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RESEARCH ARTICLE
Borneol protects against cerulein-induced oxidative stress and
inflammation in acute pancreatitis mice model
Sapana Bansod
1
| Shrilekha Chilvery
1
| Mohd Aslam Saifi
1
| Tridip Jyoti Das
2
|
Hui Tag
2
| Chandraiah Godugu
1
1
Department of Regulatory Toxicology,
National Institute of Pharmaceutical Education
and Research (NIPER), Hyderabad, Telangana,
India
2
Department of Botany, Rajiv Gandhi
University, Ron Hills, Doimukh, Arunachal
Pradesh, India
Correspondence
Dr. Chandraiah Godugu, Assistant Professor,
Department of Regulatory Toxicology,
National Institute of Pharmaceutical Education
and Research (NIPER), Balanagar, Hyderabad,
Telangana 500037, India.
Email: chandragodugu@gmail.com
Funding information
North East-Twinning, Grant/Award Number:
MAP/2015/58
Abstract
Borneol is a commonly used flavouring substance in traditional Chinese medicine,
which possesses several pharmacological activities including analgesic,
antiinflammatory, and antioxidant properties. The aim of this study was to investigate
the effects of borneol on cerulein-induced acute pancreatitis (AP) model. Swiss albino
mice were pretreated with borneol (100 and 300 mg/kg) daily for 7 days, before six
consecutive injections of cerulein (50 μg/kg/hr, intraperitoneally). The protective
effect of borneol was studied by biochemical, enzyme linked immunosorbent assay,
histological, immunoblotting, and immunohistochemical analysis. Oral administration
of borneol significantly attenuated pancreatic damage by reducing amylase, lipase
levels and histological changes. Borneol attenuated cerulein-induced oxidative-
nitrosative stress by decreasing malondialdehyde, nitrite levels, and elevating reduced
glutathione levels. Pancreatic inflammation was ameliorated by inhibiting
myeloperoxidase activity and pro-inflammatory cytokine (Interleukins and TNF-α)
levels. Furthermore, borneol administration significantly increased nuclear factor
E2-related factor 2 (Nrf2), superoxide dismutase (SOD1) expression and reduced
phospho-NF-κB p65 expression. Treatment with borneol significantly inhibited TNF-
α, IL-1β, IL-6, and inducible nitric oxide synthase expression in cerulein-induced AP
mouse model. Together, these results indicate that borneol which is currently used as
US-FDA approved food adjuvant has the potential to attenuate cerulein-induced AP
possibly by reducing the oxidative damage and pancreatic inflammation by modulat-
ing Nrf2/NF-κB pathway.
KEYWORDS
acute pancreatitis, borneol, inflammation, Nrf2/NF-κB signaling pathway, oxidative stress
1|INTRODUCTION
Acute pancreatitis (AP) is the most common gastrointestinal problems
with increasing incidences of morbidity and mortality over the recent
years.
1
AP is mainly characterized by acinar cells atrophy, necrosis,
activation of digestive enzymes (amylase and lipase), and inflamma-
tory cell aggregation in the pancreas.
2,3
The actual molecular mecha-
nism behind the pathophysiology of AP is poorly understood. Based
on the literature, oxidative stress and activation of inflammatory cas-
cades are majorly involved in the pathophysiology of AP.
4
Oxidative
stress is widely studied pathological event which plays a central role
in AP development.
5
During AP, over production of oxygen radicals
results in the downregulation of endogenous antioxidant defense sys-
tem.
6
Under normal conditions, activation of endogenous defense sys-
tem namely superoxide dismutase (SOD), glutathione (GSH), and
catalase enzymes scavenge the oxygen free radicals and protect the
Received: 7 November 2019 Revised: 9 September 2020 Accepted: 22 October 2020
DOI: 10.1002/tox.23058
Environmental Toxicology. 2020;1–10. wileyonlinelibrary.com/journal/tox © 2020 Wiley Periodicals LLC 1
cells against oxidative stress and external stimuli.
7
Nuclear transcrip-
tion factor, nuclear factor E2-related factor 2 (Nrf2) is a master regula-
tor of the variety of antioxidant and cytoprotective enzymes. Several
studies reported that the downregulation of Nrf2 expression in
cerulein-induced AP.
8,9
In nucleus, it forms complex with antioxidant
responsive element (ARE) and regulates the gene expression of sev-
eral antioxidant and cytoprotective enzymes including SOD, catalase,
and heme oxygenase-1 to detoxify the oxidative stress.
10,11
In addi-
tion to oxidative stress, inflammatory signaling also equally contrib-
utes to the pathogenesis of AP. Oxidative stress developed in
pancreas also triggers the release of large number of inflammatory
cells and activation of nuclear factor kappa-light-chain-enhancer of
activated B cells (NF-κB) which upregulates the transcription of
inflammatory cytokines (tumor necrosis factor-alpha (TNF-α), interleu-
kins (IL)-6 and IL-1β) and causes pancreatic and systemic inflamma-
tion. Based on the AP pathogenesis, there is a need to find safe and
effective therapeutic treatment against this severe inflammatory dis-
ease condition.
Recently, natural products have gained popularity for their
unrevealed antioxidant and antiinflammatory properties for treating
various acute and chronic inflammatory diseases.
12,13
(+) -Borneol is
a naturally occurring bicyclic monoterpenoid alcohol-based aromatic
flavoring substance of plant Acorus calamus, which possesses differ-
ent biological effects, such as analgesic and neuroprotective activi-
ties.
14,15
Moreover, it is the most commonly used ancient herb in
traditional Chinese medicine, which has several clinical applica-
tions.
