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Citation: Xu, S.; Zhou, Y.; Yu, L.;
Huang, X.; Huang, J.; Wang, K.; Liu,
Z. Protective Effect of Eurotium
cristatum Fermented Loose Dark Tea
and Eurotium cristatum Particle on
MAPK and PXR/AhR Signaling
Pathways Induced by Electronic
Cigarette Exposure in Mice. Nutrients
2022,14, 2843. https://doi.org/
10.3390/nu14142843
Academic Editors: Lindsay Brown
and Anna Gramza-Michałowska
Received: 20 May 2022
Accepted: 2 July 2022
Published: 11 July 2022
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nutrients
Article
Protective Effect of Eurotium cristatum Fermented Loose Dark
Tea and Eurotium cristatum Particle on MAPK and PXR/AhR
Signaling Pathways Induced by Electronic Cigarette Exposure
in Mice
Shuai Xu 1, Yufei Zhou 1, Lijun Yu 1 ,2 ,*, Xiangxiang Huang 1, Jianan Huang 1,2, Kunbo Wang 1,2
and Zhonghua Liu 1, 2, *
1Key Laboratory of Tea Science of Ministry of Education, National Research Center of Engineering
Technology for Utilization of Functional Ingredients from Botanicals, College of Horticulture,
Hunan Agricultural University, Changsha 410128, China; xushuai199884@163.com (S.X.);
zhouyufeittea@stu.hunau.edu.cn (Y.Z.); hxx70345@outlook.com (X.H.); jian7513@hunau.edu.cn (J.H.);
wkboo163@163.com (K.W.)
2Hunan Provincial Key Laboratory for Germplasm Innovation and Utilization of Crop,
Hunan Agricultural University, Changsha 410128, China
*Correspondence: yulijun@hunau.edu.cn (L.Y.); larkin-liu@163.com (Z.L.)
Abstract:
Electronic-cigarette smoke (eCS) has been shown to cause a degree of oxidative stress and
inflammatory damage in lung tissue. The aim of this study was to evaluate the repair mechanism
of Eurotium cristatum fermented loose dark tea (ECT) and Eurotium cristatum particle metabolites
(ECP) sifted from ECT after eCS-induced injury in mice. Sixty C57BL/6 mice were randomly divided
into a blank control group, an eCS model group, an eCS + 600 mg/kg ECP treatment group, an
eCS + 600 mg/kg ECT treatment group, an eCS + 600 mg/kg ECP prevention group, and an eCS
+ 600 mg/kg ECT prevention group. The results show that ECP and ECT significantly reduced the
eCS-induced oxidative stress and inflammation and improved histopathological changes in the lungs
in mice with eCS-induced liver injury. Western blot analysis further revealed that ECP and ECT
significantly inhibited the eCS-induced upregulation of the phosphorylation levels of the extracellular
Regulated protein Kinases (ERK), c-Jun N-terminal kinase (JNK) and p38mitogen-activated protein
kinases (p38MAPK) proteins, and significantly increased the eCS-induced downregulation of the
expression levels of the pregnane X receptor (PXR) and aryl hydrocarbon receptor (AhR) proteins.
Conclusively, these findings show that ECP and ECT have a significant repairing effect on the damage
caused by eCS exposure through the MAPK and PXR/AhR signaling pathways; ECT has a better
effect on preventing eCS-induced injury and is suitable as a daily healthcare drink; ECP has a better
therapeutic effect after eCS-induced injury, and might be a potential therapeutic candidate for the
treatment of eCS-induced injury.
Keywords:
electronic-cigarette smoke; Eurotium cristatum;Eurotium cristatum particle; Eurotium
cristatum fermented loose dark tea; MAPK; PXR; AhR; metabonomics
1. Introduction
Electronic-cigarette (eC) liquids are mainly composed of tobacco extract, glycerin,
propylene glycol and nicotine. eC liquids are relatively harmless to human health, have
a wide range of fashionable flavors, and are becoming popular among young smokers as
an alternative to traditional cigarettes [
1
,
2
]. However, after high temperature atomization,
eC liquid will still produce a small amount of formaldehyde, acetaldehyde, acrolein and
glyoxal and other harmful substances [
3
–
5
]. Related studies have shown that electronic
cigarette smoke (eCS) can increase the level of malondialdehyde(MDA) in serum [
6
], and
Nutrients 2022,14, 2843. https://doi.org/10.3390/nu14142843 https://www.mdpi.com/journal/nutrients
Nutrients 2022,14, 2843 2 of 17
decrease the activity of superoxide dismutase (SOD), catalase (CAT) and glutathione-S-
transferase (GST) [
7
], and increase the content of interleukin-1
β
(IL-1
β
), interleukin-6
(IL-6) and tumor necrosis factor alpha (TNF-
α
) [
8
]. The mitogen-activated protein kinase
(MAPK) signaling pathway is involved in the regulation of cell growth and differenti-
ation, inflammation and apoptosis through ERK, JKN and p38. Guan’s study showed
that cigarette smoke exposure could activate the expression of the ERK/JNK/p38MAPK
signaling pathway and induce the upregulation of TNF-
α
, IL-1
β
and IL-6 [
9
]. However,
although electronic cigarettes contain fewer harmful components than cigarettes, glyoxal
and methylglyoxal produced after high-temperature atomization can induce the release of
the pro-inflammatory cytokines IL-1
β
and IL-6 through the extracellular signal-regulated
kinase ERK1/2, P38 and nuclear factor kappa B (NF-
κ
B) pathways [
10
]. Moreover, related
studies have shown that electronic cigarettes with or without nicotine can activate ERK1/2
and p38 [
11
]. In addition, pregnane X receptor (PXR) is an upstream regulator of many
metabolic enzymes and transporters. It plays a role in drug metabolism and detoxification
by regulating the expression of downstream genes Cytochrome P450 3A4 (CYP3A4) [
12
].
Aromatic hydrocarbon receptor (AhR) is a nuclear and cytoplasmic shuttle protein, which
can regulate the biochemical and toxicological reactions of environmental chemicals [
13
],
and mainly mediates the expression regulation of the Cytochrome P450 1A1 (CYP1A1)
and Cytochrome P450 1A2 (CYP1A2) genes [
14
,
15
]. Therefore, inhibiting the activation
of the MAPK signaling pathway, and while promoting the activation of the PXR/AhR
signaling pathways, may be helpful in reducing the harm induced by eCS, and it is of great
significance for the treatment of eCS-induced injury to find an agent which could improve
these pathways.
Recent studies showed that Eurotium cristatum is a unique probiotic in Fu brick tea
(FBT) [
16
], possessing anti-tumor and anti-oxidation effects and the ability to regulate
intestinal microorganisms [
17
,
18
]. FBT is a reprocessed tea from primary loose dark tea
(PDT) that is fermented by Eurotium cristatum after being pressed. The amount of Eurotium
cristatum has been considered an important indicator of FBT quality [
19
]. The water extract
of FBT can inhibit the MAPK and nuclear factor-erythroid 2-related factor-2 (Nrf2) signaling
pathway in human keratinocytes (HaCaT) and reduce oxidative stress levels [
20
]. ECT is
made from the primary loose dark tea (PDT) fermented by Eurotium cristatum, and the
number of Eurotium cristatum on the surface of ECT is more than that of compressed FBT.
