dysfunction and loss of cellular and metabolic
homeostasis are conditioned by the patient’s response to the infection and are associated
with a high mortality [
3
–
5
]. Since viruses, bacteria, and fungi cause infections leading to
these conditions, the immune response must be rapid and coordinated; however, when the
immune response is dysregulated or excessively triggered, a state of hyperinflammation
occurs [
6
–
8
]. Several damage mechanisms interact, and it is vital to control the inflamma-
tory response and the pro-oxidant state during treatment [
9
], because their deregulation
leads to endothelial dysfunction, capillary permeability alterations, and variations in the
coagulation system [10].
Immunoregulation by cytokines is vital; interferons mediate natural immunity, acti-
vate mononuclear phagocytes and neutrophils, and increase the molecules of the major
histocompatibility complex. During septic shock, the participation of cytokines, such as IL-2
and IL-4, regulate the activation, proliferation, and differentiation of leukocytes [
11
–
13
].
Chemokines promote the adhesion of monocytes and neutrophils [
14
], and interferon-
gamma (IFN
γ
) acts as a potent activator of mononuclear phagocytes and neutrophils
to increase the expression of MHC class I and class II molecules [
15
]. TNF-
α
activates
mononuclear neutrophils, and eosinophils, it acts on hepatocytes to induce the synthesis of
acute-phase proteins and constitutes a central mediator of responses of the host against
Gram-negative bacteria [16].
Similarly, IL-6 mediates natural immunity since it induces the synthesis of acute-phase
proteins in the liver and is a proliferation factor for activated B cells. Its levels increase in
patients with infection and are associated with mortality in patients with sepsis [
17
–
19
].
Likewise, elevated IL-6, IL-8, and TNF-
α
concentration are associated with septic shock
and multiple organ failure [
20
–
25
]. The measurement of IL-8 identifies serious infections in
patients with neutropenia, disseminated intravascular coagulation (DIC), lactic acidosis,
severe hypoxemia, and high risk of mortality [26,27].
The endothelium actively participates in the pathophysiology and damage associated
with sepsis, leading to systemic inflammation [
28
]. Interleukin 1 (IL-1) acts on the vascular
endothelium and mononuclear phagocytes, activating endothelial functions, and it can
lead to loss of anticoagulant properties and vaso permeability [
20
]. In perfusion mod-
els, elevated levels of Il-2 are associated with capillary leak syndrome where it activates
neutrophils adhered to endothelial cells leading to the production of oxygen radicals [
20
].
Similarly, intercellular adhesion molecule 1 (ICAM-1) promotes diapedesis and participates
in endothelial damage. High levels are present in patients with septic shock [28].
The deregulation of the host response and multiorgan failure due to infection is
attributed to the interaction of various mechanisms, where oxidative stress (OS) participates.
Mitochondrial dysfunction plays a vital role in the pathogenesis of septic shock [
29
].
Modulation of mitochondrial function could be a possible therapeutic strategy in the
management of sepsis in the future because mitochondrial dysfunction leads to MOF in
septic patients [30].
Some cytokines regulate inflammation, such as interleukin 10 (IL-10), which inhibits
the activity and production of TNF-
α
, IL-1, IL-2, and chemokines [
20
,
21
]. A change in
the ratio of IL-6 to IL-10 levels might be a marker to evaluate the systemic inflammatory
response [31].
Immune cells express pattern recognition receptors (PRRs) that rapidly initiate host
defense responses upon detecting tissue damage or microbial infection. Intracellular
damage-associated molecular proteins (DAMPs) mediate immune recognition of damaged
tissue. Toll-like receptors (TLRs), a subfamily of PRRs, have emerged as crucial receptors
for DAMP recognition and initiation of the inflammatory response. During sepsis, immune
Int. J. Mol. Sci. 2023,24, 16610 3 of 26
response activation occurs due to the release of high levels of DAMPs originating from
invading microorganisms and damaged host tissue, leading to overstimulation of immune
cells. This unbalanced response is known as a cytokine storm and transforms its function
from fighting excess infection into damage due to inflammation [32].
Furthermore, the stimuli produced by the infection lead to cell apoptosis through
the intracellular or extracellular pathway (intrinsic or extrinsic pathway). Intrinsically,
proapoptotic members of the Bcl family regulate the exit of cytochrome c from mitochondria,
such as Bid and Bax. Other Bcl members (Bcl-XL and Bcl-2) are anti-apoptotic and are
critical in signaling pathways leading to apoptosis or cell survival. Endoplasmic reticulum
stress is another mechanism of apoptosis triggered by intracellular stimuli and mediated
by caspase 12. In the extrinsic way, activation occurs due to binding a ligand, a polypeptide
of the TNF family, with its receptor. This leads to activating initiator caspases 2, 8, and 10.
Once activated, the caspases are cleaved and activate other effector caspases (3, 6, and 7),
which enhance the cleavage of cytoskeletal and nuclear proteins, leading to apoptosis cells.
The immune system’s responsiveness is impaired during sepsis, and increased apoptosis
of immune cells may explain the development of immune dysfunction. Therefore, it is
necessary to eliminate a more significant number of apoptotic cells. Despite critical efforts
in septic shock research, no therapy significantly modifies its outcome, and new treatments
have led to several experimental studies focusing on the apoptotic process [33].
Cytokines [
34
] and reactive oxygen species (ROS) [
35
] play a catastrophic role in
sepsis. Cell respiration, protein folding, or by-products of metabolism mainly produce
some reactive oxygen species (ROS); others are mainly generated by NADPH oxidase [
36
].
Also, leukocytes are attracted to the affected sites after infection and release cytokines and
ROS [37].
Recently, antagonistic therapies anticytokine therapy targeting (tumor necrosis factor
alpha [TNF-alpha], interleukin-1 [IL-1]) and anti-endotoxin strategies have been proposed
as important therapeutic targets in sepsis. They use antibodies against endotoxins or
endotoxin receptor/carrier molecules (anti-CD14 or anti-LPS binding proteins) [
38
]. These
therapies are based on the presence of an exponential increase in many cytokines in
inflammatory conditions where a cytokine storm is present. Recent studies demonstrate the
effectiveness and usefulness of an early start of comprehensive therapeutic management in
sepsis [39].
On the other hand, many experimental studies of
in vitro
models of sepsis using mouse
hepatocytes AML12 have been published. These cells were treated with lipopolysaccharide
(LPS) to induce sepsis by hepatocyte injury. In cells pretreated with melatonin, the effects
on oxidative stress, inflammation, mitophagy, mitochondrial biogenesis, and adenosine
triphosphate (ATP) levels were reduced, and the mitochondrial quality was improved. This
finding demonstrated that the antioxidant melatonin had potential benefic therapeutic
effects in sepsis induced in the liver by injury, promoting mitophagy and stimulating the
biogenesis of mitochondrial activity. These findings justify research to explore the precise
effects, the underlying mechanisms, and the effectiveness of melatonin in the clinical
setting [40].
In animal models of sepsis, there were significant anti-inflammatory, antioxidant,
and anti-apoptotic effects with the use of Sulforaphane, which inhibits the TLR4/NF-
κ
B
signaling pathway. There was a reduction in the cardiac damage caused by sepsis [
41
].
This finding supports the idea that the use of antioxidant therapy together with standard
therapy in sepsis might be beneficial.
During septic shock, the cytokine storm is caused by the invasion of pathogens and a
sustained high level of cytokines, which alter homeostasis and cause potentially fatal organ
dysfunction [42,43].
Hyperinflammation is a prooxidant state with increased expression of reactive oxygen
species (ROS). [Superoxide (O
2−
), Hydroxyl radical (OH), Hydroperoxyl radical (OOH),
Peroxyl radical (ROO)], RNS [Nitric oxide (NO), Nitrogen dioxide (NO
2
) radical] are
present in the circulating immune cells and in the affected organs, [
44
,
45
]. Elevated levels of
Int. J. Mol. Sci. 2023,24, 16610 4 of 26
intracellular superoxide are produced by NADPH oxidase, cyclooxygenase-2 (COX-2), and
xanthine oxidase in the mitochondria during sepsis. SOD neutralizes superoxides under
physiological conditions, but not during sepsis. In mouse models, the inhibition of NOX-2
prevents organ damage induced by sepsis. The inhibition of COX-2 also leads to a decrease
in peroxynitrite in the experimental model of sepsis [
46
–
48
]. These findings suggest the
importance of limiting excessive ROS generation with antioxidants rather than their effect
on hyperinflammation to prevent sepsis. The clinical value of this strategy could be better
if tested in the clinical environment.
Clinical trials to control only inflammation in septic shock have not had overwhelming
success for many years. However, new immunomodulatory therapies and cytokine blockers
have shown great success in severe cases of COVID-19 [
49
]. Standard management in
intensive care units solves many fundamental conditions associated with septic shock, such
as the cardiometabolic conditions and hemodynamic imbalances. Many studies in animal
and experimental models use antioxidant therapy in septic shock; however, few studies
demonstrate that the use of antioxidant therapy added to standard management in human
patients with septic shock reduces organ dysfunction, improves clinical conditions, and
reduces oxidative stress [
50
]. For this reason, our objective was to analyze whether adding
antioxidant therapy in a randomized fashion to the standard treatment applied to patients
with septic shock helps control cytokine levels and oxidant stress markers, and improves
organ dysfunction.
2. Results
2.1. Population Studied
A total of 131 patients were included in this study, of which 61 (47%) were male and
70 (53%) female. The median age was 68 (58–78) years. Table 1shows the demographic
characteristics of the patients according to their assigned antioxidant treatment.
Table 1. Demographic characteristics of patients in each group of treatment.
Vit C
n= 25
Vit E
n= 27
NAC
n= 24
MT.
n= 26
Control
n= 29 p
Age 62 (58–78) 70 (51–77) 68.5 (58.5–78) 62.5 (58–69) 75 (65–81) 0.10
Weight, kg 70 (65–80) 71 (60–82) 69.5 (56.5–80) 66 (60–78) 70 (61–80) 0.80
Height 1.7 (1.5–1.7) 1.7 (1.6–1.7) 1.7 (1.6–1.8) 1.7 (1.6–1.71) 1.7 (1.6–1.7) 0.39
Body mass index 24.9 (23–30.1) 24.6 (22.8–29) 23 (20.7–6.1) 25.35 (21–28) 25 (23.4–28.6) 0.41
Male/Female 10 (40)/15 (60) 17 (62.96)/10 (37.04) 14 (58.33)/10 (41.67) 13 (50)/13 (50) 16 (55.17)/13 (44.83) 0.53 §
SAPSII 39.44 ±14.10 45.70 ±16.37 42.41 ±19.84 40.88 ±16.80 47.20 ±7.11 0.60 ¥
APACHEII 14 (12–19) 20 (15–24) 15.5 (11–20.5) 16.5 (10–21) 17 (15–25) 0.15
SOFA 8 (6–9) 9 (7–11) 8 (4–10) 8 (6–9) 9 (7–11) 0.42
NUTRIC 4.16 ±2.21 4.81 ±1.64 4.08 ±1.83 3.88 ±1.79 5.10 ±1.54 0.41 ¥
Diabetes mellitus 7 (28.00) 5 (18.52) 5 (20.83) 6 (23.08) 8 (27.59) 0.90 *
Arterial hypertension 10 (40.00) 11 (40.74) 12 (50.00) 8 (30.77) 15 (51.72) 0.53 §
COPD 1 (4.00) 5 (18.52) 4 (16.67) 2 (7.69) 0 (0.00) 0.05 *
Smoking 17 (68.00) 12 (44.44) 9 (37.50) 15 (57.69) 14 (48.28) 0.22 §
Cancer 6 (24.00) 11 (40.74) 8 (33.33) 8 (30.77) 14 (48.28) 0.39 §
Cirrhosis 2 (8.00) 2 (7.41) 1 (4.17) 1 (3.85) 4 (13.79) 0.71 *
CKD 2 (8.00) 3 (11.11) 4 (16.67) 3 (11.54) 3 (10.34) 0.92 *
Hypothyroidism 4 (16.00) 4 (14.81) 2 (8.33) 6 (23.08) 7 (24.14) 0.56 *
CVD 3 (12.00) 0 (0.00) 1 (4.17) 2 (7.69) 3 (10.34) 0.41 *
Heart stroke 1 (4.00) 0 (0.00) 3 (12.50) 2 (7.69) 2 (6.90) 0.43 *
Atrial fibrillation 3 (12.00) 2 (7.41) 3 (12.50) 5 (19.23) 4 (13.79) 0.79 *
DVT 0 (0.00) 0 (0.00) 1 (4.17) 0 (0.00) 2 (6.90) 0.39 *
PE 0 (0.00) 0 (0.00) 2 (8.33) 1 (3.85) 2 (6.90) 0.42 *
Vit C: vitamin C; Vit E: vitamin E; NAC: N-acetylcysteine; MT: melatonin; TX: treatment; COPD: chronic obstructive
pulmonary disease; CKD: chronic kidney disease; CVD: cerebral vascular dis-ease; DVT: Deep venous thrombosis;
PE: Pulmonary embolism. Values are expressed as median (p25–p75); Statistical test: Kruskal–Wallis test,
¥ one-way ANOVA, § chi-squared test, and * Fisher’s exact test.
According to the different conditions that patients may present when entering an
Intensive Care Unit, such as the reason for admission, place of access, diagnosis, and loca-
tion of the initial infection, we compare these variables between the antioxidant treatment
groups to evaluate if they were homogeneous. We show no differences, which reveals the
homogeneity of these clinical conditions (Table 2).
Int. J. Mol. Sci. 2023,24, 16610 5 of 26
Table 2. Demographic characteristics of patients according to treatment group.