16
Besides, borneol also used in the treatment of wound
healing, treating burns, sore throat, and skin infections in folk reme-
dies.
17
Borneol exhibited significant protective effect against global
cerebral ischemia-reperfusion injury model by inhibiting oxidative
injury, apoptosis, and inflammatory reaction.
18
Moreover, a recent
study has found that borneol ameliorated cerebral ischemia by
downregulating the production of pro-inflammatory mediators, such
as inducible nitric oxide synthase (iNOS) and TNF-α,inrats.
19
Fur-
ther, the neuroprotective effect of borneol was reported in an ische-
mic model of oxygen-glucose deprivation followed by reperfusion.
20
Borneol was found to be a promising molecule for the treatment of
neuropathic pain in the hyperalgesia model.
21
Agrowingnumberof
evidences have shown that borneol is a very good penetration
enhancer. Borneol binds with the lipid parts of cell membranes and
modulates the activity of variety of enzymes, ion channels, carriers,
and receptors.
22
Additionally, borneol also penetrates into the
blood-brain barrier (BBB) and enhance bioavailability of other
drugs.
22
Other evidence showed that borneol protects against oxi-
dative DNA damage in primary rat hepatocytes.
23
To the best of our
knowledge, there are no reports on borneol as an antipancreatitis
agent till date. So based on these literatures, we hypothesize that
borneol may alleviate cerulein-induced AP. The protective effect of
borneol was assessed by amylase, lipase and alanine aminotransfer-
ase (ALT) levels; oxidative-nitrosative stress parameters, pro-
inflammatory cytokine (IL-1β, IL-6, and TNF-α) levels and histopath-
ological changes in the pancreas, liver and lungs were evaluated.
Further, molecular mechanism was investigated by immunoblot
analysis of Nrf2, SOD1, IL-1β, IL-6, TNF-α, iNOS, and NFκBp65
expression in the pancreatic tissues.
2|MATERIALS AND METHODS
2.1 |Experimental animals and study design
Male Swiss albino mice (25-30 g) were acclimatized for 4 to 5 days
before the beginning of the experiment. Obtained experimental mice
were approved by Institutional Animal Ethics Committee (IAEC),
NIPER-Hyderabad. All animals were provided controlled environmen-
tal temperature range (24 ± 2C) with 55 ± 10% relative humidity and
12 hours light/dark cycle with free access to water and laboratory
chow throughout the experimental conditions.
Mice were randomized into five groups. The normal control mice
were administered saline via intraperitoneal route. AP was induced via
six intraperitoneal (ip) injections of cerulein (50 μg/kg/hour). Borneol
alone group animals received borneol (300 mg/kg, orally) suspended
in 2% tween 80 daily for seven consecutive days. Borneol low dose
group mice received borneol (100 mg/kg, orally) while borneol high
dose group animals received borneol (300 mg/kg, orally), daily for
seven consecutive days. After 7 days pre-treatment of borneol low
dose and high dose animals were induced AP by six injections of
cerulein (50 μg/kg, ip) at an interval of 1 hour apart and animals were
sacrificed after 6 hours of cerulein last injection (Figure 1). Like many
natural products due to their poor bioavailability issues, oral dosing
requires significantly higher doses compared to other routes of admin-
istrations mainly parenteral routes. In the present study, we have
selected the borneol doses based on the previously published
FIGURE 1 Experimental design and intervention of borneol in
cerulein-induced AP. Mice were randomized into five groups. The
normal control mice were administered saline via intraperitoneal
route. AP was induced via six intraperitoneal (ip) injections of cerulein
(50 μg/kg/hr). Borneol alone group animals received borneol
(300 mg/kg, orally) suspended in 2% tween 80 daily for seven
consecutive days. Borneol low dose group mice received borneol
(100 mg/kg, orally) while borneol high dose group animals received
borneol (300 mg/kg, orally), daily for seven consecutive days. After
7 days pretreatment of borneol low dose and high dose animals were
induced AP by six injections of cerulein (50 μg/kg, ip) at interval of
1 hour and animals were sacrificed after 6 hours of cerulein last
injection [Color figure can be viewed at wileyonlinelibrary.com]
2BANSOD ET AL.
literature.
21
Moreover, we have selected the duration of borneol pre-
treatment on the basis of our earlier studies where protective agent
was given 7 days prior to the AP induction and found promising pre-
ventive effects against severe pancreatic damage.
4,24
Blood and vital
organs including pancreas, liver, and lung were collected for further
biochemical and histological assessment.
2.2 |Biomarkers estimation
Blood was collected before the sacrifice in heparin containing tubes
and centrifuged at 9168gfor 10 minutes at 4C and plasma was sepa-
rated for further analysis. Plasma amylase, lipase, and ALT activities
were determined by using commercially available kinetic assay kits
(Accurex Biomedical Pvt. Ltd, India).
2.3 |Myeloperoxidase assay
Neutrophil sequestration in the pancreas was quantified by measuring
pancreatic myeloperoxidase (MPO) levels.
25,26
Briefly, pancreatic tis-
sues were homogenized in chilled 50 mM potassium phosphate buffer
(pH 6.0) containing 0.5% hexadecyl trimethyl ammonium bromide and
centrifuged at 9168gfor 10 minutes at 4C. The resulting pellet was
resuspended in the same buffer followed by centrifugation at 9168g
for 10 minutes at 4C and the supernatant was used to measure MPO
activity by using o-dianisidine. The absorbance was measured at
460 nm and expressed in units per mg of protein. The protein estima-
tion was done by using Bradford assay.
2.4 |Determination of oxidative stress parameters
Malondialdehyde (MDA), nitrite, and GSH contents were measured in
pancreatic tissue by following the previously described method.
