p38 MAPK inhibition alleviates experimental acute pancreatitis in mice

Shanghai, China

p38 MAPK inhibition alleviates experimental acute pancreatitis in mice

Ming-Hua Cao, Jing Xu, Hai-Dong Cai, Zhong-Wei Lv, Ya-Jing Feng, Kun Li, Chun-Qiu Chen and Yong-Yu Li

Shanghai, China

BACKGROUND:The mitogen-activated protein kinases (MAPKs) signaling pathway is involved in infammatory process. However, the mechanism is not clear. The present study was to investigate the role of p38 MAPK in acute pancreatitis in mice.

METHODS:Mice were divided into 4 groups: saline control; acute pancreatitis induced with repeated injections of cerulein; control plus p38 MAPK inhibitor SB203580; and acute pancreatitis plus SB203580. The pancreatic histology, pancreatic enzymes, cytokines, myeloperoxidase activity, p38 MAPK and heat shock protein (HSP) 60 and 70 were evaluated.

RESULTS:Repeated injections of cerulein resulted in acute pancreatitis in mice, accompanying with the activation of p38 MAPK and overexpression of HSP60 and HSP70 in the pancreatic tissues. Treatment with SB203580 signifcantly inhibited the activation of p38 MAPK, and furthermore, inhibited the expression of HSP60 and HSP70 in the pancreas, the infammatory cytokines in the serum, and myeloperoxidase activity in the lung.

CONCLUSION:The p38 MAPK signaling pathway is involved in the regulation of infammatory response and the expression of HSP60 and HSP70 in acute pancreatitis.

(Hepatobiliary Pancreat Dis Int 2015;14：101-106)

acute pancreatitis; p38 MAPK; heat shock protein

Introduction

Acute pancreatitis (AP) is a potentially lethal disease. Pro-infammatory cytokines and stress stimuli during progress of AP induce mitogen-activated protein kinase (MAPK) signaling cascades, mediating most of their effects on the immune response.[1]In mammalian cells, three major families of MAPKs have been found so far, including p38 MAPK, extracellular signalregulated kinase (Erk 1/2), and c-Jun N-terminal protein kinase (JNK). Among them, p38 MAPK is involved in the severity of pancreatitis and in the respiratory distress syndrome associated with AP.[2]However, the mechanism of p38 MAPK on AP is not clear.

Heat shock protein (HSP) 60 and HSP70, molecular chaperones in the HSP family, constitute the important components of the cell, and participate in a variety of physiological functions, such as protein synthesis, degradation, folding and transportation.[3-5]HSPs are overexpressed under stress such as AP and this increase protects the pancreas from infammatory injury.[6-8]It is known that the production of HSPs is regulated by heat shock transcription factor-1 (HSF1). In resting cells, HSF1 is inactive and is typically associated with preexisting HSPs in the cytoplasm. However, when stressed, HSPs are dissociated from HSF1 which is in turn binding consensus sites on DNA and activates HSPs transcription.[9]It is appreciated that HSF1 activity is modulated by phosphorylation which is related to MAPKs,[10,11]but the role of p38 MAPK in regulating HSP60 and HSP70 expression in AP is still not fully understood.

This study induced AP in mice by repeated injections of cerulein. The parameters of AP were assessed as described by Kubisch and colleagues,[12]and systemic infammation was determined by the serum infammatory mediator levels and lung myeloperoxidase (MPO) activity. Importantly, the effects of p38 MAPK on HSP60 and HSP70 expression and the possible roles in AP were studied by using a selected p38 MAPK inhibitor (SB203580).

Methods

Animals

Twenty-eight C57BL mice (purchased from Slater, Shanghai, China) weighing 18-22 g with an equal number for each sex were used in this study. The mice were housed for one week under standard condition with free access to water and laboratory chow. Prior to the experiment, the mice were fasted overnight with only water allowed. The animals' welfare and the experimental procedures were approved by the Animal Ethics Committee of Tongji University, Shanghai, China.

