Molecular Medicine Reports Molecular Medicine Reports 1791-2997 1791-3004 D.A. Spandidos 10.3892/mmr.2018.9595 mmr-18-06-5345 Articles Dexamethasone inhibits NF-кBp65 and HMGB1 expression in the pancreas of rats with severe acute pancreatitis ZhaoShangping 1 YangJinming 2 LiuTing 2 ZengJuanxian 3 MiLiangliang 2 XiangKaimin 2 ICU, The Third Xiangya Hospital of Central South University, Changsha, Hunan 410013, P.R. China Department of Gastrointestinal Surgery, The Third Xiangya Hospital of Central South University, Changsha, Hunan 410013, P.R. China Department of General Surgery, The Third Xiangya Hospital of Central South University, Changsha, Hunan 410013, P.R. China Correspondence to: Dr Kaimin Xiang, Department of Gastrointestinal Surgery, The Third Xiangya Hospital of Central South University, 138 Tongzipo Road, Yuelu, Changsha, Hunan 410013, P.R. China, E-mail: xiangkaiming2017@163.com 122018 25102018 18 6 5345 5352 02012018 23082018 Copyright: © Zhao et al. 2018 This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

Severe acute pancreatitis (SAP) starts as a local inflammation of pancreatic tissue that induces the development of multiple extra-pancreatic organ dysfunction; however, the underlying mechanisms remain unclear. The present study was designed to evaluate the effect of dexamethasone (DXM) on pancreatic damage and to investigate the role of high-mobility group box-1 (HMGB1) and nuclear factor-κB (NF-κBp65) in the development of SAP in animal and cell models. For the in vivo experiment, 35 Sprague-Dawley rats were randomly assigned to three groups: The sham-operation control group, the SAP group and the DXM treatment group. Histological analysis revealed that, when DXM was infused into SAP rats, edema formation and structural alterations with necrosis were reduced, and the number of apoptotic cells was markedly reduced. In addition, compared with the SAP group, the expression level of HMGB1 was significantly decreased in the nucleus and the expression level of NF-κBp65 was significantly decreased in the cytoplasm from rats treated with DXM. In vitro, DXM was able to suppress the apoptosis and cell death induced by caerulein (CAE), and DXM could suppress the expression of NF-κBp65 and HMGB1 induced by CAE, as demonstrated by western blotting and immunofluorescence analysis. Therefore, these results provide an experimental basis for investigating the underlying therapeutic mechanisms of DXM treatment for SAP.

 dexamethasone severe acute pancreatitis high-mobility group box-1 nuclear factor-κB Introduction

Acute pancreatitis is a relatively common disease and severe acute pancreatitis (SAP) is associated with a high mortality rate, ranging from 15–40% (1). SAP starts as a local inflammation of pancreatic tissue and is characterized by the development of systemic inflammatory response syndrome (SIRS) and multiple organ dysfunction (2,3). Unfortunately, despite several years of experimental and clinical research, the precise pathophysiology of SAP, particularly in the clinical context remains unclear. This has precluded the development of definitive treatment modalities for this potentially life-threatening illness.

Experimental and clinical studies over the past decade have reported the profiles of intrapancreatic and circulating cytokines, chemokines, adhesion molecules, transcription factors including nuclear factor (NF)-κB and high-mobility group box 1 (HMGB1) (4,5), the nucleotide-binding oligomerization domain receptor, Toll-like receptor (TLR)9 (6) and protective pathways, including the heme oxygenase-1 pathway and the peroxisome proliferator-activated receptor-γ which are involved in SAP (7).

NF-κB has increasingly gained attention over the past several years as a factor in human inflammation, immune regulation and cancer biology. Studies from the last two decades have suggested that NF-κB is an early, major activator of pro-inflammatory mediators in the pathogenesis of SAP (8–10). The HMGB1 protein, as a late mediator of endotoxin lethality, was demonstrated to exhibit a delayed release by cultured macrophages of more than 8 h following stimulation with endotoxin, tumor necrosis factor (TNF), or interleukin (IL)-15 (11,12). Furthermore, previous studies have demonstrated that HMGB1 may serve an important role in SAP (9,11) and it may act as a key inflammatory mediator that participates in the development of SIRS and multiple organ damage in SAP (10). HMGB1, TLR4 and NFκB are possible therapeutic targets for SAP treatment; therefore, HMGB1 antagonists, TLR4 antagonists and NFκB inhibitors should be considered. TLR4 antagonists are the closest to being used in a clinical setting for SAP treatment. Two TLR4 antagonists, namely VGX-1027 and eritoran are already in clinical development (13–16).

