The Effect Of Toll-like Receptors 4 On Acute Pancreatitis Associated With Intestinal Injury In Rats

Acute pancreatitis (AP) is one of the common clinical acute abdomen. Especially severe acute pancreatitis (SAP) has a ferocious disease progression and the co-infection case-fatality rate can be up to 25%. Substantial studies have shown that the important reason in the pathogenesis of AP is the injury of intestinal mucosal barrier and it mediating pathological changes. SAP can lead to damages of intestinal barrier function, which will directly cause bacterial and endotoxin translocation, gut-derived infections and endotoxemia, further stimulates SIRS (systemic inflammatory response syndrome, SIRS) and multiple organ dysfunction syndrome (MODS).Toll-like receptors (Toll-like receptors, TLRs) are a class of cell surface receptors, which play an important role in the inflammation,immunity, identification of pathogens. Toll-like receptor 4 (Toll-like receptor 4, TLR4) as the LPS receptor can identify the LPS of G-bacteria, whose association with acute pancreatitis associated with intestinal injury has now been confirmed and gradually attention. As far as I know, the TLR4 is one of the first found subtype TLRs, it was widely studied, and its signal transduction pathway is also the more clear in the past few years. Some factors activating TLR4, through a series of signal transduction, amplify the inflammation.TLR4-mediated signal transduction, includs MyD88- dependent and non-independent pathway and its main mode is through the MyD88-independent pathway, MyD88-dependent pathway mainly mediates nuclear factor-κΒ(NF-κΒ) activation and cytokine production. Based on researches, the expression of TLR4 obviously increases in the intestinal microvascular endothelial cells and mucosal lamina propria of leukocytes in AP. Ji-min Ma found that it shows the significant cowelation between the TLR4 expression levels of intestinal mucosal and intestinal mucosa permeability index and the histopathological changes of intestinal mucosa also exists significant correlation. Now the TLR4mAb has been developed which was applied to ulcerative colitis mice and found that it can alleviate the condition of ulcerative colitis. Thereby, we speculate that admistrating TLR4mAb therapy also alleviate the acute pancreatitis associated with intestinal injury in AP rats.Objective:To investigate the pathology and DAO activity of intestinal tissue, the expression of TLR4, NF-κΒp65, TNF-αand IL-6 in intestinal tissue and demonstrate the pathogenesis of TLR4 signal transduction pathway on acute pancreatitis associated with intestinal injury by establishing AP model and admistrating TLR4mAb therapy, to explore the protective effect of TLR4mAb on experimental AP in rats.Method: 1 Animal model: The experiment was performed in wistar rats under anesthesia injected intra-peritoneally with 10% chloral hydrate. Then, the abdomen median incision was performed. The pancreatic gland was exposed, bile duct was injected antidromicly by microinjector with 5% sodium cholate (0.1 ml/100g) at the speed of 0.2ml/min. Then the bile duct entering duodenum was pinched. After 3minutes, the ligature was cut off and abdomen was sutured.2 Animal groups: All wistar rats (n=18) were divided into three groups at random: sham-operation group (n=6), AP group (n=6), and TLR4mAb group (n=6). TLR4mAb(50μg) was immdiately administrated via abdominal cavity injection in TLR4mAb group after making the animal models.3 Mensuration of biochemical parameters: The levels of serum amylase and limpse were determined respectively by automatic biochemical analyzer.4 Pancreatic gland and intestinal histopathology: Paraffin section was stained by hematoxylin-eosin .5 Mensuration of DAO activity: Take the homogenate of intestinal tissue, according to the method that was invented by jun-you li etal.6 Mensuration of TNF-αand IL-6: Take the homogenate of intestinal tissue, the assay of TNF-αand IL-6 in intestinal tissue according to the instruction of radiative immunity kit.7 Immunohistochemical staining: After dehydration and antigen heat plerosis, the sections were incubated with 3% hydrogen peroxide for 15min. The sections were dripped with 5%goat serum for 30min, then incubated with first antibody at 4°C overnight. In negative control group, PBS replaced first antibody. Dripping successively the second antibody and the third antibody, which