The Role And Regulation Mechanism Of Autophagy For Acute Necrotizing Pancreatitis In Rats

Acute pancreatitis (AP) is an acute inflammatory condition of the pancreas that mayinvolve other regional or remote digestion tissues. It is often caused the prematuretrysinogen activation, which induced by activating other digestive enzymes. According tothe clinical severity of AP, it is broadly classified as mild acute pancreatitis (MAP) andsevere acute pancreatitis (SAP). Although the majority of patients have good prognos withmild episode of AP, some still develop SAP and suffer systemic inflammatory responsesyndrome (SIRS), with multiple organ dysfunction syndromes (MODS), even lead to death.However, the pathogenic mechanism is not completely understood in SAP, and there is nospecific treatment for SAP currently.Autophagy, which has been proposed as a mode of cell death, is a process in whichcells generate energy and metabolites by digesting their own organelles. Autophagy isrelated to many diseases. However, the role of autophagy has just begun in AP. Basically,the impaired autophagy should be considered as the key pathological responses in acutepancreatitis. Therefore, we hypothesize that the inhibition of autophagy, or blocking theautophagic process may become a potential target for the treatment of AP. In this study, weestablish the animal models of acute necrotizing pancreatitis (ANP) and observe theexpression of autophagy in pancreatic tissues in ANP.3-methyladenine (3-MA,autophagy inhibitor) interferes the process of autophagy and clarifies the mechanism ofautophagy in pancreatitis. Pyrrolidine dithiocarbamate (PDTC, NF-κB inhibitor),interferes the NF-κB signaling channel to observe the regulation mechanism of NF-κBsignaling pathway in autophagy. Further more, we explore the imposible molecularmechanism and the pathogenesis in AP, especially the occurrence and development of SAP to improve the prognosis of patients with new treatment strategies.Part1The significance and expression of autophagy after rat′s acute necrotizing pancreatitisObjective: To observe the significance and expression of autophagy on the models ofthe acute necrotizing pancreatitis rats this induces to the Sodium Taurocholic-acid.Methods: Thirty-six SD rats were randomized to sham operation (SO) group andacute necrotizing pancreatitis (ANP) group．The model of ANP was induced by retrogradeinjection of5%sodium taurocholate into the bili-pancreatic duct. Rats were killed at3,6,and12h after sodium taurocholate injection. The histopathological changes of pancreatictissue and serum inflammatory factors were obversed. Enzyme histochemical assay detectMPO concentration of pancreatic tissue. The formation of autophagy was observed byelectron microscopy. The expressions of LC3and Beclin1in the pancreas after acutenecrotizing pancreatitis were detected by immunohistochemistry staining and western blotanalysis at each time point.Results: The pathological changes of the ANP groups were exacerbated to differentdegrees. Levels of amylase, TNF-α and MPO were significantly increased, compared withSO groups (SO group vs. ANP group, P<0.05). Therefore, in our study, a rat model ofANP was well induced by standard retrograde infusion of bilio-panereatic duct with5%sodium taurocholate solution. The structure of normal pancreatic acinar cells is clear, andautophagic vacuole is rare. The number of autophagosome is increased at each time pointin ANP group. Expression of LC3and Beclin1in rat pancreas maintained at a very lowlevel in SO group, but their level started to elevate at3h after ANP and showed theincreased strong expression at time of6h and12h (SO group vs. ANP group, P<0.05).There was a considerable association of LC3protein with pathological scores of thelevel of pancreas injure at3h and6h (p<0.05).Conclusion: Acute necrotizing pancreatitis induced the increased level of LC3andBeclin1expressions in rat pancreas. Their expression correlated with inflammatory changes, which demonstrated up-regulation of the autophagic activity and the response toinjury in rat pancreas after ANP. Part2The influence of3-MA in autophagy and apoptosis after acutenecrotizing pancreatitis in rats．Objective: To investgate the role and machanism of3-methyladenine (3-MA,autophagy inhibitor) and the effect of acinar cell apoptosis in acute necrotizing pancreatitis(ANP).Methods: Fifty-four SD rats were randomized into sham operation (SO) group, ANPgroup and3-MA group.3-MA was administrated before the injection of sodiumtaurocholate. Rats were killed at3,6, and12h after sodium taurocholate injection. ELISAassay detect serum trypsin activation peptide (TAP). The histopathological changes ofpancreatic tissue were evaluated by microscope. The level of apoptosis after ANP in thethree groups were meeasured by the TUNEL.The expression of LC3and Bcl-2expressionwere assessed by immunofluorescence staining and Caspase-3expression was assessed byimmunohistochemistry method. Bcl-2, Caspase-3mRNA were measured by reversetranscriptase polymerase chain reaction (RT-PCR).Results: Compared with the SO group, ANP group and3-MA serum TAP wereelevated at all time points. TAP decreased at all time points in3-MA group compared withthe ANP group,but there is no significant difference between12h (P>0.05). Pathologicaldamage and score the ANP group and3-MA for12h, no difference (P>0.05). Pathologicalscore of3h and6h is decreased in3-MA group compared with the ANP group. theTUNEL positive cell were much more in3-MA group than ANP group at3h and6h, thedifferences had statistical significance(P<0.05)．The expression of LC3and Bcl-2aredecreased in the pancreatic cells in3-MA compared with ANP group byimmunofluorescence. RT-PCR results showed that Bcl-2mRNA content at all time pointsalso are reduced. Compared with the ANP group, Caspase-3activity in rat pancreatic tissue increased at3h and6h in3-MA group, with agreement Caspase-3mRNA pancreatic tissuecontent also increased (P <0.05).Conclusion:3-MA inhibits autophagy can reduce the original activation of ratpancreatic injury of ANP and trypsin, Inhibition of ANP autophagy to promote theformation of acinar cell apoptosis, An increase in apoptosis may be related Caspase-3andBcl-2.3-MA inhibits autophagy through the increase in apoptosis to reduce pancreaticpathological damage and plasminogen activation. Part3Autophagy regulation by the NF-κB signal axis in acutenecrotizing pancreatitisObjectives: To investigate the significance of Beclin1and LC3in the pancrea of ratafter acute necrotizing pancreatitis (ANP) and whether NF-κB signal regulate autophagyduring sodium taurocholate-induced ANP.Methods: Acute necrotizing pancreatitis was induced in rat by sodium taurocholateinjection in the pancreaticobiliary duct. PDTC was administrated before the injection ofsodium taurocholate. Then, serum amylase activity, trypsinogen activation peptide (TAP)and serum tumor necrosis factor-α concentrations, and morphological signs of pancreatitiswere measured. The formation of autophagosome and the activation of lysosome wereobserved in pancreas after ANP by using electron microscope. Meanwhile, the pancreaticlevels of NF-κB and essential proteins involved in formation of the autophagosome(Beclin1and LC3) were assessed by western blot and immunohistochemistry analysis.Results: Sodium taurocholate increased the levels of serum amylase, TAP, tumornecrosis factor-α and pancreatitic NF-κB, compared to sham-operated rats. Sodiumtaurocholate also increased levels of Beclin1and LC3-II. Inhibition of the NF-κB signalaxis with PDTC reduced serum amylase levels, blocked pancreatic NF-κB activation, andinhibited the sodium taurocholate–induced increases in Beclin1and the cleavage of LC3.Conclusions: NF-κB pathway activation stimulates autophagy during induction of ANP. Targeted inhibition of the NF-κB pathway may reduce levels of Beclin1and LC3inpancreatic tissue and provide novel therapeutic strategies for reducing the severity of ANP.