27,28
Pancreatic tissue samples were homogenized in phosphate buffer and
homogenates were used for the estimation of MDA levels and
remaining tissue homogenate were centrifuged at 9168gfor
10 minutes at 4C and the supernatant was used for the estimation of
nitrite and GSH content in the pancreas. The MDA, GSH, and nitrite
content were expressed as μM/mg protein. The protein estimation in
the tissue homogenates and supernatants was done by Bradford
assay.
2.5 |Measurement of IL-6 and IL-1βlevels by
ELISA
Enzyme linked immunosorbent assay (ELISA) for inflammatory inter-
leukins including IL-6 and IL-1βwas performed as per the manufac-
turer's protocols (eBioscience, USA catalogue no. 88-7064 and
88-7013). Cytokine levels in the supernatants were measured and
expressed as picogram/mg protein.
2.6 |Histological and immunohistochemistry
evaluation
For histological evaluation, pancreatic tissues were fixed in 10% neu-
tral formalin buffer, dehydrated in gradient alcohols and cleared by
using xylene. Further, pancreatic tissues were embedded in paraffin
wax and sections were taken at 5 μm by using microtome (Leica, Ger-
many). Hematoxylin and eosin (H&E) staining was performed as per
the previously described methods.
27
Next, immunohistochemical
(IHC) staining was performed according to the manufacturer's proto-
col by Poly Excel HRP/DAB Detection System (PathnSitu, USA). Anti-
gen retrieval was carried out in deparaffinized and hydrated tissue
sections using citrate buffer (pH-6.0) at 95C for 20 minutes. Tissue
sections were kept in 3% H
2
O
2
for 15 minutes followed by blocking
with 3% bovine serum albumin for 1 hour. Further, sections were
incubated with primary antibodies for NF-κB p65 (1:200, Santa Cruz
Biotechnology, USA), overnight at 4C. Next day, sections were
washed with immuno buffer and incubated in the PolyExcel target
binder followed by PolyExcel HRP labeled polymer for 15 minutes
each. Sections were stained by using (DAB) substrate-chromogen
followed by counter staining with hematoxylin. Slides were
dehydrated, cleared, and mounted by using DPX. Stained slides were
observed under the light microscope (Olympus CX23, Japan) at ×40
magnification. The histological alterations in the pancreas, liver, and
lungs from each group were examined and scored semi-quantitatively
on the basis of previous literature in a blinded manner.
24,29,30
Extent
of damage in the pancreas, liver and lungs were scored on a scale of
0 to 3 (0: normal and 3: severe). On the other hand, IHC slides were
examined under the light microscope (Olympus, USA) and 10-15
visual fields from each group were observed, captured, and quantified
by using the ImageJ software (NIH, USA).
2.7 |Western blot analysis
Pancreatic tissues were homogenized in radioimmunoprecipitation
assay lysis buffer containing protease inhibitors followed by sonica-
tion and centrifugation. Then, supernatant was collected and protein
concentrations were estimated by Bicinchoninic acid assay. An equal
amount of protein were run on 10% sodium dodecyl sulfate polyacryl-
amide gel electrophoresis and transferred to a nitrocellulose mem-
brane following the standard protocol.
28,31
The primary antibodies,
such as NFκB p65 (Catalogue no.-D14E12, #8242) and p-NFκB p65
(Catalogue no.-93H1, #3033) were procured from Cell Signaling Tech-
nology, USA, whereas iNOS (Catalogue no.-N9657) were purchased
from Sigma–Aldrich, USA. Nrf-2 (Catalogue no.-sc-722), SOD1
(Catalogue no.-sc-17 767), TNF-α(Catalogue no.-sc-1350), IL-1β
(Catalogue no.-sc-7884), IL-6 (Catalogue no.-sc-1265), β-actin
(Catalogue no.-sc-47 778) and HRP conjugated secondary anti-mouse,
anti-rabbit, and anti-goat antibodies were purchased from Santa Cruz
Biotechnologies, CA, USA. The protein bands were visualized using an
enhanced chemiluminescence (ECL- Bio-Rad India Pvt. Ltd.) and
detected by chemiluminescence detector FusionFx (Vilber Lourmat,
BANSOD ET AL.3
France). Densitometric analysis was performed using the ImageJ soft-
ware (NIH, USA).
27
β-actin was used as a loading standard.
2.8 |Statistical analysis
All calculations were represented as mean ± SEM. Statistical compari-
sons between more than two groups were done by One-way ANOVA
followed by Tukey's multiple comparisons test by using the Graph Pad
Prism version 6.01 software. P-value <.05 was considered as statisti-
cally significant.
3|RESULTS
3.1 |Effect of borneol on body weight, organ
weight, plasma amylase, lipase, and ALT levels
Cerulein-treated AP mice observed a significant (P< .001) increase in
the pancreas weights, pancreatic weight/body weight ratio but no sig-
nificant difference was found in body weights reduction as compared
to the control group animals (Figure 2A-C). Pretreatment with borneol
in cerulein-treated AP mice significantly (P< .001) restored the
pancreas weights and pancreatic weight/body weight ratio. Amylase
and lipase are the gold standard for the diagnosis of AP. Therefore, to
examine the effect of borneol in AP, we assessed plasma amylase and
lipase levels. Our study results revealed that drastic increase in the
amylase and lipase levels in cerulein-induced AP mice, which indicate
successful induction of AP in mice. However, oral administration of
borneol markedly reduced the plasma amylase and lipase levels in
cerulein-exposed AP mice (Figure. 2D-E). Additionally, we also
assessed the pancreatitis-associated liver injury by measuring the
plasma ALT levels. Cerulein exposed mice showed increase in plasma
ALT levels while, borneol treatment reduced the plasma ALT levels as
compared to cerulein-treated AP mice (Figure 2F). Borneol alone
treated mice did not show any changes in the organ weights, plasma
amylase, lipase and ALT levels when compared to the normal control
mice indicating its safety.
