The Mechanical Injury Of Intestinal Mucosal Barrier In Rat Models With Severe Acute Pancreatitis And The Intervene Of Different Factors

Objective:To observe the the changes of the secreted phospholipase A 2(s PLA2), Occludin-1 and ZO-1 in rats intestinal in severe acute pancreatitis(SAP) model; To ex plore th e roles of the epithelial cell apoptosis and the tight junction fracture in SAP; To explore the relationship between myo sin light chain kinase(M LC K) and tight junction protein in intestinal mucosal mechanical barrier, so as to further ex plore the intestin al barrier d ysfunction in severe acute pancreatitis pathogenesis; To ex plore the traditional Chinese medicine theor y in severe acute pancreatitis with intestinal barrier injury on patho genesis; To observe the function of Qing yi decoction, Dex amethasone, M L-7 and dendritic cells(DC) in intestinal mucosal epithelial cell apoptosis; To observe the changes of tight junction structure and the inflammatory factor ex pression levels for exploring the protective effect and mechanism of Chinese medicine Qing yi decoc tion on intestinal mechanical barrier injury; To test the combinational effects of traditional Chinese medicine and western medicine; To improve the clinical results of severe acute pancreatitis associated intestinal injury. Methods:Part one: To investi gate the effect of Qingyi decoction on the ex pression of Occludin-1 and ZO-1 in intestinal barrier injury. One hundred and twent y ei ght health y male Spragu e-Dawley rats(weight, 180 ~220 g; age, 8 wk) were purchased from the Specific Pathogen Free(SPF) Ani mal Center of Dalian Medical Universit y. The animals were randoml y divided into 4 groups(n =32 per group): The control(Sham operation) group, SAP model group, SAP plus Qing yi deco ction treatment(QYT) group and SAP plus dex amethasone treat ment(DEX) grou p. The SAP model was induced b y retrograd e infusion of 1.5% sodium deox ycholate into the biliopancreatic duct of the rats. Eight rats per group rad omley selected were euthanized with 10% chloral h yd rate at 2 h, 6 h, 12 h, 24 h after treatment. Pacreas and intestinal were removed, and prepared b y following our published protocol. HE staining were used to observe pathological changes of the p ancreas and intestinal tissues; Transmission electron microscop y(ECM) was applied to observe the change of the tight j unction between epithelial cells; Serum am yl ase level was det ermined using automatic biochemical analyzer; Serum endotox in and TNF alpha were determined b y ELIS A assa y; Within 24 h ours after the SAP rat model was established, the apoptosis of intestinal mu cosa epithelial cells was tested b y using TUNEL assa y; Real-time PCR and Western blot methods were used to detect the ex pression of Occludin-1, ZO-1 and s P LA2 in rats.Part two: To investigate the role of myosin light chain kinase on the ex pression of Occlu din-1 and ZO-1 in intestinal barrier injury. This part included both in vitro and in vivo models. In animal ex periments, Fort y eight health y male Sprague-Dawle y rats were randoml y divided into four groups: the control group, SAP group, SAP plus the Qing yi deco ction group and SAP plus M L-7(dose: 1 mg/kg, concentration: 1 mg/ml) group. At 24 h post-operation, animals were euthanized, blood and tissue samples were b e removed. Serum am ylase content was determined using automatic biochemical anal yz er. Serum end otox in and TNF alpha contents were determined b y ELIS A assay. Real-time PCR and Western blot methods were used to detect the ex pression of Occludin-1, ZO-1 and M LCK in rats. In c ell ex perimental section, intestinal mucosal epithelial cell lines(Caco2) wer e cultured in containing 20% fetal bovine serum(FBS), 1%penicillin(PN) of the total nutrient medium(DMEM). Caco2 cell cultured after 48 h, at different concentrations of lipopolysaccharide(with 100ng/ml, 1μg/ml, 10μg/ml and 100μg/ml LPS in culture medium) stimulated intestinal mucosal epithelial cells. In flammatory cytokine and chemokine profiles were detected with Lumninex multiplex assay. Western blot was used to detecte the ex pression levels of Occludin-1, ZO-1 and M