Characteristics Vit C
n= 25
Vit E
n= 27
NAC
n= 24
MT.
n= 26
Control
n= 29 p
Reason for admission, n(%)
Surgical 7 (28.00) 6 (22.22) 5 (20.83) 4 (15.38) 13 (44.83) 0.14
Non-surgical 18 (72.00) 21 (77.78) 19 (79.17) 22 (84.62) 16 (55.17)
Place of admission, n(%)
Emergency 14 (58.33) 15 (55.56) 13 (54.17) 18 (69.23) 14 (48.28)
0.76
Operating room 4 (16.67) 5 (18.52) 3 (12.50) 3 (11.54) 7 (24.14)
Hospitalization 4 (16.67) 7 (25.93) 8 (33.33) 5 (19.23) 7 (24.14)
Diagnoses at admission, n(%)
Cardiovascular 0 (0.00) 0 (0.00) 0 (0.00) 2 (7.69) 2 (6.90)
0.82
Respiratory 6 (25.00) 6 (22.22) 7 (29.17) 4 (15.38) 5 (17.24)
Gastrointestinal 9 (37.50) 5 (18.52) 4 (16.67) 5 (19.23) 6 (20.69)
Neurological 1 (4.17) 1 (3.70) 1 (4.17) 2 (7.69) 2 (6.90)
Sepsis 6 (25.00) 1 (3.70) 7 (29.17) 9 (34.62) 12 (41.38)
Trauma 0 (0.00) 1 (3.70) 1 (4.17) 0 (0.00) 0 (0.00)
Metabolic 1 (4.17) 1 (3.70) 0 (0.00) 0 (0.00) 1 (3.45)
Hematologic 0 (0.00) 2 (7.41) 0 (0.00) 1 (3.85) 1 (3.45)
Renal/Genitourinary 0 (0.00) 1 (3.70) 3 (12.50) 3 (11.54) 0 (0.00)
Site of infection, n(%)
Pulmonary 7 (29.17) 11 (40.74) 9 (39.13) 11 (42.31) 10 (34.48)
0.83
Gastrointestinal 10 (41.67) 8 (29.63) 5 (21.74) 5 (19.23) 11 (37.93)
Nefrourinary 3 (12.50) 3 (11.11) 6 (26.09) 6 (23.08) 3 (10.34)
CNS 0 (0.00) 2 (7.41) 0 (0.00) 0 (0.00) 1 (3.45)
Skin and soft tissues 2 (8.33) 2 (7.41) 2 (8.70) 2 (7.69) 2 (6.90)
Endocarditis 0 (0.00) 0 (0.00) 0 (0.00) 0 (0.00) 1 (3.45)
Gastrointestinal 0 (0.00) 1 (3.70) 0 (0.00) 2 (7.69) 1 (3.45)
Vit C: vitamin C; Vit E: vitamin E; NAC: N-acetylcysteine; MT: melatonin.
2.2. SOFA Score Assessment
The SOFA score was assessed in patients during the five days of antioxidant treatment
to identify the impact of the addition of antioxidant therapy on organ damage. Supple-
mentary Material S1 shows the reduction in the score. Statistical analysis using repeated
measures (which evaluates changes over time) showed that there was a statistically signifi-
cant reduction in the score in patients treated with Vit C from 8 to 3.5, (p= 0.001) between
day 0 and day 5. The reduction with NAC was from 7 to 4, (p= 0.003) and with melatonin
from 8 to 2 (p= 0.001). The patients who received Vit E and the control group also showed a
decrease in the score; however, the difference was not statistically significant, and the final
SOFA score remained high [50].
In patients treated with antioxidants, selenium levels were maintained at the same
level as at admission; however, in the control group, they decreased (Figure 1A–C).
Int. J. Mol. Sci. 2023,24, 16610 6 of 26
Int.J.Mol.Sci.2023,24,xFORPEERREVIEW6of28
melatoninfrom8to2(p=0.001).ThepatientswhoreceivedVitEandthecontrolgroup
alsoshowedadecreaseinthescore;however,thedifferencewasnotstatisticallysignifi-
cant,andthefinalSOFAscoreremainedhigh[50].
Inpatientstreatedwithantioxidants,seleniumlevelsweremaintainedatthesame
levelasatadmission;however,inthecontrolgroup,theydecreased(Figure1A–C).
Int.J.Mol.Sci.2023,24,xFORPEERREVIEW7of28
Figure 1. Cont.
Int. J. Mol. Sci. 2023,24, 16610 7 of 26
Int.J.Mol.Sci.2023,24,xFORPEERREVIEW8of28
Figure1.(A)Val ue sofoxidativestressmarkersbeforeandafterantioxidanttherapy.Groups:VitC
(n=25),VitE(n=27),NAC(n=24),MT(n=26),andcontrol(n=29).VitC:vitaminC;LPO:
lipoperoxidation(nmolMDA/mL);TAC:totalantioxidantcapacity(TroloxnM/mL);Thiol(µM/mL);
NitratesandNitrites(HNO3−/NO2−nmol/mLofserum).Carbonylation(ngcarbonyl/mL);(B)Enzy-
maticpathwaybeforeandafterantioxidanttherapy.Peroxida ses,(U/L)SOD:superoxidedismutase
(U/mgL);(C)Enzymaticpathwaybeforeandafterantioxidanttherapy.GPX:glutathioneperoxidase
(µmolofNADPH/min/mL);GSH:glutathione(µM/mLofserum);GST:(µM/mg/protein);GR:glu-
tathionereductase(U/min/mLThioredoxin(µM/mg/protein).
2.3.LipoperoxidationLevels:EnzymaticandNon‐EnzymaticAntioxidantPathways
Lipidperoxidationandoxidativestressmarkerswereincreasedatthebeginningof
thetreatmentinpatientswithsepticshock,andadecreasewasobservedinallpatients
whoreceivedantioxidanttherapy.However,onlyMTshowedastatisticallysignificant
change.NO3−andNO2−levelswereelevatedbeforetreatment,andtherewasastatistically
significantdecreaseinpatientstreatedwithVitC.Theantioxidantcapacityofthepatients
showedlowlevelsbeforetreatment,andthelevelsincreasedwiththeuseofVitE,NAC,
andMT;nevertheless,thedifferencewasnotstatisticallysignificant.
Regardingtheenzymaticantioxidantpathway,weevaluatedglutathioneandfound
increasedlevelsinpatientstreatedwithNACp=0.05.Glutathioneperoxidaseshowed
changeswiththeuseofMTp=0.02.SODsignificantlydecreasedwiththeuseofVitEand
MTp=0.004andp=0.001,respectively.Glutathionereductaseshowedelevatedlevels
Figure 1.
(
A
) Values of oxidative stress markers before and after antioxidant therapy. Groups: Vit
C (
n= 25
), Vit E (n= 27), NAC (n= 24), MT (n= 26), and control (n= 29). Vit C: vitamin C; LPO:
lipoperoxidation (nmol MDA/mL); TAC: total antioxidant capacity (TroloxnM/mL); Thiol (
µ
M/mL);
Nitrates and Nitrites (HNO
3−/
NO
2−
nmol/mL of serum). Carbonylation (ngcarbonyl/mL); (
B
) Enzy-
matic pathway before and after antioxidant therapy. Peroxidases, (U/L) SOD: superoxide dismutase
(U/mgL); (
C
) Enzymatic pathway before and after antioxidant therapy. GPX: glutathione peroxi-
dase (
µ
mol of NADPH/min/mL); GSH: glutathione (
µ
M/mL of serum); GST: (
µ
M/mg/protein);
GR: glutathione reductase (U/min/mL Thioredoxin (µM/mg/protein).
2.3. Lipoperoxidation Levels: Enzymatic and Non-Enzymatic Antioxidant Pathways
Lipid peroxidation and oxidative stress markers were increased at the beginning of
the treatment in patients with septic shock, and a decrease was observed in all patients
who received antioxidant therapy. However, only MT showed a statistically significant
change. NO
3−
and NO
2−
levels were elevated before treatment, and there was a statistically
significant decrease in patients treated with Vit C. The antioxidant capacity of the patients
showed low levels before treatment, and the levels increased with the use of Vit E, NAC,
and MT; nevertheless, the difference was not statistically significant.
Regarding the enzymatic antioxidant pathway, we evaluated glutathione and found
increased levels in patients treated with NAC p= 0.05. Glutathione peroxidase showed
changes with the use of MT p= 0.02. SOD significantly decreased with the use of Vit E and
MT p= 0.004 and p= 0.001, respectively. Glutathione reductase showed elevated levels
before treatment and were decreased with the use of Vit C p= 0.02. Thioredoxins did
not increase in the groups treated with antioxidants and the control group. All patients
who received antioxidants had increased peroxidases, and the differences were statistically
significant. The level decreased in the control group, but there was no statistical difference.
The levels of non-enzymatic antioxidants showed that there was an increase in thiols
in patients treated with Vit E. All patients had low levels of Vit C but the only group that
showed an increase was the one treated with Vit C p= 0.003.
2.4. Cytokine Quantification
IL-1
β
, IL-2, IL4, IL-6, IL-8, IL-10, IL-12p70, IL-17A, TNF-
α
, INF
γ
, IP-10, MCP-1, and
TGF
β
-1 were quantified using the ELISA method. Table 3shows the levels of proinflam-
matory and anti-inflammatory cytokines before and after treatment with the antioxidant
therapy. Paired-sample t-tests showed significant differences between the pretreatment and
posttreatment values. With the use of Vit C, IL-6 decreased (p= 0.006), while Vit E increased
IL-2 and IL-12 (p= 0.01 for both), as well as IFN
γ
(p= 0.03). NAC decreased TNF
α
(
p= 0.004
)
Int. J. Mol. Sci. 2023,24, 16610 8 of 26
and increased IL-12 (p= 0.04), while MT increased MCP-1 (p= 0.01) and decreased IL-6,
IL-8, and IL-4 (p= 0.001, p= 0.001, and p= 0.04, respectively). In the control group, there
was an increase in MCP-1 (p= 0.002) and a decrease in IL-6 and IL-8 (
p= 0.002
and p= 0.001,
respectively). We also found that all antioxidants increased TGF-
β
; Vit C before treatment
44.7 (8.8–414.2) after 46.9 (0–532.2) p= 0.80, MT
45.09 (8.8–626.3)
to
51.4 (8.8–359.6)
p= 0.53,
NAC 27.6 (8.8–411.6) to 52.9 (8.8–789.7) p= 0.08, control
45.1 (8.8–1033)
to 75.8 (8.8–356-9)
0.67 except Vit E 63 (8.8–452.8) to 52.9 (0–826.7). The changes are shown in Figure 2.
Table 3. Levels of cytokines in patients from the different treatment groups.
Proinflammatory Anti-Inflammatory
Before After Before After
Group IL-1βIL-1βpIL-4 IL-4 p
Vit C 9.8 (8.12–5700) 8.1 (6.4–178.79) 0.16 3.3 (0.39–328) 2.2 (0.39–65.2) 0.53
Vit E 8.1 (8.1–360) 8.1 (8.1–170.7) 0.66 5.35 (0.39–275.5) 13.7 (0.39–484.9) 0.21
NAC 10.8 (8.1–139.5) 8.1 (8.1–172.7) 0.27 4.05 (0.39–4262.7) 6.4 (0.39–844.4) 0.39
MT 8.1 (8.1–691) 8.1 (7.1–1050) 0.34 3.8 (0.39–184) 2.09 (0.39–117) 0.04
Control 8.1 (8.1–4110) 8.2 (8.1–1337) 0.28 3.7 (0.39–1307.7) 1.5 (0.39–1000) 0.52
IL-6 IL-6 IL-10 IL-10 p
Vit C 124.89 (22.35–546.49) 59.82 (14.93–148.1) 0.006 73.3 (2.97–1094.30) 66 (2.97–2762.5) 0.56
Vit E 106.345 (5.64–512.885) 16.965 (0.94–233.24) 0.07 106.3 (2.9–1322) 74 (2.9–362.9) 0.17
NAC 127.48 (31.27–997.595) 69.85 (3.805–231.895) 0.90 56.5 (4.6–1007.97) 54.7 (7.34–1370.7) 0.88
MT 344.87 (15.13–564.88) 23.25 (7.45–238.45) 0.001 70.5 (6.7–1815) 57.4 (4.1–5.6) 0.11
Control 236.24 (22.92–2197.01) 35.97 (6.91–112.95) 0.002 107 (2.97–1683) 55.1 (1.9–3960) 0.08
IL-8 IL-8 pTGF-βTGF-βp
Vit C 12.6 (1.45–83.6) 6.1 (1.45–46.34) 0.14 44.7 (8.8–414.2) 46.9 (0–535.2) 0.80
Vit E 19.05 (1.45–241) 8.6 (1.45–269) 0.16 63 (8.8–452.8) 52.9 (0–826.7) 0.41
NAC 14.8 (1.45–283) 9.5 (1.84–269.9) 0.73 27.6 (8.8–411.6) 52.9 (8.8–789.7) 0.08
MT 13.8 (1.45–227.17) 8.9 (1.45–367) 0.001 45.09 (8.8–616.3) 51.4 (8.8–359.06) 0.53
Control 21.9 (1.45–688.8) 7.6 (1.45–100) 0.001 45.1 (8.8–1033) 75.8 (8.8–356.9) 0.67
Proinflammatory Regulatory cytokines
TNF-αTNF-αIL-2 IL-2 p
Vit C 0.80 (0.41–99.6) 0.65 (0.41–18.9) 0.26 1.4 (0.45–100.4) 0.45 (0.45–10.6) 0.36
Vit E 0.74 (0.41–119) 0.8 (0.41–144.9) 0.85 0.56 (0.30–51.4) 1.13 (0.45–102.7) 0.01
NAC 1.06 (0.41–76.6) 0.1 (0.41–144.9) 0.04 0.58 (0.45–51.4) 0.66 (0.45–102.7) 0.15
MT 0.64 (0.41–20.4) 0.80 (0.41–49.6) 0.84 0.45 (0.45–8.9) 0.45 (0.45–18.4) 0.34
Control 0.41 (0.41–32.6) 0.54 (0.41–65) 0.79 0.51 (0.45–3.80) 0.45 (0.45–15.3) 0.54
IP-10 IP-10 pIL-12 IL-12 p
Vit C 73.3 (2.97–1094.3) 66.05 (2.97–2762.53) 0.57 1.2 (0.85–21.8) 1.06 (0.85–32.1) 0.12
Vit E 106.3 (2.97–1322.2) 74.5 (2.9–362.9) 0.17 1.49 (0.85–176.4) 1.45 (0.85–222.7) 0.01
NAC 56.5 (4.68–1007.9) 54.7 (7.34–1370.7) 0.88 1.49 (0.85–62.36) 1.4 (0.85–187.02) 0.04
MT 69.6 (6.7–1001.7) 57.4 (4.1–565.8) 0.11 1.1 (0.85–27.8) 0.92 (0.85–49.1) 0.11
Control 107.6 (2.9–1683.8) 55.1 (1.9–3960.96) 0.08 1.2 (0.85–46.7) 1.2 (0.85–59.4) 0.75
MCP-1 MCP-1 pIFN-γIFN-γp
Vit C 324 (15.1–4321) 216 (5.3–1641) 0.11 3.08 (3.05–31.40) 3.05 (3.05–23.01) 0.46
Vit E 189.1 (1.6–2700) 119 (1.6–2866) 0.45 3.15 (3.05–20.77) 3.05 (3.05–96.16) 0.03
NAC 257.6 (78.08–2954.7) 152.8 (23.5–2569.9) 0.44 3.49 (3.05–23.98) 3.93 (3.05–36.71) 0.15
MT 295 (23.2–3479) 210.7 (31.2–2504.4) 0.01 3.05 (3.05–18.18) 3.05 (3.05–14.09) 0.53
Control 316 (38–4110) 185. (11.03–1000) 0.002 3.2 (3.05–67.44) 3.05 (3.05–33.24) 0.21
Vit C: vitamin C. Vit E: vitamin E. NAC: N-acetylcysteine. MT: melatonin. MCP-1: chemotactic protein from
monocytes 1: range test signaled by Wilcoxon. Values are expressed in pg/mL.