Induction of AP and pharmacological treatment

Cerulein (50 μg/kg) or saline (control) was given intraperitoneally for six times at hourly intervals to the mice to induce AP.[13]SB203580 (5 μmol/kg) was administered intraperitoneally[14]at 30 minutes before and 4 hours after the frst injection of cerulein or saline. The mice were divided into 4 groups: (1) saline (control,n=7); (2) cerulein (AP,n=7); (3) saline+SB203580 (SB,n=7) and (4) cerulein+SB203580 (AP+SB,n=7). Three hours after the fnal injection, the animals were euthanized by decapitation under isofurane anesthesia, and blood was collected. All of the blood samples were centrifuged and the serum was collected, aliquoted, and stored at -20 ℃ for measurement of pancreatic enzymes and infammatory mediators. The pancreas and lung tissues were quickly removed, rinsed with normal saline, weighed and divided into several portions. One portion of the pancreas was fxed in 10% formalin immediately, whereas others were frozen in liquid nitrogen and stored at -80 ℃ for later analysis.

Pathological examination

The pancreas was fxed in 10% formalin, paraffn embedded, sectioned and hematoxylin and eosin stained. The sections were studied under a light microscope. Multiple randomly chosen microscopic felds from at least four mice from each treatment group were examined and scored for semi-quantitative assessment on a scale of 0-3 (0 normal and 3 severe) blindly by a pathologist. The scoring was based on the number of acinar cell necroses, presence of vacuolization, interstitial edema, interstitial infammation as represented by infltrating infammatory cells, congestion of blood vessels, and extent to which these characteristics affected the organ as has been previously described.[12]

Amylase and lipase activity in supernatant or serum

The assays of amylase and lipase activity in serum were performed according to the manufacturer's protocol of the amylase and lipase assay kits (Jiancheng Technology, China). Results were measured as units per liter.

Levels of infammatory mediators in serum

The levels of interleukin-6 (IL-6) and cytokine-inducible neutrophil-chemoattractant 1 (CINC1/KC) in serum were quantifed using the mouse IL-6 and KC enzymelinked immunosorbent assay (ELISA) kits based on the manufacturer recommended protocols (H-Y Biological Co. Ltd., China). The optical density was determined at 490 nm for absorbance in an ELISA instrument (BioTek, USA). Each specimen was measured twice and data were calculated as picogram per milliliter.

MPO activity in the lung

MPO activity in the lung tissue was measured as described previously.[8]Tissue samples were homogenized with a homogenizer (PRO Scientifc Inc., USA) at 2400 rpm. An aliquot of this homogenate was taken for protein determination using a protein detection kit (BCA, Thermo); the rest was centrifuged at 10 000 g at 4 ℃ for 10 minutes. The supernatant was used for MPO measurement with the MPO assay kits according to the manufacturer recommended protocols (Jiancheng Technology, China). Each specimen was measured twice. The data were calculated as the units per milligram protein.

Levels of HSP60 and HSP70 in pancreatic tissues

Pancreatic tissues were homogenized and then centrifuged with the method described above. The supernatant was used for HSP60 and HSP70 measurement with mouse HSP60 and HSP70 ELISA kits (R&D Systems, USA). Meanwhile the protein content of the tissue was measured with the method described above, each specimen was measured twice and averaged, and the data were calculated as picogram per milligram protein.

Expression of p38 and phospho p38 in pancreas

The pancreatic tissues from the different treatment groups were homogenized, and lysed using the lysate buffer and the lysate was analyzed by Western blotting according to the method described previously.[15]Primary antibodies used in this study included anti-p38 and anti-phospho p38 (p-p38) in a 1:500 dilution (Santa Cruze Biotechnology, USA). After washing with TBST, the nitrocellulose membranes (Whatman GmbH, Germany) was incubated with the appropriate secondary antibody conjugated to horseradish peroxidase in a 1:5000 dilution at room temperature for 1 hour, washed with TBST. Finally, antibody binding was detected by enhanced chemiluminescent detection system (Santa CruzeBiotechnology, USA). A monoclonal GAPDH (1:1000 dilution) (Santa Cruze Biotechnology, USA) was used as an internal reference. All samples were measured in double and averaged, and experiments were repeated in at least four mice in each group. The band densities were analyzed with the ImageJ analysis system.

Statistical analysis

Results were presented as mean±SEM, and statistical analysis was performed using the SPSS package, version 19.0 for Windows. All data were analyzed by one-way ANOVA. Differences were considered as signifcant atPvalues <0.05.

Results

SB203580 attenuated cerulein-induced pancreatic morphological changes

No morphology changes were found in saline or saline plus SB203580 group (Fig. 1A, B). Cerulein-treated mice displayed histological signs of AP characterized by interstitial edema, necrosis of acinar cells and infltration of infammatory cells in pancreatic tissue (Fig. 1C). Treatment with SB203580 markedly attenuated these infammatory changes (Fig. 1D).