On the basis of these investigations, we speculated that ECP and ECT have protective
effects on eCS-induced tissue damage. However, studies on the application of ECP and
ECT to eCS have not been reported. Thus, the aim of this work was to explore the ability of
ECP and ECT to improve lung injury and hepatotoxicity induced by eCS in order to provide
alternative anti-eCS drugs. Therefore, in this study, we established an eCS-damaged model
of C57BL/6 female mice; ECP and ECT were used to prevent and treat mice, respectively,
and the effects of oxidative stress and inflammatory factor levels in serum were investigated.
In addition, the underlying potential mechanism was partially elucidated using Western
blot analysis of the MAPK, PXR and AhR signaling pathways.
2. Materials and Methods
2.1. Preparation of ECP and ECT Extract
The primary loose dark tea (PDT) was sterilized at 121
◦
C for 20 min, and was
inoculated with Eurotium cristatum after cooling. Fermentation was carried out in an
incubator at 28
◦
C and 80% humidity. After drying at 70–80
◦
C for 120 min, we collected
ECT and stored it at
−
20
◦
C in a refrigerator. The processed ECT was sifted through a
100-mesh
sieve, and the ECP at the bottom of the sieve and the ECT on the sieve surface
were collected. They were was extracted by ultrasonic extraction (40 kHz) in ultrapure
water for 30 min at 100
◦
C and then concentrated, freeze-dried and decompressed to
obtain ECT and ECP powders. The dosages of ECP and ECT depends on the body weight
(600 mg/kg) of mice. The dosages of ECP and ECT were selected based on references and
preliminary experiments [21].
Nutrients 2022,14, 2843 3 of 17
2.2. Analysis of Physicochemical Components of ECP and ECT
The contents of the water extract, tea polyphenols and free amino acids were deter-
mined according to GB/T 8305-2013, GB/T 8313-2008 and GB/T8314-2013, respectively.
The contents of catechin, gallic acid and caffeine were analyzed by HPLC according to the
method of Huang [
21
]. The HPLC analysis of quercetin and kaempferol was carried out
according to the method of Samanidou [22].
2.3. Metabolomic Analysis of ECP and ECT
2.3.1. Sample Preparation and Extraction
The 150 mg ECP and ECT samples were placed in a 2 mL grinding thickening tube, and
1 mL of extraction solution (methanol/water = 7:3, pre-cooled at
−
20
◦
C), and two small
steel balls were added to the test tube, and grinded in a tissue grinder (50 Hz for 5 min).
The samples were placed at 4
◦
C, vortexed once every 10 min for a total of 3 times, and then
4◦C overnight. The next day, they were vortexed and centrifuged at 13,000×gfor 10 min
at 4
◦
C. After centrifugation, 800
µ
L of the supernatant was taken and passed through
a 0.22
µ
m filter membrane, and the filtered samples were placed in a sample bottle for
LC-MS analysis.
2.3.2. UPLC-MRM Analysis
In this experiment, a Waters ACQUITY UPLC I-Class (waters, USA) tandem QTRAP6500
Plus high-sensitivity mass spectrometer (SCIEX, USA) was used for the separation and
quantitative detection of metabolites. The chromatographic column was an ACQUITY
UPLC HSS T3 column (100
×
2.1 mm, 1.8
µ
m, Waters). The mobile phase was an aqueous
solution containing 0.1% formic acid (liquid A) and 100% acetonitrile (liquid B) containing
0.1% formic acid. Elution B was carried out with the following gradient: 0~2 min, 5%;
2~22 min
, 5~95%; 22~27 min, 95%; 27~30 min, 5%. An injection volume of 8
µ
L, solvent
flow rate of 0.3 mL/min, and column temperature of 40
◦
C were used. For the QTRAP
6500 Plus system with the EST Turbo ion spray interface, the ion source parameters were
set as follows: the ion source temperature was set to 500
◦
C; the ion spray voltage (IS) was
set to 4500 V (positive mode)/
−
4500 V (negative mode), ion source gas I (GS1), gas II (GS2)
and air curtain gas (CUR) were set to 40, 40 and 20 psi, respectively. The MRM detection
window was set to 120 s and the target scanning time was set to 0.5 s.
2.4. eC Liquid Preparation
eCS liquid was provided by the China Tobacco Hunan Industry Co., Ltd. (Changsha,
China). It consists of 95% aerosol and 5% cigarette essential oil. The atomizer consists of
propylene glycol (PG), vegetable glycerin (VG), water and nicotine. Tobacco essential oil is
extracted from tobacco by supercritical carbon dioxide. Its main components are nicotine,
anthracene-D10, phenanthrene-D10, cyclohexene, glycerin and other substances.
2.5. Chemicals
Analytical kits for SOD, GSH-Px and MDA were purchased from the Nanjing Jiancheng
Bioengineering Institute (Nanjing, China). ELISA kits for the analysis of tumor necrosis
factor alpha (TNF-
α
), interleukin-6 (IL-6), IL-8, and IL-1
β
were purchased from Wuhan
Hualianke Biotechnology Co. Ltd. (Wuhan, China). Extracellular signal-regulated kinases
(ERK), phospho-ERK (p-ERK), c-Jun N-terminal kinase (JNK), phosphor-JNK (p-JNK),
p38 and phosphor-p38 (p-p38) were purchased from Cell Signaling Technology (Danvers,
MA, USA). Monoclonal antibodies against PXR (ab192579), AhR (ab84833), and GAPDH
(ab181602) were purchased from Abcam (Cambridge, UK). All other chemicals and reagents
were of analytical grade.
2.6. Animals and Experimental Design
C57BL/6SPF female mice (15
±
1 g) were purchased from Changsha Slakejingda
Experimental Animal Co., Ltd., and with the experimental animal production license
Nutrients 2022,14, 2843 4 of 17
number: SCXK (Hunan) 2016-0002. Approved by the Animal Test Committee of Hunan
Agricultural University, the mice were raised in the Animal Experimental Center of the
Tea Research Institute of Hunan Agricultural University. The temperature of the feeding
environment was (25
±
1)
◦
C, the humidity was 40–70%, and the light time was 12 h day and
night. After two weeks of adaptive feeding, 60 mice were randomly divided into 6 groups:
(1) blank control group; (2) eCS exposure model group; (3) eCS + ECP treatment group;
(4) eCS + ECT treatment group; (5) eCS + ECP prevention group; (6)
eCS + ECT
prevention
group. Mice in the blank control group and the eCS group received an equal volume of
distilled water by gavage every day for 12 weeks; meanwhile, mice in the eCS group were
exposed to eCS from the 5th week. In the first 8 weeks, the mice in the treatment group
received an equal volume of distilled water by gavage and were exposed to eCS. From
the 9th week to the 12th week, the mice in the treatment group were no longer exposed to
eCS and were fed with ECT and ECP of 600 mg/kg each of ECT and ECP, respectively. In
the first 4 weeks, the mice in the prevention group received an equal volume of distilled
water by gavage. From the 5th week to the 12th week, the mice in the prevention group
were fed with ECT and ECP at the concentration of 600 mg/kg immediately after daily
eCS exposure (Figure 1). The mice in the eCS exposure group were exposed to a self-made
passive smoking box on the first day for 12 min, increased by 5 min every day until 60 min
was reached. The self-made passive smoking device for mice (0.9 m
×
0.6 m
×
0.5 m plastic
box) evenly distributes 20 vents with a radius of 2 cm around and at the top. Vacuum
diaphragm pumps were purchased from Kamoer, KVP15-KL-1 (Shanghai, China). The
rubber tube at the air inlet was connected to the electronic cigarette smoker, and the rubber
tube at the air outlet extended into the box to release smoke. For the smoking frequency,
GB/T16450-2004 was referred to in order to establish the smoke damage model.