3.2 |Borneol reduces MDA, nitrite, GSH and MPO
levels
The levels of MDA (Figure 3A,P< .05) and nitrite (Figure 3B) were sig-
nificantly increased in cerulein challenged AP animals when compared
to the normal control mice. Treatment with borneol resulted in
FIGURE 2 Effects of borneol on cerulein-induced AP. A, Body weight; B, Pancreas weight; C, Ratio of pancreatic weight to body weight
(mg of pancreatic weight/g body weight) and levels of, D, Plasma lipase; E, Plasma amylase and F, Plasma ALT. All values are given as mean ± SEM
(n = 5). ***P< .001 as compared to control group; #P< .05, ##P< .01, and ###P< .001, as compared to AP group. Oneway ANOVA followed by
Tukey's multiple comparisons test [Color figure can be viewed at wileyonlinelibrary.com]
4BANSOD ET AL.
significant decrease in levels of MDA and nitrite in the pancreatic tis-
sue. The content of endogenous antioxidant GSH (Figure 3C) was sig-
nificantly reduced in cerulein-treated AP mice compared to the
normal control group. Borneol treatment restored the GSH levels as
compared to AP control group. Further, the extent of neutrophil infil-
tration and accumulation in pancreatic tissues was measured by MPO
activity (Figure 3D). The AP group showed markedly increased in
MPO activity compared to normal control animals, while borneol
treatment significantly decreased pancreatic MPO activity.
3.3 |Borneol inhibits inflammatory cytokines IL-1β
and IL-6 levels
Effect of borneol on inflammatory cytokine levels were measured by
using ELISA method. The levels of IL-1βand IL-6 in pancreatic and
lung tissue were increased significantly in the AP group as compared
to the control mice (Figure 3E-H). As shown in Figure 3, treatment
with borneol significantly decreased IL-1βand IL-6 levels in pancreas
and lungs. Whereas, borneol alone treated mice did not show any
alteration in the studied inflammatory cytokines.
3.4 |Borneol activates Nrf2 and SOD1
In order to examine the effect of borneol on redox transcription fac-
tors, such as Nrf2 and antioxidant enzyme, SOD1 protein expression
was analyzed via western blot (Figure 4A). Our data showed a signifi-
cant decrease in Nrf2 and SOD1 expressions in the AP mice as com-
pared to the normal control mice. Interestingly, our intervention with
borneol led to significant increase in the Nrf2 and SOD1 expression
compared to the AP group (Figure 4C,D). These results indicated that
the borneol showed the potential antioxidant effect via activating
Nrf2 and SOD1 expression in the pancreatic tissue.
FIGURE 3 Effect of borneol on cerulein-induced changes in oxidative-nitrosative stress, MPO activity, pancreas and lungs IL-1βand IL-6
levels in cerulein-induced AP mice. A, MDA levels; B, Nitrite levels; C, GSH levels; D, MPO activity; E, Pancreas IL-1β; F, Pancreas IL-6; G, Lung
IL-1β; and H, Lung IL-6 levels. All values are given as mean ± SEM (n = 5). **P< .01, ***P< .001 as compared to control group; #P< .05, ##P< .01
and ###P< .001, as compared to AP group. Oneway ANOVA followed by Tukey's multiple comparisons test
BANSOD ET AL.5
3.5 |Borneol downregulates inflammatory
markers, such as iNOS, IL-1β, NF-κB, TNF-α, and IL-6
Effect of borneol on inflammatory mediators including iNOS, IL-1β,
TNF-α, and IL-6 expression was evaluated by immunoblotting and
the respective blots were shown in Figure 4B. We observed that
protein expression pattern of inflammatory mediators, such as
iNOS, IL-1β,TNF-α, and IL-6 were significantly (P< .001) increased
in the cerulein-treated AP animals when compared to the normal
healthy animals. The treatment of animals with borneol in AP ani-
mals significantly (<0.001) decreased the expression of iNOS, IL-1β
TNF-αand IL-6 (Figure 4E-I). Further, NF-κBp65expressionin
pancreatic tissue was performed by IHC and western blot analysis.
Our western blot results revealed that borneol oral administration
significantly inhibited NF-κB p65 expression in cerulein-treated AP
mice (Figure 4G). In addition to western blotting, IHC results also
revealed the expression of NF-κB p65 markedly upregulated in the
cerulein-induced AP animals. However, treatment with borneol
significantly inhibited NF-κB p65 expression (Supplementary
Figure 1A-B).
3.6 |Histological evaluation of the pancreas, lungs,
and liver
The effect of borneol on the histological architecture of the pan-
creas, liver, and lungs in cerulein-induced AP was performed by
H & E staining (Figure 5A-F). In normal control mice, no alteration
in the histological architecture of the pancreas was observed.