The inhibition of HMGB1 by sodium butyrate has been reported to have beneficial effects on SAP development; however, sodium butyrate is not a specific HMGB1 inhibitor (17).

Dexamethasone (DXM), a type of steroid medication, can improve microcirculation and inhibit enzymes and inflammatory mediators (18). It has been used to treat SAP, but its protective effects and associated mechanisms on pancreatic injury remain unclear. In the present study, it was hypothesized that HMGB1 and NF-κBp65 are involved in the therapeutic mechanism through which DXM acts on SAP. Experiments were performed to investigate the influence of DXM on the expression levels of NF-κBp65 and HMGB1, as well as on apoptosis, in pancreatic cells of SAP rats, to observe the therapeutic efficacy and investigate the underlying therapeutic mechanisms of DXM treatment.

 Materials and methods <sec> <title>Materials and reagents

Sodium taurocholate solution (TCA) was obtained from Beijing Solarbio Science & Technology Co., Ltd., (Beijing, China) and DXM injections were obtained from Hubei Tianyao Pharmaceutical Co., Ltd., (Fancheng, China) chloral hydrate was purchased from Sangon Biotech Co., Ltd., (Shanghai, China). The HMGB1 antibody was from Abcam (Cambridge, UK) and the NF-κB p65 antibody was from Cell Signaling Technology, Inc., (Danvers, MA, USA).

 Animals

In total, 35 male Sprague Dawley rats, 6–8 weeks old and weighing between 200–250 g, were purchased from Hunan Slake Jingda Experimental Animal Co. Ltd., (Changsa, China). The study was approved by the Ethics Committee of Central South University (Changsa, China).

 Experimental model and groups

Rats were raised in rooms with a 12-h light/dark cycle at 25°C for 1 week prior to the experiment. Rats were fasted for 12 h and given food and fresh tap water ad libitum up to the experiment. Anesthesia was administered by intraperitoneal injection of 10% chloralic hydras (0.3 ml/100 g). The SAP model was induced by the standard retrograde infusion of a freshly prepared 5% TCA into the biliopancreatic duct following laparotomy. The 35 rats were randomly divided into three groups: (i) Sham-operation control group (sham group, n=5), where rats received an equivalent volume of normal saline following a successful sham operation; (ii) SAP model group (SAP group, n=15), where rats received an equivalent volume of 5% TCA; and (iii) DXM treatment group (DXM group, n=15), where rats were administered one dose of DXM (0.5 mg/100 g) intravenously in the tail following successful SAP induction. The rats were sacrificed by dislocation of the neck in the state of anesthesia at designated time-points following the induction of pancreatitis.

 Histological examination

For histological analysis, pancreas tissue specimens were fixed in 4% formaldehyde at room temperature for 3 days, embedded in paraffin, sectioned at 4 µm and treated with hematoxylin-eosin (HE) staining for 10 min at room temperature. The terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) assay was used to examine the apoptotic cells in the pancreas. Following staining with 0.5% hematoxylin for 15 min at room temperature, the nuclei of healthy cells were stained blue, while those in apoptotic cells presented brown/yellow staining. Integrated optical density (IOD) analysis was used to indirect reaction the apoptosis. The expression levels of HMGB1 and NF-κBp65 in the pancreas were examined by immunohistochemistry (IHC). Specimens were mounted in Permount and examined using routine light microscopy.

 Cell culture

AR42J pancreatoma cells were obtained from American Type Culture Collection, (Manassas, VA, USA) and cultured in a humidified atmosphere with 5% CO2 at 37°C. The cells were treated with 10−8 mol/L caerulein (CAE) and/or DXM. Experiments were assigned to three groups: CK group, CAE group and DXM group. In the DXM group, cells were co-treated with CAE and 10−6, 10−7 or 10−8 mol/L DXM for 24 h. In the CK group, which acted as the control group, the AR42J cells were only treated with PBS. Cells in the CAE group were treated with CAE alone.