Nutrients 2022, 14, x FOR PEER REVIEW 4 of 18
2.6. Animals and Experimental Design
C57BL/6SPF female mice (15 ± 1 g) were purchased from Changsha Slakejingda Ex-
perimental Animal Co., Ltd., and with the experimental animal production license num-
ber: SCXK (Hunan) 2016-0002. Approved by the Animal Test Committee of Hunan Agri-
cultural University, the mice were raised in the Animal Experimental Center of the Tea
Research Institute of Hunan Agricultural University. The temperature of the feeding en-
vironment was (25 ± 1) °C, the humidity was 40–70%, and the light time was 12 h day and
night. After two weeks of adaptive feeding, 60 mice were randomly divided into 6 groups:
(1) blank control group; (2) eCS exposure model group; (3) eCS + ECP treatment group;
(4) eCS + ECT treatment group; (5) eCS + ECP prevention group; (6) eCS + ECT prevention
group. Mice in the blank control group and the eCS group received an equal volume of
distilled water by gavage every day for 12 weeks; meanwhile, mice in the eCS group were
exposed to eCS from the 5th week. In the first 8 weeks, the mice in the treatment group
received an equal volume of distilled water by gavage and were exposed to eCS. From the
9th week to the 12th week, the mice in the treatment group were no longer exposed to eCS
and were fed with ECT and ECP of 600 mg/kg each of ECT and ECP, respectively. In the
first 4 weeks, the mice in the prevention group received an equal volume of distilled water
by gavage. From the 5th week to the 12th week, the mice in the prevention group were
fed with ECT and ECP at the concentration of 600 mg/kg immediately after daily eCS
exposure (Figure 1). The mice in the eCS exposure group were exposed to a self-made
passive smoking box on the first day for 12 min, increased by 5 min every day until 60
min was reached. The self-made passive smoking device for mice (0.9 m × 0.6 m × 0.5 m
plastic box) evenly distributes 20 vents with a radius of 2 cm around and at the top. Vac-
uum diaphragm pumps were purchased from Kamoer, KVP15-KL-1 (Shanghai, China).
The rubber tube at the air inlet was connected to the electronic cigarette smoker, and the
rubber tube at the air outlet extended into the box to release smoke. For the smoking fre-
quency, GB/T16450-2004 was referred to in order to establish the smoke damage model.
Figure 1. The schematic diagram of experimental groupings.
2.7. Collection of Serum and Lung and Liver Tissues in Mice
The mice fasted for 12 h before death and were anesthetized with pentobarbital so-
dium. The serum was collected from the eyeballs, left at room temperature for 1 h, centri-
fuged for 10 min at 4 °C for 2500 r/min, and stored at −80 °C. The lung and liver tissues
Figure 1. The schematic diagram of experimental groupings.
2.7. Collection of Serum and Lung and Liver Tissues in Mice
The mice fasted for 12 h before death and were anesthetized with pentobarbital sodium.
The serum was collected from the eyeballs, left at room temperature for 1 h, centrifuged
for 10 min at 4
◦
C for 2500 r/min, and stored at
−
80
◦
C. The lung and liver tissues were
removed and washed 3 times in normal saline at 4
◦
C. The surface water and blood stains
were dried using sterile filter paper. The right lung tissues were placed in formalin to
prepare pathological sections; the remaining lung and liver tissues were stored at
−
80
◦
C
for follow-up testing.
Nutrients 2022,14, 2843 5 of 17
2.8. Histological Evaluation
The right lung tissue was fixed in formalin for 3 days, embedded in paraffin, and
stained with hematoxylin-eosin solution. Optical microscopy was used to assess the
morphological changes in the lung tissue.
2.9. Biochemical Analysis
The oxidative stress index in the serum of mice was evaluated by measuring the
activity of GSH-Px and SOD and the content of MDA in the serum using a biochemical kit.
The serum levels of TNF-
α
, IL-8, IL-6 and IL-1
β
were determined by ELISA kits to evaluate
the inflammatory reaction. All the tests were carried out according to the instructions of
the reagent.
2.10. Western Blot Analysis
The lung and liver tissue proteins were extracted using a total protein kit (Solarbio,
Beijing, China), and tissue protein concentration was quantified to 1
µ
g/
µ
L using a BCA kit
(Solarbio, Beijing, China). An amount of 20
µ
g of denatured proteins was separated in 10%
polyacrylamide gel electrophoresis (80 V, 25 min, 120 V, 60 min); then, the target protein was
transferred to PVDF membranes (300 mA, 90 min). Afterwards, the membranes were sealed
in TBS-Tween (TBST) containing 5% skim milk powder for 1 h. The closed membranes
were washed in a shaker with TBST 3 times, for 10 min each time. The membranes were
incubated overnight in a primary antibody (used at a dilution of 1:10,000) at 4
◦
C, and the
membranes were washed three times with TBST and incubated with a secondary antibody
(used at a dilution of 1:10,000) at room temperature for 1.5 h. After the membranes were
washed with TBST three times, the membranes were added to a chemiluminescent mixture,
and were exposed to a chemiluminescence imager to display the target protein. The image
was transformed by imaging software, the gray ratio of the target strip to the GAPDH
internal reference strip was evaluated and the experiment was repeated 3 times.
2.11. Statistical Analysis
The metabolites were identified and quantitatively analyzed using the MRM quanti-
tative software MultiQuant (AB Sciex, Framingham, MA, USA), combined with a widely
targeted metabolic standard database (BGI-WideTarget-Library) independently established
by the BGI (Shenzhen, China). Bioinformatics analysis was carried out using the OmicStu-
dio tool available at https://www.omicstudio.cn/tool (accessed on 11 February 2022). The
data were analyzed and processed by IBM SPSS Statistics 22.0 software, and the significant
differences among groups were analyzed by single-factor analysis of variance (one-way
ANOVA). According to the homogeneity of variance, the LSD method and Tamhane method
were adopted. All experimental data were expressed as the mean
±
SD, and plotted with
GraphPad Prism 7, Adobe Acrobat DC and Photoshop CS6. Statistical significance was set
at p< 0.05 or p< 0.01.
3. Results
3.1. Analysis of Physicochemical Components of ECT and ECP
It can be seen from Table 1that, except for ECG, the contents of the other components
in ECT were much higher than those in ECP, especial for tea polyphenols, caffeine and
gallic acid. There is no specific standard for the analysis of physicochemical components
of ECP, so the current HPLC detection method was only able to detect a small amount of
ECP content. However, the similar content of the two water extracts indicates that the ECP
extract may contain undetected chemical substances, but further detection and research
are needed.
Nutrients 2022,14, 2843 6 of 17
Table 1. Change of physicochemical components of ECT and ECP.
