| Porcine rotavirus(PoRV)is one of the main viruses causing diarrhea in suckling piglets.It mainly infects intestinal epithelial cells and destroys intestinal epithelial barrier(IEB).In the process of infection,rotavirus can stimulate enterochromaffin cells to produce5-hydroxytryptamine(5-HT),activate enteric glial cells(EGC)to secrete glial cell derived neurotrophic factor(GDNF),increase the expression of ZO-1 in intestinal epithelial cells,change the distribution of ZO-1 in intestinal epithelial cells,and destroy the integrity of IEB.EGCS secrete S-nitrosoglutathione(GSNO)to promote the expression of tight junction related proteins,improve the localization of tight junction proteins,and participate in maintaining the integrity of IEB.IEB is an important intestinal barrier against pathogens,including the physical and chemical barrier on the surface of intestinal epithelial cells,epithelial layer and tight junction protein between epithelial cells.ZO-1 protein,as the cytoskeleton,plays the role of intercellular connection and adhesion.Lactobacillus amylovorus is one of the lactic acid bacteria,which can colonize and form stable flora in the intestinal tract.It is known that Lactobacillus amylovorus from the intestinal tract can inhibit the destruction of epithelial integrity caused by pathogens and maintain the normal distribution of tight junction proteins in the epithelial barrier.But whether it can improve the intestinal epithelial barrier by activating EGCS to secrete GDNF and GSNO and promoting the expression of ZO-1,The mechanism of interfering with rotavirus to destroy intestinal epithelial barrier is unknown.In this study,ipec-j2 cells and 2-day-old suckling Kunming mice were used to establish models,respectively,to explore the protective effect or repair ability of Lactobacillus amylovorus and its secretion supernatant on PoRV damaged intestinal epithelial barrier at cell level and jejunum level.The results of regulating GDNF by Lactobacillus amylovorus and its secretion supernatant showed that the content of 5-HT in L.a group decreased with time,but the expression level was the highest at each time period.The content of 5-HT in BI group and AI group was higher than that in RV group.The content of 5-HT in BI group decreased with time.The content of 5-HT in AI group increased first and then decreased.The content of 5-HT in RV group was higher than that in control group.The results showed that the relative expression of GDNF mRNA in L.a group was always on the rise and the highest in each time period.The expression of GDNF mRNA in BI group and AI group increased first and then decreased.The expression of GDNF mRNA in RV group was always on the decline.The overall level of GDNF mRNA in BI group was higher than that in AI group and RV group,and the overall level of GDNF mRNA in RV group was lower than that in control group.The overall trend of relative expression of ZO-1 mRNA was consistent with that of GDNF mRNA.At the protein level,the overall trend of ZO-1,GFAP and GDNF protein expression was similar.The expression of ZO-1 protein in L.a group was the highest at each time period,and the expression of GFAP and GDNF protein was the highest except the third day after PoRV infection;The expression of GFAP protein was the highest in BI group and GDNF protein was the highest in AI group on the 3rd day after PoRV infection.Immunohistochemical results showed that there was no significant difference in the average light intensity of GDNF protein between L.a group and BI group(P > 0.05),which was higher than that of RV group and AI group.The expression of ZO-1 protein in L.a group was the highest,and that in RV group was higher than that in control group(P > 0.05).The expression of ZO-1 protein in BI group was higher than that in AI group and RV group;The expression of GFAP protein in BI group was the highest,and that in L.a group was higher than that in AI group and RV group.The overall trend of relative expression of ZO-1 and GDNF mRNA in cell experiment was similar to that in animal experiment.At the protein level,the expression of ZO-1 protein and GDNF protein in S.BI group,L.a.S group and S.AI group was higher than that in RV group in the early stage of PoRV infection,and the expression of ZO-1 protein and GDNF protein in RV group was increased in the late stage.Immunofluorescence results showed that the fluorescence intensity of GDNF protein in S.BI group and S.AI group was significantly higher than that in RV group(P < 0.01),and there was no significant difference in the fluorescence intensity of GDNF protein between L.a.S group and control group(P > 0.05).There was no significant difference in the fluorescence intensity of ZO-1protein.The protein expression level of L.a.S group was higher than that of other groups,while that of RV group was the lowest.The results of the investigation on the regulation of GSNO by Lactobacillus amylovorus on ZO-1 are divided into two parts: 1.the regulation of endogenous GSNO on ZO-1.The GSNO content in L.a group was significantly higher than that of other groups(P < 0.05).The overall change trend of GSNO content in RV group,BI group and AI group was similar,which was both rising first and then decreasing,and the overall level of GSNO content in RV group was lower than that of control group.2.Exogenous GSNO could regulate the expression of ZO-1.In cell experiment and animal experiment,there was no significant difference in the relative expression of ZO-1 mRNA between GSNO treatment group(G.AI group)and Lactobacillus amylovorus and its secretion supernatant treatment group(P > 0.05).At the protein level,the trend of ZO-1 protein expression in G.AI group and S.AI Group was similar,and both decreased with time;In the animal experiment,the expression of ZO-1 protein in g.ai group and RV group decreased with time,while that in AI group increased at first and then decreased.The expression of ZO-1 protein in G.AI group was higher than that in AI group in the early stage,and on the contrary in the later stage.The expression of ZO-1 protein in AI group was higher than that in RV group except the first day after PoRV infection.The expression of GDNF,GFAP and ZO-1 protein in G.AI group was higher than that in AI group and RV group.The results of antioxidant activity of Lactobacillus amylovorus showed that the content of NO and MDA in L.a group had no significant change,but the expression level was low.The content of CAT,SOD,GSH and GSH-Px in L.a group was high,which was basically higher than that in other groups.The content of NO and MDA in RV group was high,which was significantly higher than that in other groups(P < 0.05).The content of CAT,SOD,GSH and GSH-Px in L.a group was low,which had no significant change.The contents of NO and MDA in BI group and AI group were lower than those in RV group and higher than those in L.a group,while the contents of CAT,SOD,GSH and GSH-Px in BI group and AI group were lower than those in L.a group and higher than those in RV group.In conclusion,Lactobacillus amylovorus and its secretion supernatant can promote the release of GDNF and GSNO,and enhance the autocrine of GDNF in ipec-j2 cells,thereby promoting and adjusting the expression and distribution of ZO-1;Lactobacillus amylovorus reduced the contents of NO and MDA,increased the contents of SOD,CAT,GSH and GSH-Px,enhanced the antioxidant capacity,and indirectly enhanced the expression of ZO-1 in intestinal epithelial cells.The combination of the two can further improve the tight junction between jejunal epithelial cells and ipec-j2 cells,and interfere with PoRV damage of intestinal epithelial barrier.This study provides a certain reference and basis for the follow-up study of probiotics and their secreted products to regulate the integrity of intestinal epithelial cell barrier and intervene virus infection. |
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