Int. J. Mol. Sci. 2023,24, 16610 9 of 26
Int.J.Mol.Sci.2023,24,xFORPEERREVIEW10of28
VitC324(15.1–4321)216(5.3–1641)0.113.08(3.05–31.40)3.05(3.05–23.01)0.46
VitE189.1(1.6–2700)119(1.6–2866)0.453.15(3.05–20.77)3.05(3.05–96.16)0.03
NAC257.6(78.08–2954.7)152.8(23.5–2569.9)0.443.49(3.05–23.98)3.93(3.05–36.71)0.15
MT295(23.2–3479)210.7(31.2–2504.4)0.013.05(3.05–18.18)3.05(3.05–14.09)0.53
Control316(38–4110)185.(11.03–1000)0.0023.2(3.05–67.44)3.05(3.05–33.24)0.21
VitC:vitaminC.VitE:vitaminE.NAC:N-acetylcysteine.MT:melatonin.MCP-1:chemotacticpro-
teinfrommonocytes1:rangetestsignaledbyWilcoxon.Valuesareexpressedinpg/mL.
Figure2.Proinflammatoryinterleukinsshowdecreaseswithasignificantdeltawhenevaluatingthe
pretreatmentandposreatmentlevels.WithVitC,thereisadecreaseinIL-6(p=0.006);withVitE
thereisanincreaseinIL-2,IL-12,andIFNγ(p=0.01,p=0.01,andp=0.03,respectively);withMT,
thereisanincreaseinMCP-1andadecreaseinIL-6,IL-8,andIL-4(p=0.001,p=0.001,p=0.01,and
p=0.04,respectively);andwithNAC,thereisadecreaseinTNFα(p=0.04)andanincreaseinIL-12
(p=0.04).IndividualswithoutantioxidanttherapyhaddecreasedlevelsofIL-6(p=0.002),IL-8(p=
0.001),andMCP-1(p=0.002).
2.5.CanonicalCorrelation
Acanonicalcorrelationanalysiswasconducted.Itincludedclinicalvariablessuchas
SOFAscore,PCTPCRlaboratory,andoxidantstressbiomarkers,andconsideredexplan-
atoryvariables(u1,u2,u3)andeffectvariables(v1,v2,v3).
Thefirstevaluationofthecanonicalcorrelationcarriedoutwaswithlaboratorypa-
rametersandbiomarkers.Weincludedtheexplanatoryvariablesandthoseofpossible
Figure 2.
Proinflammatory interleukins show decreases with a significant delta when evaluating the
pretreatment and posttreatment levels. With Vit C, there is a decrease in IL-6 (p= 0.006); with Vit
E there is an increase in IL-2, IL-12, and IFN
γ
(p= 0.01, p= 0.01, and p= 0.03, respectively); with
MT, there is an increase in MCP-1 and a decrease in IL-6, IL-8, and IL-4 (p= 0.001, p= 0.001, p= 0.01,
and
p= 0.04
, respectively); and with NAC, there is a decrease in TNF
α
(p= 0.04) and an increase in
IL-12 (p= 0.04). Individuals without antioxidant therapy had decreased levels of IL-6 (p= 0.002), IL-8
(p= 0.001), and MCP-1 (p= 0.002).
2.5. Canonical Correlation
A canonical correlation analysis was conducted. It included clinical variables such as
SOFA score, PCT PCR laboratory, and oxidant stress biomarkers, and considered explana-
tory variables (u1, u2, u3) and effect variables (v1, v2, v3).
The first evaluation of the canonical correlation carried out was with laboratory
parameters and biomarkers. We included the explanatory variables and those of possible
effect (cytocines). We found that in patients with septic shock, there is a high correlation of
0.95 when there are high levels of procalcitonin and carbonylation and low levels of total
antioxidant capacity (TAC), glutathione, and vitamin C. This correlates with low levels of
IL4 and elevated levels of IP10, IL1B, MCP1, IL-6, IL-17, and TNFα.
In the second evaluation of the canonical correlation, we found a correlation of 0.86
when there are high levels of LPO, carbonylation, and low levels of GSH, selenium, and
thiols; this correlates with low levels of IL-4, il12p70, and IFNy and high levels of IL1B,
Int. J. Mol. Sci. 2023,24, 16610 10 of 26
mcp1, and IL-6. There was a high correlation of 0.82 (although lower than the two previous
correlations) which shows us that high levels of the SOFA score, CRP, and CRP, and low
levels of GSH and GSH peroxidase correlate with low levels of IL-4, TGFB1, and Il12p70
and high levels of IL1B and IL8 (Table 4).
Table 4. Canonical correlation analysis between cases and controls with septic shock pretreatment.
Variable Level Variable Coef. Std. Err. t p-Value IC95% Corr.
u1
High PCT 0.0172182 0.0042318 4.07 0.0001 0.0085873 0.0258491
0.9529
Carbonyl −0.0068545 0.0030781 −2.23 0.033 −0.0131324 −0.0005766
Low
TAC −0.0002907 0.0000967 −3.01 0.005 −0.000488 −0.0000935
GSH 5.295229 1.522535 3.48 0.002 2.189998 8.400459
Vit C 6.144225 0.6809717 9.02 0.001 4.755374 7.533075
v1
Low IL4 −0.0073968 0.0025368 −2.92 0.007 −0.0125706 −0.002223
High
IP10 0.0009865 0.0001994 4.95 0.001 0.0005798 0.0013932
IL1B 0.0002558 0.000123 2.08 0.046 5.01 ×10−60.0005067
MCP 1 −0.0006084 0.0001506 −4.04 0.001 −0.0009155 −0.0003012
IL6 0.0003399 0.0000436 7.8 0.001 0.0002511 0.0004288
ILl17a −0.0098983 0.0025994 −3.81 0.001 −0.0151998 −0.0045967
TNFα0.049159 0.0144382 3.4 0.002 0.0197122 0.0786059
u2
High LPO −0.1764843 0.0744333 −2.37 0.024 −0.328292 −0.0246766 0.8643
Carbonyl 0.0164766 0.0056307 2.93 0.006 0.0049928 0.0279604
Low
GSH −11.67688 2.785091 −4.19 0.001 −17.35711 −5.996647
Selenium 492.1174 139.0012 3.54 0.001 208.6226 775.6122
Thiol −0.1522415 0.0385513 −3.95 0.001 −0.2308675 −0.0736155
v2
Low
il4 0.0124842 0.0046404 2.69 0.011 0.00302 0.0219484
IL12p70 0.0367106 0.0155704 2.36 0.025 0.0049544 0.0684667
IFNy −0.0140323 0.0035662 −3.93 0.001 −0.0213055 −0.006759
TGFB1 0.0031482 0.00154 2.04 0.049 7.40 ×10−60.006289
High IL1B −0.0007156 0.000225 −3.18 0.003 −0.0011744 −0.0002568
MCP1 0.0013014 0.0002755 4.72 0.001 0.0007396 0.0018632
IL6a 0.0002323 0.0000797 2.92 0.007 0.0000698 0.0003947
u3
High SOFA −0.3846204 0.1060993 −3.63 0.001 −0.6010113 −0.1682295 0.8218
PCT −0.0219292 0.009223 −2.38 0.024 −0.0407397 −0.0031188
PCR 0.0441247 0.0167844 2.63 0.013 0.0098926 0.0783568
Low GSH 6.547262 3.318249 1.97 0.057 −0.2203513 13.31488
GSHpx −6.29239 1.747836 −3.6 0.001 −9.857125 −2.727655
v3 Low
IL4 0.0070789 0.0055288 1.28 0.21 −0.004197 0.0183549
TGFB1 0.0038936 0.0018348 2.12 0.042 0.0001515 0.0076356
IL12p70 −0.0400625 0.0185511 −2.16 0.039 −0.0778978 −0.0022272
High IL1B −0.0007372 0.000268 −2.75 0.01 −0.0012838 −0.0001905
IL8 0.0300677 0.0085392 3.52 0.001 0.0126518 0.0474836
The canonical correlation revealed a significantly high correlation between the first (0.9529), the second (0.8643),
and third (0.8218). Lambda test with an F = 0.001; the Lawley–Hotteling trace and Loy’s largest root yielded the
same statistical significance.
2.6. Protein–Protein Interaction Network
To present the participation of different mechanisms of damage in sepsis, we show
the protein–protein interactions of the cytokines evaluated in this work; a diagram was
created based on the Kyoto Encyclopedia of Genes and Genomes (KEGG), as well as the
different signaling pathways that could be detected (Supplementary Material S2). The
quantified interleukins might be involved in the presence of viral or bacterial infections that
can lead to the development of septic shock. The interaction network was generated using
the ShinyGO 0.76 server [
51
]. Also, we show in Figure 3that in sepsis, the intrinsic and
extrinsic pathways participate in the different mechanisms involved in damage to tissues
and organs. And, we also explain our work on a hypothesis of inflammatory damage and
oxidative stress through a graphical abstract.
Int. J. Mol. Sci. 2023,24, 16610 11 of 26
Int.J.Mol.Sci.2023,24,xFORPEERREVIEW12of28
2.6.Protein–ProteinInteractionNetwork
Topresenttheparticipationofdifferentmechanismsofdamageinsepsis,weshow
theprotein–proteininteractionsofthecytokinesevaluatedinthiswork;adiagramwas
createdbasedontheKyotoEncyclopediaofGenesandGenomes(KEGG),aswellasthe
differentsignalingpathwaysthatcouldbedetected(SupplementaryMaterialS2).The
quantifiedinterleukinsmightbeinvolvedinthepresenceofviralorbacterialinfections
thatcanleadtothedevelopmentofsepticshock.Theinteractionnetworkwasgenerated
usingtheShinyGO0.76server[51].Also,weshowinFigure3thatinsepsis,theintrinsic
andextrinsicpathwaysparticipateinthedifferentmechanismsinvolvedindamageto
tissuesandorgans.And,wealsoexplainourworkonahypothesisofinflammatorydam-
ageandoxidativestressthroughagraphicalabstract.
Figure3.Intrinsicandextrinsicpathwayforinductionofapoptosis.Intrinsicandextrinsicpathway
forinductionofapoptosis.(1)TheextrinsicpathwayofapoptosiscanbeactivatedbyTNF-depen-
dentApo2L/TRAILreceptors.(2)Insomeextionalcases,Apo2L/TRAILcanbeintrinsicallypathway
activatedbyBid.(3)TheintrinsicpathwaycanbeactivatedbyDNAormicrotubuledamage,invol-
vingtheinteractionofsomeproteinssuchasBAXandBAK.(4)Apoptosiscanalsobepromotedby
activationofp53.Abbreviations:Apaf-1=apoptoticprotease-activatingfactor1,Apo2L/Trail=Re-
combinanthumanapoptosisligand2/inducingtumornecrosisfactor-relatedapoptosisligand2,
FADD=thefas-associateddeathdomainprotein,GR=glutathionereductase,GSH=glutathione,
GSSG=oxidizedglutathione,H2O2=hydrogenperoxide,IAP=apoptosisinhibitor,NF-KB=kappa
nuclearfactor,O2−=superoxideanion,TNF=tumornecrosisfactor.
3.Discussion
Thecontroloftheactivationofpathophysiologicalmechanismssuchasoxidative
stress,andinnateandadaptiveinflammatoryresponseinsepticshockisvital[52],Dete-
riorationandorgandamageresultfromderegulationofphysiologicalfunctionsthatlead
toproteolysis[53,54]activationofthecomplementsystem,thecoagulationpathway,the
fibrinolyticsystem,lipidpathways,oxidativestress,andcytokineproduction[43].
Figure 3.
Intrinsic and extrinsic pathway for induction of apoptosis. Intrinsic and extrinsic path-
way for induction of apoptosis. (1) The extrinsic pathway of apoptosis can be activated by TNF-
dependent Apo2L/TRAIL receptors. (2) In some extional cases, Apo2L/TRAIL can be intrinsically
pathway activated by Bid. (3) The intrinsic pathway can be activated by DNA or microtubule
damage, involving the interaction of some proteins such as BAX and BAK. (4) Apoptosis can also
be promoted by activation of p53. Abbreviations: Apaf-1 = apoptotic protease-activating factor
1,
Apo2L/Trail = Recombinant
human apoptosis ligand 2/inducing tumor necrosis factor-related
apoptosis ligand 2, FADD = the fas-associated death domain protein, GR = glutathione reductase,
GSH = glutathione, GSSG = oxidized glutathione, H
2
O
2
= hydrogen peroxide, IAP = apoptosis
inhibitor, NF-KB = kappa nuclear factor, O2−= superoxide anion, TNF = tumor necrosis factor.
3. Discussion
The control of the activation of pathophysiological mechanisms such as oxidative
stress, and innate and adaptive inflammatory response in septic shock is vital [
52
], Deteri-
oration and organ damage result from deregulation of physiological functions that lead
to proteolysis [
53
,
54
] activation of the complement system, the coagulation pathway, the
fibrinolytic system, lipid pathways, oxidative stress, and cytokine production [43].
On the other hand, the participation of pathogen-derived molecular patterns (PAMPs)
and damage-associated molecular patterns (DAMPs), recognized by specific receptors on
the cell surface, initiate the transcription cascade of inflammatory molecules due to the
translocation of the nuclear factor NF-kβin activated cells.
Imbalance requires early therapeutic management [
55
–
57
] since the cytokine storm
leads to a potentially fatal result [42,57].
Cytokines activate and inhibit cellular functions by negatively regulating effector
mechanisms and restoring homeostasis through the release of cytokines IL-10 and trans-
forming growth factor-
β
. They also regulate protein deposition in the extracellular matrix
and angiogenesis [58].
The effects of the use of antioxidant therapy in patients with septic shock and its effect
on cytokines were among the objectives of this study since we have previously reported
reduced biomarkers of oxidant stress with the use of this therapy. Furthermore, there was
Int. J. Mol. Sci. 2023,24, 16610 12 of 26
better disease control in patients with COVID-19. Although the results in previous works
are good, experimental and clinical studies are still required to confirm this hypothesis [
59
].