Physicochemical Components ECT ECP
Tea polyphenols (%) 14.80 ±0.22 3.40 ±0.21 *
Water extract (%) 41.55 ±0.37 39.13 ±1.71
Free amino acid (%) 3.02 ±0.02 2.62 ±0.01 *
Gallic acid (mg/g) 13.23 ±0.66 2.14 ±0.20 *
Theobromine (mg/g) 1.82 ±0.06 1.42 ±0.32
Theophylline (mg/g) 0.41 ±0.12 0.08 ±0.02 *
Caffeine (mg/g) 39.40 ±1.25 5.65 ±0.208 *
EGC (mg/g) 5.12 ±0.32 2.74 ±0.10 *
C (mg/g) 6.41 ±0.15 0.17 ±0.01 *
EC (mg/g) 3.12 ±0.05 0.08 ±0.01 *
EGCG (mg/g) 9.86 ±0.17 5.39 ±0.57 *
GCG (mg/g) 7.87 ±0.23 2.27 ±0.34 *
ECG (mg/g) 1.68 ±0.11 4.89 ±0.43 *
Quercetin (mg/g) 0.82 ±0.08 0.18 ±0.09 *
Kaempferol (mg/g) 1.49 ±0.217 0.20 ±0.04 *
(+)-catechin (C), (
−
)-epicatechin (EC), (
−
)-gallocatechin gallate (GCG), (
−
)-epigallocatechin (EGC),
(−)-epicatechin gallate (ECG), (−)-epigallocatechin gallate (EGCG). *: compared with the ECT group, p< 0.05.
3.2. Metabolomic Analysis of ECP and ECT
3.2.1. Principal Component Analysis (PCA) and Partial Least Squares-Discriminant
Analysis (PLS-DA) of ECT and ECP
In the unsupervised PCA score chart (Figure 2A), the first and second principal
components explained 89.31% and 2.32% of the variation, respectively. In the supervised
PLS-DA score chart (Figure 2B), the first and second principal components explained
90.78% and 1.85% of the variation, respectively. There are obvious differences in the
distribution of Figure 2A,B between the ECT and ECP samples, indicating that there are
significant differences between them. Emergency aggregation appeared in the ECT and
ECP samples, which confirmed the stability and reproducibility of the analysis method.
The interpretation rate R2 and predictive power Q2 of PLS-DA both exceeded 0.9. Cross-
validation of 100 permutation tests showed that the R2 and Q2 intercepts were 0.97 and
−
0.75, respectively, showing that the Q2 intercept was less than 0, which indicates that the
PLS-DA model is reliable (Figure 2C).
Nutrients 2022, 14, x FOR PEER REVIEW 7 of 18
Figure 2. (A) PCA score chart of ECT and ECP; (B) PLS−DA score chart of ECT and ECP; and (C)
response sequencing test diagram of PLS−DA analysis model.
3.2.2. Heatmap Analysis of the Differential Metabolites of ECT and ECP
Heatmaps were drawn to visualize the difference between the 212 compounds in
ECT and ECP, with each column representing a sample and each row representing a me-
tabolite (Figure 3). Red indicates that the metabolite content is above the sample average,
while blue indicates that the metabolite content does not reach the average level. It can be
seen from the heatmaps that, comparing ECT and ECP, ECT contains more phenols and
their derivatives, phenolic acids, glycosides, flavonoids, lignans, terpenes and other sub-
stances, while ECP contains more amino acids and their derivatives, organic acids, alka-
loids, carbohydrates and other substances, and the amounts of esters and coumarin and
its derivatives are equal.
Figure 2.
(
A
) PCA score chart of ECT and ECP; (
B
) PLS
−
DA score chart of ECT and ECP; and
(C) response sequencing test diagram of PLS−DA analysis model.
3.2.2. Heatmap Analysis of the Differential Metabolites of ECT and ECP
Heatmaps were drawn to visualize the difference between the 212 compounds in ECT
and ECP, with each column representing a sample and each row representing a metabolite
(Figure 3). Red indicates that the metabolite content is above the sample average, while blue
Nutrients 2022,14, 2843 7 of 17
indicates that the metabolite content does not reach the average level. It can be seen from the
heatmaps that, comparing ECT and ECP, ECT contains more phenols and their derivatives,
phenolic acids, glycosides, flavonoids, lignans, terpenes and other substances, while ECP
contains more amino acids and their derivatives, organic acids, alkaloids, carbohydrates
and other substances, and the amounts of esters and coumarin and its derivatives are equal.
Nutrients 2022, 14, x FOR PEER REVIEW 7 of 18
Figure 2. (A) PCA score chart of ECT and ECP; (B) PLS−DA score chart of ECT and ECP; and (C)
response sequencing test diagram of PLS−DA analysis model.
3.2.2. Heatmap Analysis of the Differential Metabolites of ECT and ECP
Heatmaps were drawn to visualize the difference between the 212 compounds in
ECT and ECP, with each column representing a sample and each row representing a me-
tabolite (Figure 3). Red indicates that the metabolite content is above the sample average,
while blue indicates that the metabolite content does not reach the average level. It can be
seen from the heatmaps that, comparing ECT and ECP, ECT contains more phenols and
their derivatives, phenolic acids, glycosides, flavonoids, lignans, terpenes and other sub-
stances, while ECP contains more amino acids and their derivatives, organic acids, alka-
loids, carbohydrates and other substances, and the amounts of esters and coumarin and
its derivatives are equal.
Figure 3.
(
A
) Heat map of amino acids and derivates, phenols and derivatives, phenolic acids,
glycosides, organic acids and esters in ECT and ECP; (
B
) Heat map of flavonoids, alkaloids and
carbohydrates in ECT and ECP; (
C
) Heat map of lignans, coumarins and derivatives, terpenoids and
other differential metabolites in ECT and ECP.
3.2.3. Boxplot Analysis of the Differential Metabolites of ECT and ECP
Based on functional pharmacology, 39 bioactive metabolites with the highest content
of ECT and ECP were selected from the differential metabolites’ heatmaps to draw the
boxplots, and the content difference was analyzed in detail. There were 18 metabolites
with a higher content in ECT and 21 metabolites with a higher content in ECP. It can be
seen from Figure 4that the contents of C, EC, EGC, ECG and GCG in ECT are higher than
those in ECP, but the difference is not very significant, being only 1–5 times higher than
those of ECP. The contents of eight flavonoid metabolites such as morin, vitexin, tricetin,
licochalcone B, eriodictyol, dihydrokaempferol, hesperetin and naringenin7-O-glucoside,
in ECT were higher than those in ECP. Apart from that, the content of vitexin in ECT, which
was about 5 times higher than that in ECP, the content of seven other flavonoids was more
than 10 times higher than that in ECP. The two glycosides oroxin A and sophoroside in
ECT were two and three times higher than those in ECP, respectively. The three organic
acids gallic acid, 5-acetylsalicylic acid and isochlorogenic acid B in ECT were two, three
and five times higher than those in ECP, respectively.
Nutrients 2022,14, 2843 8 of 17
Nutrients 2022, 14, x FOR PEER REVIEW 9 of 18
Figure 4. 39 differential metabolites box diagram of ECT and ECP. (A): Catechin; (B): flavonoids;
(C): flavonoids; (D): flavonoids; (E): flavonoids; (F): amino acids; (G): alkaloids; (H): other metabo-
lites.
