Protective Mechanisms Of Emodin Alleviates Pancreatic Acinar Cell Injury In Rats With Severe Acute Pancreatitis

Background: Acute pancreatitis(AP)is one of the most frequent gastrointestinal diseases leading to total hospital stays.Approximately 20% of patients with AP suffer a severe attack with progression to systemic inflammatory response syndrome(SIRS)and multiple organ dysfunction syndrome(MODS).Although various related concepts and techniques for diagnosis and treatment continue to improve,SAP is still the most serious acute abdomen with the mortality is up to 20-30%.Acute biliary disease is the main pathogenic factor of SAP in our country,accounting for ~50-60%.According to Chinese medicine theory,“Yangming fu-organ syndrome” plays an important role in the pathogenesis of SAP.Base on “the six hollow organs must keep its dredging function”,“Tong li gong xia” is the preferred treatment in the early stage of SAP.Rheum palmatum L.integrates the effects of “Tongxia” and dispersing blood stasis,which is an important drug for SAP administration.Emodin(1,3,8-trihydroxy-6-methylanthraquinone)is a natural anthraquinone derivative isolated from the Chinese herb Rheum palmatum L.,and is also an important lead compound for drug synthesis.The pharmacological effects of emodin have many similarities with Rheum palmatum L.,which has various biological activities,including anti-inflammatory,laxative,microcirculation improvement and immune regulation.The clinical application value of emodin is great.Early animal experiments found that emodin has a good protective effect on SAP,however,the unknown drug targets and molecular mechanisms obstruct the further promotion and application of emodin in clinical treatment.Therefore,exploring the pharmacological actions and molecular mechanisms of emodin against SAP may provide a theoretical basis for the development of new drugs for clinical treatment of this disease.Objective: To explore the characteristic abnormal protein markers in pancreatic tissue of early SAP rats after emodin administration based on i TRAQ proteomics technique,as well as the targets and intervention routes of emodin in the SAP therapy.Methods:(1)In the study related to the action of emodin against rats with SAP induced by administering 5% sodium taurocholate(STC),Sprague Dawley(SD)rats were randomly divided into sham operation(SO)group,SAP model group and high,medium and low dose emodin groups(60,30 and 15 mg/kg).Emodin was given by intragastric administration once every 6 h after modeling surgery,and 12 h after surgery,animals were anesthetized before sacrificing.Enzyme-linked immunosorbent assay(ELISA)was performed to measure the plasma levels of amylase,lipase,IL-6,IL-1β and TNF-α.The protective effect of emodin on SAP rats was evaluated by the pancreatic histopathology and ultrastructural changes of pancreatic cells.Myooperoxidase(MPO)and IL-6,IL-1β and TNF-α m RNA expressions were observed to investigate the anti-inflammatory activity of emodin.The abnormal protein markers in pancreatic tissue of early SAP rats after emodin administration were identified through comparing the differentially expressed proteins between SO,SAP and emodin groups using i TRAQ proteomics technique,and the molecular mechanism of emodin in the treatment of SAP was further explored by western boltting assay.(2)In vitro study of emodin against acinar cell injury,STC was used to induce rat pancreatic acinar AR42 J cell injury.After pretreating with emodin(40,20 and 10 μM)for 2 h,MTT assay and LDH release were used to evaluate the protective effect and cytotoxicity of different concentrations of emodin on STC injured AR42 J cells.The amylase and trypsin activity,as well as the levels of IL-6,IL-1β and TNF-α in the culture medium were measured to investigate the anti-inflammatory effect of emodin in vitro.The effect of emodin on the protein expression of HTRA1/TGF-β1 signaling pathway in AR42 J cells was investigated by immunofluorescence and western blotting.Finally,the HTRA1 gene overexpression plasmid was constructed according to the m RNA sequence of rat HTRA1 gene for cell transfection experiments,which can determine whether emodin protects pancreatic acinar cells from injuring by inhibiting HTRA1/TGF-β1 signaling pathway.