In this sense, cytokines involved in innate immunity are attractive targets for thera-
peutic intervention [
60
]. They are essential in diseases whose pathogenesis is the result of a
defective regulation of the cytokine network; therefore, cytokine-targeted therapies with
monoclonal antibodies, soluble receptors, or small molecule inhibitors promise new possi-
bilities for the treatment of patients resistant to standard drug regimens. The proposal to
add antioxidants to standard therapy in septic shock is based on the reduction in oxidative
stress and the increase in antioxidant capacity, and our group was one of the first to carry
out studies in humans employing this therapy [50,60–62].
In this study, we support the hypothesis raised by us and other researchers that the
function of cytokines changes with the use of antioxidant therapy. We found an increase in
the level of IL-2 after treatment with Vit E, NAC, and MT. Only Vit E showed a statistically
significant difference. This finding coincides with a previous one in children undergoing
dialysis treatment, where they combined NAC with Vit E and demonstrated regulation of
the cellular redox state and modulation of the cytokine profile. That study also found a
reduction in organ damage at the level of the kidney in patients with septic shock [
63
]. IL-2
stimulates the proliferation of NK cells, increases their cytolytic function, and acts on B cells
as a proliferation factor. Its increase is relevant since it acts as a stimulus for synthesizing
antibodies and has a regulatory function.
The combined use of Vit E and NAC to stimulate the production of IL4 had a regulatory
effect on oxidative stress, inflammation, and organ failure in experimental studies with
an animal model [
64
]. In patients with septic shock, we found that, in addition to the
attenuation of oxidative stress, there was an improvement in organic damage, and IL-4
levels increased in patients who received vitamin E. However, the delta did not reach
statistical significance (p= 0.06).
NAC increased IL-10, which has regulatory properties. The increase in IL-10 levels
due to the use of antioxidants is an important finding since there is multiorgan dysfunction
that involves uncontrolled production of reactive oxygen species (ROS) and a cytokine
storm in septic shock. At the same time, exhaustion of antioxidants that contribute to
the progression to septic shock occurs. Macrophages secrete proinflammatory and anti-
inflammatory mediators during the infectious process. The binding of lipopolysaccharide
(LPS) to Toll-like receptor 4 (TLR4) releases TNF-
α
, which initiates proinflammatory events
through tumor necrosis factor receptor 1 (TNFR1) signaling, which is an effect of IL-10 [
65
].
In humans with septic shock, the effect achieved with MT may be related to improving
IL-10 levels.
IL-12 has a regulatory function and there was no association between its levels and
severity of the disease in a study of children with septic shock [
66
]. In another study, IL12
levels were not modified in patients in a septic state after surgery [
67
]. The increase or
decrease in IL-12 levels during septic shock could be measured longitudinally to determine
its relevance in this condition. Unlike other studies, we found that Il-12 levels increased with
Vit E and NAC therapy p= 0.001. This finding is relevant because this cytokine promotes
cellular immunity by inducing the maturation of T cells into Th1 cells and stimulating the
secretion of gamma interferon (IFN-
γ
), which activates natural killer cells, macrophages,
and cytotoxic T cells. Therefore, its deficiency leads to deterioration of immune functions,
favoring the spread of the infection.
The increase in IL-12 found in this study has particular importance given that the most
important function of IL-12 is the induction of gamma interferon (IFN), which is a mediator
in viral, fungal, bacterial, and parasitic resistance. The induction of IL-12 triggered by
microbial products is a potent stimulus for IFNy, which leads to the quick control of the
infection by these agents. However, if the response is not controlled, the synthesis of IL-12
can result in an excessive activation of the immune system which causes damage to the
host tissue and even death. This occurs in autoimmune diseases and/or septic shock.
Int. J. Mol. Sci. 2023,24, 16610 13 of 26
The Induction of IL-12 during acute infection is crucial to initiate the synthesis of INFy
by NK cells and T lymphocytes, and mediators of resistance against viruses, bacteria, fungi,
and parasites. In several animal models, IL-12 has been used as immunotherapy to protect
cellular immunity and to control or reject inappropriate responses to pathogenic infections
in organisms with immune disorders. In any case, increasing its concentration can be
beneficial. However, the excess IL-12 response appears to be responsible for some problems
observed during severe microbial infections. Under these conditions, IL-12 antagonists
have attenuated immunopathology and prevented death. The toxic effects of the proposed
therapy with IL-12 were observed in mice, and therefore its use as therapy in humans
is controversial. A study with different doses is still necessary, or its combined use with
antimicrobials with other molecules or with antioxidants [68].
In this study, IL-6 showed elevated levels before antioxidant therapy. Levels decreased
in patients treated with Vit C, MT, and NAC. However, this effect was similar in the control
group. This finding coincides with that from a systematic cohort study of patients with
sepsis, in which the therapy was only with antibiotics and considered Il6 as a possible
predictor of response to this therapy. The study also documented the substantial interindi-
vidual variability in the induction of IL-6 during sepsis since it depends on the type of
infection, type of bacteria, and origin of the organ involved. All patients in our study
received antibiotics. Therefore, the change and difference observed with antioxidants based
on this cytokine will require an appropriate methodological strategy to discern whether the
antioxidant effect creates synergy with the impact or is only related to the antibiotic [69].
Another regulatory cytokine involved in controlling the infectious process is TGF-
β
since it inhibits viral replication and cell proliferation. We found that all our patients
had low levels of TGF-
β
before treatment, and levels increased in the group treated with
antioxidants and in the control group. However, none showed a significant statistical
difference; only the group treated with NAC had a clear trend of p= 0.08. Other studies
have reported that its increase requires nutritional products such as sesame seeds and that
it has synergistic properties with antioxidants such as vitamin E [70].
There is evidence for the complex pathogenesis of septic shock and LPS-induced
re-actions. In recent years, mediators such as cytokines have aroused great interest for their
role in inflammatory responses induced by LPS, TNFα, and IL-1 [71–74].
The relevance of TGFB1 in septic shock has been studied in mouse macrophages, and
it inhibits JNK activity stimulated by lipopolysaccharide (LPS). It might have a possible
inhibitory mechanism on TGF-beta in the signaling of inflammatory responses induced by
LPS [
75
]. We found increased TGF-beta levels using all the antioxidants studied (Vit C, Vit
E, NAC). In the control group, the levels decreased; it is precisely in this group of patients
where SOFA remained elevated.
Several studies have demonstrated the involvement of proinflammatory cytokines
in septic shock, and patients receiving standard treatment improve in intensive care
units [76,77]
; However, this response to treatment depends on the number of organs
affected since admission [
78
] or if it was associated with surgical intervention, site of
infection [
79
,
80
], burn [
81
,
82
], endocarditis [
83
,
84
], necrotizing fasciitis [
85
,
86
], meningi-
tis [
87
,
88
], and septic arthritis [
89
–
91
]. The active participation of cytokines correlates with
severity and prognosis [76].
High amounts of IL-1 and TNF-
α
are released during systemic inflammation, resulting
in hypotension and shock. However, IL-1 is frequently undetectable and, when present,
has little predictive value [
92
]. This study confirms this observation since the cytokines’
IL-1
β
and TNF
α
levels did not show relevant changes after treatment. In this regard, we
consider that with our findings, we show that there are favorable changes in some cytokines
with standard comprehensive management and antioxidant therapy. Comprehensive
management probably requires exploring the synergistic therapeutic link of these pathways
since there is information that with the use of some specific biologicals, the beneficial effect
has not been fully proven in humans with infectious processes. During the COVID-19
Int. J. Mol. Sci. 2023,24, 16610 14 of 26
pandemic, the use of anakinra, an IL-1 antagonist, or IL-6 antagonists were not wholly
conclusive [93].
On the other hand, results using antioxidants at the experimental level have shown that
some have regulatory capacity on oxidative stress. MT has radical scavenging properties
and protects cell membrane lipids, cytosol proteins, nuclear proteins, and mitochondrial
DNA. In our research, it reduced LPO, similar to Galley’s findings [
94
]. Also, the beneficial
effect of MT has been demonstrated more widely in experimental cells, plants, and animals,
although its mechanism of action remains unknown. The effects of MT could be related
to its detoxifying capacity, thus protecting molecules from the destructive effects of OS in
various conditions, such as ischemia/reperfusion (cerebrovascular accident and myocardial
infarction), ionizing radiation, and drug toxicity. In sepsis, the protective effects of MT
are associated with the inhibition of apoptosis and the reduction in OS [
95
]. MT has
been shown to increase total antioxidant capacity (TAC) in experimental studies, equally
with the associated use of Vit E and NAC [
95
–
97
]. On the other hand, the use of MT
decreased the SOFA score, confirming the same effect found in multiorgan lesions induced
by sepsis [98,99].
After release, plasma melatonin is rapidly distributed and transported to the mi-
tochondria, acting as an antioxidant. In mammals, MT is an agonist of MT1 and MT2
receptors [
100
,
101
], and has pharmacological and physiological anti-inflammatory effects
through receptor-dependent and non-receptor-dependent pathways [
102
]. It reduces levels
of lipid peroxidation [
103
]. Also, we found an increase in GR activity in patients before
treatment, which may lead to a decrease in GSH concentrations and an increase in ROS. GR
activity decreased in patients treated with vitamin C, and GST activity remained unchanged
in patients treated with Vit C and other antioxidants, but in the control they decreased.
These findings on the activity of these enzymes with antioxidant treatment suggest that
they could contribute to the reduced levels of LPO, and the SOFA score that evaluates MOF.
GPx uses selenoprotein P as a cofactor and decreases during septic shock [
104
]. We
found low levels of Se in patients with septic shock and MOF. Although Se levels did not
increase with the administration of antioxidant therapy, they were maintained, contrasting
with the decreased levels found in untreated patients, who also showed no improvement in
the SOFA score. The exact mechanism is not yet understood; however, our results suggest
that the antioxidant therapy can maintain Se levels in septic shock patients, leading to
improved GPx activity.
One of the inducers of sepsis is endotoxin or lipopolysaccharide (LPS), which comes
from Gram-negative bacteria and leads to sepsis and multiorgan dysfunction [
105
]. LPS
increases heart rate and decreases systemic vascular resistance, leading to hypotension.
LPS also induces the synthesis of cytokines and elevates neutrophils, free radicals, and
proteolytic enzymes that lead to endothelial dysfunction [106].
The glutathione peroxidase enzyme metabolizes intermediates formed by aerobic
metabolism, such as hydrogen peroxide; this reaction occurs in the cytosol and mitochon-
dria, resulting in oxidized glutathione, which is reduced by glutathione reductase. GR
employs NADPH as a cofactor, helping to recycle an oxidation–reduction [107,108].
Glutathione is a tripeptide composed of three amino acids: glutamic acid, glycine, and
cysteine. It plays a vital role in septic shock since it protects against damage caused by
free radicals. Like glutathione, Vit E is a fat-soluble antioxidant [
109
]. It reacts with free
radicals generated in the lipid phase and protects the lipids in the membranes. Vit C has
radical scavenging activity, stabilizes membrane structures, oxidizes vitamin E, and has a
synergistic mechanism [110].
In this study, Vit C and Vit E decreased LPO. Vit E and MT increased the total antiox-
idant capacity. NAC, a direct precursor of glutathione, increased glutathione levels and
improved multiorgan failure (MOF), in accordance with previous research [111].
The effect of various antioxidants, such as NAC, MT, vitamins A, C, and E, enzyme
cofactors (selenium and zinc), endogenous compounds (ubiquinone,
α
-lipoic acid, bilirubin,
albumin, ferritin), and quercetin, can inhibit reactive nitrogen, oxygen species (ROS), and
Int. J. Mol. Sci. 2023,24, 16610 15 of 26
RNS [
61
]. The effect NAC has on improving antioxidant capacity is due to the replenish-
ment of glutathione (GSH) and the sequestration of ROS. It also improves hemodynamic
variables, cardiac index, oxygenation, and static lung compliance [
62
], and reduces the
levels of IL-6 and ICAM-1 [60].
Innate cytokines are attractive targets for therapeutic intervention [
63
]. They are es-
sential in diseases whose pathogenesis is the result of a defective regulation of the cytokine
network; therefore, cytokine-targeted therapies with monoclonal antibodies, soluble recep-
tors, or small molecule inhibitors promise new possibilities for the treatment of patients
resistant to standard drug regimens [64,65]. The proposal to add antioxidants to standard
therapy in septic shock has been previously proposed for its benefits in terms of reduction
in oxidative stress and an increase in antioxidant capacity. Our group was one of the first
to propose this [50,61].
In septic shock, interleukins are the first to increase, reaching a maximum peak at
2 h, when procalcitonin increases, and IL-6 and IL-8 levels decrease [
112
]. This aspect
limits the interleukins’ determination during clinical practice; therefore, having little use
in emergencies [
112
]. Moreover, as the deterioration of the organ progresses, determining
cytokines, considering their lifespan, could add more information to clinical treatment.
C-reactive protein (CRP) and procalcitonin (PCT) could be of help in the guidance of
management. Determining oxidative stress markers is not routinely feasible today; however,
this study and the experimental ones that show the participation of oxidative stress in the
progression of damage in patients with septic shock allow us to suggest using antioxidant
therapy associated with standard management.
In sepsis, organ dysfunction is potentially fatal, and it is caused by deregulation of
the host response to infection. The pathological process is complex, and several cytokines
participate in the damage mechanism, which, when dysfunctional, lead to an intertwined
storm whose outcome worsens function and causes structural damage at the cellular,
tissue, and organ level. The uncontrolled inflammation cascade requires regulation of both
the cytokines involved and other intertwined mechanisms, such as the deregulation of
oxidative stress, which requires simultaneous treatment and modulation to repair tissues
and improve organ function.
Standard therapy in sepsis includes hydroelectrolytic hemodynamic management
and antibiotic therapy that essentially control the initial inflammatory pathophysiological
damage. However, deregulation of oxidative stress must also participate and is necessary
to improve organ dysfunction and interact in the modulation of some cytokines.
Although there are limitations with using oxidative stress markers in emergency
conditions, in this research we show that there is deregulation of oxidative stress in septic
shock, which is related to cytokine storm, and that the complex interaction produced by
treatment with antioxidants can improve organic dysfunction.
A limitation of this study is that the sample size would have to be larger to confirm
the statistical power for all cytokines.
4. Methods and Materials
This study was a prospective, longitudinal, aleatorized, and blind clinical trial with a
cohort of patients who were treated between April 2018 and January 2022. This dataset
corresponds to the same population used in another study of our group and the study was
completed with a new study of cytokines, which were not previously analyzed [50].
4.1. Study Population
Patients of any gender, over 18 years of age who were admitted to the intensive care
unit of the ABC Observatory Medical Center and Santa Fe campus with a diagnosis of septic
shock, according to the Third International Consensus on Sepsis and Septic Shock, were
included [
73
]. The characterization of septic shock included refractory hypotension requir-
ing the use of vasopressors despite resuscitation with sufficient fluids (20 mL/kg colloid
or 40 mL/kg crystalloid) to maintain blood pressure
≥
65 mmHg and
lactate > 2 mmol/L
.
