Effects Of Rho Kinase Inhibitors On Intestinal Barrier Dysfunction In Septic Rats

Objective: The effect of Rho kinase（ROCK）inhibitor Y27632 on the expression and permeability of tight junction proteins（ZO-1 and Occludin）in the intestinal tissues of rats with sepsis was investigated by using the cecum ligation and puncture（CLP）method to establish a rat model of sepsis,and to investigate whether Y27632 plays a protective role on the intestinal barrier function of rats with sepsis and its mechanism.Methods: Thirty-two healthy,adult male SD rats were randomly divided into four groups（n=8）: sham-operated group（Sham group）,sham-operated+Y27632group（Sham+Y27632 group）,sepsis group（Sepsis group）and sepsis + Y27632 group（Sepsis+Y27632 group）.The sepsis rat model was established by the CLP method,and only the intestinal canal was turned without ligation and perforation in the Sham and Sham+Y27632 groups.5mg/kg of Y27632 solution was injected intraperitoneally15 min before modeling in the Sham+Y27632 and Sepsis+Y27632 groups,and an equal amount of Y27632 solution was injected intraperitoneally at the same time point in the Sham and Sepsis groups.PBS solution.After 24 h of modeling,the rats were euthanized.Histopathological changes in the small intestine were observed by hematoxylin-eosin（HE）staining and the small intestinal mucosal injury score（Chiu’s score）was performed;the ultrastructure of intestinal epithelial cells was observed by transmission electron microscopy（TEM）;serum D-lactate（D-Lac）,an index of intestinal permeability,was measured by enzyme-linked immunosorbent assay（ELISA）;the expression levels of ROCK1,phosphorylated MLC（p-MLC）,tight junction protein ZO-1 and Occludin in the small intestine were determined by immunohistochemistry（IHC）.Result:（1）Under light microscopy,the small intestinal villi in the Sham and Sham+Y27632 groups had normal structure and regular arrangement without congestion and fracture;in the Sepsis group,the small intestinal villi were disorganized,some of them were fractured and detached,and the mucosal vessels were hemorrhaged;in the Sepsis+Y27632 group,the histopathological damage of the small intestine was significantly reduced,and the small intestinal villi were still regularly arranged and the continuity was more complete.The small intestinal mucosal injury score showed that the Chiu’s score was significantly higher in the Sepsis group compared with the Sham group（P<0.001）;the Chiu’s score was significantly lower in the Sepsis+Y27632 rats compared with the Sepsis group（P<0.001）.（2）The TJ structure between intestinal epithelial cells in the Sham and Sham+Y27632 groups was clear under electron microscopy,and the cell gap was not significantly widened;the electron-dense material between intestinal epithelial cells in the Sepsis group was reduced,indicating that the TJ structure was damaged;compared with the Sepsis group,the TJ structure damage in the Sepsis+Y27632 group was reduced.（3）The serum D-Lac content of rats in the Sepsis and Sepsis+Y27632groups was significantly increased compared with the Sham group（P<0.001）;the serum D-Lac content of rats in the Sepsis+Y27632 group was significantly decreased compared with the Sepsis group（P<0.001）;the serum D-Lac content of rats in the Sepsis+Y27632 group was significantly decreased compared with the Sepsis group（P<0.001）.D-Lac content was significantly reduced in the Sepsis+Y27632 group compared with the Sepsis group（P<0.001）.（4）The protein expression of ROCK1 and p-MLC in intestinal tissues of rats in the Sepsis group was significantly up-regulated and that of ZO-1 and Occludin was significantly down-regulated compared with the Sham group（P<0.001）;the protein expression of ROCK1 and p-MLC in intestinal tissues of rats in the Sepsis+Y27632 group was significantly down-regulated compared with the Sepsis group,while the protein expression of ZO-1 and Occludin were significantly up-regulated（P<0.001）.The differences between the Sham and Sham+Y27632 groups for each of the above tests were not statistically significant（P>0.05）.Conclusion: Y27632 ameliorates intestinal barrier dysfunction in septic rats,probably by inhibiting the ROCK1/MLC signaling pathway,upregulating intestinal tight junction protein expression and reducing intestinal permeability.