 Detection of cellular apoptosis assay

For this experiment, 2×106 cells were plated into 60-mm dishes and then treated with or without CAE/DXM for 24 h. Cellular apoptosis was detected using the AnnexinV-fluorescein isothiocyanate Apoptosis kit (BD Biosciences, Franklin Lakes, NJ, USA) and a FACSCanto II flow cytometer (BD Biosciences), following the manufacturers protocol.

 Cell survival assay

AR42J cells (5×103 cells/well) were seeded into a 96-well plate and a MTT-based assay was performed after 24 h. DMSO (150 µl) was used to dissolve the purple formazan and the absorbance at 570 nm was measured to determine the cell survival rates.

 Immunofluorescence assay

Immunofluorescence was performed on AR42J cells following blocking with 1X PBS/5% Normal Goat Serum (005-000-121; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA)/0.3% Triton X-100 at room temperature for 60 min using anti-HMGB1 (1:500; cat. no. ab79823; Abcam), and anti-NF-κBp65 (1:500; cat. no. 8242; Cell Signaling Technology, Inc.) primary antibodies and cy3-conjugated secondary antibodies (1:500; Abcam). Nuclei were counterstained with DAPI (1:1,000; Sigma Aldrich; Merck KGaA, Darmstadt, Germany) for 1 h at room temperature and cover slips were mounted with Fluorescence Mounting Medium (Dako; Agilent Technologies, Inc., Santa Clara, CA, USA) and examined using a fluorescence microscope.

 Western blotting

AR42J cells were harvested using RIPA buffer (P002A; Auragene; Hunan Aijia Biotechnology Co., Ltd., Hunan, China) and a BCA Protein Quantitation kit used for protein determination. Proteins were subjected to SDS-PAGE and immunoblotting, as previously described (19). A total of 30 µg protein per lane was separated by 10% SDS-PAGE, transferred onto polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA), blocked with 5% skimmed milk for 1 h at room temperature and incubated with primary antibodies at 4°C overnight: Anti-HMGB1 (1:1,0000; cat. no. ab79823; Abcam), anti-NF-κBp65 (1:1,1000; cat. no. 8242; Cell Signaling Technology, Inc.) and anti-β-actin (1:1,1000; cat. no. 3700; Cell Signaling Technology, Inc.). Then the blots were incubated with the corresponding secondary antibodies (1:5,000; sheep anti-rat; SA001 and sheep anti rabbit; SA009 Auragene; Hunan Aijia Biotechnology Co., Ltd.) at room temperature for 2 h. An AuraECL Chemiluminescence detection kit (Auragene; Hunan Aijia Biotechnology Co., Ltd.) was used for visualization.

 Statistical analysis

All results are expressed as the mean ± standard deviation from 3 independent experiments. One-way analysis of variance was used for statistical analysis and Tukey post hoc test was used for verification. P<0.05 was considered to indicate a statistically significant difference and SPSS version 17 (SPSS, Inc., Chicago, IL, USA) was used for analysis.

 Results <sec> <title>Histological alterations in the pancreas

Histological alterations in pancreatic tissues were observed under a light microscope by HE staining, as presented in Fig. 1A. In the sham group, pancreatic interstitial cells demonstrated slight hemorrhage and mild edema. Pancreatic tissue in the SAP group demonstrated increased levels of hemorrhage, edema and structural distortion with necrosis compared with the sham group (data not shown). Notably, when DXM was infused into the SAP rats, edema formation and structural alterations with necrosis were reduced. In addition, the therapeutic influence of DXM was time-dependent and differences between the DXM group, and the SAP group were especially noticeable after 12 h. (data not shown). The number of apoptotic acinar cells increased in the SAP group compared with the sham group. Similarly, following DXM infusion, the number of apoptotic cells was markedly reduced compared with the SAP group and this reduction was time-dependent, with a significant reduction observed after 6 h (P<0.05; Fig. 1B and C).

 IHC analysis of NF-κBp65 and HMGB1 expression in the pancreas

Inflammatory mediators serve key roles in the pathogenesis of SAP. In the present study, alterations in NF-κBp65 and HMGB1 expression were examined in the pancreas. In the sham group, the detection of HMGB1 was very weak in the nucleus (Fig. 2A) and a low level of NF-κBp65 was concentrated in the cytoplasm (Fig. 2B). In the SAP group, intense immunoreactivity with HMGB1 and NF-κBp65 was detected in the nucleus and cytoplasm (Fig. 2). In the DXM group, the expression levels of HMGB1 and NF-κBp65 were decreased in the nucleus and the cytoplasm, respectively (Fig. 2), compared with the SAP group.