3.3. Pathological Changes in Lung Tissue Sections in Mice
The pathological section of the lung tissue of mice is shown in Figure 5. Compared
with the blank control group, the eCS group showed an irregular arrangement of tracheal
cilia, thickening of alveolar wall, rupture of the alveolar septum, dilation of the cavity,
infiltration of inflammatory cells and obvious blood osmosis. Compared with the eCS ex-
posure model group, the lung tissue trachea of mice in the prevention group and treat-
ment group of ECP and ECT was more regular, the alveolar wall became thinner, the de-
gree of alveolar septum injury was attenuated, the wall expansion became smaller, the
Figure 4.
39 differential metabolites box diagram of ECT and ECP. (
A
): Catechin; (
B
): flavonoids;
(
C
): flavonoids; (
D
): flavonoids; (
E
): flavonoids; (
F
): amino acids; (
G
): alkaloids; (
H
): other metabolites.
The contents of D-mannose, deoxyglucose, L-fucose, mannitol and lactose in ECP were
higher than those in ECT. Except for mannose, the other four sugars were more than 10 times
higher than ECT. The contents of L-hydroxyproline, L-leucine, L-tyrosine, L-phenylalanine
and their derivatives in ECP were higher than those in ECT, and the lowest difference was
more than 30 times. The contents of five flavonoid metabolites in ECP, namely, isobavachin,
isobavachalcone, licochalcone A, 7,8-dihydroxyflavone and dihydromyricetin, were higher
than those in ECT. Apart from the content of dihydromyricetin, which was only slightly
higher than that in ECT, the contents of the other four flavonoids were more than 10 times
higher than that in ECT. The contents of heobromine, theophylline, caffeine and betaine in
ECP were higher than those in ECT. Apart from the content of betaine, which was nearly
Nutrients 2022,14, 2843 9 of 17
40 times higher than that in ECT, the contents of the other three alkaloids were only about
2 times those in ECT. The content of protocatechuic acid in ECP was more than 10 times
higher than that in ECT. It is worth noting that the content of three other metabolites in
ECP, namely, steviolbioside, kahweol and ginkgolide K, was more than 50 times higher
than that in ECT.
3.3. Pathological Changes in Lung Tissue Sections in Mice
The pathological section of the lung tissue of mice is shown in Figure 5. Compared with
the blank control group, the eCS group showed an irregular arrangement of tracheal cilia,
thickening of alveolar wall, rupture of the alveolar septum, dilation of the cavity, infiltration
of inflammatory cells and obvious blood osmosis. Compared with the eCS exposure model
group, the lung tissue trachea of mice in the prevention group and treatment group of
ECP and ECT was more regular, the alveolar wall became thinner, the degree of alveolar
septum injury was attenuated, the wall expansion became smaller, the inflammatory cell
infiltration decreased and there was no obvious blood osmosis. ECP and ECT intragastric
intervention improved lung tissue injury, the alveolar tissue morphology nearly returned
to the normal level, and the treatment group had a better anti-inflammatory potential than
the preventive treatment group.
Nutrients 2022, 14, x FOR PEER REVIEW 10 of 18
inflammatory cell infiltration decreased and there was no obvious blood osmosis. ECP
and ECT intragastric intervention improved lung tissue injury, the alveolar tissue mor-
phology nearly returned to the normal level, and the treatment group had a better anti-
inflammatory potential than the preventive treatment group.
Figure 5. The effects of ECP and ECT on lung histopathology in mice with eCS exposure-induced
lung injury (200×). (A): Control; (B): eCS; (C): eCS + ECP T; (D): eCS + ECT T; (E): eCS + ECP P; (F):
eCS + ECT P.
3.4. Changes in Serum Oxidative Stress Indexes and Inflammatory Factors in Mice
Whether ECT and ECP can alleviate oxidative stress and the inflammatory response
caused by eCS exposure is the key to whether ECT and ECP have preventive and thera-
peutic effects. According to the results in Figure 6, Compared with the blank control
group, the contents of TNF-α, IL-8, IL-6, IL-1β and MDA in the serum of the eCS model
group were significantly increased (p < 0.01), while the activities of GSH-Px and SOD were
significantly decreased (p < 0.01). Compared with the eCS model group, the serum oxida-
tive stress index of mice treated with ECP and ECT were improved. The ECP and ECT
treatment groups had a significantly reduced level of MDA in the serum and increased
activities of GSH and SOD (p < 0.05). The ECP and ECT prevention groups also had a
reduced level of MDA in the serum, and increased activities of GSH and SOD, and there
were significant differences in the ECT prevention group (p < 0.05). Compared with the
eCS model group, the inflammatory factors in serum of mice treated with ECP and ECT
were improved. The contents of TNF-α, IL-6 and IL-8 in the ECP prevention group were
significantly decreased (p < 0.05), but the contents of TNF-α, IL-1β, IL-6 and IL-8 in the
ECP and ECT treatment groups and the ECT prevention group were significantly de-
creased (p < 0.01).
Figure 5.
The effects of ECP and ECT on lung histopathology in mice with eCS exposure-induced
lung injury (200
×
). (
A
): Control; (
B
): eCS; (
C
): eCS + ECP T; (
D
): eCS + ECT T; (
E
): eCS + ECP P;
(F): eCS + ECT P.
3.4. Changes in Serum Oxidative Stress Indexes and Inflammatory Factors in Mice
Whether ECT and ECP can alleviate oxidative stress and the inflammatory response
caused by eCS exposure is the key to whether ECT and ECP have preventive and therapeutic
effects. According to the results in Figure 6, Compared with the blank control group, the
contents of TNF-
α
, IL-8, IL-6, IL-1
β
and MDA in the serum of the eCS model group were
significantly increased (p < 0.01), while the activities of GSH-Px and SOD were significantly
decreased (p < 0.01). Compared with the eCS model group, the serum oxidative stress
index of mice treated with ECP and ECT were improved. The ECP and ECT treatment
groups had a significantly reduced level of MDA in the serum and increased activities of
GSH and SOD (p < 0.05). The ECP and ECT prevention groups also had a reduced level of
Nutrients 2022,14, 2843 10 of 17
MDA in the serum, and increased activities of GSH and SOD, and there were significant
differences in the ECT prevention group (p < 0.05). Compared with the eCS model group,
the inflammatory factors in serum of mice treated with ECP and ECT were improved. The
contents of TNF-
α
, IL-6 and IL-8 in the ECP prevention group were significantly decreased
(p < 0.05), but the contents of TNF-
α
, IL-1
β
, IL-6 and IL-8 in the ECP and ECT treatment
groups and the ECT prevention group were significantly decreased (p < 0.01).
Nutrients 2022, 14, x FOR PEER REVIEW 11 of 18
Figure 6. The effects of ECP and ECT on GSH-Px and SOD activities and the expression of MDA,
TNF-α, IL-6, IL-8, and IL-1β levels in serum of mice exposed to eCS. (A) Active unit of GSH-Px. (B)
Activity of SOD. (C) Concentration of MDA. (D) Concentration of TNF-α. (E) Concentration of IL-
1β. (F) Concentration of IL-6. (G) Concentration of IL-8. The measures represent mean ± SD. **: com-
pared with the blank control group, p < 0.01; #: compared with the eCS model group, p < 0.05; ##:
compared with the eCS model group, p < 0.01.
