Protective Effects Of Probiotics On Alcohol-Induced Liver Injury In Mice

Objective The effects of Lactobacillus rhamnosus GG(LGG),Bifidobacterium lactis(Bifi)and Lactobacillus rhamnosus-Lactobacillus dibacterium(Mix)on alcohol-induced liver injury in mice were investigated to evaluate the effectiveness of their protective effects on alcoholic liver injury in mice,and to provide a basis for further understanding of their mechanisms of action in vivo.Methods 80 6-week-old male C57BL/6J mice were randomly divided into control group(Ctrl),Lactobacillus rhamnosus GG group(LGG),Bifidobacterium lactis group(Bifi),Lactobacillus rhamnosus GG-Bifidobacterium lactis dibacterium complex group(Mix),alcohol group(EtOH),alcohol + Lactobacillus rhamnosus GG group(EtOH + LGG),alcohol + Bifidobacterium lactis group(EtOH + Bifi),alcohol + Lactobacillus rhamnosus GG-Bifidobacterium lactis dibacterium complex group(EtOH + Mix),10 animals in each group.Ctrl group,LGG group,Bifi group and Mix group feed Lieber-De Carli control liquid diet.EtOH group,EtOH+LGG group,EtOH+Bifi group and EtOH+Mix group feed Lieber-De Carli alcohol liquid diet containing 5%(w/v)alcohol.At the same time,the corresponding probiotic solution was intragastric.After 9 h of final gavage,mice were executed.The activities of alanine aminotransferase(ALT)and aspartate aminotransferase(AST)in serum were detected.The histopathological changes of liver were observed by H&E staining and oil red O staining.The contents of Alcohol dehydrogenase(ADH),Acetaldehyde dehydrogenase(ALDH)and Malonaldehyde(MDA)in liver were tested by the aldehyde kit.The mRNA expression levels of inflammatory factors,oxidative stress and bile acid(BA)signaling pathway were detected by real-time quantitative PCR.Western blotting was used to detect the expression of proteins related to hepatic BA signaling pathway.The changes of intestinal microbiota in mice were analyzed by 16 SrDNA sequencing.Results(1)Effects of probiotics on alcohol-induced liver injury in mice: The liver index,serum ALT and AST levels in EtOH group were significantly higher than those in Ctrl group,while the liver index,serum ALT and AST levels in EtOH+LGG group,EtOH+Bifi and EtOH+Mix groups were significantly lower than those in EtOH group,and the effects were similar among the three groups.H&E staining showed that EtOH+LGG group,EtOH+Bifi and EtOH+Mix group all reduced the number of inflammatory cell infiltration and fat vacuole of liver cells,restored the normal cell structure of liver,and the histopathologic score was significantly reduced.The results of oil red O staining showed that EtOH+LGG group,EtOH+Bifi and EtOH+Mix group significantly reduced the red staining area and the positive area rate in hepatocytes.(2)Effects of probiotics on hepatic inflammatory cytokine in mice: The Tnf-α,Il-6,Il-1β,Tgf-β,Mcp1 and Il-10 mRNA in EtOH group were significantly higher than those in Ctrl group.The Tnf-α,Il-6,Il-1β,Tgf-β and Mcp1 mRNA in EtOH+LGG group,EtOH+Bifi and EtOH+Mix groups were significantly lower than those in EtOH+Mix group,and the effect was most prominent in EtOH+Mix group,followed by EtOH+Bifi group.EtOH+LGG group,EtOH+Bifi group and EtOH+Mix group further improved Il-10 mRNA expression compared with EtOH group,and the effect was more prominent in EtOH+LGG group.(3)Effects of probiotics on hepatic ADH and ALDH activity in mice: The activity of hepatic ADH of EtOH group was significantly lower than that of Ctrl group and the difference was statistically significant,while ALDH activity was not significantly different.The ADH and ALDH activities increased significantly in EtOH+LGG,EtOH+Bifi and EtOH+Mix groups,and the ADH activities increased better in EtOH+LGG and EtOH+Mix groups.The increase of ALDH activity was similar in EtOH+LGG group,EtOH+Bifi group and EtOH+Mix group.(4)Effects of probiotics on alcohol-induced oxidative stress in mice: Hepatic MDA content in EtOH group was significantly higher than that in Ctrl group,while MDA content in EtOH+LGG group,EtOH+Bifi group and EtOH+Mix group was significantly lower than that in EtOH group with statistical difference,and the effect was the best in EtOH+LGG group.Compared with Ctrl group,EtOH group Nox4,P47 phox,P67 phox and Gp91 phox mRNA levels were significantly increased.Compared with EtOH+Mix group,EtOH+Bifi group and EtOH+Mix group,Nox4,P47 phox,P67 phox and Gp91 phox mRNA levels in EtOH+LGG group are significantly reduced,and the reduction of Nox4 mRNA level is more prominent in EtOH+Mix group.The P47 phox,P67 phox and Gp91 phox mRNA were significantly decreased in EtOH+Bifi group.The Sod1,Catalase and Gsh-px mRNA in EtOH group were higher than those in Ctrl group,and the Sod1 and Gsh-px mRNA in EtOH+LGG and EtOH+Bifi groups were further higher than those in EtOH group,and the difference was statistically significant.The Catalase mRNA level in EtOH+Bifi group was higher than that in EtOH group with statistical difference,while the Sod1,Catalase and Gsh-px mRNA levels in EtOH+Mix group were lower than those in EtOH group with statistical difference.The Cyp2e1 and Ho-1 mRNA in EtOH group were significantly higher than those of Ctrl group,and the Nrf2 mRNA was not significantly different.Compared with EtOH+LGG group,EtOH+Bifi group and EtOH+Mix group,Cyp2e1 mRNA levels in EtOH+LGG group and EtOH+Bifi group were significantly decreased,while Nrf2 and Ho-1 mRNA levels in liver of mice in EtOH+LGG group and Etoh +Bifi group were further increased with statistical difference.The Ho-1 mRNA levels in EtOH+Mix group were lower than those in EtOH group with statistical difference.