Int. J. Mol. Sci. 2023,24, 16610 16 of 26
Patients were required to give a signed consent or an informed assent of the patient or re-
sponsible caregiver. Patients with a history of having signed an advance directive, chronic
use of steroids and/or statins in the last six months, or with recent use of antioxidant
treatment before the moment of septic shock, and who had any contraindication for the
use of Vit C, Vit E, NAC, or MT, as well as pregnant women, were excluded and patients
who, having already been included, withdrew their informed consent were removed from
the study.
An advance directive is a legal instrument through which a competent person deter-
mines in writing their will regarding the treatment that they would and would not like to
receive in a situation where they cannot express their will.
In standard therapy, patients receive various therapies; in general, they receive an-
tibiotics, blood products, sedation and analgesia, steroids, and intravenous solutions.
Hemodynamic support is monitored, and goals related to lactate control, glycemic control,
cultures, mechanical ventilator withdrawal test, stress ulcer prophylaxis, and nutritional
care are performed [72].
4.2. Sample Size
The sample size was calculated considering the mean difference between low levels
of ascorbic acid and improvement in treatment with antioxidants. The test suggested the
inclusion of 11 patients in each group for a desired power of 80% and an alpha error of
<0.05 based on a previous study [
74
]. To achieve greater statistical power, the sample size
was increased to >24 patients per group, and a power of 97% was reached with an alpha
error of 0.03.
4.3. Randomization
Computerized electronic selection was used to divide the patients into blocks; Ran-
domization was 1:1 in balanced blocks. There were 6 blocks, with an approximate number
of 25 patients per block. Personnel who were not involved in the study participated in
the blinding and placed the indicated therapy in identical opaque envelopes numbered
from
1 to 125
, and these were applied consecutively. Group 1 received Vit C, Group 2
received Vit E, Group 3 received NAC, Group 4 received MT, and Group 5 was the control
group with antioxidant treatment. Of 3745 patients admitted to the ICU during this study,
only 131 had septic shock. Once the diagnosis of septic shock was established (inclusion
criterion), we applied treatment with antioxidants to patients according to randomization.
We show the number of patients who entered each group and the type of treatment they
received. Additionally, we provide the conditions that occurred during follow-up (inter-
vention discontinuation, adverse effects, death) in each group and show the number of
patients finally included in the analysis (Supplementary Material S3).
4.4. Masking and Drug Administration
The random assignment sequence for the administration of antioxidants was gen-
erated at the coordination center using a computer-generated randomization program.
The pharmacy maintained the blinding. Blinding was also carried out from the study’s
beginning until the results’ analysis. A pharmaceutical professional maintains surveil-
lance and care of the form of application of each of the antioxidants according to the type
of absorption and interactions with other medications and the most appropriate way to
administer them under the conditions that each patient requires.
All antioxidants were administered orally or through a nasogastric tube for 5 days in
addition to standard therapy. NAC was administered in 1200 mg tablets every 12 h. In
addition, patients were given 50 mg of MT in 5 mg tablets once a day and vitamin C in 1 g ef-
fervescent tablets every 6 h. Vit E capsules of 400 IU were administered every 8 h. The prepa-
ration and administration of these drugs is presented in
Supplementary Material S4
. The
doses of antioxidants were chosen according to what is reported in the
literature [113–116]
.
Int. J. Mol. Sci. 2023,24, 16610 17 of 26
The pharmacotherapeutic follow-up of the antioxidants used in the clinical trial
had strict surveillance of the administration of medications by the CMABC Pharmacy
Department to establish that antioxidants were administered as much as possible un-
der the same conditions, both in the preparation and preparation administration hours
(Supplementary Material S4).
4.5. Data Collection Method
Upon admission to the intensive care unit, a complete medical history and physical
examination of the patients were performed to obtain the demographic data of the patients.
APACHE II and SAPS II prognostic scores were calculated upon admission to the intensive
care unit, and the SOFA score for organ dysfunction (neurological, respiratory, hemody-
namic, hepatic, and hematological) was performed. Laboratory measurements were taken
upon admission to the ICU and for every day of hospital stay, which included complete
blood count, blood chemistry, serum electrolytes (sodium, potassium, chloride, calcium,
and magnesium), liver function tests, C-reactive protein, procalcitonin, and venous and
arterial blood gases.
The SOFA score was evaluated using 6 important systems, including respiration
(PaO
2
/FiO
2
), coagulation (platelet count), liver (bilirubin), cardiovascular (mean arterial
pressure), central nervous system (Glasgow Coma Scale, GCS), and renal system (creatinine
and/or urine output) [
117
]. A detailed definition of the SOFA criteria including the relevant
thresholds is shown in Supplementary Material S5.
4.6. Standard Therapy in the ICU
The patients were treated according to the recommendation of the International Guide-
lines for the Management of Sepsis and Septic Shock. This management includes fluid
replacement, hemodynamic support, lactate goals, control of the infectious focus, antibi-
otic cultures, use of blood products, sedation and test for withdrawal from mechanical
ventilation, use of steroids, glycemic control, prophylaxis of stress ulcers, and nutrition [
77
].
4.7. Sample Collection and Storage
Blood samples were obtained from each patient that entered the draw, before initiation
of the treatment, and 48 h after its administration. The blood samples were centrifuged for
20 min at 936
×
gand 4
◦
C. The plasma of the samples was placed in 3 or 4 aliquots and
stored at −30 ◦C.
4.8. Oxidative Stress Markers in Plasma
4.8.1. NO3−/NO2−Ratio
The NO
3−
was reduced to NO
2−
by the nitrate reductase enzyme reaction. A quantity
of 100
µ
L of plasma previously deproteinized with 0.5 N, NaOH and 10%, ZnSO
4
was
mixed, and the supernatant was incubated for 30 min at 37
◦
C in presence of nitrate
reductase (5 units). At the end of the incubation period, 200
µ
L of sulfanilamide 1% and
200
µ
L of N-naphthyl-ethyldiamine 0.1% were added and the total volume was adjusted to
1 mL. The absorbance was measured at 540 [118].
4.8.2. GSH Concentration
A total of 100
µ
L of plasma previously deproteinized with 20% trichloroacetic acid
(vol/vol) and centrifugated to 10,000
×
gfor 5 min was added to 800
µ
L of phosphate buffer
50 mM, pH 7.3, plus 100
µ
L of 5, 50-dithiobis-2-nitrobenzoic acid 1 M. The mixture was
incubated at room temperature for 5 min and absorbance was read at 412 nm [119].
4.8.3. Evaluation Total Antioxidant Capacity (TAC)
Briefly, 100
µ
L of plasma were suspended in 1.5 mL of a reaction mixture prepared as
follows: 300 mM acetate buffer pH 3.6, 20 mM hexahydrate of ferric chloride, and 10 mM
of 2,4,6-Tris-2-pyridil-s-triazine dissolved in 40 mM HCl. These reactives were added in
Int. J. Mol. Sci. 2023,24, 16610 18 of 26
a relation of 10:1:1 v/v, respectively. After mixing, samples were incubated at 37
◦
C for
15 min in the dark. The absorbance was measured at 593 nm [120].
4.8.4. Lipid Peroxidation (LPO)
A quantity of 50
µ
L CH
3
-OH with 4% butylated hydroxytoluene plus phosphate buffer
pH 7.4 was added to 100
µ
L of plasma. It was incubated and centrifuged at 4000 rpm in
room temperature for 2 min. Then, the n-butanol phase was extracted, and absorbance was
measured at 532 nm [118].
4.8.5. Carbonylation Protein Concentration
Briefly, 100
µ
L of plasma were added to 500
µ
L of HCl 2.5 N in parallel with another
sample with 500
µ
L of 2, 4-dinitrophenylhydrazine (DNPH) and incubated. At the end of
the incubation period, they were centrifuged at 15,000
×
gfor 5 min. The supernatant was
discarded. Two washings were performed. The mixture was incubated again at 37
◦
C for
30 min. Absorbance was read in a spectrophotometer at 370 nm, using bi-distilled water as
blank and a molar absorption coefficient of 22,000 M−1cm−1[118].
4.8.6. Determination of Selenium (Se)
Selenium (Se) determination was performed using 200
µ
L of serum according to the
method described by Soto et al., and the absorbance was read at 600 nm [118].
4.8.7. Thiols
This test consists of Ellman’s reagent reaction with a thiol group, commonly a thio-
late, producing thiol-nitrobenzoate through potassium hydride. The technique used was
previously described by Erel and Neselioglu [
121
], with some modifications carried out
in our laboratory as previously reported [
122
]. A quantity of 50
µ
L of serum was used for
the determination and the absorbance was measured at 415 nm. The calibration curve was
obtained with solution GSSG 1 mg/1 mL and the absorbance was measured at 415 nm.
4.8.8. GPx Activity
A total of 100
µ
L of serum was suspended in 1.6 mL of 50 mM phosphate buffer
(KH
2
PO
4
, pH 7.3), 0.2 mM NADPH, 1 mM GSH, and 1 IU/mL glutathione reductase. The
mixture was incubated for 3 min at 37
◦
C; then, 100
µ
L of 0.25 mM H
2
O
2
was added to
start the reaction and the absorbance was monitored for 7 min at 340 nm [
123
]. The units
are expressed in
µ
mol NADPH oxidized/min/mL in serum with an extinction coefficient
of 6220 M−1cm−1at 340 nm of NADPH [124].
4.8.9. GST Activity
A total of 100
µ
L of serum was added to 700
µ
L of phosphate buffer (KH
2
PO
4
, 0.1 M,
pH 6.5) with 100
µ
L of 0.1 mM GSH and 100
µ
L of 0.1 mM 1-chloro-2,4-dinitrobenzene
(CDNB). The sample was incubated and monitored for 7 min at 37 ◦C and read at 340 nm.
GST activity was expressed in units of GS-DNB
µ
mo/min/mL of serum with an extinction
coefficient of 14,150 M−1cm−1.
4.8.10. Extracellular Superoxide Dismutase (ecSOD)
ecSOD activity was determined via electrophoresis using native 10% polyacrylamide
gels. Electrophoresis was carried out at 120 V for 4 h, as previously described by Pérez-
Torres et al. [
39
]. In brief, 100 L of serum were used; the gel was incubated in 2.45 mM NBT
solution for 20 min. The liquid was discarded, and then the gel was incubated in a TEMED
solution with 36 mM potassium phosphate (pH 7.8) and 0.028 mM riboflavin. The gel was
exposed to a UV lamp for 10 min and washed with distilled water to stop the reaction. A
standardized curve was obtained using a serial dilution (2.5, 5, 10, 15, 30, and 60 ng) with
SOD from bovine erythrocytes (Sigma Aldrich Chemical SA de RL de C.V., Toluca, México).
SOD activity was calculated.
Int. J. Mol. Sci. 2023,24, 16610 19 of 26
4.8.11. Vitamin C Levels
Briefly, 100
µ
L of 20% trichloroacetic acid were added to 100
µ
L of plasma and cen-
trifuged at 5000 rpm for 5 min. Then, 200
µ
L of Folin–Ciocalteu reagent 0.20 mM was
added to the supernatant. The mixture was incubated for 10 min. The absorbance was
measured at 760 nm [103,125].
4.9. Cytokine Measurement
Serum cytokine levels were determined using a flow cytometry bead-based LEGEND-
plex
™
HU Essential Immune Response Panel (BioLegend, San Diego, CA, USA), a 13-plex
human panel to analyze the following cytokines and chemokines: IL-1
β
, IL-2, IL-4, IL-6,
IL-8, IL-10, IL-12p70, IL-17A CCL2 (MCP-1) CXCL10 (IP-10), IFN-
γ
, TGF-
β
1, and TNF-
α
.
The assay was performed following the manufacturer’s protocol; briefly, samples were
diluted 2-fold with assay buffer and 50 mL of sample or standard was mixed with 25
µ
L of
previously vortexed mixed beads in a V-bottom plate, then incubated for 2 h, centrifuged
at 250 rpm for 5 min, and washed two times with 200
µ
L wash buffer, 25
µ
L of detection
antibody were added and incubated as above, and without washing, 25
µ
L of SA-PE were
added and incubated for 30 min, and afterward, centrifugation and washing steps were
repeated, adding a final 150
µ
L of wash buffer to each well before being individually
transferred to microtubes for the acquisition in a BD FACS AriaTM Fusion (BD Biosciences,
San Jose, CA, USA). Analytes concentrations were calculated using the LEGENDplex Data
Analysis SoftwareCatalog 740932 Version 9. Software GraphPad Prism version 9.4.0.
4.10. Statistical Analysis
Continuous variables are expressed as mean
±
standard deviation or median with the
minimum and maximum values. Categorical variables, such as frequencies and percentages,
are reported. Normality distribution was evaluated using the Shapiro–France test. Non-
parametric tests (Mann–Whitney) or Student’s tests, according to the Gaussian distribution,
were performed to detect significant independent variables.
Some variables were standardized and Bonferroni correction was adjusted for multiple
comparisons. For the paired analyses (before–after), the Friedman or Wilcoxon test was
used with the signed-rank test according to the distribution of the data. Pearson’s Chi-
square (
χ2
) test or Fisher’s exact test were used to compare proportions between two groups.
For the multivariate analysis with the confounding factors, binary logistic regression was
used. For time and group analyses, repeated-samples analysis and panel data tests of
different models (pooled model, model for longitudinal data, marginal approximation
model, and multilevel model) were performed. A general adjustment was made to select
by random age using propensity matching between treated and untreated patients.
We perform a canonical correlation analysis (CC) to analyze the correlation of two
subsets of variables. In the first set, we include the x expressed as U (p) (X1, X2, .... XP) in
which the variables that they made up were (IL-1
β
, IL-2, IL4, IL-6, IL-8, IL-10, IL-12p70,
IL-17A, TNF-
α
, INF
γ
, IP-10, MCP-1, and TGF
β
-1). In the second set of variables, expressed
by V(q) (Y1, Y2, .... Yq). We included the SOFA score, C-reactive protein, procalcitonin,
lipoperoxidation, selenium, thiols, vitamin C, carbonylation, glutathione, Gpx, GST, GR,
SOD, and antioxidant capacity (CAT). We include more than two variables on both sides
of the equation in this type of analysis. The main objective of CC is to identify the linear
combinations and the value of the response of that correlation.