3.5. Changes in Relative Expression of ERK, JNK and p38 MAPK Phosphorylated Proteins in
Mousee Lung
The phosphorylation of the MAPK pathway is one of the key characteristics of oxi-
dative stress and inflammation caused by eCS. According to the results in Figure 7, com-
pared with the blank control group, the phosphorylation levels of the ERK, JNK and p38
proteins in the lungs of the eCS model group were significantly increased (p < 0.01). Com-
pared with the eCS model group, the phosphorylation level of the ERK and JNK proteins
in the ECP and ECT treatment groups decreased significantly (p < 0.01). The phosphory-
lation level of the p38 protein in the ECP treatment group decreased significantly (p <
0.05), and the phosphorylation level of the p38 protein in the ECT treatment group de-
creased, but there was no significant difference (p > 0.05). The phosphorylation levels of
the JNK protein significantly decreased in the ECP prevention group (p < 0.05), and the
phosphorylation levels of the ERK and P38 protein were decreased but there was no sig-
Figure 6.
The effects of ECP and ECT on GSH-Px and SOD activities and the expression of MDA,
TNF-
α
, IL-6, IL-8, and IL-1
β
levels in serum of mice exposed to eCS. (
A
) Active unit of GSH-Px.
(
B
) Activity of SOD. (
C
) Concentration of MDA. (
D
) Concentration of TNF-
α
. (
E
) Concentration of
IL-1
β
. (
F
) Concentration of IL-6. (
G
) Concentration of IL-8. The measures represent mean
±
SD.
**: compared
with the blank control group, p < 0.01; #: compared with the eCS model group, p< 0.05;
##: compared with the eCS model group, p< 0.01.
3.5. Changes in Relative Expression of ERK, JNK and p38 MAPK Phosphorylated Proteins in
Mousee Lung
The phosphorylation of the MAPK pathway is one of the key characteristics of oxida-
tive stress and inflammation caused by eCS. According to the results in Figure 7, compared
with the blank control group, the phosphorylation levels of the ERK, JNK and p38 proteins
in the lungs of the eCS model group were significantly increased (p< 0.01). Compared
with the eCS model group, the phosphorylation level of the ERK and JNK proteins in the
ECP and ECT treatment groups decreased significantly (p< 0.01). The phosphorylation
level of the p38 protein in the ECP treatment group decreased significantly (p< 0.05), and
the phosphorylation level of the p38 protein in the ECT treatment group decreased, but
there was no significant difference (p> 0.05). The phosphorylation levels of the JNK protein
significantly decreased in the ECP prevention group (p< 0.05), and the phosphorylation
Nutrients 2022,14, 2843 11 of 17
levels of the ERK and P38 protein were decreased but there was no significant difference
(p> 0.05). The phosphorylation levels of ERK and P38 significantly decreased in the ECT
prevention group (p< 0.05), while the phosphorylation levels of JNK decreased, but there
was no significant difference (p> 0.05).
Nutrients 2022, 14, x FOR PEER REVIEW 12 of 18
nificant difference (p > 0.05). The phosphorylation levels of ERK and P38 significantly de-
creased in the ECT prevention group (p < 0.05), while the phosphorylation levels of JNK
decreased, but there was no significant difference (p > 0.05).
Figure 7. The effects of ECP and ECT on the expression of the p38, p-p38, JNK, p-JNK, ERK and p-
ERK proteins in the lung of mice exposed to eCS. Western blot analysis for the expression of phos-
phorylated (A) p-ERK and total ERK, (B) p-JNK and total JNK, (C) p-p38 and total p38 in lung tis-
sues of mice induced by eCS. The measures represent mean ± SD. **: compared with the blank con-
trol group, p < 0.01; #: compared with the eCS model group, p < 0.05; ##: compared with the eCS
model group, p < 0.01.
3.6. Changes in Relative Expression of PXR and AhR Proteins in Liver Tissue of Mice
To assess the preventive and therapeutic ability of ECT and ECP against eCS-induced
liver damage, the PXR/AhR signaling pathway was investigated by Western blot. Accord-
ing to the results in Figure 8, the protein expression of PXR in the liver of the eCS model
group was lower than those of the normal control group (p > 0.05); the protein expression
of AhR in the liver of the eCS model group was significantly lower than those of the nor-
mal control group (p < 0.01). Compared with the eCS model group, the protein expression
of PXR and AhR in the ECP and ECT treatment groups significantly increased (p < 0.05),
and the protein expression of PXR and AhR in the ECP prevention group increased, but
there was no significant difference (p > 0.05). The protein expression of PXR and AhR in
the ECT prevention group significantly increased (p < 0.05).
Figure 8. The effects of ECP and ECT on the expression of the PXR and AhR protein in the liver of
mice exposed to eCS. Western blot analysis for the expression of (A) PXR and (B) AhR in liver tissues
Figure 7.
The effects of ECP and ECT on the expression of the p38, p-p38, JNK, p-JNK, ERK and
p-ERK proteins in the lung of mice exposed to eCS. Western blot analysis for the expression of
phosphorylated (
A
) p-ERK and total ERK, (
B
) p-JNK and total JNK, (
C
) p-p38 and total p38 in lung
tissues of mice induced by eCS. The measures represent mean
±
SD. **: compared with the blank
control group, p < 0.01; #: compared with the eCS model group, p< 0.05; ##: compared with the eCS
model group, p< 0.01.
3.6. Changes in Relative Expression of PXR and AhR Proteins in Liver Tissue of Mice
To assess the preventive and therapeutic ability of ECT and ECP against eCS-induced
liver damage, the PXR/AhR signaling pathway was investigated by Western blot. Accord-
ing to the results in Figure 8, the protein expression of PXR in the liver of the eCS model
group was lower than those of the normal control group (p> 0.05); the protein expression
of AhR in the liver of the eCS model group was significantly lower than those of the normal
control group (p< 0.01). Compared with the eCS model group, the protein expression of
PXR and AhR in the ECP and ECT treatment groups significantly increased (p< 0.05), and
the protein expression of PXR and AhR in the ECP prevention group increased, but there
was no significant difference (p> 0.05). The protein expression of PXR and AhR in the ECT
prevention group significantly increased (p< 0.05).
Nutrients 2022, 14, x FOR PEER REVIEW 12 of 18
nificant difference (p > 0.05). The phosphorylation levels of ERK and P38 significantly de-
creased in the ECT prevention group (p < 0.05), while the phosphorylation levels of JNK
decreased, but there was no significant difference (p > 0.05).
Figure 7. The effects of ECP and ECT on the expression of the p38, p-p38, JNK, p-JNK, ERK and p-
ERK proteins in the lung of mice exposed to eCS. Western blot analysis for the expression of phos-
phorylated (A) p-ERK and total ERK, (B) p-JNK and total JNK, (C) p-p38 and total p38 in lung tis-
sues of mice induced by eCS. The measures represent mean ± SD. **: compared with the blank con-
trol group, p < 0.01; #: compared with the eCS model group, p < 0.05; ##: compared with the eCS
model group, p < 0.01.