 DXM regulates cell apoptosis in AR42J cells treated with CAE

To confirm that DXM is involved in the regulation of cellular apoptosis of AR42J cells treated with CAE, flow cytometry was performed. As presented in Fig. 3, compared with the control group, apoptosis was significantly increased in AR42J cells following treatment with CAE (P<0.001; Fig. 3A and B). DXM could significantly suppress apoptosis induced by CAE (P<0.01) and the reduction in apoptosis was associated with the DXM concentration (Fig. 3B).

 Cell survival assay in AR42J cells treated with CAE and DXM

Compared with the CK group, cell survival was significantly inhibited in the CAE group (P<0.001) and could be significantly improved by DXM treatment (P<0.05). Additionally, a positive association was demonstrated between cell viability and DXM concentration (Fig. 3C).

 Effect of DXM on HMGB1 and NF-κBp65 expression in AR42J cells

Immunofluorescence (Fig. 4A) and western blotting (Fig. 4B) analyses demonstrated that HMGB1 and NF-κBp65 were expressed at low levels in AR42J cells treated with PBS (CK group), were expressed at a high level in the CAE group, and were expressed at an intermediate level in the DXM and CAE co-treated group. The expression of HMGB1 and NF-κBp65 decreased in a dose-dependent manner with DXM treatment and reached the lowest levels when treated with 10−6 M DXM, compared with the CAE group (Fig. 4).

 Discussion

SAP is an acute, critical illness with rapid onset, long duration and rapid progress that results in massive necrosis of pancreatic tissue, extrapancreatic multiple organ failure, and a high mortality rate. Despite advances in treatment techniques, the mortality of SAP has improved slowly over the past several decades and the pathogenesis of SAP has not been completely clarified (20,21). However, experts have focused on inflammatory mediators and their corresponding antagonists as potential therapeutic targets, due to their contribution to the injury of the pancreas and other organs, frequently resulting in patients succumbing to the condition.

DXM is a long-acting corticosteroid. Its therapeutic effects on SAP are primarily associated with suppressing the production and/or actions of inflammatory mediators, enhancing the anti-stress capacity of the body, reducing endotoxemia, and scavenging oxygen free radicals (22,23). In the present study, to investigate the protective effects of DXM on the pancreas of SAP rats the effect of DXM treatment on the expression levels of NF-κBp65 and HMGB1, as well as on the histopathology of the pancreas in rats with SAP, was investigated.

NF-κBp65 is a transcription factor that regulates various genes involved in inflammatory and immune responses, including cytokine and adhesion molecules (24). TNF-α expression is directly regulated by NF-κBp65, as there are NF-κBp65 binding sites on the TNF-α promoter. Research has demonstrated that the inhibition of NF-κBp65 can result in decreased expression levels of cytokines, including TNF-α, reducing the inflammatory response in organisms. The role of NF-κBp65 activation in the pathogenesis of SAP was previously reviewed (25) and in a later investigation, this laboratory demonstrated evidence that the upregulation of NF-κBp65 could aggravate SAP-induced pancreatic injury (26). NF-κBp65 inhibitors in in vitro and in vivo models of SAP have already been studied (27,28). In the present study, DXM was also demonstrated as an inhibitor of NF-κBp65 and that DXM can downregulate the expression of NF-κBp65 protein in the pancreatic tissue of SAP rats and in AR42J cells treated with CAE. Previous studies have demonstrated that HMGB1 acts through multiple mechanisms, including through the NF-κBp65 pathway (29). However, it is not certain whether there are binding sites for NF-κBp65 on the HMGB1 promoter. Therefore, it remains unclear whether HMGB1 expression is directly regulated by NF-κBp65.

