Mechanism Of APD In Alleviating Severe Acute Pancreatitis Associated Intestinal Injury

Severe acute pancreatitis(SAP)is a type of emergency characterized by extensive inflammation of the pancreas with tissue necrosis,rapid onset,severe disease,and high mortality.In the early stage of SAP,multiple organ damage caused by uncontrolled systemic inflammatory response is the main pathophysiological basis of patient death.Among them,pancreatitis-related intestinal injury(PAII)is the most common pancreatic disease in the early course of SAP.External complications are closely related to the severity of SAP.According to reports,the intestine,especially the small intestine,is not only an organ involved in the SAP process,but also a booster of systemic inflammation.Therefore,actively preventing the occurrence of PAII or reducing its damage is an important part of SAP treatment.Previous studies have confirmed that in the early stage of SAP,abdominal paracentensis drainage(APD)is used to discharge pancreatitis associated ascite fluid(PAAF),a fluid containing a variety of toxic substances,thus having an obvious positive effect on both alleviating the degree of systemic inflammation and improving the intestinal barrier function.However,the understanding of the protective mechanism of this intestinal tract is still very limited and needs to be further studied.Toll-like receptor 4(TLR4)is a member of the mammalian pattern recognition receptor family,and it is believed to be a key transmembrane protein that transmits extracellular antigen recognition information to cells and triggers an inflammatory response.Recent studies have shown that TLR4 plays a vital role in inflammatory initiation and organ injury during SAP.Through the myeloid differentiation primary response gene 88(My D88)-dependent pathway,TLR4 can mediate the activation of nuclear factor(NF)-κ B,which further induces the expression and release of inflammatory cytokines including tumor necrosis factor(TNF)-αand interleukin(IL)-6,resulting in a vicious cycle of positive feedback of inflammation.The TLR4/NF-κ B proinflammatory pathway has been reported to be involved in intestinal inflammation and apoptosis induced by SAP,but TLR4 can mediate downstream signal transduction only by binding to its specific ligand.High mobility group box protein 1(HMGB1)as a member of damage-associated molecular patterns family has been confirmed to be the most important endogenous ligand for TLR4,particularly in SAP.Especially,extracellular HMGB1 as a late mediator of lethality in systemic inflammation participates in the development of multiple organ injury caused by SAP.Active neutralization with anti-HMGB1 antibodies can prevent intestinal mucosal barrier dysfunction in mice with SAP.Accordingly,our previous research reported that APD can significantly reduce the level of HMGB1 by draining PAAF rich in high concentrations of HMGB1.Therefore,we speculate that the reduced effect of HMGB1 by early APD treatment may have a protective role in SAP-induced intestinal injury.However,little is known about how this treatment strategy triggers intestinal protection and its specific mechanism of action,and further research is urgently needed.we explored the effect of APD on intestinal inflammation and accompanying apoptosis in rats with SAP.Furthermore,experiments were conducted to determine whether HMGB1 in PAAF had a regulatory role in the intestinal TLR4 signaling pathway to confirm the protective effect via inhibiting HMGB1-mediated TLR4 signaling.methods and resultsPart Ⅰ Adult male Sprague-Dawley rats(weight,190-210 g)were randomly divided into Sham group,SAP group and APD group,with 15 rats in each group.SAP model was established by retrograde injection of 5% sodium taurocholate into pancreatic bile duct and APD model was established by placing drainage tube in right lower abdomen of rats at the basis of SAP.Part Ⅱ Another rats were injected intraperitoneally with cerulein(six consecutive injections,20 μg/kg body weight,at 1-h intervals)to induce mild acute pancreatitis(MAP),and then were randomly divided into the following four groups of 6 rats each: Controls(CN group),PAAF injection(PI group),PAAF + anti-HMGB1 neutralizing antibody(PIH group)and PAAF + control lg Y(PIC group).CN group rats received an intraperitoneal injection of 8m L saline solution;PI group rats received an intraperitoneal injection of 8 m L sterile supernatant of PAAF;PIH group rats received an intraperitoneal injection of 8 m L sterile supernatant of PAAF containing 200 μg anti-HMGB1 neutralizing antibody;PIC group rats received an intraperitoneal injection of 8 m L sterile supernatant of PAAF containing 200 μg control lg Y.After modeling,pathological changes of small intestine and pancreas were observed by HE staining,serum amylase and lipase activities were detected by automatic biochemical analyzer,serum endotoxin levels were detected by limulus amebocyte lysate method,serum DAO activity was detected by ultraviolet colorimetry,TNF-α,IL-6、D-lactic acid and HMGB1 levels were detected by ELISA method,apoptosis of intestinal mucosal epithelial cells was detected by TUNEL assay,m RNA expression of TLR4 was detected by real-time PCR,the expression of TLR4 signaling pathway and apoptosis-related proteins were detected by immunohistochemistry and Western blot.Compared with SAP group,the pathological damage of small intestine and pancreas of rats in APD group was significantly reduced,the pathological score was decreased(P<0.05),serum amylase,lipase and DAO activity,serum TNF-α,IL-6,D-lactic acid and endotoxin levels were also declined(P<0.05).The expression of TLR4,My D88,NF-κ B p65,TNF-α and IL-6 in the small intestine were significantly lower in APD group than those in SAP group(P<0.05).In addition,compared with SAP group,apoptotic index,cleaved caspase-3,and Bax expression were significantly decreased in the APD group,while Bcl-2 expression was increased(P<0.05).Compared with the control group,the serum level of HMGB1 in the PI group was increased obviously(P < 0.05).With the blockade of HMGB1 in the PAAF,the level of HMBG1 in serum was significantly decreased compared with that in the PI group(P < 0.05).Eight hours after injection,compared with the MAP control group,expression of TLR4,My D88 and NF-κ B p65 phosphorylation level were significantly increased in the PI group.When treated with the anti-HMGB1 neutralizing antibody,the TLR4 signaling pathway was significantly inhibited compared with that in the PI group.The results showed that early implementation of APD can significantly suppress overactivation of the intestinal TLR4 signaling pathway through draining HMGB1 from PAAF,thereby reducing the release of proinflammatory cytokines and the incidence of apoptosis,and ultimately reducing the severity of SAP-induced intestinal injury.