LCK. The time point was ultimatel y selected as 48 hours post-LPS stimulated as the epithelial cell damage model, a control group was set up to detect the structure change of intestinal mucosal epithelial cell’s skeleton, at the same time observe the intervention of emodin(40μM) and myosin light streptokinase inhibitors M L-7(10μM).Part three: To study the effect of dendritic cells on damaged epithelial cells. The intestinal mucosal epithelial cell injury model is established b y LPS stimulated intestinal mucosal epithelial cell lines Caco2. Then divided into control group, LPS group, 10% of dendritic cells(dendritic cells and Caco2 trained ratio is at 1:10) intervention group, 5% of dendritic cells(dendritic cells and Caco2 trained ratio is at 1:20) intervention group and 2.5%(dendritic cells and Caco2 trained ratio is at 1:40) intervention group. Fort y eight hours after treatment. Inflammatory factors were det ected using Luminex multiplex assay. The ex pression level of Occludin-1, ZO-1 and M LCK were determined b y Western blot. The change of intestinal mucosal epithelial cell skeleton structure was determined using a laser scanning confocal microscope. Results:1. Serum am ylase, endotox in, TNF alpha content, s P LA 2, and M LCK protein levels in SAP model group were significantl y increased compared to control group. However, the tight junction protein in intestinal tissue is decreased si gnificantly. Th e pathological d amage of pancreas an d intestinal is obvious. The gap of tight junction between intestinal mucosal epithelial cells widened significantl y. Intestinal mucosa epithelial cells apoptosis were significantl y in creased( P < 0.01). Compared with SAP model group, serum am ylase, endo toxin and TNF alpha content, s P LA 2, M LCK protein content in intestinal tissue in the Qingyi decoction, dex am ethasone, M L-7 groups were significantl y decreased. The content of Occludin-1 and ZO-1 in intestinal tissue increased significantl y. The pathological damage o f pancreas and intestine is minor. Minor structure damage of tight junction was discovered between the intestinal mucosa epithelial cells. Intestinal mucosa epithelial cells apoptosis among three treatment groups were significantl y decreased( P < 0.05).2. Compared with control group, In the LPS stimulation group, Caco2 cells’ MLCK content increased obviously(P < 0.05), intercellular tight junction protein Occl udin-1 and ZO-1 con tent decreased significantl y( P < 0.05), cytoskeleton structure damage is apparent, with LPS stimulation after 48 h of intestinal mucosal epithelial cell damage is the most si gnificant. Compared with LPS stimulation group, Caco2 cells’ MLCK content in emodin and M L-7 i ntervention group were decreased signifi cantl y( P < 0.05), intercellular tight junction protein, Occludin-1 and ZO-1 content were increased si gn ificantl y( P < 0.05), the damage o f cytoskeleton structure was reduced. Compared with that of LPS stimulation group, Caco2 intercellular tight junction protein, Occludin-1 and ZO-1 content increased significantl y( P < 0.0 5), M LCK content had no significant change( P < 0.05) in the dendritic cells intervention group. Conclusion:1. The SAP model was successful induced b y retrograde infu sion of 1.5% sodium deox ycholate into the biliopancreatic duct of the rats. Pancreas and intestinal tissue injury chan ges significantl y, ser u m endotox in and TNF alpha levels were increased signi ficantl y in SAP model rats.2. s P LA2 plays an important role in intestinal mucosal mechanical barrier injury. It can promote the intestinal mucosa epithelial cells apoptosis.3. The high ex pression of M LCK can deduce the ex pression of Occludin-1 and ZO-1, lead to tight junction gap widenin g, plays an important role in intestinal mucosal mechanical barrier injury in SAP.4. The dex amethasone and dendritic cells can reduce the levels of inflammatory med iat ors and cytokines, M L-7 can inhibit the ex cessive ex pression of M LCK. Qing yi deco ction can promote intestinal peristalsis, reduce intestinal permeabilit y, reduce intestinal bacterial translocation. It has a unique advantage in the treatment of SAP.