HMGB1, a DNA-binding intranuclear protein, is a late activator of the inflammatory cascade when released into the extracellular space. HMGB1 is released from necrotic cells or secreted by activated monocytes or macrophages. HMGB1 induces pro-inflammatory cytokines in human monocytes via TLR4 and NF-κB activation (30). HMGB1 can mediate cell-to-cell signaling by binding to the receptor for advanced glycation end products and toll-like receptors (TLR), especially TLR-2 and TLR-4, to enhance the inflammatory response (29,31). As a late-phase mediator, HMGB1 was previously discovered to be upregulated in a number of acute and chronic inflammatory conditions, including sepsis, acute lung injury and rheumatoid arthritis (32–34). In contrast to other known pro-inflammatory cytokines, the delayed kinetics of HMGB1 provide a wide window of opportunity for therapeutic approaches (35). The present results also highlight the possible beneficial role of anti-inflammatory cytokines, including IL-10 and IL-13, in the pathogenesis of SAP. These cytokines may counteract the type-1 pro-inflammatory cytokines secreted in response to the activation of the TLR4-NF-κB pathway induced by HMGB1.

As a result, HMGB1 offers the hope of developing an anti-inflammatory therapy that is practical and effective. In a previous study serum HMGB1 levels, were demonstrated to be significantly elevated in patients with SAP and were correlated with disease severity (36). Therefore, it has been speculated that HMGB1 may be a target for anti-inflammatory treatment in SAP. In the present study, it was demonstrated that HMGB1 expression levels increased in the pancreas with SAP and decreased following treatment with DXM, suggesting that HMGB1 may be pivotal for the inflammatory response and organ injury observed in SAP and may be a therapeutic target for SAP. It was hypothesized that elevated HMGB1 levels may represent an aggravating factor in SAP and the associated multiple organ damage. However, HMGB1 affects a number of angiogenesis-associated conditions, including cancer, proliferative diabetic retinopathy and wound healing through the p53 pathway, and it is a promising therapeutic target in a number of tumors, including epidermal tumors, prostate cancer and colon cancer (37–39). Therefore, the exact function of HMGB1 and its mechanism still need to be elucidated. Notably, while investigating the relevance of HMGB1-TLR4-NF-κB-induced pro-inflammatory cytokines in SAP, it was demonstrated that the antibiotic and immunomodulatory agent fusidic acid may prevent pancreatitis through the reduction of TNF-α and IL-8. This is of interest because sodium fusidate is clinically available and could be immediately considered for the treatment of SAP (40,41).

In conclusion, DXM can reduce the levels of NF-κBp65 and HMGB1 and mitigate the pathological alterations in the pancreas of rats with SAP and in AR42J cells treated with CAE. In in vivo and vitro experiments, DXM was identified to have a therapeutic effect on SAP. Therefore, NF-κBp65 and HMGB1 may serve auxiliary roles in the treatment of SAP. Overall, each of these inflammatory mediators has benefits and should be used appropriately in future clinical practice.

 Acknowledgements

Not applicable.

 Funding

The present study was supported by Key project of Hunan science and technology plan (grant no: 2013FJ4093).

 Availability of data and materials

The datasets used during the present study are available from the corresponding author upon reasonable request.

 Authors contributions

SZ, LM and KX conceived and designed the study. SZ, TL, JZ and JY performed the experiments. SZ and JY wrote the manuscript. KX reviewed and edited the manuscript. All authors read and approved the manuscript and agree to be accountable for all aspects of the research in ensuring that the accuracy or integrity of any part of the work were appropriately investigated and resolved.

 Ethics approval and consent to participate

The present study was approved by the Ethics Committee of Central South University (Changsa, China).

 Patient consent for publication

Not applicable.

 Competing interests

The authors declare they have no competing interests.