U1 1
4a11X1 þ a12X2 þ. ... . . þ a1pXp
U2 1
4a21X1 þ a22X2 þ. ... . . þ a2pXp
Ur 1
4ar1X1 þ ar2X2 þ. ... . . þ arpXp
Int. J. Mol. Sci. 2023,24, 16610 20 of 26
And with the predictive variable, it is expressed as follows:
V1 1
4b11Y1 þ b12Y2 þ. ... . . þ b1pYp
V2 1
4b21Y1 þ b22Y2 þ. ... . . þ b2pYp
Vr 1
4br1Y1 þ br2Y2 þ. ... . . þ brpYp
The largest correlation was identified between U1 and V1; the second largest corre-
lation was between U2 and V2, as long as there was no correlation between U1 and U2,
nor between V1 and V2; and the third largest correlation was between U3 and V3 and, as
described above. There could be no alteration between U3, and U1 and U2, nor between
V3, and V1 and V2. These combinations are known as canonical variables. Differences were
considered statistically significant when the p-value was <0.05. Statistical analyses were
performed using the STATA V.16 Software and Sigma Software Plot 14 (Jendel Corporation,
1986–2017, New York, NY, USA).
4.11. Ethical Aspects
A signed informed consent form was obtained from each participant in accordance
with the Declaration of Helsinki, as amended at the Congress in Tokyo, Japan. This research
was approved by the Ethics, Biosafety and Research Committees of the National Institute
of Cardiology (INCICh registry number: PT 10-0-76) and the ABC Campus Observatory
Medical Center, approval number ABC-18-19; Registry of Trial: Clinical-Trials.gov Identifier:
NCT 03557229.
5. Conclusions
Patients treated with antioxidants such as Vit C, Vit E, NAC, or MT, in addition to
standard therapy, show reduced levels of proinflammatory cytokines and improved regu-
latory function in Il-2, IL-12, and IFN. There is improvement of the antioxidant capacity
and reduction in biomarkers of oxidative stress with evidence of decreased organ damage
measured by SOFA score. Combining antioxidants such as Vit C, MT, and NAC associated
with standard therapy is a potential perspective in investigation through randomized
clinical trials since it could improve other clinical conditions and pathophysiological mech-
anisms that converge in septic shock. Regarding the damage mechanisms involved, there
is no evidence of improvement with the use of anticytokine biologicals and anti-apoptosis
therapy, which evidences the need of research studies to follow for the comprehensive
management of sepsis.
Supplementary Materials:
The supporting information can be downloaded at: https://www.mdpi.
com/article/10.3390/ijms242316610/s1. References [
126
–
133
] are cited in the supplementary materials.
Author Contributions:
Conception and design of the research: M.E.S., A.A.-Á. and G.B.; acquisition
of data: A.A.-Á., R.C.-S., I.P.-T., O.G.-M. and J.F.-G.; participation in patient enrolment, patient UCI,
procedures, and sample collection: A.A.-Á., R.C.-S., R.M.-V., S.C.-A., L.M.-P. and J.F.-G.; statistical
analysis and interpretation of data: M.E.S., A.A.-Á., H.S.-O. and R.G.; writing of the manuscript:
M.E.S., A.A.-Á., I.P.-T. and G.F.-Á.; critical revision of the manuscript for intellectual content: M.E.S.,
I.P.-T., G.F.-Á. and V.G.-L. All authors have read and agreed to the published version of the manuscript.
Funding:
This work was supported by Centro Medico A.B.C. Observatorio Project (ABC number
18-19) and Instituto Nacional de Cardiologia Ignacio Chavez.
Institutional Review Board Statement:
The study was conducted in accordance with the Declaration
of Helsinki and approved by the Instituto Nacional de Cardiologia Ignacio Chavez, registration
number INCICh: PT 10-0-76, and Centro Medico A.B.C. Campus Observatory, approval number
ABC-18-19. Trial Registration: ClinicalTrials.gov Identifier: NCT 03557229.
Int. J. Mol. Sci. 2023,24, 16610 21 of 26
Informed Consent Statement: Informed consent was obtained from all patients involved in the study.
Data Availability Statement: Data are contained within the article and Supplementary Materials.
Acknowledgments: We thank the patients and their relatives for their collaboration in this project.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
Singer, M.; Deutschman, C.S.; Seymour, C.W.; Shankar-Hari, M.; Annane, D.; Bauer, M. The Third International Consensus
Definitions for Sepsis and Septic Shock (Sepsis-3). JAMA 2016,315, 801. [CrossRef]
2.
Shankar-Hari, M.; Phillips, G.S.; Levy, M.L.; Seymour, C.W.; Liu, V.X.; Deutschman, C.S.; Angus, D.C.; Rubenfeld, G.D.; Singer,
M.; for the Sepsis Definitions Task Force. Developing a New Definition and Assessing New Clinical Criteria for Septic Shock.
JAMA 2016,315, 775. [CrossRef]
3.
Marshall, J.C.; Vincent, J.-L.; Guyatt, G.; Angus, D.C.; Abraham, E.; Bernard, G.; Bombardier, C.; Calandra, T.; Jørgensen,
H.S.; Sylvester, R.; et al. Outcome measures for clinical research in sepsis: A report of the 2nd Cambridge Colloquium of the
International Sepsis Forum. Crit. Care Med. 2005,33, 1708–1716. [CrossRef]
4.
Angus, D.C.; Linde-Zwirble, W.T.; Lidicker, J.; Clermont, G.; Carcillo, J.; Pinsky, M.R. Epidemiology of severe sepsis in the United
States: Analysis of incidence, outcome, and associated costs of care. Crit. Care Med. 2001,29, 1303–1310. [CrossRef]
5.
Seymour, C.W.; Liu, V.X.; Iwashyna, T.J.; Brunkhorst, F.M.; Rea, T.D.; Scherag, A.; Rubenfeld, G.; Kahn, J.M.; Shankar-Hari, M.;
Singer, M.; et al. Assessment of Clinical Criteria for Sepsis. JAMA 2016,315, 762. [CrossRef]
6.
Alunno, A.; Carubbi, F.; Rodríguez-Carrio, J. Storm, typhoon, cyclone or hurricane in patients with COVID-19? Beware of the
same storm that has a different origin. RMD Open 2020,6, e001295. [CrossRef]
7.
Levin, M. Childhood Multisystem Inflammatory Syndrome—A New Challenge in the Pandemic. N. Engl. J. Med.
2020
,
383, 393–395. [CrossRef]
8.
Castagnoli, R.; Votto, M.; Licari, A.; Brambilla, I.; Bruno, R.; Perlini, S.; Rovida, F.; Baldanti, F.; Marseglia, G.L. Severe Acute
Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Infection in Children and Adolescents. JAMA Pediatr.
2020
,174, 882.
[CrossRef]
9.
Crimi, E.; Sica, V.; Williams-Ignarro, S.; Zhang, H.; Slutsky, A.S.; Ignarro, L.J.; Napoli, C. The role of oxidative stress in adult
critical care. Free Radic. Biol. Med. 2006,40, 398–406. [CrossRef]
10. Volk, T.; Kox, W.J. Endothelium function in sepsis. Inflamm. Res. 2000,49, 185–198. [CrossRef]
11.
Letessier, W.; Demaret, J.; Gossez, M.; Allam, C.; Venet, F.; Rimmelé, T.; Monneret, G. Decreased intra-lymphocyte cytokines
measurement in septic shock patients: A proof of concept study in whole blood. Cytokine 2018,104, 78–84. [CrossRef]
12.
Roth, G.; Moser, B.; Krenn, C.; Brunner, M.; Haisjackl, M.; Almer, G.; Gerlitz, S.; Wolner, E.; Boltz-Nitulescu, G.; Ankersmit,
H.J. Susceptibility to programmed cell death in T-lymphocytes from septic patients: A mechanism for lymphopenia and Th2
predominance. Biochem. Biophys. Res. Commun. 2003,308, 840–846. [CrossRef]
13. Cohen, J. The immunopathogenesis of sepsis. Nature 2002,420, 885–891. [CrossRef]
14.
Chao, J.; Cui, S.; Liu, C.; Liu, S.; Liu, S.; Han, Y.; Gao, Y.; Ge, D.; Yu, A.; Yang, R. Detection of early cytokine storm in patients with
septic shock after abdominal surgery. J. Transl. Int. Med. 2020,8, 91–98. [CrossRef]
15.
Liu, X.; Zhan, Z.; Li, D.; Xu, L.; Ma, F.; Zhang, P.; Yao, H.; Cao, X. Intracellular MHC class II molecules promote TLR-triggered
innate immune responses by maintaining activation of the kinase Btk. Nat. Immunol. 2011,12, 416–424. [CrossRef]
16.
Liang, Y.; Li, X.; Zhang, X.; Li, Z.; Wang, L.; Sun, Y.; Liu, Z.; Ma, X. Elevated Levels of Plasma TNF-
α
Are Associated with
Microvascular Endothelial Dysfunction in Patients with Sepsis Through Activating the NF-
κ
B and p38 Mitogen-Activated Protein
Kinase in Endothelial Cells. Shock 2014,41, 275–281. [CrossRef]
17.
Hack, C.E.; De Groot, E.R.; Felt-Bersma, R.J.; Nuijens, J.H.; Strack Van Schijndel, R.J.; Eerenberg-Belmer, A.J.; Thijs, L.G.; Aarden,
L.A. Increased plasma levels of interleukin-6 in sepsis. Blood 1989,74, 1704–1710. [CrossRef]
18.
Miguel-Bayarri, V.; Casanoves-Laparra, E.B.; Pallás-Beneyto, L.; Sancho-Chinesta, S.; Martín-Osorio, L.F.; Tormo-Calandín, C.;
Bautista-Rentero, D. Valor pronóstico de los biomarcadores procalcitonina, interleukina 6 y proteína C reactiva en la sepsis grave.
Med. Intensiv. 2012,36, 556–562. [CrossRef]
19.
Xie, Y.; Li, B.; Lin, Y.; Shi, F.; Chen, W.; Wu, W.; Zhang, W.; Fei, Y.; Zou, S.; Yao, C. Combining Blood-Based Biomarkers to Predict
Mortality of Sepsis at Arrival at the Emergency Department. Med. Sci. Monit. 2020,27, e929527-1. [CrossRef]
20.
Hack, C.E.; Zeerleder, S. The endothelium in sepsis: Source of and a target for inflammation. Crit. Care Med.
2001
,29, S21–S27.
[CrossRef]
21.
Marshall, J.C. Inflammation, coagulopathy, and the pathogenesis of multiple organ dysfunction syndrome. Crit. Care Med.
2001
,
29, S99–S106. [CrossRef]
22.
Lehrnbecher, T.; Venzon, D.; de Haas, M.; Chanock, S.J.; Kühl, J. Assessment of Measuring Circulating Levels of Interleukin-6,
Interleukin-8, C-Reactive Protein, Soluble Fcy Receptor Type III, and Mannose-Binding Protein in Febrile Children with Cancer
and Neutropenia. Clin. Infect. Dis. 1999,29, 414–419. [CrossRef]
Int. J. Mol. Sci. 2023,24, 16610 22 of 26
23.
Spasova, M.I.; Terzieva, D.D.; Tzvetkova, T.Z.; Stoyanova, A.A.; Mumdzhiev, I.N.; Yanev, I.B.; Genev, E.D. Interleukin-6,
interleukin-8, interleukin-10, and C-reactive protein in febrile neutropenia in children with malignant diseases. Folia Med.
2005
,
47, 46–52.
24.
Borrelli, E.; Roux-Lombard, P.; Grau, G.E.; Girardin, E.; Ricou, B.; Dayer, J.-M.; Suter, P.M. Plasma concentrations of cytokines,
their soluble receptors, and antioxidant vitamins can predict the development of multiple organ failure in patients at risk. Crit.
Care Med. 1996,24, 392–397. [CrossRef]
25.
Maier, B.; Lefering, R.; Lehnert, M.; Laurer, H.L.; Steudel, W.I.; Neugebauer, E.A.; Marzi, I. Early versus late onset of multiple
organ failure is associated with differing patterns of plasma cytokine biomarker expression and outcome after severe trauma.
Shock 2007,28, 668–674. [CrossRef]
26.
Damas, P.; Canivet, J.L.; de Groote, D.; Vrindts, Y.; Albert, A.; Franchimont, P.; Lamy, M.M. Sepsis and serum cytokine
concentrations. Crit. Care Med. 1997,25, 405–412. [CrossRef]
27.
Razazi, K.; Boissier, F.; Surenaud, M.; Bedet, A.; Seemann, A.; Carteaux, G.; de Prost, N.; Brun-Buisson, C.; Hue, S.; Dessap, A.M.
A multiplex analysis of sepsis mediators during human septic shock: A preliminary study on myocardial depression and organ
failures. Ann. Intensive Care 2019,9, 64. [CrossRef]
28.
Reinhart, K.; Bayer, O.; Brunkhorst, F.; Meisner, M. Markers of endothelial damage in organ dysfunction and sepsis. Crit. Care
Med. 2002,30, S302–S312. [CrossRef]
29.
Nedel, W.L.; Strogulski, N.R.; Rodolphi, M.S.; Kopczynski, A.; Montes, T.H.M.; Portela, L.V. Short-term inflammatory biomarker
profiles are associated with deficient mitochondrial bioenergetics in lymphocytes of septic shock patients—A prospective cohort
study. Shock 2023,59, 288–293. [CrossRef]
30.
Donoso, A.; Arriagada-Santis, D. Síndrome de disfunción de órganos y adaptación mitocondrial en el paciente séptico. Bol. Med.
Hosp. Infant. Mex. 2021,78, 597–611. [CrossRef]
31.
Taniguchi, T.; Koido, Y.; Aiboshi, J.; Yamashita, T.; Suzaki, S.; Kurokawa, A. Change in the ratio of interleukin-6 to interleukin-10
predicts a poor outcome in patients with systemic inflammatory response syndrome. Crit. Care Med.
1999
,27, 1262–1264.
[CrossRef]
32. Bianchi, M.E. DAMPs, PAMPs and alarmins: All we need to know about danger. J. Leukoc. Biol. 2007,81, 1–5. [CrossRef]
33.
Ayala, A.; Wesche-Soldato, D.E.; Perl, M.; Lomas-Neira, J.L.; Swan, R.; Chung, C.-S. Blockade of apoptosis as a rational therapeutic
strategy for the treatment of sepsis. Novartis Found. Symp. 2007,280, 37–49; discussion 49–52, 160–164. [CrossRef]
34. Rittirsch, D.; Flierl, M.A.; Ward, P.A. Harmful molecular mechanisms in sepsis. Nat. Rev. Immunol. 2008,8, 776–787. [CrossRef]
35.
Mittal, M.; Siddiqui, M.R.; Tran, K.; Reddy, S.P.; Malik, A.B. Reactive Oxygen Species in Inflammation and Tissue Injury. Antioxid.
