Effects Of Folic Acid On Liver Injury In Alcohol-exposed Mice Via Regulating Gut Microecology

ObjectiveTo investigate the ameliorative effect of folic acid（FA）on liver injury in alcohol-exposed mice and the potential mechanism of its effect via regulating gut microecology.Method1.Animal grouping and intervention methods:Twenty-four 7-week-old healthy male C57BL/6J mice with SPF grade were randomly divided into 3 groups（each group consisted of 8 mice and there were no significant differences in baseline body weight）after adaptive feeding for 1 week,which were control group（CON）,alcohol model group（MOD）and folic acid intervention group（FA）.The CON group was given 10.0 ml·kg^-1·d^-1normal saline by gavage.The MOD group was given 56%（v/v）alcohol by gavage(the dose was 2.5,5.0 and7.5 ml·kg^-1·d^-1in the first 3 weeks and then 10.0 ml·kg^-1·d^-1for the last 7 weeks).The dose of alcohol in the FA group was the same as that in the MOD group,and folic acid was given with5.0 ml·kg^-1·d^-1 to intervention which 1 hour before alcohol gavage,the experiment was lasting for 10 weeks.2.During the experiment,the mice in each group ate and drank freely,and their body weight were measured every week.3.Hematoxylin-Eosin（H&E）staining was used to observe pathological changes of liver and small intestine in mice.4.The automatic biochemical analyzer was used to detect the levels of serum glutamic-pyruvic transaminase（ALT）,glutamic oxalacetic transaminase（AST）,triglyceride（TG）and total cholesterol（TC）.5.Enzyme-linked immunosorbent assay（ELISA）was adopted to determine the levels of lipopolysaccharide（LPS）in serum and interleukin-1β（IL-1β）,interleukin-6（IL-6）and tumor necrosis factor-α（TNF-α）in liver.6.Western-blot（WB）was performed to test the expression levels of tight junction protein（ZO-1,Claudin 1 and Occludin）in small intestine and Toll-like receptor 4（TLR4）,Myeloid differentiation primary response 88（My D88）,Interleukin receptor-associated kinase 1（IRAK1）,Tumor necrosis factor receptor-associated factor 6（TRAF6）and Nuclear factor kappa-B（NF-κB）in liver.7.16S r DNA gene sequencing technology was used to analyze the differences of gut microbiota in each group.8.Spearman correlation analysis was used to explore the potential relationship between the indexes related alcohol-induced liver injury（serum parameters and hepatic inflammatory cytokines）and gut microbiota.Results1.The results of body weight and liver index showed:there were no significant difference in body weight among all groups（P>0.05）.Compared with the CON group,liver index of mice in the MOD group was significantly higher（P<0.05）.In the FA group,liver index of mice was significantly lower than the MOD group（P<0.05）,but compared with the CON group,it was still significantly higher（P<0.05）.2.The results of pathological observation showed:in liver tissues of mice,the structure of liver lobules in the CON group was complete and clear,liver cells were arranged in orderly manner,and there was no obvious accumulation of lipid droplets or inflammatory cell infiltration.The liver cord of the MOD group was disordered,and a large number of lipid droplets were gathered which accompanied by inflammatory cell infiltration.Compared with the MOD group,liver cord arrangement of mice in the FA group was more orderly,lipid droplet aggregation and inflammatory cell infiltration were improved,but there were still great differences compared with the CON group.In small intestine tissues,intestinal structure of the CON group was complete and morphology of intestinal villi was normal.Compared with the CON group,intestinal villi in the MOD group were disorganized,broken and shortened,the submucosa and muscular layer were partially detached.Compared with the MOD group,the intestinal villi of mice in the FA group were more neatly arranged,the height of villi and the feature and shedding of the lower layer were improved,but there were still great differences compared with the CON group.3.The results of serum biochemical analysis showed:compared with the CON group,serum ALT,AST,TG and TC levels were significantly higher in the MOD group（P<0.05）.While compared with the MOD group,serum ALT,AST and TG levels in the FA group were significantly lower（P<0.05）,but compared with the CON group,these were still significantly higher（P<0.05）.4.ELISA results showed:compared with the CON group,the serum LPS level was significantly higher in the MOD group（P<0.05）.While compared with the MOD group,LPS level in the FA group was significantly lower（P<0.05）,but it was still significantly higher than the CON group（P<0.05）.In liver tissues,the levels of IL-1β,IL-6 and TNF-αwere significantly higher in the MOD group than those of the CON group（P<0.05）.Compared with the MOD group,the levels of inflammatory cytokines in the FA group were significantly lower（P<0.05）,but compared with the CON group,the levels of these inflammatory cytokines were still significantly higher（P<0.05）.5.WB results showed that in small intestine tissues,the expression levels of tight junction protein（ZO-1,Claudin 1 and Occludin）in the MOD group were significantly decreased than those of the CON group（P<0.05）.While the expression levels of these three proteins were significantly increased in the FA group than those in the CON group（P<0.05）.But compared with the CON group,the expression levels of these three proteins were still decreased in the FA group（P<0.05）.In liver tissues,the protein expression levels of TLR4,My D88,IRAK1,TRAF6,p-IκBα,the ratio of p-IκBα/IκBαand NF-κB were significantly increased in the MOD group than those of the CON group（P<0.05）,while compared with the MOD group,these proteins in the FA group were significantly decreased（P<0.05）.However,the expression levels of TLR4,My D88,IRAK1,TRAF6,the ratio of p-IκBα/IκBαand NF-κB in the FA group were significantly increased than those in the CON group（P<0.05）.6.16S r DNA sequencing results showed:compared with the CON group,the structure of gut microbiota in the MOD group was significantly changed,and folic acid intervention showed a certain regulatory effect on it.The MOD group had a significantly higher relative abundance of Bacteroidota than that in the CON group（P<0.05）,and compared with the CON group,the relative abundance of unclassified＿Lachnospiraceae was significantly decreased（P<0.05）.And the relative abundance of Verrucomicrobia,Lachnospiraceae＿NK4A136＿group and Akkermansia in the FA group was significantly increased than those of the MOD group（P<0.05）,while the relative abundance of Proteobacteria was significantly decreased（P<0.05）.LEf Se analysis showed that Prevotellaceae and Ileibacterium were the species with significant differences in the MOD group,and Bifidobacterium＿animals were the species with significant differences in the FA group.7.Spearman correlation analysis showed:alcohol-induced liver injury was correlated with the relative abundance of some species in gut microbiota.Bacteroidota and Proteobacteria were positively correlated with serum parameters and hepatic inflammatory cytokines,while Firmicutes,Verrucomicrobiota and Akkermansia were negatively correlated with those.ConclusionFolic acid could ameliorate liver injury in alcohol-exposed mice by regulating gut microecology.The specific mechanism may be related to regulating gut microbiota,improving intestinal barrier,reducing LPS leakage and inhibiting LPS-mediated activation of liver TLR4/NF-κB inflammatory pathway.