The Research On The Function And Related Mechanism Of TLR4 In Pancreatic Encephalopathy Of Rats

Part ? Establishment of rat model of pancreatic encephalopathy and expression of related indicators OBJECTIVE: In this part,a rat model of severe acute pancreatitis complicated with brain injury was established,which was reproducible and easy to replicate.Pathology,brain-dry-wet-wet weight ratio(W/D)of pancreas and brain tissue,and amylase(AMY),lipase(LIPA),interleukin-6(IL-6),and Toll-like receptors 4(TLR4)in the pancreas and brain during SAP observation Changes in body,preliminary exploration of pathological changes in pancreatic tissue and brain injury in patients with acute pancreatitis,to explore whether it is possible to establish this model to induce acute pancreatitis associated brain injury in rats and the expression of TLR4 in severe acute pancreatitis complicated with brain injury.METHODS: The male SD rats were randomly divided into 3 groups(16 rats in each group): control group with sham operation,control group with normal saline,group with severe acute pancreatitis.The rat model of acute pancreatitis was established by retrograde cholangiopancreatic injection.The expression levels of AMY,LIPA,IL-6 and TLR4 in the serum of each group were detected by enzyme linked immunosorbent assay(ELISA).The pancreatic tissue and acute severe disease were observed under light microscope after HE staining.Pathological changes in the brain tissue of the pancreatitis(SAP)group were histopathologically scored.Western blot(WB)and real-time quantitative PCR(qRT-PCR)were used to detect the expression of TLR4 in brain tissue of severe acute pancreatitis(SAP).The correlation between pathological scores of pancreatic tissue and brain tissue was analyzed statistically,and the expression of TLR4 in acute pancreatitis and the modeling method of brain injury in severe acute pancreatitis were discussed.RESULTS: There was no hemorrhage and edema in the control group of sham-operation.Mild peripancreatic edema and a small amount of ascites were observed in the saline group.Under light microscope,the interstitial tissue of the pancreas was significantly widened,the interlobular space was enlarged,and the pancreatic cell edema was observed.Increased pancreatic cell edema;pancreatic hemorrhage and necrosis occurred in half of the rats in the SAP model group established by retrograde injection of 5% sodium taurocholate(T8510,Solarbio)under the pancreaticobiliary duct;Under light microscope,the interstitial tissue of the pancreas was significantly enlarged,a large number of inflammatory cells infiltrated,and pancreatic focal or flake necrosis was observed.The pathological histological score of the pancreas was significantly different between the SAP group and the Nacl group and the control group.The difference between the Nacl group and the control group was significant.The rats of the SAP group were consistent with the pathological changes of the hemorrhagic pancreatitis.After 24 hours of modeling,observing the pathological changes of the brain,the brain tissue edema was observed by light microscopy of some rats in the SAP group,mainly in the gaps around the neuronal cells,there was a large lacunae around the invaginated vascular endothelial cells,and the light microscope was also visible.The nucleus dissolves and shrinks into a few neurons.In the saline group(Nacl group),there was no hemorrhage and edema in the brain tissue,and there was no obvious change;in the sham control group,there was no obvious abnormality in the brain tissue.Western blot was used to detect the relative expression level of TLR4 protein in brain tissue of each group,which was 4.53±1.081 in SAP group and 1.83±0.949 in Nacl group.There was a significant difference between the two groups(P<0.01).The relative expression of TLR4 mRNA in brain tissue of each group was detected by RT-PCR method.The relative expression of TLR4 mRNA in SAP group was 3.06 ±1.161,in Nacl group was 1.31 ±0.849.There was significant difference between SAP group and Nacl group.(P< 0.01).CONCLUSION: 1.The expression of TLR4,amylase,lipase and IL-6 in serum of rats with severe acute pancreatitis suggests that TLR4 may be involved in the pathogenesis of acute pancreatitis in rats and may be related to its severity.2.Changes in the pathology and brain index of the pancreas and brain tissue after modeling indicated that rat pancreatitis-related brain injury can be induced by establishing a model of severe acute pancreatitis in rats.3.The expression of TLR4 in brain injury tissues of rats with severe acute pancreatitis may be one of the pathogenetic mechanisms of brain injury in rats with acute pancreatitis.Part ? Expression and Mechanism of related Indexes in Rats after si RNA targeted interference with TLR4 Gene OBJECTIVE: In the first part,the rat model of brain injury induced by severe acute pancreatitis was successfully established,and the up-regulation of tlr4 expression in the brain injury tissue of acute necrotizing pancreatitis suggested that it might be related to the pathogenesis of brain injury in acute necrotizing pancreatitis.This section will use the previous method to establish a severe acute pancreatitis brain injury model,and use RNA interference(RNAi)technology,the use of rat brain stereotaxic apparatus local target intracerebroventricular injection of TLR4 si RNA,to interfere with the expression of the target gene,To detect the expression of TLR4,myeloid differentiation factor 88(My D88)and IL-6 in the brain tissue of severe acute pancreatitis,and to verify whether the injection of si RNA into the lateral ventricle of the rat stereotaxis is effective for the intervention of TLR4.To further explore the expression of TLR4 in brain injury of acute pancreatitis and related mechanisms of action,and seek possible therapeutic targets.METHODS: Sixty-four SD rats were randomly divided into 4 groups: normal saline control group(Nacl),acute severe pancreatitis group(SAP),TLR4 agonist group(LPS),and TLR4 si RNA group(si RNA).The rat model of acute pancreatitis was established by retrograde cholangiopancreatic injection injection with the same method as the first part.The TLR4 agonist group and the TLR4 si RNA group were injected with lipopolysaccharide(LPS)using the local intracerebroventricular local injection of the rat brain stereotaxic apparatus.Intervention with TLR4 si RNA.After modeling and intervention,HE staining was performed under light microscope to observe the pathological changes of brain tissue in each group.Histological histological scores were used to statistically analyze the correlation between histopathological scores of brain tissue in each group.Western blot(WB)and real-time quantitative PCR(q RT-PCR)were used to detect and analyze the relative expression of TLR4,My D88 and IL-6 in brain tissue of each group.RESULTS: In the Nacl group,SAP group,LPS group and si RNA group,there was a significant difference in the relative expression of TLR4 protein and m RNA in brain tissue.The expressions of TLR4,My D88,and IL-6 were significantly increased in SAP group and Nacl group.The expression of LPS in TLR4-specific agonist group was significantly increased compared with other groups,and the difference was statistically significant(P<0.05).However,the expression of TLR4,My D88 and IL-6 in the si RNA group was significantly lower than that in the SAP group and LPS group after the administration of TLR4 specific inhibitor.The difference was statistically significant.(P<0.05).CONCLUSION: 1.Using RNA interference technology to directly apply TLR4 si RNA carrying si RNA vector to local target site can effectively regulate the expression of target gene.2.It was further demonstrated that the release of IL-6 and other inflammatory factors through the My D88-dependent pathway may be one of the pathogenesis of rat pancreatitis.3.The expression of TLR4 is related to the severity of brain injury in rats with pancreatitis,which provide a possible therapeutic target for brain injury in pancreatitis.