The cerulein-induced AP mice showed the significant alteration
in the histological architecture of pancreatic tissue such as acinar
cells atrophy, necrosis and inflammatory cell infiltration, while
treatment with borneol markedly reduced pancreatic atrophy and
inflammation (Figure 5A and 4D). Additionally, we also assessed
the cerulein-induced alterations in the liver and lungs histopa-
thology by H & E staining. AP mice showed the increase in the
extent of Kupffer cells, sinusoidal spaces between hepatocytes
and destruction of central vein architecture in the liver (Figure 5B
and 4E). Borneol administration markedly decreased the inflam-
matory cells infiltration and restored the liver histology. Further-
more, concurrent cerulein exposures increased the alveolar
thickening and number of inflammatory cell aggregation in the
FIGURE 4 Effect of borneol on immunoblot of A, Nrf2 and SOD1; B, Inflammatory mediators including iNOS, IL-1β, NF-κB, TNF-α, and IL-6
expression in the pancreas of cerulein-induced AP mice. C-I, The graph shows the densitometric analysis of Nrf2, SOD1, iNOS, IL-1β, NF-κB,
TNF-α, and IL-6 expression in pancreas. All values are given as mean ± SEM (n = 3-5). ***P< .001 as compared to control group; ##P< .01 and
###P< .001, as compared to AP group. Oneway ANOVA followed by Tukey's multiple comparisons test
6BANSOD ET AL.
lungs, whereas treatment with borneol significantly restored
the alveolar thickening and inflammation (Figure 5C and 4F).
Thus, these results strongly indicate the protective effect of bor-
neol against cerulein-induced AP and associated liver and lungs
injury.
4|DISCUSSION
AP is a common gastrointestinal disease, characterized by acute and
systemic inflammation associated with multiple organ failure.
32
Till
date very few pharmacological agents have reached the clinical trials
FIGURE 5 Effect of borneol on pancreatic damage and AP-associated liver and lung injury during cerulein-induced AP. A-F, Quantitative total
histology score and representative photomicrographs of H&E-stained pancreas, liver and lung tissue sections of control mice and mice pre-
treated with borneol (100-300 mg/kg) 7 days before the cerulein (50 μg/kg/hrs*6 injections) induction of AP at ×40 magnification. Red arrows-
inflammatory cell infiltration, blue arrows-sinusoidal spaces between hepatocytes and star (*)-destruction of central vein architecture in the liver.
All values are given as mean ± SEM. ***P< .001 as compared to control group; ###P< .001, as compared to AP group. Oneway ANOVA followed
by Tukey's multiple comparisons test. G, A schematic sketch shows the underlying mechanisms of borneol against cerulein-induced AP. Repetitive
cerulein exposure damaged acinar cell which results in the generation of oxidative stress and activation of inflammatory signaling in the pancreas.
Borneol inhibited cerulein-induced pancreatic injury, oxidative-nitrosative stress and pancreatic inflammation via activation of Nrf2 and inhibition
of NF-κB signaling pathway in AP mice model [Color figure can be viewed at wileyonlinelibrary.com]
BANSOD ET AL.7
for the intervention of AP, thus there is a need to find effective and
safe treatment options for AP.
33
Naturally occurring
phytoconstituents offer an attractive option for the prevention and
treatment of various diseases if incorporated in daily diet as supple-
ments.
34
Various studies have shown that phytoconstituents like cur-
cumin, berberine, withaferin, and visnagin found effective against
cerulein-induced AP.
4,8,35,36
Borneol is one of the promising
phytoconstituent with well-known antioxidant and antiinflammatory
activity. However, its role in AP is yet to be established. Therefore, in
this study, we have investigated the antipancreatitic effects of bor-
neol on cerulein-induced AP model. Our results showed that oral
administration of borneol at the doses of (100 and 300 mg/kg) ame-
liorated cerulein-induced AP and associated liver and lungs injury. The
promising pancreatic protective effect of borneol was found at the
dose of 300 mg/kg in cerulein-induced AP. In addition, borneol did
not induce any kind of adverse effects to animals as evident from the
borneol alone treated animal data and which is very much similar to
the normal control mice, indicating it is safe and effective for
therapeutic use.
Pancreatic biomarkers such as amylase and lipase are used for the
detection of AP.
37
Increased levels of these pancreatic digestive
enzymes are responsible for early-stage destruction of pancreatic acinar
cells and activation of inflammatory processes in the pancreas.
38
It is
reported that inhibition of these digestive enzymes would decrease the
AP and associated organ injury.
39
In the present study, cerulein-induced
AP mice resulted in a significant elevation of plasma lipase and amylase
levels. However, this increased levels of digestive enzymes were
inhibited by borneol treatment, which suggest the pancreatic protective
effects of borneol against cerulein-induced AP. Pancreatic acinar cell
damage and inflammatory cells aggregation are hallmark features of
AP. Cerulein-induced AP observed severe alterations in the pancreatic
histology including acinar cells atrophy, inflammatory cell accumulation
and pancreatic necrosis, these results were in correlation with earlier
reports.
40
Oral administration of borneol showed the protective effect
against cerulein-induced pancreatitis damage by attenuating acinar cells
atrophy, pancreatic necrosis and reduced inflammatory cell infiltrations.
Further, we observed significant alteration in the liver and lung histol-
ogy, which indicate the AP associated multiple organ dysfunctions and
these results were in line with the previous literature.
4
Treatment with
borneol prevented the histological damage to liver and lungs in
cerulein-induced AP. In addition, the elevated levels of pancreatic MPO
are significantly attenuated by borneol treatment, which suggests that
borneol inhibited leukocyte infiltration in the cerulein treated AP mice.
These results strongly suggest that daily consumption of food that con-
tains borneol might be helpful to maintain pancreatic health and attenu-
ate cerulein-induced AP.
Oxidative stress is one of the crucial events involve in the patho-
genesis of AP.
41
ROS directly bind with the lipids and proteins of the
cell membranes, which led to oxidative degradation and subsequently
result in the generation of lipid peroxide byproducts in the pancreatic
tissues.
42
The intrinsic defense is provided by the activation of Nrf2/
ARE signaling pathways by increasing transcription of endogenous
antioxidant moieties including GSH, SOD1, and catalase. It was
reported that borneol produced promising antioxidant effects in hepa-
tocytes and cortical neuronal cells by decreasing the ROS produc-
tion.
