Role And Mechanism Of EHD2 In Nonalcoholic Fatty Liver Disease

Objective:Non-alcoholic fatty liver disease(NAFLD)is the most prevalent chronic liver disease worldwide,which can progress to steatohepatitis,liver fibrosis,cirrhosis and hepatocellular carcinoma,and the incidence of metabolic liver diseases,including alcoholic liver disease,is rapidly increasing due to the improvement of living standards.Despite their high prevalence and incidence,their complex pathogenesis remains unclear and there are no effective therapeutic approaches.Eps15 Homology Domain-containing protein 2(EHD2)is the least conserved molecule in the EHD protein family and is expressed in mammals,and EHD2 is associated with the regulation of cell membrane transport,signal transduction regulation,and transcriptional regulation of endocytosis,and is involved in the development of a variety of diseases and tumors.The aim of this project was to investigate the expression level of EHD2 in NAFLD,to initially explore the role of EHD2 in the development of NAFLD and the possible molecular mechanisms,and to provide a potential marker for the early diagnosis and treatment of NAFLD.Methods:1.NAFLD gene expression data and clinical characteristics information were downloaded from the Gene Expression Omnibus(GEO)database,and patients were grouped into high and low risk groups based on risk scores.Differential gene and enrichment pathway analysis of EHD2 was performed using bioinformatics software to observe which pathways are enriched by differential genes and to predict the biological behavior of EHD2 that may be involved in regulating the development of NAFLD..2.Serum specimens were collected from patients with nonalcoholic fatty liver disease and healthy population at Nantong University Hospital,and the enzyme-linked immunosorbent assay was applied to determine the expression of EHD2 in serum and further analyze the correlation between the expression of EHD2 and clinical parameters of patients with NAFLD.3.In vivo and in vitro models of NAFLD were constructed by feeding mice with a high-fat diet and treating HepG2 cells with oleic acid,and oil red O staining,hematoxylin and eosin staining were applied to detect the degree of hepatic steatosis,and immunoblotting,immunohistochemistry and transmission electron microscopy were used to verify the EHD2 in the fatty liver model.Expression and autophagic status were verified.4.EHD2 cell overexpression groups and control groups were constructed by cell transfection,and the mechanism of action of EHD2 affecting the development of NAFLD was verified by immunoblotting and oil red O staining.5.Based on the hints in gene enrichment analysis,immunoblotting was used to explore the possible involvement of EHD2 in the development of NAFLD by related pathways.Results:1.The biological effects of EHD2 on NAFLD were mainly focused on functional pathways such as autophagy-related genes,fatty acid degradation,ester exchange reaction and endocrine-related signaling pathways,as suggested by gene enrichment analysis;2.Serum EHD2 levels were significantly reduced in patients with NAFLD compared with healthy population controls,and analysis of clinical parameters showed that EHD2 was significantly associated with NAFLD;3.In The expression levels of EHD2 and autophagy-related protein LC3-II/I were significantly reduced in the livers of mice fed a high-fat diet and in oleic acid-treated HepG2 cells,and the expression levels of autophagy-specific substrate p62 were elevated,and the expression of lipid metabolism-related transcription factor SREBP1 was significantly reduced.4.The overexpression of EHD2 cells was reversed after LC3-II/I and p62 protein expression,and oil red O staining showed a significant reduction in lipid droplets,suggesting that EHD2 attenuates lipid accumulation associated with autophagy;5.In the constructed in vivo and in vitro models of NAFLD,phosphorylated AMPK expression was significantly elevated and phosphorylated mTOR expression was decreased in high-fat mouse liver and oleic acid-treated HepG2 cells after EHD2 overexpression.Conclusion:1.Serum EHD2 was significantly lower in NAFLD patients than in normal healthy populations,and the results of clinical analysis suggest that EHD2 may be an independent risk factor;2.EHD2 can regulate hepatic autophagy and affect the development of NAFLD;3.EHD2 triggers lipid autophagy by activating the AMPK/mTOR signaling pathway,and may be a potential target for promoting nonalcoholic hepatic lipid EHD2 triggers lipid autophagy through activation of AMPK/mTOR signaling pathway and may be a potential therapeutic target for improving lipid metabolism in NAFLD.