SB203580 attenuated cerulein-induced increase of amylase and lipase activities in serum

Compared to the control group, cerulein increased the levels of amylase and lipase (P<0.05), and administration of SB203580 signifcantly decreased these levels in cerulein-treated mice (Fig. 2).

SB203580 attenuated cerulein-induced increase of IL-6 and KC levels in serum, and MPO activity in the lung

The serum levels of IL-6 and KC in cerulein-treated mice were signifcantly elevated compared with the control group. Administration of SB203580 signifcantly decreased the levels of IL-6 and KC in the cerulein group (P<0.05) (Fig. 3).

Compared to the control group, cerulein signifcantly increased MPO activity in lung tissues (P<0.05), and SB203580 signifcantly decreased the MPO activity in the cerulein-treated mice (Fig. 4).

SB203580 mitigated cerulein-induced increase of HSP60 and HSP70 levels in the pancreas

Fig. 1.SB203580 attenuated cerulein-induced pancreatic morphological changes in mice (hematoxylin and eosin, original magnifcation ×200, scale bar =50 μm).A: A representative fgure from control group;B: A representative figure from SB group;C: A representative fgure from AP group;D: A representative fgure from AP+SB group;E: Histological scores of pancreas figures, and results are expressed as mean±SEM (n=4). *:P<0.01, vs the control group; #:P<0.01, vs the AP group.

Fig. 2.SB203580 attenuated cerulein-induced increase of amylase (A) and lipase (B) activities in serum of mice. The data were presented as mean±SEM (n=7). *:P<0.05, vs the control group; #:P<0.05, vs the AP group.

Fig. 3.SB203580 attenuated cerulein-induced increase of IL-6 (A) and KC (B) levels in serum of mice. The data were presented as mean±SEM (n=7). *:P<0.05, vs the control group; #:P<0.05, vs the AP group.

Fig. 4.SB203580 attenuated cerulein-induced increase of MPO activity in the lung of mice. The data were presented as mean±SEM (n=7). *:P<0.05, vs the control group; #:P<0.05, vs the AP group.

Fig. 5.SB203580 mitigated cerulein-induced increase of HSP60 (A) and HSP70 (B) levels in the pancreas of mice. The data were presented as mean±SEM (n=7). *:P<0.01, vs the control group; #:P<0.01 and ##:P<0.05, vs the AP group.

Fig. 6.SB203580 reduced the elevated expression of p38 and phospho p38 in pancreatic tissues of mice induced by cerulein.A: Profling of p38 and phospho p38 MAPK protein expression;BandC: Densitometric quantifcation of p38 and phospho p38 MAPK protein expression;D: Ratios of densitometric quantifcation between p-p38 and p38, and results were shown as mean± SEM (n=7). *:P<0.05, vs the control group; #:P<0.05, vs the AP group.

Repeated injection of cerulein signifcantly increased the levels of HSP60 and HSP70 in the pancreas (Fig. 5,P<0.05), and SB203580 signifcantly reduced the levels of pancreatic HSP60 and HSP70 in cerulein-treated mice.

SB203580 reduced the elevated expression of p38 and phospho p38 in pancreatic tissues induced by cerulein

After repeated injection of cerulein, Western blotting analysis showed that the expression of p38 and its phosphorylated form (p-p38) in the pancreatic tissues of mice was signifcanty increased compared to the control group (P<0.05). Treatment with SB203580 signifcantly decreased the expression of both p38 and p-p38 (P<0.05).

Discussion

The pathogenesis of AP is that the abnormal activation of pancreatic enzyme results in the early injury of pancreatic acinar cells, which leads to a local infammatory reaction and subsequent systemic infammatory response. During the progress of AP, the p38 MAPK signaling pathway is involved in the regulation of NF-κB activation that plays a crucial role in the infammatory cascade.[16-18]Activation of p38 MAPK results in phosphorylation of MK2, a downstream protein kinase of p38 MAPK, and triggers the NF- κB and exaggerates infammation.[19]Our previous study[8]showed that MK2 gene deletion ameliorates the systemic infammatory responses in experimental AP. The present study revealed that the specifc p38 inhibitor SB203580 reduced p38 MAPK activity in cerulein-induced AP, attenuated the infammatory response and cytokine release in the pancreas of AP mice. In another study,[20]we found that SB203580 had similar effects in isolated pancreatic fragments stimulated by cerulein, further demonstrating that the p38 MAPK signaling pathway is involved in AP.