3.6. Changes in Relative Expression of PXR and AhR Proteins in Liver Tissue of Mice
To assess the preventive and therapeutic ability of ECT and ECP against eCS-induced
liver damage, the PXR/AhR signaling pathway was investigated by Western blot. Accord-
ing to the results in Figure 8, the protein expression of PXR in the liver of the eCS model
group was lower than those of the normal control group (p > 0.05); the protein expression
of AhR in the liver of the eCS model group was significantly lower than those of the nor-
mal control group (p < 0.01). Compared with the eCS model group, the protein expression
of PXR and AhR in the ECP and ECT treatment groups significantly increased (p < 0.05),
and the protein expression of PXR and AhR in the ECP prevention group increased, but
there was no significant difference (p > 0.05). The protein expression of PXR and AhR in
the ECT prevention group significantly increased (p < 0.05).
Figure 8. The effects of ECP and ECT on the expression of the PXR and AhR protein in the liver of
mice exposed to eCS. Western blot analysis for the expression of (A) PXR and (B) AhR in liver tissues
Figure 8.
The effects of ECP and ECT on the expression of the PXR and AhR protein in the liver of
mice exposed to eCS. Western blot analysis for the expression of (
A
) PXR and (
B
) AhR in liver tissues
of mice induced by eCS. The measures represent mean
±
SD. **: compared with the blank control
group, p < 0.01; #: compared with the eCS model group, p< 0.05; ##: compared with the eCS model
group, p< 0.01.
Nutrients 2022,14, 2843 12 of 17
4. Discussion
The results show that both ECP and ECT can have antioxidant and anti-inflammatory
effects in the lung through the MAPK pathway and detoxification roles in liver through
the PXR/AhR pathway to decrease eCS-induced injury. Oxidative stress induced by eCS
plays an important role in the process of causing lung and liver injury. ECP and ECT
could improve oxidative stress and increase the activity of antioxidant enzymes in serum,
which may play an antioxidant role in eCS-induced oxidative damage by inhibiting MDA
and promoting the production of GSH and SOD. Overall, the antioxidant capacity of the
treatment group was better than that of the prevention group, which may be due to the
mice in the treatment group not being exposed to eCS in the third month. It is worth noting
that the therapeutic effect of ECP is better than that of ECT, while the preventive effect of
ECT is better than that of ECP. The other feature of damage caused by eCS is inflammation,
and oxidative stress can also amplify the degree of inflammatory damage [
23
,
24
]. eCS and
its atomized harmful substances can induce the release of pro-inflammatory cytokines,
such as TNF-
α
, IL-1
β
, IL-6 and IL-8, thus aggravating inflammation and apoptosis [
9
,
25
].
The results of this study show that the levels of the TNF-
α
, IL-1
β
, IL-6 and IL-8 pro-
inflammatory factors in mice exposed to eCS increased significantly, but both ECP and ECT
reversed this situation; the trend of the anti-inflammatory effect was that the therapeutic
effect was better than the preventive effect, and the therapeutic effect of ECP was better
than that of ECT, while the preventive effect of ECT was better than that of ECP. This result
is also consistent with the lung histopathological observation.
To further explore the mechanism of oxidative stress and anti-inflammation induced
by ECP and ECT on eCS exposure through the MAPK pathway, studies have shown that
cigarette smoke is a potential ERK trigger that can induce inflammation and oxidative stress
by activating ERK signaling cascades in the lungs [
26
]. In chronic obstructive pulmonary
disease (COPD), activation of ERK produces pro-inflammatory cytokines, such as TNF-
α
,
IL-1
β
, IL-6 and IL-8 [
27
,
28
]. JNK and p38 are also two important signaling pathways in the
MAPK family. Phosphorylated by signaling factors, they can act on a variety of downstream
transcription factors and produce stress responses such as
inflammation [29,30]
. eCS can
activate the p38 MAPK pathway and promote inflammation, which may be caused by
acrolein produced by the heating of glycerol in eCS [
31
,
32
]. Glyoxal and methyl glyoxal in
electronic cigarette smoke induce the expression of proinflammatory cytokines in human
nasal epithelial cells throughERK1/2 and p38MAPK [
10
]. In addition, some studies have
shown that nicotine can cause oxidative stress and activate p38 MAPK, but it is not the
only factor that activates the MAPK pathway [
11
,
33
]. In our experimental study, long-term
exposure to eCS significantly increased the level of ERK, JNK and p38 MAPK phosphoryla-
tion in the lung tissue of mice, and the treatment group showed a good therapeutic effect in
reducing the level of MAPK phosphorylation, except that the ECT treatment group could
not effectively reduce the level of p38 phosphorylation. ECT treatment also decreased
the phosphorylation levels of ERK and p38 MAPK in the prevention group, but the effect
was not as good as that in the treatment group, which may be due to the fact that the
phosphorylation ability of MPAK caused by daily exposure to eCS was better than the
repair ability of ECP and ECT, and the effect of ECP prevention group on reducing the
phosphorylation level was less obvious. It is worth noting that ECP treatment tends to
normalize the level of MAPK phosphorylation caused by eCS. In addition to the immune
repair ability of the body, according to the previous metabolomics data, ECP’s excellent
therapeutic effect may be related to its high content of amino acids and their derivatives,
organic acids, alkaloids and sugars. In addition, other high levels of active substances
in ECP also play an important role in the MAPK pathway, such as protocatechuic acid,
ginkgolide K and kahweol. Protocatechuic acid can inhibit the inflammation induced by
lipopolysaccharide in primary keratinocytes by reducing the activation of the Toll-like
receptor (TLR4)/NF-
κ
B signaling pathway and of the JNK and p38-MAPK pathways [
34
].
Ginkgolide K can reduce oxygen-glucose-induced SH-SY5Y cell injury by inhibiting the p38
and JNK signaling pathways [
35
]. Kahweol may inhibit the phosphorylation of JNK1/2 and
Nutrients 2022,14, 2843 13 of 17
p38 MAPK and the activation of NF-
κ
B to reduce the expression of phorbol 12-myristate
13-acetate (PMA)-enhanced matrix metalloproteinase-9 (MMP-9) to promote an anti-tumor
effect [
36
]. In addition, studies have shown that the extracellular polysaccharide secreted
by Eurotium cristatum, which is composed of mannose, glucose and galactose, has a strong
immunomodulatory ability. The polysaccharide controls the activation of phagocytes,
and indirectly kills pathogens by improving the levels of TNF-
α
, IL-1
β
, IL-6, IFN-
γ
and
NO, and it has shown effective immunomodulatory activity [
37
,
38
]. It is also associated
with the ability of the cell surface receptor TLR4 to bind, which mediates the activation
of the signal-regulated protein kinases ERK, JNK and p38 MAPK, thus inducing the ex-
pression of immune factor genes [
39
]. In addition, in Eurotium cristatum, an unreported
allyl benzaldehyde derivative, cristaldehyde A, and an unreported quinone derivative,
cristaquinone A, have been found, and its five known compounds also have significant
anti-inflammatory and free radical scavenging activity [
40
]. This is consistent with the
results of previous oxidative stress and inflammatory factor level tests, which suggests that
the protective mechanism of ECP and ECT against eCS-induced lung injury may be at least
partly attributed to the enhancement of antioxidant and anti-inflammatory defense systems
by inhibiting the MAPK signaling pathway.
Pregnane X receptor (PXR) and aromatic hydrocarbon receptor (AhR) are mainly
expressed in the liver and participate in regulating drug metabolism enzymes in the liver.
