Effect Of Lactobacillus Casei On Intestinal Tissue And Intestinal Cell Tight Tunction Protein With Rotavirus Infection

Rotavirus?RV?is one of the main causes of diarrhea among young animals and human beings,widely exists in the world and it's harmful to the breeding industry and human health.As an important component of intestinal health,probiotics and their metabolites are able to inhibit RV infection.In addition,they play an important role in maintaining the tight connection between intestinal epithelial cells,the stability of intestinal tissue barrier and the protection of intestinal mucosa.In order to investigate the effect of Lactobacillus casei on the morphology,function and its corresponding mechanism of porcine intestinal epithelial IPEC-J2 cells and small intestinal barrier of Kunming suckling mice infected with porcine rotavirus OSU strain,this study explored the intervention or antagonistic effect of probiotics on RV infection at the cellular level in vitro and in vivo.The inhibiting effects and mechanisms of pretreatment and post-treatment of Lactobacillus casei on RV infected cells were explored on the platform of IPEC-J2 cells.The protein secreted by Lactobacillus casei for 6 hours was segmented by molecular sieve?<3 KD,3-10 KD,10-50 KD,>50 KD and total protein?,using CCK-8 method to determine the maximum non-toxic dose of each segmented protein.The experiment was divided into protein pretreatment group,post-treatment group and treatment group according to the sequence of the action of secretory protein and RV and select the secretory protein with the best inhibitory effect on RV.Real time fluorescent quantitative PCR?q PCR?was used to detect each group:blank control group,RV group,pretreatment group,post-treatment group and treatment group.Cell samples of each group were collected at 0 h,12 h,24 h,36 h,48 h,60 h and 72 h,afterward detect the m RNA changes of occludin and claudin-1.In vivo test,Kunming mouse milk?2-day-old?was used as experimental animal,which was divided into five groups:blank control group?control group for short?,probiotic group,virus group,prevention group?first feeding Lactobacillus casei and then attacking RV?,treatment group?first attacking RV and then feeding Lactobacillus casei?,(each suckling mouse fed Lactobacillus casei50?L,with virulence of 10^-5.28 TCID₅₀/0.1m L porcine rotavirus 50?L).On the 1 d,2 d,3d,4 d,6d,8 d and 10 d,the jejunal tissue samples of each group were taken.The body weight,RV content in feces,changes of jejunal villi,expression of tight junction protein and distribution of SIg A content were detected by ELISA,he staining of paraffin section,immunohistochemistry,indirect immunofluorescence of frozen section,q PCR and other technologies,and expound the regulating effect of the probiotics before or after the small intestine infecting RV.1.In vitro test.?1?Lactobacillus casei secreted protein to reduce RV infection in IPEC-J2 cells.From the point of view of treatment,the effect of virus inhibition:pretreatment group>control treatment group>post treatment group?P<0.05?.In terms of molecular weight,the secretory proteins of<3 KD,3-10KD,10-50 KD,>50 KD and total protein all inhibited RV to a certain extent.The secretory proteins of molecular weight<3 KD had the strongest effect on RV infectivity?P<0.05?,but the effect of>50 KD was not significant?P>0.05?.?2?The results of real-time fluorescent quantitative PCR of occludin and claudin-1 showed that occludin in RV group The relative expression of m RNA began to decrease gradually at 12 h,and then decreased to the lowest level at 60H;it was significantly lower from 24 h than that of the control group and the pretreatment group?P<0.05?;at 24 h,48 h and 60 h,it was significantly lower than that of the control treatment group?P<0.05?,and the difference in the rest time was not significant?P>0.05?;compared with the post-treatment group,the difference in each time period was not significant?P>0.05?.The relative expression of claudin-1 m RNA in the post-treatment group was significantly lower than that in the control group?P<0.05?from 24 h,significantly lower than that in the control treatment group?P<0.05?at 24 h and 60 h,and significantly lower than that in the pre-treatment group?P<0.05?at 12 h,24 h,48 h and 60 h,with no significant difference between the rest?P>0.05?.The change of claudin-1 m RNA showed that the relative expression of claudin-1 m RNA in RV group decreased gradually from 12 h to the lowest and then became stable after 60 h;it was significantly lower in RV group from 24 h to the control group?P<0.05?,from 12 h to the pretreatment group?P<0.05?,and from 36 h to the control treatment group?P<0.05?.The relative expression of claudin-1 m RNA in the post-treatment group was lower than that in the RV group at12 h?P>0.05?,higher at 24 h,36 