20,23
Consistent with the earlier literatures, we measured the
MDA levels in pancreatic tissue, which is the by-products of lipid per-
oxide, and antioxidant enzymes including GSH levels in the pancreas.
Borneol exhibited its antioxidant potential via significant reduction in
the MDA levels, whereas, elevation in the pancreatic GSH content.
Nrf2 is an antioxidant transcription protein that increases the tran-
scription of endogenous antioxidant enzymes and protects cells
against oxidative stress. After activation of Nrf2 upon nuclear translo-
cation, Nrf2 binds to the ARE and stimulates the transcription of cyto-
protective and antioxidant proteins.
43
Therefore, to study the
antioxidant effect of borneol on Nrf2/ARE signaling pathway in
cerulein-induced AP, we investigated the protein expression of Nrf2
and SOD1 via Western blot analysis. Cerulein-induced production of
oxidative stress is associated with the decreased expression of Nrf2
and SOD1 signaling pathway proteins.
11
Our study results revealed
that borneol activated Nrf2 signaling and increased the expression of
intracellular antioxidants GSH and SOD1 and attenuated cerulein-
induced oxidative stress in AP mice. These results were in line with
the previous literature where borneol reduced oxidative stress burden
in the cerebral ischemic model.
15
Hence, from our results we can pre-
sume that borneol conferred amelioration against cerulein-induced AP
via an antioxidative pathway.
Cerulein-induced oxidative stress in acinar cells triggers the acti-
vation of inflammatory signaling and release of inflammatory markers
like IL-1β, IL-6, and TNF-α.
44
NF-κB is a well-studied inflammatory
signaling mediator, which plays crucial role in variety of cellular pro-
cesses including cell survival. In cytoplasm, NF-κB is present in quies-
cent form with its subunit I-κB. Upon its activation, I-κB gets
phosphorylated and releases NF-κB, which increases NF-κB nuclear
translocation and stimulates the transcription of inflammatory media-
tors including IL-1β, IL-6 and TNF-α. The major trigger for the activa-
tion of NF-κB is the elevated oxidative stress, which further proceeds
to activation of inflammatory cascades.
20
Upregulation of NF-κB
expression was observed in variety of diseases and it is a key inflam-
matory signaling involved in the progression of AP.
45
The results of
present study revealed that cerulein-induced oxidative-nitrosative
stress, activated NF-κB p65 signaling and subsequently increased the
expression of inflammatory markers including TNF-α, IL-1β, IL-6 and
iNOS in pancreatic tissues were controlled by borneol. Our data is in
line with previous reports, where ROS regulate the activation of
inflammatory cascades in the cerulein-induced AP.
46,47
Treatment
with borneol effectively inhibited NF-κB p65 expression, an important
regulator of the inflammation and oxidative stress and led to the
downregulation of the inflammatory mediator's, such as TNF-α, IL-1β,
IL-6, and iNOS expression in the pancreatic tissues. Borneol-mediated
inhibition of NF-κB p65 expression was also associated with the ele-
vated intracellular antioxidant levels in the cerulein-induced AP. These
results are in accordance with the earlier studies where borneol
showed antioxidant and anti-inflammatory activity in experimental
cerebral ischemia injury and lung injury models via downregulation of
NF-κB signaling pathway.
14
These results indicate that borneol
8BANSOD ET AL.
showed promising antioxidant and anti-inflammatory effects against
cerulein-induced AP model (Figure 5G). Based on the above results,
we consider that the protective effect of borneol could be due to first
the reduction of oxidative stress via activation of Nrf2 activity and
second being highly hydrophobic agent; it easily penetrates into the
inflammatory macrophages and decreases cytokines production. The
third possible reason could be inhibition of NF-κB p65 expression,
which modulates the cytokines production and it may help in the acti-
vation of antioxidant machinery through Nrf2 activation.
5|CONCLUSION
Our data indicate that oral administration of borneol effectively atten-
uated cerulein-induced pancreatic injury, oxidative-nitrosative stress,
and inflammation by activation of Nrf2 and inhibition of NF-κB signal-
ing pathway in AP mice. Our results clearly suggested that borneol
could be a promising candidate for the treatment of AP. The natural
compounds like borneol can be given to people who are at high risk of
AP due to their chronic alcoholism or other risk factors. Therefore,
supplementing these protective agents may prevent or treat the
development of AP. In addition, future studies may be designed to
increase the efficiency of this orally active compound by reducing the
dose through novel oral drug delivery systems. Studies may also be
warranted to understand the more detailed mechanisms of protection
by using novel molecular techniques.
ACKNOWLEDGMENTS
The authors would like to thank Mr. Amit Khurana for his kind support
in experimental work. The authors acknowledge the Department of
Pharmaceuticals, Ministry of Chemicals and Fertilizers, Government of
India for supporting the research activities at NIPER-Hyderabad and
Department of Biotechnology (DBT), Govt. of India, for the financial
support to Dr. Chandraiah Godugu by North East-Twinning Grant:
MAP/2015/58.
CONFLICT OF INTEREST
The authors declare no potential conflict of interest.
ORCID
Chandraiah Godugu https://orcid.org/0000-0001-5904-3134
REFERENCES
1. Yuan J, Tan T, Geng M, Tan G, Chheda C, Pandol SJ. Novel small mol-
ecule inhibitors of protein kinase D suppress NF-kappaB activation
and attenuate the severity of rat cerulein pancreatitis. Front Physiol.
2017;8:1014.