Redox Signal. 2014,20, 1126–1167. [CrossRef]
36. Reczek, C.R.; Chandel, N.S. ROS-dependent signal transduction. Curr. Opin. Cell Biol. 2015,33, 8–13. [CrossRef]
37.
Li, F.; Yan, R.; Wu, J.; Han, Z.; Qin, M.; Liu, C.; Lu, Y. An Antioxidant Enzyme Therapeutic for Sepsis. Front. Bioeng. Biotechnol.
2021,9, 800684. [CrossRef]
38.
Wesche-Soldato, D.E.; Swan, R.Z.; Chung, C.-S.; Ayala, A. The apoptotic pathway as a therapeutic target in sepsis. Curr. Drug
Targets 2007,8, 493–500. [CrossRef]
39.
Manchikalapati, R.; Schening, J.; Farias, A.J.; Sacco, K. Clinical Utility of Interleukin-1 Inhibitors in Pediatric Sepsis. Shock
2023
.
[CrossRef]
40.
Hu, B.; Chen, Z.; Liang, L.; Zheng, M.; Chen, X.; Zeng, Q. Melatonin Promotes Mitochondrial Biogenesis and Mitochondrial
Degradation in Hepatocytes During Sepsis. Altern. Ther. Health Med. 2023,29, 284–289.
41.
Hadi, S.M.H.; Majeed, S.; Ghafil, F.A.; Altoraihi, K.; Hadi, N.R. Effect of Sulforaphane on cardiac injury induced by sepsis in a
mouse model: Role of toll-like receptor 4. J. Med. Life 2023,16, 1120–1126. [CrossRef]
42. Tisoncik, J.R.; Korth, M.J.; Simmons, C.P.; Farrar, J.; Martin, T.R.; Katze, M.G. Into the Eye of the Cytokine Storm. Microbiol. Mol.
Biol. Rev. 2012,76, 16–32. [CrossRef]
43.
Chousterman, B.G.; Swirski, F.K.; Weber, G.F. Cytokine storm and sepsis disease pathogenesis. Semin. Immunopathol.
2017
,
39, 517–528. [CrossRef]
44.
Dare, A.J.; Phillips, A.R.J.; Hickey, A.J.R.; Mittal, A.; Loveday, B.; Thompson, N.; Windsor, J.A. A systematic reviewof experimental
treatments for mitochondrial dysfunction in sepsis and multiple organ dysfunction syndrome. Free Radic. Biol. Med.
2009
,
47, 1517–1525. [CrossRef]
45. Halliwell, B.; Gutteridge, J.M.C. Free Radicals in Biology and Medicine; Oxford University Press: Oxford, UK, 2015. [CrossRef]
46.
Joseph, L.C.; Kokkinaki, D.; Valenti, M.-C.; Kim, G.J.; Barca, E.; Tomar, D.; Hoffman, N.E.; Subramanyam, P.; Colecraft, H.M.;
Hirano, M.; et al. Inhibition of NADPH oxidase 2 (NOX2) prevents sepsis-induced cardiomyopathy by improving calcium
handling and mitochondrial function. JCI Insight 2017,2, e94248. [CrossRef]
47.
Virdis, A.; Colucci, R.; Fornai, M.; Blandizzi, C.; Duranti, E.; Pinto, S.; Bernardini, N.; Segnani, C.; Antonioli, L.; Taddei, S.; et al.
Cyclooxygenase-2 Inhibition Improves Vascular Endothelial Dysfunction in a Rat Model of Endotoxic Shock: Role of Inducible
Nitric-Oxide Synthase and Oxidative Stress. J. Pharmacol. Exp. Ther. 2005,312, 945–953. [CrossRef]
48.
Jacobi, J.; Kristal, B.; Chezar, J.; Shaul, S.M.; Sela, S. Exogenous superoxide mediates pro-oxidative, proinflammatory, and
procoagulatory changes in primary endothelial cell cultures. Free Radic. Biol. Med. 2005,39, 1238–1248. [CrossRef]
Int. J. Mol. Sci. 2023,24, 16610 23 of 26
49.
Rondovic, G.; Djordjevic, D.; Udovicic, I.; Stanojevic, I.; Zeba, S.; Abazovic, T.; Vojvodic, D.; Abazovic, D.; Khan, W.; Surbatovic,
M. From Cytokine Storm to Cytokine Breeze: Did Lessons Learned from Immunopathogenesis Improve Immunomodulatory
Treatment of Moderate-to-Severe COVID-19? Biomedicines 2022,10, 2620. [CrossRef]
50.
Aisa-Álvarez, A.; Pérez-Torres, I.; Guarner-Lans, V.; Manzano-Pech, L.; Cruz-Soto, R.; Márquez-Velasco, R.;
Casarez-Alvarado, S.
;
Franco-Granillo, J.; Núñez-Martínez, M.E.; Soto, M.E. Randomized Clinical Trial of Antioxidant Therapy Patients with Septic
Shock and Organ Dysfunction in the ICU: SOFA Score Reduction by Improvement of the Enzymatic and Non-Enzymatic
Antioxidant System. Cells 2023,12, 1330. [CrossRef]
51.
Ge, S.X.; Jung, D.; Yao, R. ShinyGO: A graphical gene-set enrichment tool for animals and plants. Bioinformatics
2020
,36, 2628–2629.
[CrossRef]
52. Chen, G.Y.; Nuñez, G. Sterile inflammation: Sensing and reacting to damage. Nat. Rev. Immunol. 2010,10, 826–837. [CrossRef]
53.
Ruiz-Sanmartín, A.; Ribas, V.; Suñol, D.; Chiscano-Camón, L.; Palmada, C.; Bajaña, I.; Larrosa, N.; González, J.J.; Canela, N.;
Ferrer, R.; et al. Characterization of a proteomic profile associated with organ dysfunction and mortality of sepsis and septic
shock. PLoS ONE 2022,17, e0278708. [CrossRef]
54.
Bauzá-Martinez, J.; Aletti, F.; Pinto, B.B.; Ribas, V.; Odena, M.A.; Díaz, R.; Romay, E.; Ferrer, R.; Kistler, E.; Tedeschi, G.; et al.
Proteolysis in septic shock patients: Plasma peptidomic patterns are associated with mortality. Br. J. Anaesth.
2018
,121, 1065–1074.
[CrossRef]
55.
Reinhart, K.; Daniels, R.; Kissoon, N.; Machado, F.R.; Schachter, R.D.; Finfer, S. Recognizing Sepsis as a Global Health Priority—A
WHO Resolution. N. Engl. J. Med. 2017,377, 414–417. [CrossRef]
56.
Cohen, J.; Vincent, J.-L.; Adhikari, N.K.J.; Machado,F.R.; Angus, D.C.; Calandra, T.; Jaton, K.; Giulieri, S.; Delaloye, J.;
Opal, S.; et al
.
Sepsis: A roadmap for future research. Lancet Infect. Dis. 2015,15, 581–614. [CrossRef]
57. Cavaillon, J.-M. Exotoxins and endotoxins: Inducers of inflammatory cytokines. Toxicon 2018,149, 45–53. [CrossRef]
58.
Striz, I.; Brabcova, E.; Kolesar, L.; Sekerkova, A. Cytokine networking of innate immunity cells: A potential target of therapy. Clin.
Sci. 2014,126, 593–612. [CrossRef]
59.
Anderson, G.; Reiter, R.J. Melatonin: Roles in influenza, COVID-19, and other viral infections. Rev. Med. Virol.
2020
,30, e2109.
[CrossRef]
60.
Paterson, R.L.; Galley, H.F.; Webster, N.R. The effect of N-acetylcysteine on nuclear factor-
κ
B activation, interleukin-6, interleukin-
8, and intercellular adhesion molecule-1 expression in patients with sepsis*. Crit. Care Med. 2003,31, 2574–2578. [CrossRef]
61.
Soto, M.E.; Guarner-Lans, V.; Soria-Castro, E.; Manzano Pech, L.; Pérez-Torres, I. Is Antioxidant Therapy a Useful Complementary
Measure for COVID-19 Treatment? An Algorithm for Its Application. Medicina 2020,56, 386. [CrossRef]
62.
Rank, N.; Michel, C.; Haertel, C.; Med, C.; Lenhart, A.; Welte, M.; Meier-Hellmann, A.; Spies, C. N-acetylcysteine increases liver
blood flow and improves liver function in septic shock patients: Results of a prospective, randomized, double-blind study. Crit.
Care Med. 2000,28, 3799–3807. [CrossRef]
63.
Zaniew, M.; Zachwieja, J.; Lewandowska-Stachowiak, M.; Sobczyk, D.; Siwi´nska, A. The antioxidant therapy modulates
intracellular lymphokine expression in children on dialysis. Przegl. Lek. 2006,63 (Suppl. S3), 63–67.
64.
Abdelrazik, E.; Hassan, H.M.; Abdallah, Z.; Magdy, A.; Farrag, E.A. Renoprotective effect of N-acetylcystein and vitamin E in
bisphenol A-induced rat nephrotoxicity; Modulators of Nrf2/NF-
κ
B and ROS signaling pathway. Acta Biomed.
2022
,93, e2022301.
[CrossRef]
65.
Sawoo, R.; Dey, R.; Ghosh, R.; Bishayi, B. Exogenous IL-10 posttreatment along with TLR4 and TNFR1 blockade improves tissue
antioxidant status by modulating sepsis-induced macrophage polarization. J. Appl. Toxicol. 2023,43, 1549–1572. [CrossRef]
66.
Martin, J.G.; Kurokawa, C.S.; Carpi, M.F.; Bonatto, R.C.; Moraes, M.A.; Fioretto, J.R. Interleukin-12 in children with sepsis and
septic shock. Rev. Bras. Ter. Intensiv. 2012,24, 130–136. [CrossRef]
67. Nansen, A.; Randrup Thomsen, A. Viral Infection Causes Rapid Sensitization to Lipopolysaccharide: Central Role of IFN-αβ.J.
Immunol. 2001,166, 982–988. [CrossRef]
68.
Gaignage, M.; Uyttenhove, C.; Jones, L.L.; Bourdeaux, C.; Chéou, P.; Mandour, M.F.; Coutelier, J.; Vignali, D.A.; Van Snick, J. Novel
antibodies that selectively block mouse IL-12 enable the re-evaluation of the role of IL-12 in immune protection and pathology.
Eur. J. Immunol. 2021,51, 1482–1493. [CrossRef]
69.
Weidhase, L.; Wellhöfer, D.; Schulze, G.; Kaiser, T.; Drogies, T.; Wurst, U.; Petros, S. Is Interleukin-6 a better predictor of successful
antibiotic therapy than procalcitonin and C-reactive protein? A single center study in critically ill adults. BMC Infect. Dis.
2019
,
19, 150. [CrossRef]
70.
Visavadiya, N.P.; Soni, B.; Dalwadi, N. Free radical scavenging and antiatherogenic activities of Sesamum indicum seed extracts
in chemical and biological model systems. Food Chem. Toxicol. 2009,47, 2507–2515. [CrossRef]
71.
Hesse, D.G.; Tracey, K.J.; Fong, Y.; Manogue, K.R.; Palladino, M.A.; Cerami, A.; Shires, G.T.; Lowry, S.F. Cytokine appearance in
human endotoxemia and primate bacteremia. Surg. Gynecol. Obstet. 1988,166, 147–153.
72.
Beutler, B.A.; Milsark, I.W.; Cerami, A. Cachectin/tumor necrosis factor: Production, distribution, and metabolic fate
in vivo
.J.
Immunol. 1985,135, 3972–3977. [CrossRef]
73.
Michie, H.R.; Manogue, K.R.; Spriggs, D.R.; Revhaug, A.; O’Dwyer, S.; Dinarello, C.A.; Cerami, A.; Wolff, S.M.; Wilmore, D.W.
Detection of Circulating Tumor Necrosis Factor after Endotoxin Administration. N. Engl. J. Med.
1988
,318, 1481–1486. [CrossRef]
Int. J. Mol. Sci. 2023,24, 16610 24 of 26
74.
Waage, A.; Brandtzaeg, P.; Halstensen, A.; Kierulf, P.; Espevik, T. The complex pattern of cytokines in serum from patients with
meningococcal septic shock. Association between interleukin 6, interleukin 1, and fatal outcome. J. Exp. Med.
1989
,169, 333–338.
[CrossRef]
75.
Kang, Y.-H.; Brummel, S.E.; Lee, C.-H. Differential effects of transforming growth factor-
±
1 on lipopolysaccharide induction of
endothelial adhesion molecules. Shock 1996,6, 118–125. [CrossRef]
76.
Basodan, N.; Al Mehmadi, A.E.; Al Mehmadi, A.E.; Aldawood, S.M.; Hawsawi, A.; Fatini, F.; Mulla, Z.M.; Nawwab, W.; Alshareef,
A.; Almhmadi, A.H.; et al. Septic Shock: Management and Outcomes. Cureus 2022,14, e32158. [CrossRef]
77.
Evans, L.; Rhodes, A.; Alhazzani, W.; Antonelli, M.; Coopersmith, C.M.; French, C.; Machado, F.R.; Mcintyre, L.; Ostermann,
M.; Prescott, H.C.; et al. Surviving sepsis campaign: International guidelines for management of sepsis and septic shock 2021.
Intensive Care Med. 2021,47, 1181–1247. [CrossRef]
78. Angus, D.C.; van der Poll, T. Severe Sepsis and Septic Shock. N. Engl. J. Med. 2013,369, 840–851. [CrossRef]
79.
Gabriel, V.; Grigorian, A.; Nahmias, J.; Pejcinovska, M.; Smith, M.; Sun, B.; Won, E.; Bernal, N.; Barrios, C.; Schubl, S.D. Risk
Factors for Post-Operative Sepsis and Septic Shock in Patients Undergoing Emergency Surgery. Surg. Infect.
2019
,20, 367–372.
[CrossRef]
80. Medam, S.; Zieleskiewicz, L.; Duclos, G.; Baumstarck, K.; Loundou, A.; Alingrin, J.; Hammad, E.; Vigne, C.; Antonini, F.; Leone,
M. Risk factors for death in septic shock. Medicine 2017,96, e9241. [CrossRef]
81.
Rech, M.A.; Mosier, M.J.; McConkey, K.; Zelisko, S.; Netzer, G.; Kovacs, E.J.; Afshar, M. Outcomes in Burn-Injured Patients Who
Develop Sepsis. J. Burn. Care Res. 2019,40, 269–273. [CrossRef]
82.
Boehm, D.; Menke, H. Sepsis in Burns—Lessons Learnt from Developments in the Management of Septic Shock. Medicina
2021
,
58, 26. [CrossRef]
83.
Nappi, F.; Martuscelli, G.; Bellomo, F.; Avtaar Singh, S.S.; Moon, M.R. Infective Endocarditis in High-Income Countries. Metabolites
2022,12, 682. [CrossRef]
84.
