Protective Effect And Mechanism Of Lactobacillus On Autophagy In Intestinal Epithelial Cells Of Mice Stimulated By Lipopolysaccharides

Lactobacilli is widely used for maintaining animal intestinal healthy.Intestine of piglet is always expose to lots of stress.Probiotics play an important role in modulation of the autophagy and stabilization of the intestinal epithelial cells.It is important to clarify what function Lactobacilli could exert on the autophagy in intestines and to analyse signaling tranduction of the autophagy.The study will provide the theoretical basis for regulation of intestinal stability,which is necessary to develop effective lactic acid bacteria to promote the healthy of the piglets intestine.The aim of this study is to evaluate the modulatory effects of different doses of Lactobacillus rhamnosus GG(LGG)and Lactobacillus with different adhesive ability on autophagy of intestine of C57BL/6 mice model stimulated by LPS.In vitro autophagy model was created with cultured mouse macrophage RAW264.7 cell stimulated by LPS to analyse the protection of lactobacillus on cell.In vivo autophagy model with mice i.p.with LPS to induce autophagy.Before the LPS stimulation,pretreatment of mice with LGG at dosage of 106CFU/d LGG or 109CFU/d for a week was used to evulate the modulation of different dose of LGG on the autophage.In another experiment,mice was pretreated with lactobacillus with differert adhesive ability at dosage of 108 CFU/d for a week before the mice was i.p.with LPS to induce autophagy with the aim to evulate the modulation of.different adhesive lactobacilli on autophage.Sera were collected immediately from mice after euthanization.The mRNA level and protein of autophagy-related molecular were measured by qRT-PCR,Western-blot and immunohistochemistry.Compared with RAW264.7 cell stimulated by LPS alone,pretreatment with LGG for RAW264.7 cells significantly decreased the level of autophagy.Compared with mouse stimulated by LPS alone,pretreatment of mouse with different doses of LGG significantly decreased level of autophagy.Compared with LPS stimulation alone group,LC3 level of in mouse fed with high or low doses of LGG significantly decreased.The level of apoptosis was highest in mouse fed with high doses of lactobacilli.Interesting,LPS stimulation decreased the level of p38 MAPK signaling molecular protein.Different doses of LGG inhibited the level of ERK signaling molecular protein and increased the level of tight-junction protein Claudin3.Compared with mouse stimulated by LPS alone,pretreatment of mouse with lactobacilli even with different adhesive ability significantly decreased the level of autophagy.The result of qRT-PCR indicated that lactobacilli with different adhesive ability significantly decreased the mRNA level of LC3 and Beclin-1.LPS stimulation increased the level of caspase-3 and facilitated the apoptosis in intestines,however,pretreatment of Lactobacillus reuteri ZJ617 with high adhesive ability decreased the level of caspase-3.Pretreatment of lactobacilli even with high or low adhesive ability increased the level of tight-junction protein occludin in the intestines of mouse.The results indicated that pretreatment of LGG with different dose or lactobacili with different adhesive ability could regulate the autophagy in the intestine of mouse stimulated by LPS.