2. Esrefoglu M, Gül M, Ates B, Yilmaz I. Ultrastructural clues for the pro-
tective effect of ascorbic acid and N-acetylcysteine against oxidative
damage on caerulein-induced pancreatitis. Pancreatology. 2006;6(5):
477-485.
3. Esrefoglu M, Gül M, Ates B, Batçio
glu K, Selimo
glu MA Antioxidative
effect of melatonin, ascorbic acid and N-acetylcysteine on caerulein-
induced pancreatitis and associated liver injury in rats. World J
Gastroenterol. 2006;12(2):259-264.
4. Tiruveedi VL, Bale S, Khurana A, Godugu C Withaferin A, a novel
compound of Indian ginseng (Withania somnifera), ameliorates
Cerulein-induced acute pancreatitis: Possible role of oxidative stress
and inflammation. Phytother Res. 2018;32(12):2586-2596.
5. Koiwai T, Oguchi H, Kawa S, Yanagisawa Y, Kobayashi T, Homma T.
The role of oxygen free radicals in experimental acute pancreatitis in
the rat. Int J Pancreatol. 1989;5(2):135-143.
6. Dabrowski A, Konturek SJ, Konturek JW, Gabryelewicz A. Role of oxi-
dative stress in the pathogenesis of caerulein-induced acute pancrea-
titis. Eur J Pharmacol. 1999;377(1):1-11.
7. Talalay P, Dinkova-Kostova AT, Holtzclaw WD. Importance of phase
2 gene regulation in protection against electrophile and reactive oxy-
gen toxicity and carcinogenesis. Adv Enzyme Regul. 2003;43:121-134.
8. Pasari LP, Khurana A, Anchi P, Aslam Saifi M, Annaldas S, Godugu C.
Visnagin attenuates acute pancreatitis via Nrf2/NFκB pathway and
abrogates associated multiple organ dysfunction. Biomed
Pharmacother. 2019;112:108629.
9. Liu X et al. isoliquiritigenin ameliorates acute pancreatitis in mice via
inhibition of oxidative stress and modulation of the Nrf2/HO-1 path-
way. Oxid Med Cell Longev. 2018;2018:7161592.
10. He X, Kan H, Cai L, Ma Q. Nrf2 is critical in defense against high
glucose-induced oxidative damage in cardiomyocytes. J Mol Cell Car-
diol. 2009;46(1):47-58.
11. Jung KH, Hong SW, Zheng HM, et al. Melatonin ameliorates cerulein-
induced pancreatitis by the modulation of nuclear erythroid 2-related
factor 2 and nuclear factor-kappaB in rats. J Pineal Res. 2010;48(3):
239-250.
12. Latruffe N. Natural products and inflammation. Molecules. 2017;
22(1).1–3.
13. Bansod S, Aslam Saifi M, Khurana A, Godugu C. Nimbolide abrogates
cerulein-induced chronic pancreatitis by modulating β-catenin/Smad
in a sirtuin-dependent way. Pharmacol Res. 2020;156:104756.
14. Zhong W, Cui Y, Yu Q, et al. Modulation of LPS-stimulated pulmonary
inflammation by Borneol in murine acute lung injury model. Inflamma-
tion. 2014;37(4):1148-1157.
15. Liu R, Zhang L, Lan X, et al. Protection by borneol on cortical neurons
against oxygen-glucose deprivation/reperfusion: involvement of anti-
oxidation and anti-inflammation through nuclear transcription factor
kappaappaB signaling pathway. Neuroscience. 2011;176:408-419.
16. Wang S, Zhang D, Hu J, Jia Q, Xu W, Su D. A clinical and mechanistic
study of topical borneol-induced analgesia. EMBO Mol Med. 2017;9
(6):802-815.
17. Almeida JRGds, Souza GR, Silva JC, Saraiva SRGdL, Júnior RGdO,
Quintans JdSS. Borneol, a bicyclic monoterpene alcohol, reduces
nociceptive behavior and inflammatory response in mice. Sci-
entificWorldJournal. 2013;2013:808460.
18. Yu B, Ruan M, Zhang Z-N, Cheng H-B, Shen X-C. Synergic effect of
borneol and ligustrazine on the neuroprotection in global cerebral
ischemia/reperfusion injury: a region-specificity study.Evid Based
Complement Alternat Med. 2016;2016:4072809.
19. Chang L et al. (+)-Borneol is neuroprotective against permanent cere-
bral ischemia in rats by suppressing production of proinflammatory
cytokines. J Biomed Res. 2017;31(4):306-314.
20. Liu R, Zhang L, Lan X, et al. Protection by borneol on cortical neurons
against oxygen-glucose deprivation/reperfusion: involvement of anti-
oxidation and anti-inflammation through nuclear transcription factor
κappaB signaling pathway. Neuroscience. 2011;176:408-419.
21. Jiang J, Shen YY, Li J, Lin YH, Luo CX, Zhu DY. (+)-Borneol alleviates
mechanical hyperalgesia in models of chronic inflammatory and neu-
ropathic pain in mice. Eur J Pharmacol. 2015;757:53-58.
22. Zhang QL, Fu BM, Zhang ZJ. Borneol, a novel agent that improves
central nervous system drug delivery by enhancing blood-brain bar-
rier permeability. Drug Deliv. 2017;24(1):1037-1044.
23. Horváthová E, Kozics K, Srancˇíková A, Hunáková L, Gálová E,
Ševcˇovicˇová A. Borneol administration protects primary rat
BANSOD ET AL.9
hepatocytes against exogenous oxidative DNA damage. Mutagenesis.
2012;27(5):581-588.