 Abbreviations SAP

severe acute pancreatitis

 DXM

dexamethasone

 HMGB1

high-mobility group box-1

 NF-κB

nuclear factor-κB

 CAE

caerulein

 SIRS

systemic inflammatory response syndrome

 TNF

tumor necrosis factor

 TCA

sodium taurocholate solution

 HE

hematoxylin-eosin

 TUNEL

terminal deoxynucleotidyl transferase, dUTP nick-end labeling

 References UnenoYTaneishiKKanaiMOkamotoKYamamotoYYoshiokaAHiramotoSNozakiANishikawaYYamaguchiDDevelopment and validation of a set of six adaptable prognosis prediction (SAP) models based on time-series real-world big data analysis for patients with cancer receiving chemotherapy: A multicenter case crossover studyPloS One12e0183291201710.1371/journal.pone.0183291288375925570326 ShenQLiZHuangSLiLGanHDuXGIntestinal mucosal barrier dysfunction in SAP patients with MODS ameliorated by continuous blood purificationInt J Artif Organs1402017 LipinskiMRydzewskaGImmature granulocytes predict severe acute pancreatitis independently of systemic inflammatory response syndromePrz Gastroenterol121401442017287021045497134 ZhangZWZhangQYZhouMTLiuNXChenTKZhuYFWuLAntioxidant inhibits HMGB1 expression and reduces pancreas injury in rats with severe acute pancreatitisDig Dis Sci5525292536201010.1007/s10620-009-1073-019997973 WangBXuXBJinXXWuXWLiMLGuoMXZhangXHEffects of ω-3 fatty acids on Toll-like receptor 4 and nuclear factor κB p56 in the pancreas of rats with severe acute pancreatitisPancreas4612671274201710.1097/MPA.000000000000093528984787 YanYLuBLiPWangJNOD receptor and TLR9 modulation in severe acute pancreatitisinduced intestinal injuryMol Med Rep1684718476201710.3892/mmr.2017.766128990073 XiongJWangKYuanCXingRNiJHuGChenFWangXLuteolin protects mice from severe acute pancreatitis by exerting HO-1-mediated anti-inflammatory and antioxidant effectsInt J Mol Med39113125201710.3892/ijmm.2016.280927878246 FengCLiBWangLLChenLIZhouXLvFQLiTSEffect of peritoneal lavage with ulinastatin on the expression of NF-κB and TNF-α in multiple organs of rats with severe acute pancreatitisExp Ther Med1020292034201510.3892/etm.2015.2802266685914665968 LiGWuXYangLHeYLiuYJinXYuanHTLR4-mediated NF-κB signaling pathway mediates HMGB1-induced pancreatic injury in mice with severe acute pancreatitisInt J Mol Med3799107201610.3892/ijmm.2015.241026719855 LinYJDingYWuJNingBTPterostilbene as treatment for severe acute pancreatitisGenet Mol Res15201610.4238/gmr.150383306097843 YangRTenhunenJTonnessenTIHMGB1 and histones play a significant role in inducing systemic inflammation and multiple organ dysfunctions in severe acute pancreatitisInt J Inflam20171817564201710.1155/2017/1817564283168605339498 KwakMSLimMLeeYJLeeHSKimYHYounJHChoiJEShinJSHMGB1 binds to lipoteichoic acid and enhances TNF-α and IL-6 production through HMGB1-mediated transfer of lipoteichoic acid to CD14 and TLR2J Innate Immun7405416201510.1159/00036997225660311 StojanovicICuzzocreaSManganoKMazzonEMiljkovicDWangMDoniaMAl AbedYKimJNicolettiFIn vitro, ex vivo and in vivo immunopharmacological activities of the isoxazoline compound VGX-1027: Modulation of cytokine synthesis and prevention of both organ-specific and systemic autoimmune diseases in murine modelsClin Immunol123311323200710.1016/j.clim.2007.03.00417449326 FagonePMuthumaniKManganoKMagroGMeroniPLKimJJSardesaiNYWeinerDBNicolettiFVGX-1027 modulates genes involved in lipopolysaccharide-induced Toll-like receptor 4 activation and in a murine model of systemic lupus erythematosusImmunology142594602201410.1111/imm.12267245277964107669 LeeJCMenacherrySDiehlMCGiffearMDWhiteCJJubaRBagarazziMLMuthumaniKBoyerJAgarwalVSafety, bioavailability, and pharmacokinetics of VGX-1027-A novel oral anti-inflammatory drug in healthy human subjectsClin Pharmacol Drug Dev591101201610.1002/cpdd.19327138022 LucasKMaesMRole of the Toll Like receptor (TLR) radical cycle in chronic inflammation: Possible treatments targeting the TLR4 pathwayMol Neurobiol48190204201310.1007/s12035-013-8425-723436141 ZhangTXiaMZhanQZhouQLuGAnFSodium butyrate reduces organ injuries in mice with severe acute pancreatitis through inhibiting HMGB1 expressionDig Dis Sci6019911999201510.1007/s10620-015-3586-z25686746 SellnerSKocabeySZhangTNekollaKHuttenSKrombachFLiedlTRehbergMDexamethasone-conjugated DNA nanotubes as anti-inflammatory agents in vivoBiomaterials1347890201710.1016/j.biomaterials.2017.04.03128458030 CenCLiJLiuJYangMZhangTZuoYLinCLiXLong noncoding RNA LINC01510 promotes the growth of colorectal cancer cells by modulating MET expressionCancer Cell Int1845201810.1186/s12935-018-0503-5295817075861636 MofidiRMadhavanKKGardenOJParksRWAn audit of the