| Objective: Severe acute pancreatitis(SAP)is accompanied by the damage of the tight junction(TJ)barrier,and the tight junction barrier can be regulated by cell division cycle 42(Cdc42)and F-actin.The purpose of this study was to investigate the effect of carbachol on the gut tight junction barrier in rats with severe acute pancreatitis and the role of Cdc42 and F-actin in it.Methods: Healthy Wistar female rats were separated into a sham-operation(SO)group(n=10),SO + carbachol group(n=10),SAP group(n=30)and SAP + carbachol group(n=30).Sodium taurocholate(5%)was retrogradely injected into the biliopancreatic duct of rats to induce SAP.The rats were injected intraperitoneally 12 hours after modeling,the mortality was observed,and the remaining rats were killed 24 hours after modeling.Subsequently,16 S rRNA sequencing was used to detect bacterial translocation(BT)in the gut of surviving animals.Hematoxylin and eosin(HE)staining was used to detect morphological changes in the pancreas and intestine.The expression of F-actin and tight junction proteins was analyzed by western blotting and immunofluorescence,and Cdc42 expression was analyzed by western blotting and immunohistochemistry.Results: The results demonstrated that the intestinal injury in SO and SO + carbachol groups was lower than that in the SAP + carbachol group(P<0.05);however,the intestinal injury was similar in the SO and SO + carbachol groups(P>0.05),and was significantly more severe in the SAP group compared with the SAP + carbachol group(P<0.05).Similarly,pancreatic injury in the SAP and SAP + carbachol groups was significantly higher compared with the SO and SO + carbachol groups(P<0.05);however,pancreatic injury was similar in the SAP and SAP + carbachol groups(P>0.05),and in the SO and SO + carbachol groups(P>0.05).The results of serum enzymology showed that the serum levels of lipase in the SAP and SAP + carbachol groups were significantly higher compared with the SO group(P<0.05).In addition,there was no difference between the SAP and SAP + carbachol groups(P>0.05),and between the SO and SO + carbachol groups(P>0.05).The results of serum lipase and amylase were similar to those of pancreatic HE.In addition,the mortality and BT rate of SAP + carbachol group were lower than that of SAP group,but there was no statistical difference(36.7% vs 46.7%,P>0.05;42.1% vs 56.3%,P>0.05).In addition,western blot results showed that the expression of Cdc42,F-actin and claudin-2 was significantly higher in the SAP and SAP + carbachol groups compared with the SO and SO +carbachol groups(P<0.05),and the expression of occludin and zonula occludens-1 were significantly higher in the SO and SO + carbachol groups compared with the SAP and SAP +carbachol groups(P<0.05).Immunohistochemistry results showed that Cdc42 expression was significantly increased in the SAP and SAP + carbachol groups compared with the SO group(P<0.01).Furthermore,Cdc42 expression,in the SO + carbachol group was the lowest(P<0.01),and expression of Cdc42 was significantly decreased in the SAP + carbachol group compared with the SAP group,(P<0.01).Immunofluorescence results showed that occludin and ZO-1 staining intensity in the SAP + carbachol group was significantly higher compared with that in the SAP group(P<0.05).However,occludin and ZO-1 staining intensity was significantly higher in the SO + carbachol group compared with the SO group(P<0.05).Conversely,claudin-2 and F-actin staining intensity was significantly higher in the SAP group compared with the SAP + carbachol group(P<0.05),and significantly lower in the SO + carbachol group compared with the SAP + carbachol group(P<0.05).The results of immunofluorescence and immunohistochemistry were consistent with those of Western blot.Conclusion: There are changes of expression of Cdc42 / F-actin and damage of tight junction barrier in the intestinal mucosa of SAP rats.Carbachol can protect the gut tight junction barrier of SAP rat model,in which Cdc42 / F-actin plays a regulatory role. |
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