24. Godugu C, Pasari LP, Khurana A, et al. Crocin, an active constituent
of Crocus sativus ameliorates cerulein induced pancreatic inflamma-
tion and oxidative stress. Phytother Res. 2020;34(4):825-835.
25. Trivedi PP, Jena GB. Dextran sulfate sodium-induced ulcerative
colitis leads to increased hematopoiesis and induces both local as
well as systemic genotoxicity in mice. Mutat Res. 2012;744(2):
172-183.
26. Trivedi PP, Jena GB, Tikoo KB, Kumar V. Melatonin modulated
autophagy and Nrf2 signaling pathways in mice with colitis-
associated colon carcinogenesis. Mol Carcinog. 2016;55(3):255-267.
27. Bansod S, Khurana A, Godugu C. Cerulein-induced chronic pan-
creatitis in Swiss albino mice: an improved short-term model for
pharmacological screening. J Pharmacol Toxicol Methods. 2019;96:
46-55.
28. Bansod S, Doijad N, Godugu C. Berberine attenuates severity of
chronic pancreatitis and fibrosis via AMPK-mediated inhibition of
TGF-β1/Smad signaling and M2 polarization. Toxicol Appl Pharmacol.
2020;403:115162.
29. Jo IJ, Bae GS, Choi SB, et al. Fisetin attenuates cerulein-induced acute
pancreatitis through down regulation of JNK and NF-κB signaling
pathways. Eur J Pharmacol. 2014;737:149-158.
30. Leema G, Tamizhselvi R. Protective effect of Scopoletin against
Cerulein-induced acute pancreatitis and associated lung injury in
mice. Pancreas. 2018;47(5):577-585.
31. Chilvery S, Bansod S, Saifi MA, Godugu C. Piperlongumine attenuates
bile duct ligation-induced liver fibrosis in mice via inhibition of TGF-
β1/Smad and EMT pathways. Int Immunopharmacol. 2020;88:
106909.
32. Tenner S et al. American College of Gastroenterology guideline: man-
agement of acute pancreatitis. Am J Gastroenterol. 2013;108(9):1400-
1415.
33. Jha RK, Ma Q, Sha H, Palikhe M. Acute pancreatitis: a literature
review. Med Sci Monit. 2009;15(7).147–156.
34. Kim DG, Bae GS, Choi SB, et al. Guggulsterone attenuates cerulein-
induced acute pancreatitis via inhibition of ERK and JNK activation.
Int Immunopharmacol. 2015;26(1):194-202.
35. Wang Y, Bu C, Wu K, Wang R, Wang J. Curcumin protects the
pancreas from acute pancreatitis via the mitogen-activated pro-
tein kinase signaling pathway. Mol Med Rep. 2019;20(4):3027-
3034.
36. Choi SB, Bae GS, Jo IJ, Wang S, Song HJ, Park SJ. Berberine inhibits
inflammatory mediators and attenuates acute pancreatitis through
deactivation of JNK signaling pathways. Mol Immunol. 2016;74:
27-38.
37. Ismail OZ, Bhayana V. Lipase or amylase for the diagnosis of acute
pancreatitis? Clin Biochem. 2017;50(18):1275-1280.
38. Ikei S, Ogawa M, Yamaguchi Y. Blood concentrations of polymorpho-
nuclear leucocyte elastase and interleukin-6 are indicators for the
occurrence of multiple organ failures at the early stage of acute pan-
creatitis. J Gastroenterol Hepatol. 1998;13(12):1274-1283.
39. Schmidt J, Lewandrowski K, Castillo CFD, et al. Histopathologic cor-
relates of serum amylase activity in acute experimental pancreatitis.
Dig Dis Sci. 1992;37(9):1426-1433.
40. Xiao W et al. Protective effect of asiatic acid in an experimental
cerulein-induced model of acute pancreatitis in mice. Am J Transl Res.
2017;9(8):3842-3852.
41. Folch E, Gelpí E, Roselló-Catafau J, Closa D. Free radicals generated
by xanthine oxidase mediate pancreatitis-associated organ failure. Dig
Dis Sci. 1998;43(11):2405-2410.
42. Dabrowski A et al. Oxygen-derived free radicals in cerulein-induced
acute pancreatitis. Scand J Gastroenterol. 1988;23(10):1245-1249.
43. Fu X, Li P, Yin W, et al. Overexpression of Nrf2 protects against lipo-
polysaccharide and Cerulein-induced pancreatitis in vitro and in vivo.
Pancreas. 2020;49(3):420-428.
44. Yu JH, Kim H. Oxidative stress and inflammatory signaling in cerulein
pancreatitis. World J Gastroenterol. 2014;20(46):17324-17329.
45. KryzhevskiĭVV Nichitaĭlo ME, MedvetskiĭEB, MoshkovskiĭG The
role of cytokines in pathogenesis of acute pancreatitis. Klin Khir.
2000;2000(1):54-57.
46. Norman JG, Fink GW, Franz MG. Acute pancreatitis induces intra-
pancreatic tumor necrosis factor gene expression. Arch Surg. 1995;
130(9):966-970.
47. Lv C, Jin Q. Maresin-1 inhibits oxidative stress and inflammation and
promotes apoptosis in a mouse model of Caerulein-induced acute
pancreatitis. Med Sci Monit. 2019;25:8181-8189.
SUPPORTING INFORMATION
Additional supporting information may be found online in the
Supporting Information section at the end of this article.
How to cite this article: Bansod S, Chilvery S, Saifi MA,
Das TJ, Tag H, Godugu C. Borneol protects against cerulein-
induced oxidative stress and inflammation in acute
pancreatitis mice model. Environmental Toxicology. 2020;1–10.
https://doi.org/10.1002/tox.23058
10 BANSOD ET AL.
