Effects Of Early Intervention Of Houpu Sanwu Decoction On Interstitial Mucosal Mechanical Barrier Injury Associated With Severe Acute Pancreatitis

Objective: By applying Houpu Sanwu decoction to rats with severe acute pancreatitis(SAP),we observed the expression variations of intestinal mucosal mechanical barrier tight junction proteins and its regulatory pathway MLCK/p-MLC,and explored the effects of Houpu Sanwu decoction on intestinal mucosal mechanical barrier in rats with severe acute pancreatitis and the possible mechanism.The aim is to provide a theoretical basis for the prevention and treatment of SAP-related intestinal-derived damage by Houpu Sanwu decoction.Methods: There were 40 SPF healthy adult male SD rats,which were randomly separated into 4 groups,They were the Sham-operation group(Sham),the severe acute pancreatitis model group(SAP),the Houpu Sanwu decoction group(HPD)and the MLCK inhibitor(ML-7)group(ML-7),with 10 rats in each group.(1)Modeling: rats were anesthetized with 1% sodium pentobarbital(35mg/Kg)before modeling.Rats in the SAP,HPD and ML-7 groups were retrogradely injected with 3.5%sodium taurocholate(1ml/kg)into the biliopancreatic duct after opening the abdominal cavity to induce the establishment of the SAP model.Closure of the abdomen in the sham-operated group after opening the abdominal cavity and then turning the pancreas slightly several times.(2)Drug administration:Rats in the HPD group were treated with Houpu Sanwu decoction(4.6g/kg)by gavage 0.5h before modeling and every 12h(12h,24 h,36h)after modeling.Rats in the ML-7 group were treated with ML-7(2μg/g)by intraperitoneal injection every 12 h.Rats in the SAP and sham-operated groups were treated with saline in equal amounts and at the same time by gavage.(3)Sampling and detecting: we observed the changes of spirit,activity and eating of rats in each group during the experimental period.All rats were anesthetized again 48 hours after modeling,and the abdominal cavity was opened.The general condition of the abdominal cavity was observed.Blood samples were collected from the abdominal aorta and all rats were uniform execution.The serum amylase(AMY)was detected by biochemical method,the histomorphological changes of pancreas were surveyed by HE,and the pathological damage was scored to definited whether the SAP model was successfully established.ELISA was performed to detect serum levels of the inflammatory factors including interleukin(IL)-1β,and tumor necrosis factor-α(TNF-α),and to detect the levels of plasma diamine oxidase(DAO)and serum D-lactate(D-lac),which are indicators of intestinal mucosal permeability.Observation of morphological changes in HE staining of ileal mucosal tissue and scoring of pathology.Western-blot and immunofluorescence were used to detect changes in the expression of intestinal barrier tight junction proteins Claudin-1,Occludin,ZO-1 and signaling molecules MLCK and p MLC.And the m RNA expression of TNF-α,IL-1β,MLCK and p-MLC in ileum tissue were tested by real-time fluorescence quantitative PCR(q-PCR).Results:1.Compared with the Sham group,serum AMY,serum inflammatory factors TNF-α and IL-1β levels,ileal tissue TNF-α and IL-1β m RNA levels,pancreatic histopathological damage score,and ileal histopathological damage score were significantly higher in the SAP group rats(p<0.001).Compared with the SAP model group,serum AMY,TNF-α and IL-1β levels,ileal tissue TNF-α and IL-1β m RNA levels,pancreatic histopathological damage score and ileal histopathological damage score of rats in the HPD and ML-7 groups were significantly lower,and the differences were statistically significant(p<0.05).2.Compared with the Sham group,the expression of serum intestinal permeability indexes DAO and D-lac,and ileal tissue tight junction barrier proteins Claudin-1,Occludin,and ZO-1 were clearly up-regulated in SAP rats(p<0.001).The serum DAO and D-lac levels and the expression of Claudin-1,Occludin and ZO-1proteins in ileal tissues of rats in the HPD and ML-7 groups were gradually increasedcompared with the SAP group,and the differences were statistically significant(p<0.05).3.Compared with the Sham group,the MLCK/p-MLC protein expression and m RNA levels in the ileum tissue of rats in the SAP group were significantly higher(p<0.01).Compared with the SAP group,the protein and m RNA expressions of MLCK/p-MLC were significantly reduced in the HPD group and the ML-7 group,and the differences were statistically significant(p<0.05).Conclusions:1.Houpu Sanwu decoction could effectively alleviate the inflammatory response of pancreas and intestine in SAP by reducing the levels of TNF-α and IL-1β in serum and intestinal tissue.2.Houpu Sanwu decoction could reduce the increase of intestinal mucosal barrier permeability by upregulating intestinal mucosal mechanical barrier tight junction proteins likewise Occludin,Claudin-1,and ZO-1,reduce SAP-related intestinal damage then protect intestinal mucosal barrier,and maintain intestinal barrier function.3.The protective effect of Houpu Sanwu decoction on SAP associated intestinal mucosal barrier injury might be related through inhibition of the MLCK/p-MLC pathway.