h,48 h,60 h and 72 h?P>0.05?,lower at 12 h,36 h,48 h,60 h and72 h?P<0.05?,lower at 12 h,48 h,60 h and 72 h?P<0.05?,lower at 48 h?P<0.05?than that in the co-treatment group,and no significant difference at the rest of the time?P<0.05?.2.In vivo test of suckling mice?1?The related effects of Lactobacillus casei on RV infected suckling mice:Lactobacillus casei can alleviate the weight loss of suckling mice infected with RV.There was no significant difference?P>0.05?in the weight of suckling mice between 1-4 days,and the weight of probiotics group was significantly higher than that of other groups?P<0.05?at 5-10 days.The weight of suckling mice in control group and prevention group was significantly higher than that of RV group?P<0.05?.The detection of RV ELSIA in the feces of suckling mice showed that the virus content of RV group and treatment group showed a wave like change,the virus content of the prevention group was the highest one day after the attack,and then decreased;the virus content of the prevention group and treatment group was significantly lower than that of RV group from three days?P<0.05?.The HE staining of jejunal paraffin section showed that the arrangement of jejunal villi in RV group was disordered,the jejunal villi in RV group were shortened and shed,the internal tissues were missing,the epithelial cells showed vacuolization and other obvious pathological changes,the intestinal villi in the prevention group and the treatment group were significantly improved compared with the virus group.By measuring the length of jejunal villi in each group,we can see that there was no significant difference between the control group,the bacteria group and the prevention group?P>0.05?,but there was no significant difference between the RV group and the treatment group?P>0.05?at 1 d,3 d,and 5 d,which was significantly lower than the treatment group?P<0.05?The difference was not significant at 5 d?P>0.05?.The results showed that the green fluorescence was the darkest and the SIg A content was the lowest in RV group,and the SIg A content was the most in probiotics group,which was significantly higher than that in RV group?P<0.05?Damage,promote the secretion of SIg A in intestine.?2?Expression of occludin and claudin-1 in intestinal tissue.The results of real-time fluorescent quantitative PCR showed that there was no significant difference in the relative expression of occludin and claudin-1 m RNA between the control group and the probiotic group?P>0.05?;the relative expression of occludin and claudin-1 m RNA in the probiotic group was significantly higher than that in the RV group?P<0.05?on the 6 d,8 d and 10d,and the relative expression of occludin m RNA in the treatment group and the prevention group was significantly higher than that in the RV group?P<0.05?The relative expression of claudin-1m RNA in the treatment group and the prevention group was higher than that in the RV group on the6 d,8 d and 10 d?P>0.05?.The results of immunohistochemistry showed that the coarse brown granules of occludin and claudin-1 were mainly distributed in the outer membrane and a small amount in the cytoplasm.The results of occludin test showed that the average optical density of probiotic group,control group and prevention group was significantly higher than that of RV group?P<0.05?;the optical density of probiotic group was the highest,significantly higher than that of control group?P<0.05?;the optical density of prevention group was slightly higher than that of treatment group,with no significant difference?P>0.05?;the optical density of treatment group was slightly higher than that of RV group,with no significant difference?P>0.05?.The results showed that the average optical density of RV group was significantly lower than that of other groups?P<0.05?,probiotic group was significantly higher than other groups?P<0.05?,the optical density of treatment group was lower than that of control group?P<0.05?,and there was no significant difference between treatment group and prevention group?P>0.05?.In conclusion,Lactobacillus casei can inhibit RV infection of IPEC-J2 cells.Pretreatment or coprocessing treatment with secretory proteins with molecular weight less than 3 KD is the best way to inhibit RV infection of IPEC-J2 cells.It can promote the m RNA expression of occludin and claudin-1 to enhance the integrity of cells.Feeding Lactobacillus casei to suckling mice after RV infection,improve the growth rate of body weight,reduce the content of RV in feces of suckling mice,protect and repair the intestinal villi damaged by RV infection,significantly increase the secretion of SIg A by intestinal mucous membrane of RV infected suckling mice,and promote the expression of occludin and claudin-1 in jejunum of suckling mice,protect the barrier homeostasis of intestinal cells.