Gut Microbiota Mediates Phosphatidylcholine Metabolism In The Development Of Liver Cirrhosis

Background:Cirrhosis is the common end-stage of chronic liver diseases caused by various etiology,characterized by diffuse fibrosis of the liver and pseudollobular formation,and finally devolepments into liver decompensation.At present,there is a lack of effective treatment throughout the world.Therefore,it is necessary to further study the pathogenesis of cirrhosis in order to find effective intervention targets.The treatment targeted at liver cannot completely block the progression of cirrhosis,so researchers gradually turn their eyes onto the extrhepatic organs.Decades of growing microbial studies,the intestinal microbiota（IM）and their metabolites play important roles in the development of cirrhosis,but the underlying molecular mechanism remain obscure.Therefore,the purpose of this study was to investigate the role and molecular mechanism of intestinal microbes and their metabolites in the development of cirrhosis.Methods:（1）CCl₄-treated mice or normal control（NC）were sacrificed after 5and 15 weeks of intervention.The disease severity was confirmed by Masson’s trichrome or Sirius red staining.Metagenomics sequencing and fecal untargeted metabolomics were performed to evaluate the composition and metabolic function of IM in parallel with the development of cirrhosis.（2）The activity of fecal phospholipase A₂（PLA₂）and the content of fecal lysophosphatidylcholine in CCl₄-treated mice and NC were detected after 15 weeks of intervention.The relationship between lysophosphatidylcholine and differential intestinal flora identified by Metagenomics sequencing was analyzed by Spearman correlation analysis.（3）On the basis of a mouse liver cirrhosis model by CCl₄ treatment,the antibiotic cocktail was used in drinking water to interfer with the normal structure of IM.After 15 weeks of CCl₄ intervention,samples were taken from normal control group（O.W）,cirrhosis group（C.W）,antibiotic control group（O.A）and cirrhosis-antibiotic group（C.A）.The intestinal phosphatidylcholine metabolism disorder was detected by determining the activity of fecal PLA₂ and the content of lysophosphatidylcholine the mice from each group.The G protein-coupled receptor 4（GPR4）and inflammatory factors in mice’s colon tissue were detected.The level of serum diamine oxidase（ABP1）and colonic tight junction protein（ZO-1）in mice from each group were detected to evaluate the damage of colonic mucosal barrier.The levels of serum inflammatory factors in mice from each group were detected.Inflammatory factors,Masson staining and Sirius red staining were detected in liver tissue of mice in each group to evaluate the levels of liver inflammation and fibrosis.（4）HMVEC was treated with lysophosphatidylcholine,and the phosphorylation levels of inflammatory signaling pathway molecules p65,ERK1/2 and p38 in cells were detected by Western Blot.The levels of inflammatory factors in cells and culture medium supernatant were detected by Real-Time PCR and ELISA.HMVEC with low GPR4expression（GPR4-si HMVEC）was constructed by transfecting of GPR4si-RNA to explore the effect of decreasing GPR4 expression on lysophosphatidylcholine-inducing inflammatory response of HMVEC.Results:（1）The CCl₄-treated mice presented liver fibrosis at 5 weeks and liver cirrhosis at 15 weeks indicated by collagen deposition and pseudo-lobule formation,respectively.Mice with liver cirrhosis showed distinct microbial composition from NC,even in the earlier fibrosis stage.Importantly,both of the liver fibrosis and cirrhosis mice were characterized with the depletion of Deltaproteobacteria and enrichment of Akkermansia.Furthermore,fecal metabolomics revealed distinguished metabolomics profiles of mice with liver fibrosis and cirrhosis from the NC.Notably,pathway enrichment analysis pointed to remarkable disturbance of nucleotide metabolic pathways during the development of liver cirrhosis.（2）There was abnormal phosphatidylcholine metabolism in the intestine of cirrhotic mice,which was manifested as significantly increased lysophosphatidylcholine（NC:6.09±1.49 pg/mg,Cirrhosis:12.64±4.85 pg/mg,P=0.0007）.In addition,lysophosphatidylcholine was positively correlated with Verrucomicrobia,Verrucomicrobiae,Verrucomicrobiales,Verrucomicrobiaceae,Akkermansia,Akkermansia muciniphila and Parabacteroides merdae,while it was negatively correlated with Sphingobacteriia,Flavobacterii,Sphingobacteriales,Sphingobacteriaceae,Flavobacteriales,Flavobacteriaceae and Bacteroides fragilis.（3）After using the antibiotic cocktail to interfering mice’s normal flora structure,intestinal phosphatidylcholine metabolism was disordered in mice,with significant increasement of lysophosphatidylcholine（O.W groups:15.02±3.22 ng/mg,C.W groups:30.87±5.87 ng/mg,O.A groups:33.55±4.47 ng/mg,C.A.group:48.31±7.17 ng/mg.O.W vs O.A:P=0.0125,C.W vs C.A:P=0.0175,O.W vs C.W:P=0.0286,O.A vs C.A:P=0.0404）.The treatment of antibiotic cocktail made the aggravatement of the intestinal barrier injury,increasement of the expression of colonic GPR4,promotion of the production and release of colon IL-6 and TNF-α,up-regulation of the liver IL-6 and TNF-α,and the deepening of liver fibrosis in cirrhotic mice.（4）After lysophospholipid treatment,the m RNA level of inflammatory cytokines in the HMVEC increased significantly（IL-6:control group:4.36±0.47,treatment group:16.80±2.43,P=0.0193;TNF-α:control group:53.43±0.43,treatment group:170.23±37.96,P=0.0490）,and the inflammatory factors in the culture medium supernatant also significantly increased（IL-6:control group:0.52±0.02pg/m L,treatment group:0.80±0.07 pg/m L,P=0.0309;TNF-α:control group:0.09±0.01pg/m L,treatment group:0.16±0.02pg/m L,P=0.0499）.At the same time,the phosphorylation levels of p65,ERK1/2 and p38 in inflammatory signaling pathways were increased.When GPR4 in HMVEC was knocked down,the prophosphorylation effect of lysophospholipid on inflammatory signaling pathway molecules（p65,ERK1/2 and p38）and the levels of IL-6 and TNF-αwere significantly decreased.Conclusions:（1）The intestinal microbiota is disturbed in the cirrhotic mice,which simultaneously leads to intestinal phosphatidylcholine and nucleotide metabolism disorder,and increases the content of intestinal lysophosphatidylcholine in the mice.（2）The using of Antibiotic cocktail can increase the content of intestinal lysophosphatidylcholine,aggravate intestinal barrier damage and inflammatory response,and accelerate the progression of liver cirrhosis.（3）Lysophosphatidylcholine can promote intestinal inflammatory responses by activating its receptor GPR4.