The Role Of HNF4? Mediating Transcriptional Regulation Of Pellino1 In Non-alcoholic Steatohepatitis

Background Nonalcoholic fatty liver disease(NAFLD)is the hepatic manifestation of the metabolic syndrome that can vary from steatosis to non-alcoholic steatohepatitis,cirrhosis and hepatocellular.The increased prevalence of diabetes,obesity,hypertension in the general population is considered to be the most common cause for NAFLD.NAFLD is by now recognized as the most frequent liver disease worldwide.In NASH,TLRs were activated by the pathogenic factors to regulate correlative reactions.Pellino1 is a E3 ubiqutin ligase that can catalyse the ubiquitylation of IRAK-1 or RIP1 and regulate TLRs signalling pathways.Pellino proteins contain a core forkhead-associated(FHA)domain and RING-like domain.,which is a defining feature of ubiquitin ligases.Hepatocyte nuclear factor 4?(HNF4?)is a member of the nuclear receptor superfamily.It is highly expressed in the liver,with lower levels in the kidney,intestine and pancreatic ? cells.Structural analysis of the LBD of HNF4? indicates the C14–C18 long-chain fatty acids are tightly bound to LBD.We analysised Pellino1 promoter found that it had some binging domain of HNF4?.In this report,we determined the role of HNF4? mediating transcriptional regulation of Pellino1 in non-alcoholic steatohepatitisObjective 1.To determine the role of Pellino1 in NASH 2.To clarify whether HNF4? mediates the transcriptional activity of Pellino1Methods To investigate the effect of Pellino1 on NASH,and to explores mechanism involving in the process,we used both in vivo and vitro experiments to investigate the issue.Mice were injected intravenously through the tail with 4x109 plaque-forming units of the Cre or GFP adenoviruses.After one week,We employed mice feeding with methionine-choline-deficient(MCD)diet for 8 weeks to induce NASH.After 8 weeks,we observed gross morphology and liver index between two groups(liver wet weight/body weight).Triglyceride(TG)was detected in plasm and liver by a enzymic method.Liver damage was evaluated by examining alanine aminotransferase(ALT).Hematoxylin-eosin(HE)staining and immunohistochemical staining for CD45 and F4/80 was employed to assess the steatosis and inflammatory cells infiltration.Quantitative real time-PCR(q RT-PCR)and Western blot were used to examine m RNA and protein,to investigate Pellino1 and Inflammatory cytokines level.To further explore the mechanism of Pellino1,we stimulated hepatocytes cultured in vitro with free fatty acid(FFA),using q RT-PCR,Western blot,reporter gene assay and CHIP to overview HNF4? and Pellino1 effect on inflammatory response in liver.Results In vivo studies,mice after 8 weeks MCD diet displayed severe steatosis and inflammation compared with Control diet mice.Consumption of the MCD diet led to increase in Pellino1 m RNA expression and protein expression.The group receiving the Cre-adenovirus exhibited decrease in accumulation of lipid droplets in hepatocytes,infiltration of inflammatory cells in the liver,ALT levels in plasma and IL-1? TNF? m RNA level in liver,relative to the group receiving the corresponding mock adenovirus.In vitro studies,the hepatocyte receiving the Cre-adenovirus treated with PA(500u M)exhibited decrease proinflammatory cytokines IL-1? and TNF-? m RNAlevel compared to GFP-adenovirus group.In addition,luciferase reporter assays and Chromatin Immunoprecipitation(Ch IP)showed that HNF4? could bind to Pellino1 promoter and enhanced the activity.Conclusions Our results suggestd that Pellino1 played a role in non-alcoholic steatohepatitis,transcription factors HNF4? regulated Pellino1 promoter to increase protein expression in NASH?...