management of patients with acute pancreatitis against national standards of practiceBr J Surg94844848200710.1002/bjs.567017330929 ButerAImrieCWCarterCREvansSMcKayCJDynamic nature of early organ dysfunction determines outcome in acute pancreatitisBr J Surg89298302200210.1046/j.0007-1323.2001.02025.x11872053 YuberoSRamudoLMansoMADe DiosIMechanisms of dexamethasone-mediated chemokine down-regulation in mild and severe acute pancreatitisBiochim Biophys Acta179212051211200910.1016/j.bbadis.2009.10.00119818401 JingminOXipingZChunWPingYQianYStudy of dexamethasone, baicalin and octreotide on brain injury of rats with severe acute pancreatitisInflamm Res61265275201210.1007/s00011-011-0408-422166920 ZhangXPZhangLChenLJChengQHWangJMCaiWShenHPCaiJInfluence of dexamethasone on inflammatory mediators and NF-kappaB expression in multiple organs of rats with severe acute pancreatitisWorld J Gastroenterol13548556200710.3748/wjg.v13.i41.5506172782204065976 ZhangXPZhangLXuHMXuYPChengQHWangJMShenHPApplication of tissue microarrays to study the influence of dexamethasone on NF-kappaB expression of pancreas in rat with severe acute pancreatitisDig Dis Sci53571580200810.1007/s10620-007-9867-417574527 OuJMZhangXPWuCJWuDJYanPEffects of dexamethasone and Salvia miltiorrhiza on multiple organs in rats with severe acute pancreatitisJ Zhejiang Univ Sci B13919931201210.1631/jzus.B1100351231250853494031 ChenYZhaoQChenQZhangYShaoBJinYWuJMelatonin attenuated inflammatory reaction by inhibiting the activation of p38 and NFκB in taurocholateinduced acute pancreatitisMol Med Rep1759345939201829484391 QianDWeiGXuCHeZHuaJLiJHuQLinSGongJMengHBone marrow-derived mesenchymal stem cells (BMSCs) repair acute necrotized pancreatitis by secreting microRNA-9 to target the NF-κB1/p50 gene in ratsSci Rep7581201710.1038/s41598-017-00629-3283736675428835 BiYZhuYZhangMZhangKHuaXFangZZhouJDaiWCuiYLiJYouTEffect of shikonin on spinal cord injury in rats via regulation of HMGB1/TLR4/NF-κB signaling pathwayCell Physiol Biochem43481491201710.1159/00048047428934735 AnderssonUWangHPalmbladKAvebergerACBloomOErlandsson-HarrisHJansonAKokkolaRZhangMYangHTraceyKJHigh mobility group 1 protein (HMG-1) stimulates proinflammatory cytokine synthesis in human monocytesJ Exp Med192565570200010.1084/jem.192.4.565109527262193240 AntonMAlenFde HerasGomez RSerranoAPavónFJLezaJCGarcía-BuenoBde FonsecaRodríguez FOrioLOleoylethanolamide prevents neuroimmune HMGB1/TLR4/NF-κB danger signaling in rat frontal cortex and depressive-like behavior induced by ethanol binge administrationAddict Biol22724741201710.1111/adb.1236526857094 LanKCChaoSCWuHYChiangCLWangCCLiuSHWengTISalidroside ameliorates sepsis-induced acute lung injury and mortality via downregulating NF-κB and HMGB1 pathways through the upregulation of SIRT1Sci Rep712026201710.1038/s41598-017-12285-8289319165607272 HuangWTangYLiLHMGB1, a potent proinflammatory cytokine in sepsisCytokine51119126201010.1016/j.cyto.2010.02.02120347329 LiYBXuPXuKCaiYSSunMYYangLSunJLuSMMethotrexate affects HMGB1 expression in rheumatoid arthritis, and the downregulation of HMGB1 prevents rheumatoid arthritis progressionMol Cell Biochem420161170201610.1007/s11010-016-2783-127522665 AbeAKuwataTYamauchiCHiguchiYOchiaiAHigh mobility group box1 (HMGB1) released from cancer cells induces the expression of pro-inflammatory cytokines in peritoneal fibroblastsPathol Int64267275201410.1111/pin.1216724965109 XuGFGuoMTianZQWuGZZouXPZhangWJIncreased of serum high-mobility group box chromosomal protein 1 correlated with intestinal mucosal barrier injury in patients with severe acute pancreatitisWorld J Emerg Surg961201410.1186/1749-7922-9-61259268624414458 WengHDengYXieYLiuHGongFExpression and significance of HMGB1, TLR4 and NF-kappaB p65 in human epidermal tumorsBMC Cancer13311201310.1186/1471-2407-13-311238031723697986 GnanasekarMKalyanasundaramRZhengGChenABoslandMCKajdacsy-BallaAHMGB1: A promising therapeutic target for prostate cancerProstate Cancer2013157103201310.1155/2013/157103237669113666291 LiWWuKZhaoEShiLLiRZhangPYinYShuaiXWangGTaoKHMGB1 recruits myeloid derived suppressor cells to promote peritoneal dissemination of colon cancer after resectionBiochem Biophys Res Commun436156161201310.1016/j.bbrc.2013.04.10923707808 WiddisonALSodium fusidate and the cytokine response in an experimental model of acute pancreatitisBr J Surg86715199910.1046/j.1365-2168.1999.1104j.x10361358 NicolettiFZacconePDi MarcoRMagroGGrassoSMorroneSSantoniATemperaGMeroniPLBendtzenKEffects of sodium fusidate in animal models of insulin-dependent diabetes mellitus and septic shockImmunology85645650199575581611383795