HSPs play important roles in protecting cells against stressful stimuli. It has been reported that many kinds of stress can activate the transcription factor HSF1, the latter binds to the heat shock element in the promoter of HSP genes[21,22]and results in HSP productions. A study[12]has shown that stimuli not only increase HSP27, but also its phosphorylation in pancreatic exocrine cells. Phosphorylated HSP27 protects the pancreas from ceruleininduced injury.[12]A wealth of fndings confrms that HSP60 and HSP70 overexpressions in response to stress protect the cells from the stress-induced injury.[23-27]Our previous study[28]showed that using small interfering RNA (siRNA) to knockdown HSP60, the cultured pancreatic tissues were more sensitive to cerulein-induced injuries, demonstrating the protective effect of HSP60 on pancreatic tissues. The present study showed that HSP60 and HSP70 expressions were increased in pancreatic tissues challenged by cerulein. It is interesting that the cerulein-induced over-expression of HSP60 and HSP70 were attenuated by administration of SB203580, an inhibitor of p38 MAPK. There are two possibilities, one is that p38 MAPKs have direct regulatory effect on HSPs and the other is that HSP60 and HSP70 parallel to the pancreatic damage, SB203580 alleviates cerulein-induced pancreatic injury and in parallel, decreases the expressions of HSP60 and HSP70. It is well documented that p38 MAPKs have direct regulatory effect on HSPs.[10,29,30]

Ko et al[31]found that the upregulation of HSP70 in cultured human corneal epithelial cells induced by PHSRN peptide could be blocked by SB203580, but was not affected by PD98059 or SP600125, inhibitors of Erk 1/2 and JNK, respectively. Additionally, SB203580 and p38 MAPK siRNA blocked HSP70 mRNA induction in human esophageal microvascular endothelial cells during heat shock response.[10]In parallel with these fndings, our studies supplied further evidence that p38 MAPK activated in the pancreatic tissues of AP mice, accompanied with the overexpression of HSP60 and HSP70, and these changes were blocked by SB203580. Collectively, our data revealed that the activation of p38 MAPK in the pancreatic tissues of AP mice resulted in the elevation of infammatory mediators and infammatory responses. Moreover, the p38 MAPK signaling pathway might be involved in the regulation of HSP60 and HSP70. Further studies are needed to explore the mechanisms, for example, what are the roles of the downstream molecules, such as MK2, played in the AP process. Further studies will be of high value in explaining the AP development and may breed new therapeutic clues for AP.

Acknowledgement:We thank Professor Pei-Lin Zhao for assistance with the expertly histological evaluation.

Contributors:CMH and XJ contributed equally to the article. LYY designed the protocols of the research and supported the research. CMH, XJ, CHD, LZW, FYJ, LK and CCQ performed the experiments. CMH, XJ and LYY analyzed the data, and wrote the manuscript. LYY is the guarantor.

Funding:This study was supported by grants from the National Natural Science Foundation of China (30971168 and 81270477).Ethical approval:The animals' welfare and the experimental procedures were approved by the Animal Ethics Committee of Tongji University, Shanghai, China.

Competing interest:No benefts in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

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Received January 1, 2014

Accepted after revision June 4, 2014

AuthorAffliations:Department of Pathophysiology, Institute of Digestive Disease, Tongji University School of Medicine, Shanghai 200092, China (Cao MH, Xu J, Feng YJ, Li K and Li YY); The Seventh People's Hospital of Shanghai, Shanghai 200137, China (Cao MH); The Tenth People's Hospital of Shanghai, Tongji University School of Medicine, Shanghai 200072, China (Cai HD, Lv ZW and Chen CQ)

Yong-Yu Li, MD, Department of Pathophysiology, Institute of Digestive Disease, Tongji University School of Medicine, 1239 Siping Road, Shanghai 200092, China (Tel: +86-21-65981021; Fax: +86- 21-65987071; Email: liyongyu@tongji.edu.cn)

© 2015, Hepatobiliary Pancreat Dis Int. All rights reserved.

10.1016/S1499-3872(15)60327-7

Published online January 2, 2015.