Experimental Study On The Involvement Of ROCK/NF-κB/AQP8 Signal Activation In Ethanol Induced Intestinal Mucosal Barrier Dysfunction

Objective: Intestinal mucosal barrier refers to when harmful substances in the intestines pass through the intestinal mucosa and invade tissues other than the midgut or blood,the intestines can prevent the existence of this process.Ethanol can damage the intestinal mucosal barrier function through oxidative stress,endotoxemia,cytokines,immune regulation and other links.As a small molecule G protein,Rho has a downstream effector,Rho associated kinase(ROCK),which currently has subtypes ROCK1 and ROCK2.Rho/ROCK is a common signaling pathway in the body whose activation promotes inflammation.Nuclear factor kappa B(NF-κB)is a key transcription factor that regulates a large number of inflammatory factors.Rho A/ROCK has been shown to be an upstream signal regulating NF-κB.Therefore,ROCK may accelerate the occurrence and development of intestinal mucosal barrier dysfunction through activation of NF-κB.Aquaporin(AQP)is a family of water transporters of the same origin in mammals.Up to now,12 species(AQP0-12)have been discovered.AQP8 is expressed in the duodenum,jejunum and colon tissues,which is closely related to water metabolism in the digestive system and may play a key role in alcohol-induced intestinal barrier dysfunction.The purpose of this study was to explore the role of ROCK/NF-κB/AQP8 signal activation in ethanol-induced intestinal mucosal barrier dysfunction.Methods: The experiment included 7 groups,which were Control group,Et OH group,Et OH+NC group,Et OH+ROCK1 siRNA group,Et OH+ROCK2 siRNA group,Et OH+ROCK1+ROCK2 siRNA group,Et OH+PDTC group.Rats in the Et OH group were intragastrically administered with 10 g/kg body weight/day of 35%(v/v)ethanol double distilled water for 2 weeks.Thereafter,the dosage was increased to 14 g/kg body weight/day,and the administration was continued for 10 weeks.According to the grouping situation,the corresponding lentivirus interference treatment was given.The intestinal mucosal barrier dysfunction model was established in rats.The urine of rats was collected to detect the concentration of nitrite and NO to verify the changes of intestinal mucosal permeability.The moisture content of colon tissue was calculated by wet/dry specific gravity method.H&E staining was used to observe the pathological changes of colonic mucosa in each group.Real-time PCR and Western blot were used to detect the mRNA and protein expressions of ROCK1,ROCK2,NF-κB and AQP8 in colon tissues of rats in each group.The binding activity of NF-κB in colon tissue of rats was detected by EMSA.The expression of ROCK1,ROCK2 and NF-κB in colon tissue of rats in each group was detected by immunohistochemistry.The expression of AQP8 in each group was detected by immunofluorescence assay.Results: 1.Colonic mucosa morphology in normal control group and no exception Long-term ethanol exposure can significantly increase the permeability of colonic mucosa,increase the contents of nitrite and NO in urine,significantly damage the structure of colonic mucosa,and make the water content significantly higher than that of normal group.Interrupting ROCK1 and/or ROCK2 or inhibiting NF-κB can partially protect the colonic mucosal structure and inhibit the increase of water content in colonic tissue induced by ethanol.Compared with other treatment group,Et OH+ROCK1+ROCK2 siRNA and Et OH+PDTC group of protection.2.The mRNA and protein expressions of ROCK1 and ROCK2 were lower in normal control group,and the expression of ROCK1 and ROCK2 was significantly increased by ethanol.However,the up-regulated expression of ROCK1 and ROCK2 induced by ethanol was partially inhibited by siRNA interference.Specific interference with ROCK1 also partially inhibited the increase of ROCK2 induced by ethanol,and the effect of simultaneous interference with ROCK1 and ROCK2 was particularly significant.3.There was no significant difference in NF-κB mRNA levels among all groups.NF-κB protein expression intensity in normal control group was weak,the expression in Et OH group was strong brown yellow signal,interference ROCK1 and/or ROCK2 or inhibition of NF-κB can partially protect the intestinal mucosal tissue structure,protein expression intensity was weakened.Ethanol decreased the protein expression of NF-κB p65 in the cytoplasm and increased its expression in the nucleus.However,interference with ROCK1 and/or ROCK2 and inhibition of NF-κB inhibit cytoplasmic transfer of NF-κB p65 to the nucleus.Ethanol also activated the activity of NF-κB p65,and inhibited the activity of NF-κB by interfering with ROCK1 and/or ROCK2 or by inhibiting NF-κB.4.AQP8 was mainly expressed in the cell membrane,and the highest expression was found in the normal control group.The mRNA and protein levels of AQP8 in the Et OH group were significantly lower than those in the normal control group.Interrupting ROCK1 and/or ROCK2 or inhibiting NF-κB could partially restore the reduction of the expression levels of AQP8 mRNA and protein induced by ethanol.Conclusion: In a rat model of ethanol-induced intestinal mucosal barrier dysfunction,ROCK/NF-κB/AQP8 signal activation is involved in the process of ethanol-induced intestinal mucosal barrier dysfunction.