Protective Effect Of Lactobacillus Amylovorus On Intestinal Mucosal Barrier And Liver Function Of PoRV Infection

Porcine rotavirus(PoRV)is one of the main causes of diarrhea in piglets.It has been proved that PoRV infection is not only limited to gastrointestinal tract,but also can infect intestinal organs through various ways.Intestinal tract and liver are closely connected by portal vein anatomically,so the liver is the most important organ to be attacked in the study of RV enteral infection.The "gut-liver axis" theory is put forward on this basis,and its main mechanism is the damage of the intestinal mucosal barrier,and the toxic substances such as bacterial toxins in the intestinal lumen enter the liver along with the portal vein,and then lead to liver damage.As a member of probiotics,Lactobacillus amylovorus has been proved to be effective in inhibiting PoRV infection.However,the changes of intestinal mucosal barrier and liver function during its intervention and the relationship between the two are still unclear.Accordingly,this study aims to use porcine group A rotavirus OSU strain infect IPEC-J2 cells and 3-day-old Kun Ming suckling mice to establish a model,and L.amylovorus was used to intervene,to preliminary explore whether L.amylovorus could exert the protective effect on the intestinal mucosal barrier and liver function of PoRV infected suckling mice by the "gut-liver axis" mechanism.It provides experimental basis and new ideas for scientific prevention and treatment of PoRV infection.In order to explore the effects of L.amylovorus on the chemical and mechanical barrier of intestinal mucosal infected with PoRV,the m RNA relative expressions of Mucoprotein 2(MUC2),Trefoil factor 3(TFF3),Zonula occludens-1(ZO-1)and Occludin were detected by real-time quantitative PCR.The contents of MUC2 and TFF3 were quantitatively detected by ELISA,the expressions of ZO-1 and Occludin were detected by Western blot,and the distributions and expressions of ZO-1 and Occludin were detected by immunohistochemistry.In order to explore the effects of L.amylovorus on the permeability and integrity of intestinal mucosal infected with PoRV,the contents of endotoxin(ET),diamine oxidase(DAO)and D-Lactate(D-Lactate)were determined by ELISA,and the morphological and structural changes of jejunum were observed by HE staining.In order to explore the effects of L.amylovorus on PoRV infected liver function,the activities of alanine aminotransferase(ALT)and aspartate aminotransferase(AST)were detected by colorimetric method,and the morphological and structural changes of liver tissue was observed by HE staining.The m RNA relative expression and content changes of tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6)were detected by real-time quantitative PCR and ELISA.The expressions of superoxide dismutase(SOD)and malondialdehyde(MDA)were determined by kits.The results showed that compared with the control group,the RV group significantly up-regulated the expressions of MUC2 and TFF3 and decreased the expressions of ZO-1 and Occludin in animal experiments.The expressions of MUC2 and TFF3 in L.amylovorus before inoculation group(BI group)and L.amylovorus after inoculation group(AI group)were lower than those in RV group,and the overall expressions were RV group >AI group >BI group.The expressions of ZO-1 and Occludin were higher than those of RV group,and the overall expressions was BI group >AI group >RV group,indicating that the intervention of L.amylovorus reduced the damage of PoRV to intestinal mucosal barrier function,and the preventive intervention was better than the therapeutic intervention.In cell experiments,the RV group decreased the expressions of ZO-1 and Occludin.The expressions of ZO-1 and Occludin in secrete supernatant before inoculation group(S.BI group)and secrete supernatant after inoculation group(S.AI group)were higher than those in RV group,suggesting that L.amylovorus secrete supernatant could repair intestinal epithelial cell barrier injury caused by PoRV infection.The contents of ET,DAO and D-Lactate in serum of RV group were significantly higher than those of control group,and pathological section results also showed that jejunum mucosa was damaged.The contents of ET,DAO and D-lactate in BI group and AI group were significantly lower than those in RV group,and the damage of intestinal mucosa was repaired to some extent compared with that in RV group,suggesting that L.amylovorus intervention can repair the damage of intestinal mucosa permeability and integrity caused by PoRV infection.Compared with control group,RV group significantly increased the activities of ALT and AST in serum,increased the expressions of TNF-α,IL-6 and MDA in liver tissue,and decreased the activity of SOD.BI group and AI group decreased the activities of ALT and AST,as well as the expressions of TNF-α,IL-6 and MDA,and increased the activity of SOD.Liver histopathological results showed that the liver cell staining in BI and AI groups was more homogeneous than that in RV group,and the liver lobule structure was more intact and clear than that in RV group,without obvious lesions.These results indicated that intervention of L.amylovorus could reduce the level of serum liver function enzymes and repair the pathological changes,inflammatory reactions and oxidative stress damage of liver tissue caused by PoRV infection.In conclusion,L.amylovorus can repair intestinal mucosal barrier function,improve intestinal mucosal permeability,reduce serum endotoxin level,reduce liver inflammation,enhance liver antioxidant capacity,and repair intestinal mucosal barrier function and liver function damage caused by PoRV infection through the "gut-liver axis".This study enriched the experimental data of L.amylovorus as probiotics,provided a preliminary reference and basis for the follow-up study of probiotics using the "gut-liver axis" mechanism to reveal the relationship between gut and liver during rotavirus infection,and provided a new idea for the scientific prevention and treatment of porcine rotavirus infection.