The Effect Of Lactobacillus Supernatant On Intestinal Immune Regulation And Liver Protection In LPS Sensitized Mice

Lactobacilli were one of the earlist applied and most studied probiotics,were extensively adopt in animal production.however,most of studies focus on bacteria selection and evaluation in practice,no more attention was paid on immune modulation of Lactobacillus especiall its components on intestinal epithelial cells of animal.The objective of this study was to analyze the effects of of the components and metabolites of lactobacillus rhamnosus(LGG)and Lactobacillus reuteri(ZJ617)on physiological regulation in intestine and liver of mice.1)In vitro study with cultured cell: Different combinations of LGG and ZJ617 components(SLP,gDNA,CpG-ODN,SLP plus gDNA,SLP plus CpG-ODN)were used to treat RAW264.7 cells before LPS stimulation.Cytokine production and toll-like receptor(TLR)expression were assessed by using RT-qPCR.MAPK and NF-κB signaling pathways were evaluated by using immunoblots and immunofluorescence.Pre-incubation of SLP,gDNA and CpG-ODN in hibited the activation of MAPK and NF-κB signaling pathways in LPS-stimulated RAW264.7 cells leading to attenuated cytokine production and TLR expression.The combination of SLP with gDNA or CpG-ODN further dampened the signaling pathways compared to SLP alone.LGGs and ZJ617 s significantly in hibited LPS-induced increase of expression of LC3,p-ERK1/2 and p-mTOR,and decrease expression of I-κBα protein.2)Modulation of LGGs and ZJ617 s on intestinal immunology and metabolism of mice stimulated by LPS: Six C57BL/6 mice per group were orally inoculated with ZJ617 s or LGGs for two weeks and intraperitoneally injected with LPS(10 mg/kg BW)for 24 h.LPS stimulation increased the intestinal permeability and decreased villiheight in the ileum of mice.LPS stimulation significantly increased the expression levels of TLR4,LC3,Atg5,Caspase-3 and SOD2.While,LGGs and ZJ617 s pretreatments restored the intestinal permeability and these protein expressions to the normal level like in the control group.Metabolomic analysis of ileum contents showed that there were 35 different metabolites(33 up-regulated and 2 down-regulated)in LPS group compared with the control.And sixteen different metabolites were significant lower in LPS+ZJ617s group than LPS group.There were 10 common metabolites among the three groups.3)Modulation of LGGs and ZJ617 s on liver of mice stimulated by LPS: T he aim of the trial was to investigate and compare the protective effects and molecular mec hanisms of Lactobacillus reuteri ZJ617 culture supernatant(ZJ617s)on acute liver injury in mice simulated by lipopolysacc haride(LPS)with Lactobacillus rhamnosus GG culture supernatant(LGGs)as a control.The treatment of mice was the same with the trial above.Oral ZJ617 s and LGGs significantly reduced serum alanine transaminase and aspartate transaminase levels to ameliorate liver injury in mice stimulated by LPS.They remarkably enhanced the intestinal mucosal barrier by upregulating the expressions of tight junction proteins(Claudin3,Occludin and ZO1).ZJ617 s and LGGs pretreatments down regulated IL-6 and TNF-α by in hibiting expression of TLR4,phosphorylation of MAPKs(JNK,p38 and ERK)and NF-κB nuclear translocation.They also decreased Bax and active caspase 3 expression to attenuate LPS-induced hepatic apoptosis.In conclusion,While CpG-ODN did not completely mimic LGG gDNA,the combination of SLP and CpG-ODN exerted effective immunomodulation on MAPK and NF-κB signaling pathways in LPS-stimulated macrophages.LGGs and ZJ617 s pretreatments restored the intestinal permeability and inflammation of LPS-stimulated mice to the normal level.Notably,ZJ617 s and LGGs suppressed liver autophagy by down regulating autophagic cascade proteins including Beclin1,Atg5 and LC3.ZJ617 s exert beneficial effects on liver like LGGs through suppression of hepatic TLR4-MAPK-NF-κB activation,apoptosis and the autophagy.