(3)In the study of emodin intervention in mi RNA-mediated HTRA1/TGF-β1 signaling pathway,bioinformatics analysis was used to filter the upstream mi RNAs involved in the regulation of HTRA1 gene expression.The wild type and mutant of HTRA1 gene were constructed,and then the effect of candidate upstream mi RNAs on HTRA1 gene expression was verified by double luciferase reporter assay.STC-induced acinar cell injury in vitro following pretreated with emodin(40,20 and 10 μM)for 2 h.The expressions of mi RNA in the control,model and emodin groups were detected by Real-time PCR.After transfected with both mi RNA inhibitor in vitro and mi RNA antagomir in vivo,the levels of amylase,lipase and IL-6,IL-1β and TNF-α in the culture medium and rat plasma were measured by ELISA.Real-time PCR and western blotting were used to observe the m RNA expressions of IL-6,IL-1β and TNF-α,and the protein expressions of HTRA1/TGF-β1 signal pathway.Results:(1)In the study related to the action of emodin against rats with SAP,emodin markedly decreased plasma amylase,lipase,IL-1β,IL-6 and TNF-α activities compared with SAP group.Immunohistochemistry and immunofluorescence results indicated that emodin down-regulated MPO protein expression.Meanwhile,emodin improved pancreatic histopathology and acinar cell ultrastructure.In addition,Real-time PCR results also proved that emodin significantly inhibited IL-1β,IL-6 and TNF-α m RNA expression.Finally,a total of 32 differentially expressed proteins from rat pancreas in response to stimulus were discovered by using i TRAQ-based quantitative proteomic analysis.These proteins were related to each other and involved in different biological processes.Among them,a new biomarker,high-temperature requirement A1(HTRA1),was found to associate with SAP,which was validated by western blotting.Further work found,for the first time,those proteins in the HTRA1/TGF-β1 signaling cascades were involved in the mechanisms of emodin against SAP.The expressions of HTRA1,IL-33,My D88,TRAF-6 and NF-κB were down-regulated by emodin,while the expression ofTGF-β1 protein was up-regulated.(2)In vitro study of emodin against STC-induced acinar cell injury,when the STC concentration was 498.2 μM,50% of AR42 J cells were induced to die.Compared with the model group,emodin(40,20,10,5 and 2.5 μM)pretreatment for 2 h significantly increased the relative cell viability of AR42 J cells in a dose-dependent manner.Meanwhile,emodin significantly reduced the release of amylase,trypsin,and IL-6,IL-1β and TNF-α in damaged cells.Molecular mechanism studies shown that emodin could significantly down-regulate the HTRA1,IL-33,My D88,TRAF-6 and NF-κB protein levels,but up-regulate the TGF-β1 protein level.These results indicated that emodin alleviated pancreatic acinar cells injury mainly through inhibiting HTRA1/ TGF-β1 signalling pathway,and this finding was further proved by the HTRA1 overexpression experiments.(3)In the study of emodin intervention in mi RNA-mediated HTRA1/TGF-β1 signaling pathway,using bioinformatic analyses,we collected the prospective target mi RNAs of HTRA1 and predicted that mi R-30a-5p might be an important transcriptional brake that modulates targeted HTRA1 m RNA translation.The RNA sequence alignment showed that the 3’-UTR of HTRA1 m RNA contained a site complementary to the seed region of mi R-30a-5p.The result was further proved by a dual luciferase reporter assay.In addition,western blotting analysis further showed that mi R-30a-5p mimic could markedly down-regulate HTRA1 protein expression,thereby resisting the inhibitory effect of HTRA1 on its downstream molecule TGF-β1.Real-time PCR showed that emodin(40,20 and 10 μM)pretreatment could significantly increase the expression of mi R-30a-5p in AR42 J cells compared with the model group.Furthermore,the pancreatic protective effects and anti-inflammatory activities of emodin were all abrogated with both mi R-30a-5p inhibitor in vitro and mi R-30a-5p antagomir in vivo.Conclusion:(1)HTRA1 is a characteristic biomarker in pancreatic tissue of early SAP rats,and a potential drug target for emodin against SAP;(2)The biological activity of emodin against SAP is partially mediated by regulation of HTRA1/TGF-β1 signaling pathway;(3)Emodin may inhibit the activation of HTRA1/TGF-β1 signaling pathway by up-regulating mi R-30a-5p expression,thus improving STC-induced acinar cell injury and alleviating SAP symptoms.Collectively,emodin has a good therapeutic effect on the SAP,which is worth to explore and research in depth.