Comprehensive Analysis Of Molecular Network Of Nonalcoholic Fatty Liver Disease And The Role Of OPTN In Nonalcoholic Fatty Liver Disease

ObjectiveNonalcoholic fatty liver disease(NAFLD)affects 25%of the world’s adult population and brings major health problems as well as economic burden.It is of great significance to explore the key molecular events and therapeutic targets of NAFLD for the treatment of disease.OPTN is related to the progress of tumors,inflammatory diseases and cardiovascular diseases,but the role of OPTN in NAFLD remains unclear.This study intends to reveal the role and regulatory mechanism of OPTN in NAFLD.MethodPart Ⅰ:This study integrated six NAFLD datasets from Gene Expression Omnibus(GEO),including GSE48452,GSE63067,GSE66676,GSE89632,GSE24807 and GSE37031.These datasets were used to analyze the differential genes and metabolic characteristics of NAFLD,and then were integrated with single-cell sequencing to explore the immune cell subgroups of NAFLD.In addition,a NAFLD prediction model was constructed by using the method of minimum depth random forest and the potential therapeutic target OPTN was screened,and this target was further verified in the NAFLD cell model induced by Palmitic Acid(PA).Part Ⅱ:NAFLD mouse model was constructed by C57BL/6 mice on high-fat diet for 12 weeks or MCD diet for 4 weeks.After injecting OPTN knockdown adeno-associated virus into tail vein of mice,the weight,liver injury index and blood lipid spectrum of mice were detected.The liver samples of mice were extracted for HE staining and oil red staining to observe the liver injury and steatosis.Additionally,the expression of liver adipogenic genes and inflammation related genes were detected by RT-qPCR,to investigate the effects of OPTN on hepatic inflammation as well as steatosis in mice;On the basis of knockdown or overexpression of OPTN in cell model of NAFLD,the expression of adipogenic gene was detected by RT-qPCR/WB,the lipid accumulation of cell was evaluated by oil red staining and triglyceride detection,and the level of inflammatory cytokine in supernatant was detected by ELISA for exploring the effect of OPTN on hepatocyte fat accumulation.ResultPart Ⅰ:The characteristic pathway of NAFLD is mainly enriched in metabolic signals such as carbohydrate and lipid metabolism.Immune infiltration analysis showed that memory B cells,regulatory T cells and Ml macrophages were significantly up-regulated in NAFLD,while follicular helper T cells were down regulated in NAFLD.In addition,the minimum depth random forest prediction model of NAFLD was established in this study,and the efficiency of NAFLD reached 100%.The candidate targets for NAFLD were then screened,including EN03,CXCL10,RAB26,IRRC31 and OFTN,and the potential role of OPTN as a target for NAFLD treatment was further confirmed.Part Ⅱ:In nonalcoholic fatty liver model induced by high fat diet,knockdown expression of OPTN helped mice combat obesity and improve the profile of blood lipid.OPTN knockdown can alleviate liver injury induced by high-fat diet,reduce liver fat accumulation and decrease the expression level of adipogenic gene.In nonalcoholic steatohepatitis model induced by MCD diet the expression of OPTN did not affect the body weight of mice.However,the knockdown of OPTN could alleviate the liver injury and liver fat accumulation,and decrease the expression of inflammatory genes and fibrosis-related genes in liver;In cell model of NAFLD,low expression of OPTN decreased the expression of adipogenic gene,the content of lipid droplets and triglycerides,and the levels of IL6,MCP1 and TNFa in cell supernatant Up regulation of OPTN could aggravate the above phenomenon in hepatocytes.Moreover,further knockdown of CXCL10 after overexpression of OPTN in hepatocyte can alleviate the increase of hepatocyte lipid deposition and inflammation caused by OPTN overexpression,indicating that OPTN-CXCL10 pathway is an important cascade signal of liver inflammatory response and participates in liver lipid accumulation.ConclusionThis study provides more in-depth insights into the key molecular events in the pathogenesis of NAFLD.OPTN can regulate the pathway of liver lipid metabolism in mice,and participate in the regulation of liver inflammation and the progress of liver fibrosis.OPTN-CXCL10 signaling pathway may be a new therapeutic target for NAFLD.These findings will help us better understand the occurrence and development of NAFLD.