Protective Effect And Mechanism Of Lactobacillus Reuteri ZJ617 On Intestine And Liver Of Piglets Induced By Lipopolysaccharides

As one of the most widely used and studied probiotics,lactobacillus has been applied in many important fields closely related to human life.Lactobacillus reuteri is one of the most well studied,but the current research mainly focuses on the evaluation of the probiotic effect of Lactobacillus reuteri on human beings,while the immune regulatory effect and mechanism of Lactobacillus reuteri on the intestinal health of livestock and poultry have not been enough studied and concerned.In order to elucidate the protective effects of Lactobacillus reuteri ZJ617 on intestinal immunity and barrier function,liver inflammation and its mechanism in piglets sensitized by lipopolysaccharide(LPS),so I started this research,so as to provide a more scientific theoretical basis for the development of probiotics for regulating animal intestinal health in the future.1)In vitro cell experiment:firstly,ZJ617 was heat killed and sonicated.The supernatant of ZJ617 was treated with proteinase K for 4 h,and then the cells were stimulated with 1 μg/ml LPS for 4 h to establish an inflammatory model.The effects of ZJ617 pretreatment on LPS sensitized cells were analyzed,and the cell components and metabolites in the supernatant of ZJ617 were determined.The results demonstrated that:compared with the CON group,ZJ617 significantly inhibited LPS-induced upregulation of IL-6 and IL-12 transcription,phosphorylation of p38 and JNK,and degradation of I-KBa(P<0.05).The supernatant of ZJ617 could significantly inhibit the upregulation of IL-6 and IL-12 transcription,the phosphorylation of p38 and JNK,and the degradation of I-κBα(P<0.05).However,compared with the untreated supernatant,the supernatant of ZJ617 treated with protease K had significantly lower anti-inflammatory ability.2)Animal experiment(gut):Piglets(Duroc×Landrace×Large White,including males and female;8.6± 1.1 kg)aged 28 d were assigned to 3 groups(n=6/group):CON group and LPS group were orally inoculated with PBS for 2 wk before intraperitoneal injection(i.p.)of physiological saline or LPS(25 μg/kg body weight),respectively;ZJ617+LPS group was orally inoculated with ZJ617 for 2wk before i.p.of LPS.Piglets were killed 4 h after LPS injection.Something were collected including serum,intestinal and liver samples.Some methods were used to analyze the immunomodulatory effect and protective mechanism of Lactobacillus reuteri ZJ617 on piglet intestine,such as ELISA,Western-blot,immunohistochemistry,metabonomics and 16S.1)The results of intestinal tissue showed that:after LPS stimulation,the intestinal permeability increased,the expression levels of tight junction proteins ZO-1,occludin and Claudin-3 were significantly decreased(P<0.05),and the expression levels of p-p38,p-JNK,LC3,ATG5,Beclin-1,Bax and SOD2 were significantly up-regulated(P<0.05);after ZJ617 intervention,the intestinal barrier function recovered,and there was no significant difference in protein level between the CON group and ZJ617+LPS group(P>0.05).2)Metabonomics results of ileum contents showed that compared with con group,4 metabolites in LPS group were significantly increased;compared with LPS group,5 metabolites in ZJ617+LPS group were significantly increased;KEGG pathway analysis showed that 8 metabolic pathways had significant changes.3)The results of ileal contents showed that ZJ617 did not significantly change the relative abundance and diversity of bacterial flora,but ZJ617 could restore the change of bacterial flora composition induced by LPS to a certain extent.3)Animal experiment(liver):the existence of gut-liver axis makes the intestinal barrier function and liver function closely linked.Therefore,the protective effect and mechanism of Lactobacillus reuteri ZJ617 on liver inflammation of LPS sensitized piglets were farther analyzed after intestinal test.The results indicated that:ZJ617 could inhibit the LPS-induced transcription of inflammatory cytokines IL-6,IL-12 and pro-IL-1β compared with the CON group;after LPS stimulation,the expression of proteins related to autophagy(Beclin-1,ATG5 and LC3)in liver increased significantly(P>0.05),leading to excessive autophagy,while ZJ617 could reduce the excessive autophagy induced by LPS stimulation;at the same time,the expression of apoptosis related proteins Bax and SOD2 in liver increased significantly(P>0.05);ZJ617 could reduce the excessive apoptosis induced by LPS.Therefore,Lactobacillus reuteri ZJ617 and its supernatant can significantly alleviate the inflammatory response and injury of porcine intestinal epithelial cells by inhibiting the transcription of pro-inflammatory cytokines and the expression of MAPK inflammatory signaling pathway proteins induced by LPS.ZJ617 mediates intestinal MAPK signaling pathway and autophagy signaling pathway,changes intestinal metabolites and metabolic pathways,restores the diversity of intestinal bacterial community,so as to alleviate LPS induced intestinal inflammatory response;at the same time,ZJ617 inhibits liver inflammatory signal transduction by affecting the transcription level of inflammatory cytokines,and inhibits excessive autophagy and apoptosis induced by LPS stimulation,so as to alleviate the liver injury of piglets induced by LPS.