Tripartite Motif 16 Ameliorates Nonalcoholic Steatohepatitis By Promoting The Degradation Of Phospho-TAK1

【Background】Nonalcoholic fatty liver disease(NAFLD)has become the leading cause of chronic liver disease worldwide.Non-alcoholic steatohepatitis(NASH)is an inflammatory subtype of NAFLD,which can progress hepatic fibrosis and even liver cirrhosis.NASH has become the main cause of liver transplantation in the United States;in China,the incidence of NAFLD is also increasing.Unfortunately,due to complex pathogenesis,there is still no clinically approved drug for NAFLD.Thus,there is an urgent need to explore the mechanism of NASH development and find effective targets that play pivotal roles in NASH.Ubiquitin modification plays important roles in many inflammatory and metabolism-related diseases.E3 ubiquitin ligase is the key enzyme in the ubiquitination system.Several adaptor proteins and kinases involved in NASH progression carry multiple sites that can be modified by E3 ligases.However,little is known about the ubiquitin modification of these adaptor proteins and kinases in NASH.【Objective】This research intends to find out pivotal ubiquitin-related molecules in regulation of inflammatory signals,and to systematically elaborate their roles in NAFLD.We focus on the central regulatory signal in the occurrence and development of NASH,which provides theoretical foundation and preliminary research for the treatment of NASH.【Methods】Firstly,an in vitro lipotoxic model was constructed,RNA-sequencing was used to screen the E3 ligases that significantly increased during lipotoxic stimulation;the expression was verified in various models.Chromatin immunoprecipitation and dual luciferase reporter assay were used to explore the upstream transcriptional regulation mechanism of TRIM16.Next,adenovirus was applied to explore the function of TRIM16 in vitro.Liver-specific TRIM16 knockdown and overexpression mice were constructed to explore the function of TRIM16 in vivo.Besides,multi-omics analysis and molecular biology techniques were applied to clarify the specific mechanism of TRIM16 in NASH.Finally,the clinical value of TRIM16 was evaluated by injection of adeno-associated virus in mice.【Results】A lipotoxic stimulation model was successfully constructed;RNA-sequencing revealed that E3 ubiquitin ligase TRIM16 was significantly increased in NASH.The expression of TRIM16 was verified in liver of NASH mice and patients.The up-regulation of TRIM16 in NASH was mainly regulated by transcription factor EGR2.Functionally,TRIM16 knockdown aggravated hepatocyte lipid and inflammatory phenotypes,while TRIM16 overexpression alleviated hepatocyte lipid deposition and inflammation in the lipotoxic model.Overexpression of TRIM16 in NASH mice alleviated lipid deposition and inflammation,while TRIM16 knockdown aggravated lipid deposition and inflammation.Multi-omics analysis and molecular experiments showed that TRIM16 could interact with TGF-β-activated kinase-1(TAK1),leading to the degradation of p-TAK1 through the ubiquitin-proteasome pathway.Thus,the downstream mitogen-activated protein kinase(MAPK)pathway was inhibited.Adeno-associated virus vector-mediated TRIM16 overexpression in the liver significantly reduced lipid deposition,inflammatory infiltration and fibrosis in NASH mice.【Conclusion】This study demonstrated the protective role of TRIM16 in NASH development.Overexpression of TRIM16 in NASH mice significantly reduced liver lipid deposition,inflammatory infiltration,and liver fibrosis in NASH mice.Mechanically,abnormally activated phosphorylated TAK1 in NASH can be selectively inhibited by protective up-regulated TRIM16.Adeno-associated viruses-based treatment suggested TRIM16 as a potential target for NASH treatment.