Pathological changes in the pancreas. Pancreases from the sham, SAP and DXM groups were harvested 3, 6 or 12 h following operations and were stained. (A) Mass hemorrhages, edema and structural alterations with necrosis were observed in the pancreas by hematoxylin and eosin staining (magnification, ×400). Scale bar represents 250 µm. (B) The apoptotic acinar cells in the pancreas were observed by TUNEL staining. (C) IOD analysis of TUNEL. Data are presented as the mean ± standard error of the mean (n=3). ##P<0.01, #P<0.05 vs. SAP group; ***P<0.001 vs. Sham group. SAP, severe acute pancreatitis; DXM, dexamethasone-treated; TUNEL, terminal deoxynucleotidyl transferase dUTP nick-end labeling; IOD, integrated optical density.

Immunohistochemical analysis. To detect the effects of DXM on HMGB1 and NF-κB protein expression levels in pancreatic tissue from sodium taurocholate solution-treated rats, HMGB1 and NF-κBp65 proteins were detected in pancreatic tissue from animals sacrificed 3, 6, or 12 h following operation in the sham, SAP and DXM groups. (A) The expression of HMGB1. (B) The expression of NF-κBp65 (magnification, ×200). Scale bar represents 500 µm. HMGB1, high-mobility group box 1; NF-κB, nuclear factor-κB; DXM, dexamethasone; SAP, severe acute pancreatitis.

Analysis of AR42J cell survival. (A) Flow cytometry plots and (B) statistical analysis of the rate of apoptosis for cell treated with CAE and DXM. The number of apoptotic AR42J cells treated with CAE is markedly increased compared to the CK and DXM groups, and the number of apoptotic AR42J cells decreased with an increase in DXM concentration. (C) DXM dose-dependently promotes AR42J cell viability, as assessed by the MTT assay. ###P<0.001, CK group vs. the CAE group. ***P<0.001, **P<0.01 and *P<0.05, CAE group vs. DXM group. Data are the mean ± standard error (n=3). CAE, caerulein; DXM, dexamethasone; FITC, fluroescein isothiocyanate.

Expression of HMGB1 and NF-κBp65 in AR42J cells. To examine the effects of DXM on HMGB and NF-κB protein expression in AR42J cells treated with CAE, the expression of HMGB1 and NF-κBp65 was detected. (A) The expression of HMGB1 and NF-κBp65 was detected by immunofluorescence using cy3-conjugated antibodies (red) and DAPI (blue) (magnification, ×200). Scale bar represents 500 µm. (B) Levels of HMGB1, NF-κBp65 and β-actin in AR42J cells with/without DXM were detected by western blotting. HMGB1, high-mobility group box 1; NF-κB, nuclear factor-κB; DXM, dexamethasone; CAE, caerulein.