were kept 15min respectively. The results were visualized by using 3,3-diaminobenzidine (DAB) as chromogen. At last, the sections were redyed with hematoxylin. The yellow granule of cytoplasm as the positive expression.8 Western blotting: The intestinal tissue about 100mg was homogenized thoroughly by ultrasonic disintegrator, then, 1ml lysis buffer was added to it for extraction of the protein. (the process according to the instruction of kit ) the protein about 100μg was mixed with loading buffer, then ,it was performed SDSdenaturing polycrylamide gelelectrophoresis, Successively it was thansferred to NC film, then, it was incubated with first antibody(1:250) at 4°C overnight, successively, the second antibody for 2h. The assay of antibody according to the instruction of kit was offered by Santa Crua company.Results:1 Serum amylase and Serum lipase: Compared with sham-operation group, the level of serum amylase and serum lipase were increased in AP group and TLR4mAb group (P<0.05). Compared with AP group, the level of serum amylase and serum lipase were decreased in TLR4mAb group (P<0.05).2 Pancreatic histological findings: It was seen by naked eyes and light microscope that the pancreatic gland and its surrounding tissue of sham-operation group was approximatly normal, there are dropsy, hemorrhage and necrosis in pancreatic gland and its surrounding tissue. a mass of bloody ascites in AP and TLR4mAb groups.3 Intestinal histological findings: It was seen by naked eyes and light microscope that the intestinal tissue of sham-operation group was approximatly normal, there are dropsy, hemorrhage, necrosis, inflammatory soakage, lymphopoiesis in Ap and TLR4 mAb groups.4 The activity of DAO: Compared with sham-operation group, the activity of DAO was decreased in AP and TLR4mAbgroups (P<0.05). Compared with AP group, the activity of DAO in TLR4mAb group was obviously increased (P<0.05).5 The expression of TNF-αand IL-6 in intestinal tissue: Compared with sham-operation group,the expression of TNF-αand IL-6 in intestinal tissue was significantly increased in AP group and TLR4mAb group (P<0.05). Compared with AP group, the expression of TNF-αand IL-6 in intestinaltissue was significantly decreased in TLR4mAb group (P<0.05).6 Immunochemistry:The expression of TLR4: Compared with sham-operation group, the expression of TLR4 in intestinal tissue was significantly increased in AP group and TLR4mAb group (P<0.05); There are extensive expression in the lymphocyte, monocyte, Neutrophil, acidophil, artery, vein, ring muscle of intestinal tissue in AP group; Compared with AP group, The expression of TLR4 was significantly decreased in TLR4mAb group(P<0.05)The activity of NF-κΒp65: Compared with sham-operation group, the activity of NF-κΒp65was significantly increased in AP group and TLR4mAb group (P<0.05),there are lots of yellow granules in epithelia and monocyte of intestinal mucous in AP group,Compared with AP group, the activity of NF-κΒp65 was significantly decreased in TLR4mAb group(P<0.05).7 Western blotting:The protein expression of TLR4 and NF-κΒp65: Compared with sham-operation group, It showed that the expression of TLR4and NF-κΒp65 was increased by Optical density analysis(P<0.05) in AP group and TLR4mAbgroup. Compared with AP group, the activity of NF-κΒp65 was significant decreased in TLR4mAb group(P<0.05).Conclusions:1 After bile duct injecting antidromic with 5% sodium cholate,it was detected that serum amylase and lipase increased and it was observed the degrees of oedema, hemorrhage and necrosis of pancreas and periph-tissues and ileum by staining with hematoxylin-eosin and the weak DAO of intestinal tissues. It was concluded that AP associated intestinal injury model group has been successfully manufactured.2 Compared with sham-operation group the expression of TLR4and NF-kBp65 was obviously high in the intestinal tissue of pancreatitic rats, it is proved that the signal transduction pathway via TLR4 may play an important role in the pathogenesis of acute pancreatitis associated with intestinal injury。3 TLR4mAb as one of the monoclonal antibody of TLR4 was invented recently .It can decrease the expression of TLR4and NF-κΒp65, prevent the drop of the intestinal mucosa, upgrade the activity of DAO and ameliorate the injury of the intestinal mucosa.So,it is concluded that TLR4mAb plays an important role in therapeutic effectiveness in AP.