(5)Effects of probiotics on BA signaling pathway in mice: Compared with Ctrl group,the Fxr,Fgfr4,Shp,Bsep and Ntcp mRNA in EtOH group were significantly increased,while Cyp7a1 mRNA was significantly decreased with statistical difference.The Fxr,Fgfr4,Shp,Bsep and Ntcp mRNA in EtOH+LGG group,EtOH+Bifi group and EtOH+Mix group were lower than those in EtOH+Mix group and the difference was statistically significant,and the effect was most prominent in EtOH+Mix group.The Cyp7a1 mRNA showed an increasing trend,but there was no statistical difference.Compared with Ctrl group,hepatic FXR protein in EtOH group was increased and CYP7A1 protein in EtOH+LGG group,EtOH+Bifi group and EtOH+Mix group was decreased compared with EtOH group.The expression of CYP7A1 protein increased with statistical difference.The ileal Fxr and Fgf15 mRNA in EtOH group were higher than those in Ctrl group,and only the increase of Fxr mRNA was statistically different.Compared with EtOH+LGG group,EtOH+Bifi group and EtOH+Mix group,the ileal Fxr and Fgf15 mRNA in EtOH+LGG group were decreased and the difference was statistically significant.The decrease of Fxr m RN level was most significant in EtOH+LGG group.Compared with Ctrl group,ileal FXR and FGF15 protein in EtOH group were significantly increased,while FXR and FGF15 protein were decreased in EtOH+LGG group,EtOH+Bifi group and EtOH+Mix group compared with EtOH group,and the difference was statistically significant.(6)Effects of probiotics on intestinal microbiota in mice: The 16 S rDNA gene sequencing found that there are no statistical significance in chao,ace,sobs,shannon and simpson indices,indicating that probiotics didn’t affect the Alpha diversity of intestinal microbiota in mice.Principal coordinate analysis,principal component analysis and partial least squares discriminant analysis showed that probiotics could affect the Beta diversity of intestinal microbiota to a certain extent.At the phylum level,Actinobacteria and Firmicutes in EtOH group were significantly higher than those in Ctrl group,while Proteobacteria and Verrucomicrobia were significantly lower.The level of Actinobacteria in EtOH+LGG group,EtOH+Bifi group and EtOH+Mix group was significantly decreased compared with that in EtOH group,while the level of Proteobacteria and Verrucomicrobia was increased.On the genus level,compared with Ctrl group,Olsenella,Bacteroides and Alloprevotella in EtOH group were significantly higher,while Akkermansia and Parasutterella were significantly lower.The levels of Olsenella and Alloprevotella in EtOH+LGG group,EtOH+Bifi group and EtOH+Mix group were significantly lower than those in EtOH group,while the levels of Akkermansia were significantly higher.LEf Se difference analysis showed that the specific differences were 16 species in the Ctrl group,7 species in the LGG group,3 species in the Bifi group,9 species in the Mix group,8 species in the EtOH group,3 species in the EtOH+LGG group,7 species in the EtOH+Bifi group and 4 species in the EtOH+Mix group.According to KEGG metabolic pathway analysis,10 major pathways were affected,namely lipid metabolism,metabolism of cofactors and vitamins,amino acid metabolism,transcription,translation,signal transduction,replication and repair,cell motility,nucleotide metabolism and energy metabolism.Correlation analysis between serum/liver indexes and intestinal microbiota at phylum and genus levels showed that,MDA,liver index,ALT and AST were positively correlated with Firmicutes,Actinobacteria,Parabacteroides,Alloprevotella,Parasutterella,Alistipes and Olsenella.And it is negatively correlated with Akkermansia,Parasutterella,Lactobacillus,Bilophila,Verrucomicrobia and Proteobacteria.ADH and ALDH are positively correlated with Akkermansia,Verrucomicrobia,Proteobacteria and Parasutterella.It was negatively correlated with Bacteroidetes,Actinobacteria,Parabacteroides,Alloprevotella,Alistipes,Olsenella,Barnesiella and Lactobacillus.Conclusion Lactobacillus rhamnosus,Bifidobacterium lactis and Lactobacillus rhamnosus-Lactobacillus dibacterium complex all showed protective effects against alcohol-induced liver injury in mice,and the mechanisms may be related to the inhibition of liver inflammation and oxidative stress,regulation of bile acid metabolism and improvement of intestinal microbiota.In addition,in inhibiting liver inflammation and regulating bile acid metabolism,Lactobacillus rhamnosus-Lactobacillus dibacterium had the best effect,and in inhibiting oxidative stress,the single bacterium had the best effect and the effects of Lactobacillus rhamnosus and bifidobacterium lactis were similar.