PXR is a primary chemical sensor, and it is known that it can induce the expression of
the CYP3A enzyme to regulate substance metabolism and detoxification function. AhR
is a ligand-activated transcription factor that mediates downstream regulatory genes of
the CYP1A enzyme to help decompose environmental toxins [
41
,
42
]. However, some
studies have shown that there is crosstalk between them in regulating the expression of
cytochrome P450 family genes in the liver [
43
]. In this study, eCS significantly decreased
the levels of PXR and AhR, and this situation was reversed after intragastric administration
of ECP and ECT; however, the effect of the treatment group was still better than that of the
prevention group. It is worth noting that the ECP treatment group tended to normalize
the decline in PXR levels caused by eCS exposure, showing an excellent detoxification
effect. This phenomenon also corresponds to the excellent regulation ability of the ECP
group in the MAPK pathway, indicating that ECP is more suitable as a therapeutic drug
for eCS exposure injury at the same concentration as ECT. However, surprisingly, like the
previous indicators of oxidative stress, inflammatory factors and the MAPK pathway, the
ECT prevention group was better than the ECP prevention group in reversing the decrease
in PXR and AhR induced by eCS exposure. Therefore, we speculate that ECT without
intentional removal of ECP contains not only phenols and their derivatives, phenolic
acids, glycosides, flavonoids, lignans, terpenes and other active substances in tea, but
also amino acids and their derivatives, organic acids, alkaloids and carbohydrates in ECP,
which activate various immune factors and trend factors and have a more comprehensive
immunomodulatory function. It can enhance the resistance of mice to electronic cigarette
smoke and reduce the degree of damage caused by daily eCS exposure. In addition to
detoxification, drug metabolic enzyme reactions can lead to biological activation, causing
inflammation, leading to tissue damage [
44
]. Some studies have shown that PXR is the
main receptor for exogenous substances, and the inflammatory environment causes the
activation of the NF-
κ
B transcription factor, which inhibits the activity and function of
the PXR protein, leading to the decline in the down-stream expressed CYP3A enzyme,
and finally down-regulating the drug metabolism ability [
45
]. Zhang [
46
] showed that
activation of PXR can inhibit NF-
κ
B activity to reduce inflammation; there is a feedback
regulation in this pathway. Related studies have reported that the AhR binding site is very
close to or overlaps with the NF-
κ
B binding site, which suggests that AhR and NF-
κ
B may
have potential intermodulation and crosstalk effects [
47
]. Experimental studies in animal
models of various inflammatory diseases have demonstrated that activation of AhR can
participate in a variety of cellular functions such as the regulation of the immune system
and the suppression of the inflammatory response [
48
–
50
]. The experimental results of
Nutrients 2022,14, 2843 14 of 17
the PXR/AhR signaling pathway are indeed consistent with the reported results and a
negative trend was observed between PXR/AhR and the level of inflammation (Figure 9).
However, how the nuclear receptor PXR/AhR and the NF-
κ
B pathway transcriptionally
regulate the inflammation induced by eCS exposure, as well as how ECP and ECT reduce
the inflammation caused by eCS exposure in this signaling pathway, remains unclear, and
we need further experiments to elucidate this.
Nutrients 2022, 14, x FOR PEER REVIEW 15 of 18
to or overlaps with the NF-κB binding site, which suggests that AhR and NF-κB may have
potential intermodulation and crosstalk effects [47]. Experimental studies in animal mod-
els of various inflammatory diseases have demonstrated that activation of AhR can par-
ticipate in a variety of cellular functions such as the regulation of the immune system and
the suppression of the inflammatory response [48–50]. The experimental results of the
PXR/AhR signaling pathway are indeed consistent with the reported results and a nega-
tive trend was observed between PXR/AhR and the level of inflammation (Figure 9). How-
ever, how the nuclear receptor PXR/AhR and the NF-κB pathway transcriptionally regu-
late the inflammation induced by eCS exposure, as well as how ECP and ECT reduce the
inflammation caused by eCS exposure in this signaling pathway, remains unclear, and we
need further experiments to elucidate this.
ECP and ECT can effectively ameliorate the lung injury and hepatotoxicity caused by
eCS. In addition, the good therapeutic effect of ECP and the good preventive effect of ECT
can be considered for synergistic use against lung tissue damage caused by eCS, but some
studies have shown that the drug–drug interaction may lead to adverse drug reactions, or
even synergistic toxicity [51]. Therefore, whether the combination of ECP and ECT will
cause hepatotoxicity or not needs to be verified through further experiments.
Figure 9. Effect of ECP and ECT on MAPK signaling pathways in eCS-induced lung injury and
PXR/AhR signaling pathway in eCS-induced liver injury in mice.
5. Conclusions
ECP and ECT down-regulated the phosphorylation levels of the ERK, JNK and p38
proteins in mouse lungs induced by eCS and reversed the decrease in the PXR and AhR
Figure 9.
Effect of ECP and ECT on MAPK signaling pathways in eCS-induced lung injury and
PXR/AhR signaling pathway in eCS-induced liver injury in mice.
ECP and ECT can effectively ameliorate the lung injury and hepatotoxicity caused by
eCS. In addition, the good therapeutic effect of ECP and the good preventive effect of ECT
can be considered for synergistic use against lung tissue damage caused by eCS, but some
studies have shown that the drug–drug interaction may lead to adverse drug reactions, or
even synergistic toxicity [
51
]. Therefore, whether the combination of ECP and ECT will
cause hepatotoxicity or not needs to be verified through further experiments.
5. Conclusions
ECP and ECT down-regulated the phosphorylation levels of the ERK, JNK and p38
proteins in mouse lungs induced by eCS and reversed the decrease in the PXR and AhR
protein levels in mouse livers induced by eCS. In conclusion, ECP and ECT reduced the
oxidative stress and inflammatory injury caused by eCS through the MAPK pathway, and
the hepatotoxicity caused by eCS through the PXR/AhR pathway. Therefore, ECT may
Nutrients 2022,14, 2843 15 of 17
be used in a healthy drink to reduce the risk of eCS-induced injury, and ECP might be a
potential therapeutic candidate for the treatment of eCS-induced injury.
Author Contributions:
S.X.: writing—original draft, data curation, formal analysis, methodology,
writing—review and editing. Y.Z.: investigation, data curation, writing—review and editing. X.H.:
supervision, formal analysis. L.Y.: methodology, supervision, funding acquisition, project administra-
tion, writing—review and editing. J.H.: resources, supervision. K.W.: formal analysis, supervision.
Z.L.: funding acquisition, project administration. All authors have read and agreed to the published
version of the manuscript.
Funding:
This research was supported by the National Key R&D Program of China (Grant no.
2018YFC1604403) and the Department of Science and Technology of Hunan Province (Grant
no. 2017NK2180).
Institutional Review Board Statement:
The animal study protocol was approved by Animal Experi-
mental Committee of Hunan Agricultural University (SCXK (Hunan) 2016-0002).
Informed Consent Statement: Not applicable.
Data Availability Statement:
The data used to support the results of this study are available from
the corresponding authors.
Acknowledgments:
Thanks to ChunLin Shi, 2019 student of School of Economics and Management,
The Chinese University of Hong Kong (Shenzhen), for the helpful discussion on this paper.
Conflicts of Interest: The authors declare no conflict of interest.
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