Babe
s
,
, E.E.; Lucu
t
,
a, D.A.; Petche
s
,
i, C.D.; Zaha, A.A.; Ilyes, C.; Jurca, A.D.; Vesa, C.M.; Zaha, D.C.; Babe
s
,
, V.V. Clinical Features
and Outcome of Infective Endocarditis in a University Hospital in Romania. Medicina 2021,57, 158. [CrossRef]
85.
Peetermans, M.; de Prost, N.; Eckmann, C.; Norrby-Teglund, A.; Skrede, S.; De Waele, J.J. Necrotizing skin and soft-tissue
infections in the intensive care unit. Clin. Microbiol. Infect. 2020,26, 8–17. [CrossRef]
86.
Bruun, T.; Rath, E.; Madsen, M.B.; Oppegaard, O.; Nekludov, M.; Arnell, P.; Karlsson, Y.; Babbar, A.; Bergey, F.; Itzek, A.; et al.
Risk Factors and Predictors of Mortality in Streptococcal Necrotizing Soft-tissue Infections: A Multicenter Prospective Study. Clin.
Infect. Dis. 2021,72, 293–300. [CrossRef]
87.
Kovacs, J.; Gurzu, S.; Jung, J.; Szederjesi, J.; Copotoiu, S.-M.; Copotoiu, R.; Azamfirei, L. Clinico-Pathological Particularities of the
Shock-Related Pancreatitis. Pathol. Oncol. Res. 2012,18, 977–981. [CrossRef]
88.
Coureuil, M.; Join-Lambert, O.; Lecuyer, H.; Bourdoulous, S.; Marullo, S.; Nassif, X. Pathogenesis of Meningococcemia. Cold
Spring Harb. Perspect. Med. 2013,3, a012393. [CrossRef]
89. Mora, A. A Complicated Case of Rheumatoid Arthritis in Septic Shock. Cureus 2022,14, e25712. [CrossRef]
90.
Jamal, M.; Bangash, H.I.; Habiba, M.; Lei, Y.; Xie, T.; Sun, J.; Wei, Z.; Hong, Z.; Shao, L.; Zhang, Q. Immune dysregulation and
system pathology in COVID-19. Virulence 2021,12, 918–936. [CrossRef]
91.
Neupane, M.; Kadri, S.S. Nangibotide for precision immunomodulation in septic shock and COVID-19. Lancet Respir. Med.
2023
,
11, 855–857. [CrossRef]
92.
Dinarello, C.A. Proinflammatory and Anti-inflammatory Cytokines as Mediators in the Pathogenesis of Septic Shock. Chest
1997
,
112, 321S–329S. [CrossRef]
93.
Shapiro, L.; Scherger, S.; Franco-Paredes, C.; Gharamti, A.; Henao-Martinez, A.F. Anakinra authorized to treat severe coronavirus
disease 2019; Sepsis breakthrough or time to reflect? Front. Microbiol. 2023,14, 1250483. [CrossRef]
94.
Zhang, R.; Wang, X.; Ni, L.; Di, X.; Ma, B.; Niu, S.; Liu, C.; Reiter, R.J. COVID-19: Melatonin as a potential adjuvant treatment. Life
Sci. 2020,250, 117583. [CrossRef]
95.
Carrillo-Vico, A.; Lardone, P.J.; Naji, L.; Fernández-Santos, J.M.; Martín-Lacave, I.; Guerrero, J.M.; Calvo, J.R. Beneficial pleiotropic
actions of melatonin in an experimental model of septic shock in mice: Regulation of pro-/anti-inflammatory cytokine network,
protection against oxidative damage and anti-apoptotic effects. J. Pineal Res. 2005,39, 400–408. [CrossRef]
96.
Wang, H. Protective effect of melatonin against liver injury in mice induced by Bacillus Calmette-Guerin plus lipopolysaccharide.
World J. Gastroenterol. 2004,10, 2690. [CrossRef]
97.
Lassnigg, A.; Punz, A.; Barker, R.; Keznickl, P.; Manhart, N.; Roth, E.; Hiesmayr, M. Influence of intravenous vitamin E
supplementation in cardiac surgery on oxidative stress: A double-blinded, randomized, controlled study. Br. J. Anaesth.
2003
,
90, 148–154. [CrossRef]
98.
Mayo, J.C.; Sainz, R.M.; González Menéndez, P.; Cepas, V.; Tan, D.-X.; Reiter, R.J. Melatonin and sirtuins: A “not-so unexpected”
relationship. J. Pineal Res. 2017,62, e12391. [CrossRef]
99.
Stefulj, J.; Hörtner, M.; Ghosh, M.; Schauenstein, K.; Rinner, I.; Wölfler, A.; Semmler, J.; Liebmann, P.M. Gene expression of the key
enzymes of melatonin synthesis in extrapineal tissues of the rat. J. Pineal Res. 2001,30, 243–247. [CrossRef]
100.
Masana, M.I.; Dubocovich, M.L. Melatonin Receptor Signaling: Finding the Path Through the Dark. Sci. STKE
2001
,2001, pe39.
[CrossRef]
Int. J. Mol. Sci. 2023,24, 16610 25 of 26
101.
Dubocovich, M.L. Molecular pharmacology regulation and function of mammalian melatonin receptors. Front. Biosci.
2003
,
8, 1089. [CrossRef]
102.
Tarocco, A.; Caroccia, N.; Morciano, G.; Wieckowski, M.R.; Ancora, G.; Garani, G.; Pinton, P. Melatonin as a master regulator of
cell death and inflammation: Molecular mechanisms and clinical implications for newborn care. Cell Death Dis.
2019
,10, 317.
[CrossRef]
103.
Aisa-Alvarez, A.; Soto, M.E.; Guarner-Lans, V.; Camarena-Alejo, G.; Franco-Granillo, J.; Martínez-Rodríguez, E.A.; Ávila, R.G.;
Pech, L.M.; Pérez-Torres, I. Usefulness of Antioxidants as Adjuvant Therapy for Septic Shock: A Randomized Clinical Trial.
Medicina 2020,56, 619. [CrossRef]
104.
Forceville, X.; Mostert, V.; Pierantoni, A.; Vitoux, D.; Le Toumelin, P.; Plouvier, E.; Dehoux, M.; Thuillier, F.; Combes, A.
Selenoprotein P, Rather than Glutathione Peroxidase, as a Potential Marker of Septic Shock and Related Syndromes. Eur. Surg.
Res. 2009,43, 338–347. [CrossRef]
105. Manthous, C.A.; Hall, J.B.; Samsel, R.W. Endotoxin in Human Disease. Chest 1993,104, 1872–1881. [CrossRef]
106. Stephens, R.; Mythen, M. Endotoxin immunization. Intensive Care Med. 2000,26, S129–S136. [CrossRef]
107.
Jena, A.B.; Samal, R.R.; Bhol, N.K.; Duttaroy, A.K. Cellular Red-Ox system in health and disease: The latest update. Biomed.
Pharmacother. 2023,162, 114606. [CrossRef]
108.
Wicha, P.; Tocharus, J.; Janyou, A.; Jittiwat, J.; Changtam, C.; Suksamrarn, A.; Tocharus, C. Hexahydrocurcumin protects against
cerebral ischemia/reperfusion injury, attenuates inflammation, and improves antioxidant defenses in a rat stroke model. PLoS
ONE 2017,12, e0189211. [CrossRef]
109.
Pita Rodríguez, G. Funciones de la Vitamina E en la nutrición humana/Role of vitamin E in human nutrition. Rev. Cuba. Aliment.
Nutr. 1997,11, 46–57.
110.
Tahir, M.; Foley, B.; Pate, G.; Crean, P.; Moore, D.; McCarroll, N.; Walsh, M. Impact of vitamin E and C supplementation on
serum adhesion molecules in chronic degenerative aortic stenosis: A randomized controlled trial. Am. Heart J.
2005
,150, 302–306.
[CrossRef]
111.
Haileselassie, B.; Joshi, A.U.; Minhas, P.S.; Mukherjee, R.; Andreasson, K.I.; Mochly-Rosen, D. Mitochondrial dysfunction
mediated through dynamin-related protein 1 (Drp1) propagates impairment in blood brain barrier in septic encephalopathy. J.
Neuroinflamm. 2020,17, 36. [CrossRef]
112. Riedel, S.; Carroll, K.C. Laboratory Detection of Sepsis. Clin. Lab. Med. 2013,33, 413–437. [CrossRef]
113.
Menon, V.; Mohamed, Z.U.; Prasannan, P.; Moni, M.; Edathadathil, F.; Prasanna, P.; Menon, A.; Nair, S.; Greeshma, C.;
Sathyapalan, D.T.; et al
. Vitamin C Therapy for Routine Care in Septic Shock (ViCTOR) Trial: Effect of Intravenous Vitamin C,
Thiamine, and Hydrocortisone Administration on Inpatient Mortality among Patients with Septic Shock. Indian J. Crit. Care Med.
2020,24, 653–661. [CrossRef]
114.
Fowler, A.A.; Truwit, J.D.; Hite, R.D.; Morris, P.E.; DeWilde, C.; Priday, A.; Fisher, B.; Thacker, L.R., 2nd; Natarajan, R.;
Brophy, D.F.; et al
. Effect of Vitamin C Infusion on Organ Failure and Biomarkers of Inflammation and Vascular Injury in Patients
With Sepsis and Severe Acute Respiratory Failure. JAMA 2019,322, 1261. [CrossRef]
115.
Lowes, D.A.; Almawash, A.M.; Webster, N.R.; Reid, V.L.; Galley, H.F. Melatonin and structurally similar compounds have
differing effects on inflammation and mitochondrial function in endothelial cells under conditions mimicking sepsis. Br. J.
Anaesth. 2011,107, 193–201. [CrossRef]
116.
Howe, K.P.; Clochesy, J.M.; Goldstein, L.S.; Owen, H. Mechanical Ventilation Antioxidant Trial. Am. J. Crit. Care
2015
,24, 440–445.
[CrossRef]
117.
Khwannimit, B. A comparison of three organ dysfunction scores: MODS, SOFA and LOD for predicting ICU mortality in critically
ill patients. J. Med. Assoc. Thail. 2007,90, 1074–1081.
118.
Soto, M.E.; Manzano-Pech, L.G.; Guarner-Lans, V.; Díaz-Galindo, J.A.; Vásquez, X.; Castrejón-Tellez, V.; Gamboa, R.; Huesca, C.;
Fuentevilla-Alvárez, G.; Pérez-Torres, I. Oxidant/Antioxidant Profile in the Thoracic Aneurysm of Patients with the Loeys-Dietz
Syndrome. Oxid. Med. Cell Longev. 2020,2020, 5392454. [CrossRef]
119.
Zúñiga-Muñoz, A.M.; Pérez-Torres, I.; Guarner-Lans, V.; Núñez-Garrido, E.; Velázquez Espejel, R.; Huesca-Gómez, C.; Gamboa-
Ávila, R.; Soto, M.E. Glutathione system participation in thoracic aneurysms from patients with Marfan syndrome. Vasa
2017
,
46, 177–186. [CrossRef]
120.
Benzie, I.F.F.; Strain, J.J. The Ferric Reducing Ability of Plasma (FRAP) as a Measure of “Antioxidant Power”: The FRAP Assay.
Anal. Biochem. 1996,239, 70–76. [CrossRef]
121.
Erel, O.; Neselioglu, S. A novel and automated assay for thiol/disulphide homeostasis. Clin. Biochem.
2014
,47, 326–332.
[CrossRef]
122.
Soto, M.E.; Guarner-Lans, V.; Díaz-Díaz, E.; Manzano-Pech, L.; Palacios-Chavarría, A.; Valdez-Vázquez, R.R.; Aisa-Álvarez, A.;
Saucedo-Orozco, H.; Pérez-Torres, I. Hyperglycemia and Loss of Redox Homeostasis in COVID-19 Patients. Cells
2022
,11, 932.
[CrossRef]
123.
Flohé, L.; Günzler, W.A. [12] Assays of Glutathione Peroxidase; Academic Press: Cambridge, MA, USA, 1984; pp. 114–120. [CrossRef]
124.
Soto, M.E.; Soria-Castro, E.; Lans, V.G.; Ontiveros, E.M.; Mejía, B.I.H.; Hernandez, H.J.M.; García, R.B.; Herrera, V.; Pérez-
Torres, I. Analysis of Oxidative Stress Enzymes and Structural and Functional Proteins on Human Aortic Tissue from Different
Aortopathies. Oxid. Med. Cell Longev. 2014,2014, 1–13. [CrossRef]
Int. J. Mol. Sci. 2023,24, 16610 26 of 26
125.
Jagota, S.K.; Dani, H.M. A new colorimetric technique for the estimation of vitamin C using Folin phenol reagent. Anal. Biochem.
1982,127, 178–182. [CrossRef]
126.
LYSOMUCIL—PLM. Available online: https://www.medicamentosplm.com/Home/productos/lysomucil_tabletas_
efervescentes/42/101/36846/218 (accessed on 26 June 2020).
127.
REDOXON—PLM. Available online: https://www.medicamentosplm.com/Home/productos/redoxon_tabletas_masticables/
21/101/9525/220 (accessed on 26 June 2020).
128.
ETERNAL CÁPSULAS 400 UI de México. Available online: https://www.vademecum.es/mexico/medicamento/1280368/
eternal+capsulas+400+ui (accessed on 2 September 2023).
129.
BENEDORM—PLM. Available online: https://www.medicamentosplm.com/Home/productos/benedorm_tabletas_
sublinguales/187/101/10598/224 (accessed on 26 June 2020).
130.
Johnson, C.E.; Cober, M.P.; Thome, T.; Rouse, E. Stability of an extemporaneous alcohol-free melatonin suspension. Am. J. Health
Pharm. 2011,68, 420–423. [CrossRef]
131.
National Center for Biotechnology Information. Vitamin E|C29H50O2; National Center for Biotechnology Information: Bethesda,
MD, USA, 2022.
132.
Galli, F.; Azzi, A.; Birringer, M.; Cook-Mills, J.M.; Eggersdorfer, M.; Frank, J.; Cruciani, G.; Lorkowski, S.; Özer, N.K. Vitamin E:
Emerging aspects and new directions. Free Radic. Biol. Med. 2017,102, 16–36. [CrossRef]
133.
Outline, C.; Concepts, A. Distribution in Foods. 2012; pp. 181–211. Available online: https://health2016.globalchange.gov/food-
safety-nutrition-and-distribution (accessed on 9 October 2023).
Disclaimer/Publisher’s Note:
The statements, opinions and data contained in all publications are solely those of the individual
author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to
people or property resulting from any ideas, methods, instructions or products referred to in the content.
