Effects Of Lactobacillus Amylovorus Combined With Rutin On Tight Junction Proteins And Related Cytokines Induced By Po Rv Infection

Porcine rotavirus(PoRV)is one of the pathogens causing severe diarrhea in suckling piglets.It infects intestinal epithelial cells,destroys the tight junction between cells and causes intestinal damage.Lactobacillus Amylovorus can colonize and form a stable flora in the intestine.It is known that Lactobacillus Amylovorus and its secretory supernatant can inhibit the destruction of epithelial integrity caused by pathogens and protect intestinal mucosal cells.Many studies have shown that polyphenols found in plant foods can improve the immune ability of the body and have biological activities such as antiviral,antioxidant and antitumor.The purpose of this study is to combat PoRV infection by probiotics combined with polyphenols,observe the effect of probiotics and polyphenols on PoRV infected intestinal tissue,and establish a theoretical basis for the research and development of microbial combined polyphenol preparations related to the prevention and treatment of porcine rotavirus infection in the future.In the early stage of this study,the virus inhibition rate was detected by CCK-8 method.Lactobacillus Amylovorus and rutin were used to intervene PoRV infected ipec-j2 cells and suckling mice.The test results showed that the virus content of each group was rotavirus infection group(RV group)> rutin prevention group(LR group)> Lactobacillus Amylovorus secretion supernatant prevention group(SR group)> Lactobacillus Amylovorus secretion supernatant combined with rutin prevention group(SLR group);The relative expression of ZO-1 mRNA was SLR group > SR group >LR group > RV group,and the relative expression of occludin mRNA was SLR group > LR group >SR group > RV group.The relative expression of claudin-4 mRNA in RV group was only significantly higher than that in control group at 24 hours,and were lower than that in other prevention groups,which was SLR group > LR group > SR group > RV group.In the experiment of suckling rats,the virus content in the feces of suckling rats in all prevention groups was lower than that in RV group,as follows: rotavirus infection group(RV group)> rutin low-dose prevention group(LR group)> Lactobacillus Amylovorus prevention group(SR group)> Lactobacillus Amylovorus combined with low-dose rutin prevention group(SLR group)> rutin high-dose prevention group(HR group).Through the morphological observation of jejunal villi,the jejunal villi of neonatal rats in RV group showed pathological changes such as disordered arrangement,shortening and falling off of villi,while the injury of jejunal villi in other prevention groups was significantly improved compared with RV group.The relative expression of ZO-1 and occludin mRNA and protein in RV group were significantly lower than those in other groups.The overall performance was SLR group >HR group > LR group > SR group > RV group.The relative expression of claudin-4 mRNA and protein were significantly higher than those in other prevention groups,and showed uneven distribution in intestinal tissues.The relative expression of claudin-4 mRNA and protein could be reduced in SR group,LR group,HR group and SLR group,The distribution of claudin-4 protein in intestinal tissue of each group was not affected.There was no significant difference between HR group and control group.By measuring the redox indexes of the intestinal tissue of the suckling mice in each group,the results showed that the contents of MDA,T-SOD and T-AOC in the Control group did not obvious change as a whole;The contents of MDA in RV group were the significantly above than the preventive group and the control group,while the contents of T-AOC and T-SOD were below than preventive group and the control group;The contents of MDA in SR group were below than this in LR group,but above than in this HR group and SLR group.The content of T-AOC and T-SOD in the SR group was above than those in the LR group,but below than those in the HR and SLR group;The content of MDA,T-AOC and T-SOD in the HR group showed a downward trend.By measuring the cytokine content in the intestinal tissue of the suckling mice in each group,the results showed that the overall level of trypsin content in the Control group did not obvious change and the content of PAR-2 showed an upward trend;The content of trypsin and PAR-2 in the RV group were above than those in prevention group and the control group;The content of trypsin and PAR-2 in the SR group were the below than those in LR group and above than those in SLR group and HR group.There was no significant change in the the content of TGF-β and MMP-9 in the the control group;The content of TGF-β and MMP-9 in the RV group were the above of than those in a prevention groups and control group;The content of TGF-β in the SR group was below than that in the RV group and it above than that in other groups,which showed an downward trend first and then increased.The content of MMP-9 was the higher than that in the other of the prevention groups,which showed an downward trend as a whole;The content of TGF-β in the LR group was the lower than that in the HR group and showed an downward trend as an whole.The content of MMP-9 had no significant change as an whole and had a little difference with the control group;The content of TGF-β in SLR group was lower than that in SR group and showed an upward trend,and the content of MMP-9 was lower than that this SR group and showed and an downward trend as an whole;The content of TGF-β in the HR group had no significant the change and was in the lowest,while the content of the MMP-9 showed an downward the trend and was in the lowest.In conclusion,Lactobacillus Amylovorus secretion supernatant and rutin intervention can repair the cell damage and uneven expression and distribution of tight junction protein caused by PoRV infection.Rutin has a concentration dependence on the intervention of PoRV infected cells and intestinal tissue,and low concentration rutin can cooperate with Lactobacillus amylovorus to enhance the ability to intervene PoRV infection.Both Lactobacillus amylovorus and rutin can reduce MDA,trypsin The contents of PAR-2,TGF-β and MMP-9 can increase the contents of T-SOD and T-AOC,enhance the antioxidant capacity,and reduce the damage of PoRV to intestinal epithelial cells.This study provides a reference for the prevention of PoRV infection and lays a theoretical foundation for the combined use of probiotics and polyphenols.