| Acute pancreatitis (AP) is a common acute abdomen in clinic. Especially severe acute pancreatitis (SAP) has multiple complications and a hazardous prognosis. It results in mortality rates ranging from 30 to 40%. In SAP, the renal dysfunction is about 14% ~ 43%, the mortality rate is as high as 71% ~ 84% after the development of acute renal failure (ARF).It is also one of The cause of death for patients with SAP in clinical.Toll-like receptor 4 (TLR4) is the main receptor of natural immune system to identify athogenic microorganisms. It can be widely adopted to identify specific pathogenic micro-organisms coupling signaling pathways and activates innate immune cells, eventually leading to a series of immune inflammatory response. TLR4 is widely distributed in animal and human liver, lung, heart, kidney pancreas and other tissues. TLR4 is the main receptor to megiated LPS (lipopolysaccharide,LPS) signal transduction transmembrane. It is essential for the gram-negative bacteria infection inflammation. In order to providing a new pathway for the therapy of acute pancreatitis we learned the role of TLR4 in acute pancreatitis with renal injury by observing the expression of TLR4 of renal changes on rats with AP.Objective: To observate the expression of TLR4 in mouse renal with severe acute pancreatitis, apply the antibodies to interfere TLR4, and explore the role of the pathogenesis of renal damage with acute pancreatitis, for providing theoretical basis of the prevention and treatment of acute pancreatitis with renal injury.Method: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 0.2 ml/min1 Animal model: The experiment was performed in wistar rats under anesthesia injected intra-peritoneally with 10% chloral hydrate.Then,theabdomen median incision was performed.The pancreatic gland was exposed, bile duct was injected conversely by microinjector with 5% sodium cholate (0.1 ml/100g) at the speed of 0.2 ml/min. Then the bile duct around duodenum was pinched. 3 minutes later, the ligature was cut off and abdomen was sutured.2 Animal groups: All male 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 lipase , urea nitrogen and creatinine were determined respectively by automatic biochemical analyzer. Pancreatic gland and renal histological analysis: paraffin section was stained by hematoxylin-eosin staining4 Immunohistochemical staining of TLR receptor 4 andNF-κB: After dehydration and antigen heat plerosis, the sections were incubated with 3% hydrogen peroxide for 15 min. The sections were dripped with goat serum for 11 min, then incubated with first antibody at 4°C overnight. In sham-operation group group, PBS replaced first antibody. Dripping successively the second antibody and the third antibody, which were kept 15 min respectively. The results were visualized by using 3,3-diaminobenzidine (DAB) as chromogen. At last, the sections were redyed with hematoxylin.5 Western blot: a piece of renal tissue about 100 mg was homogenized thoroughly by grinders, then 1ml lysis buffer was added to it for extraction of the protein(operation based on instruction).The protein about 20μg was mixed with loading buffer, then it was performed SDS denaturing polycrylamide gel electrophoresis, successively it was transferred 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 that was offered by Santa Crua company.Results:1 Pathological changes in rat pancreas: The pancreatic gland and its surrounding tissue of sham-operation group was approximatly normal, areas and degrees of dropsy, hemorrhage and necrsis of pancreatic gland and its surrounding tissue were severe, and the seroperitoneum was growing in AP group and TLR4mAb group group.2 Pathological changes in rat renal tissue:In sham-operation group the renal organizational structure were normal, edema, hyperemia, accumulation of inflammatory cell of renal tissue were severe in AP group, the organizational structure of renal was damaged relatively lighter in TLR4mAb group than the AP group.3 Serum amylase, lipase change: There was no significant difference in sham-operation group. Compared with the sham-operation group, the levels of serum amylase,lipase were increased in AP group and TLR4mAb group. Compared with AP group, a marked elevation of serum amylase,lipase were observed in TLR4mAb group4 Serum creatinine, urea nitrogen change: There was no significant difference in sham-operation group. The levels of serum creatinine, urea nitrogen were increased in AP group and TLR4mAb group. Compared with AP group,a marked elevation of serum creatinine, urea nitrogen were observed in TLR4mAb group5 Immunohistochemical staining of TLR receptor 4 and NF-κB:In sham-operation group, the TLR receptor 4 in renal tissue was weak expression. We found an overexpression of TLR4 in vascular endothelial cells, renal tubular epithelial cells and renal interstitial in AP group. Compared with AP group , it showed that the expression of TLR4 was decreased in TLR4mAb group by immunohisto-chemistry score (P<0.05).In sham-operation group, the NF-κB in renal tissue was weak expression. We found an overexpression of NF-κB in Vascular endothelial cells, renal tubular epithelial cells and renal interstitial in AP group. Compared with AP group, it showed that the expression of NF-κB was decreased in TLR4mAb group by immunohistochemistry score (P<0.05).6 Western blot:The expression of TLR4 protein: the density of TLR4 have weak expression in sham-operation group by scanning analysis, in AP and TLR4mAb group,TLR4 protein was significantly enhanced (P <0.05),Compared with AP group,a marked reduced of TLR4 protein was observed in TLR4mAb group(P <0.05).The expression of NF-kBp65 protein: the density of NF-kBp65 have weak expression in sham-operation group by scanning analysis,in AP and TLR4mAb group, NF-kBp65 protein was significantly enhanced (P <0.05),Compared with AP group,a marked reduced of NF-kBp65 protein was observed in TLR4mAb group (P <0.05).Conclusions:1 On the basing of serum amylase, lipase ,creatinine, urea nitrogen and those histological findings of areas and degrees of edema, hemorrhage and necrosis of pancreas and renal from this study, we concluded that AP associated renal injury model group has been successfully manufactured.2 Compared with sham-operation group the expression of TLR receptor 4 and NF-κB in renal tissue were conspicuously increased in acute pancreatitic rats.TLR receptor 4 as the gate of the inflammatory response may be closely related to the occurrence and development of renal injury.3 TLRmAb can improve renal histopathological damage,We thing that TLR receptor 4 antibody plays an important role in therapeutic effectiveness in AP. To block the TLR receptor 4 on systemic or local renal may alleviate the damage of acute pancreatitis.
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