A Study On The Mechanism Of NINJ2 Regulating The Alcoholic Liver Disease

Alchoholic liver disease（ALD）is one of the liver diseases with the highest incidence in the world.It refers to acute and chronic liver damage caused by long-term drinking and acute excessive alcohol intake,including simple liver steatosis,hepatitis,cirrhosis and hepatic carcinoma.The mechanism of alcoholic liver disease is complex and the current treatment methods are very limited.Early studies based on populations,animal models,and cellular models have found that some genes and pathways are involved in regulating the occurrence and development of alcoholic liver disease.For example,a genome-wide association study（GWAS）found that PNPLA3 gene variants are significantly associated with the risk of alcoholic liver disease.So far,most of genes and pathways involved in the regulation of alcoholic liver disease are still incompletely elucidted.In order to further explore the susceptible genes of alcoholic liver disease,this study conducted in-depth mining of the FINNGEN GWAS data of alcoholic liver disease,including Gene-based association analysis,expression quantitative trait loci analysis（e QTL）,GEO differential expression analysis,etc.The results showed that the mutations of NINJ2 and other genes are associated with the risk of alcoholic liver disease in the population;e QTL analysis showed that the expression of NINJ2 in the liver tissues of patients with alcoholic hepatitis was significantly higher than the control population.Combined with the above population-based research results,it is suggested that NINJ2 may be a regulatory gene of alcoholic liver disease.In order to further explore the mechanism of NINJ2 on alcoholic liver disease,this study first established an animal model in wild-type C57BL/6 mouse.It was found that the expression of Ninj2 in the liver of mice feding with ethanol was significantly higher than control group.Subsequently,we constructed Ninj2 gene knockout mice to study the effect of this gene in alcoholic liver disease.The serum biochemical analysis showed that under the same time and dose of alcohol induction,the serum levels of alanine aminotransferase,aspartate aminotransferase and triglyceride in Ninj2^-/-mice were significantly lower than wild type mice.Hematoxylin-eosin（HE）staining,Oil red O staining and Masson staining showed that compared with control mice,Ninj2^-/-mice have less swelled hepatocytes and lipid droplets.And the degree of fibrosis was significantly reduced.Tunel（Td T-mediated d UTP Nick-End Labeling）staining found that the apoptosis of hepatocytes in Ninj2^-/-mice was less than that in WT mice.These results showed that alcohol-induced liver damage is significantly inhibited in the Ninj2 knockout mouse.The mechanism of NINJ2 regulating alcoholic liver disease is currently unknown.Preliminary studies have shown that it may regulate inflammation response,which plays a key regulatory role in the development of alcoholic liver disease.This study found that after alcohol induction,knocking out Ninj2 significantly inhibited the expression of the core regulatory gene Cyp2e1 of alcohol metabolism,promoted the expression of the core regulatory gene of lipid metabolism Ppar-α,and reduced the expression of alcohol-induced inflammation factors（Tnf-α,Il-6）in the liver of mice.In addition,it was also found that knocking out Ninj2 increased the phosphorylation level of AKT,and regulated the activation of NF-κB（p65 subunit）,thereby inhibiting cell apoptosis.These results show that Ninj2 may regulate alcohol-induced inflammation,fat metabolism,cell apoptosis and other processes through CYP2E1-PPAR-αand AKT-NF-κB pathways in mouse liver.In order to further analyze the molecular and cellular mechanisms of NINJ2regulating alcoholic liver injury,this study knocked down the gene using si RNA in L02hepatocytes,finding that it can inhibit cell apoptosis induced by alcohol.Overexpression of NINJ2 can promote cell apoptosis and inhibit cell proliferation under alcohol stimulation.To further explore the relevant molecular mechanisms of NINJ2 regulating alcoholic liver disease,this study also performed transcriptome sequencing on the liver tissues of WT and Ninj2^-/-mice after alcohol induction.The results showed that the differentially expressed genes and pathways mainly include biological processes which have been found to be related to alcoholic liver disease closely,such as cellular stress,immune response with compounds stimulation,PPAR,MAPK,PI3K-AKT,cell apoptosis,proliferation and differentiation,CYP450 family,inflammation,etc.In addition,this study also found that NINJ2 interacts with FGFR1 through immunoprecipitation.It has been found that fibroblast growth factors such as FGF21 regulate AKT pathway interacting with FGFR,which can affect the process of alcoholic liver disease.These studies partially revealed the molecular and cellular mechanisms of NINJ2 regulating alcoholic liver injury.In summary,this study first discovered a new potential alcoholic liver disease susceptible gene-NINJ2 through in-depth mining of population genetic data.Subsequently,the Ninj2 knockout mouse model was used to clarify the relationship between the NINJ2gene and the phenotype of alcoholic liver disease for the first time.In addition,in mouse liver and cellular models,this study found that knockout of NINJ2 may inhibit the expression of CYP2E1,enhance the activation of AKT and NF-κB pathways,and reduce alcohol-induced liver steatosis,inflammation and cell apoptosis.These studies preliminarily analyzed the molecular and cytological mechanism of NINJ2 regulating alcoholic liver disease,and provided research foundation and theoretical basis for revealing the molecular genetic mechanism of alcoholic liver disease and discovering new therapeutic targets for alcoholic liver disease.