The Role And Molecular Mechanism Of Hepatocyte Specific Transcription Factor CREBH In Non-alcoholic Steatohepatitis

Objective: To investigate the role of CREBH in metabolic stress induced cell models and NASH mice modelsMethods: 1.Firstly,Hep G2 cells were treated with H2O2(0?125?250 and 500 ?M)or LPS(0?10?20 and 40 ?g/ml)for 16 h,respectively.AML-12 cells were also incubated with PA(0?200?400?600 and 800 ?M)for 24 h.Relative m RNA and protein levels of CREBH were determined by q RT-PCR and Western blot.2.Relative m RNA and protein levels of CREBH in MCD diet-induced NASH model were detected by q RT-PCR and Western blot.3.To upregulate hepatic Crebh expression,mice were injected by cauda vein with AAV8 Vector and AAV8 Crebh separately and fed a MCS diet or a MCD diet for 4-8 weeks.And another NCD or FFC diet-induced mice model was also applied in our experiment.Hepatic histology,lipid and fibrotic accumulation were observed and measured in these mice.Results: 1.We found that the protein levels of N-CREBH in Hep G2 cells were increased in a H2O2 dose-dependent manner and N-CREBH were also activated in LPS treated cells.In various concentrations of PA-induced lipid accumulation of AML-12 cells,the m RNA and protein levels of N-CREBH were all elevated.2.We established a NASH mice model by 4-8 weeks MCD diet.The m RNA expressions of CREBH in MCD diet were decreased approximately one half comparing with MCS group,the protein expressions of F-CREBH and N-CREBH in MCD group were also downregulated significantly.3.In the MCD diet induced mice model,compared with MCD+AAV8 Vector group,hepatic steatosis,inflammatory cell infiltration and red collagen fiber were significantly reduced in MCD+AAV8 Crebh group,suggesting that upregulation of CREBH can dramatically improve the hepatic injury in mice model induced by the MCD diet.At the same time,we established a mouse model of NAFLD by FFC diet.Lipid deposition in liver cells was severe in this model,but inflammatory cells infiltration and fibrosis were less in the model than that in MCD model.Compared with FFC+AAV8 Vector group,hepatic steatosis,inflammatory cell infiltration and red collagen fiber were significantly ameliorated in FFC+AAV8 Crebh group,suggesting that upregulation of CREBH could dramatically improve the hepatic injury in mice model induced by the FFC diet.Conclusions: Metabolic stress molecules can significantly activate the expression of CREBH,and CREBH plays a potential protective role in the metabolic stress induced injury of hepatocytes.In the MCD and FFC diets induced NASH models,upregulation of CREBH could dramatically improve the hepatic damage in mice.Objective: To explore the potential mechanisms of CREBH on improving the liver damage in the NASH mice model induced by MCD diet.Methods: 1.The contents of TG and TC in liver tissue and serum ALT and AST in each group were detected by biochemical assay kits.The expression levels of inflammatory cytokines MCP1,IL-1? and TNF? in mice serum from each group were analyzed by ELISA.Macrophage and Neutrophil influx were observed by immunohistochemistry.2.After injection AAV8 of upregulating CREBH into mice tail vein,the NASH model was then induced by MCD diet for 4W,6W and 8W,respectively.Liver tissue samples and serum were collected for molecular biological detections.The q RT-PCR method was applied to screen the lipid metabolism key molecules and inflammatory factors which may be regulated by CREBH in all the mice liver specimens.Western blot was used to verify the protein expression levels of SCD1,MCP1,SREBP-2,IL-1? and IL-6.3.We transfected CREBH c DNA into AML-12 cells by lentiviral transfection and generated a stable cell line.Moreover,Ch IP and Luciferase reporter assays were applied to investigate the transcriptional regulation of SCD1 or MCP1 by CREBH.Results: 1.Compared with the AAV8 Vector control group,the contents of TG and TC in liver tissue and serum ALT and AST in AAV8 Crebh group was significantly decreased.The expression levels of inflammatory cytokines MCP1,IL-1? and TNF? in mice serum were also significantly inhibited in AAV8 Crebh group.Immunohistochemical staining showed that Kuffer cells and neutrophils in the MCD+AAV8 Crebh group decreased dramatically compared with MCD+AAV8 Vector control group.2.In the MCD diet(4W,6W and 8W)mice model,compared with the AAV8 Vector control group,the m RNA expression of SCD1,PPAR? and CPT1 a in AAV8 Crebh group were significantly increased and SREBP-2 was decreased.At the same time,the m RNA expression levels of MCP1,IL-6 and IL-? were significantly reduced in AAV8 Crebh group than AAV8 Vector control group.But TNF? m RNA in MCD+AAV8 Crebh group at 4W was not significantly downregulated.And the m RNA expression of COX2 and HO-1 did not correlate well with the AAV8 Crebh intervention.Furthermore,after 8W of MCD diet intervention,compared with AAV8 Vector control group,the protein expression of SCD1 was significantly upregulated in AAV8 Crebh group,MCP1 was dramatically elevated.The levels of SCD1 and MCP1 correlated well with the upregulation of CREBH in both m RNA and protein levels.3.Chromatin immunoprecipitation and reporter gene luciferase assay showed that CREBH could bind to the promoters of SCD1 and MCP1.CREBH overexpression significantly increased promoter activity of SCD1 and decreased activity of MCP1.Conclusions: CREBH could decrease abnormal lipid deposition in liver,reduce lipotoxicity through transcriptional activating desaturase SCD1 and improve hepatic inflammatory injury through transcriptional inhibiting inflammatory factor MCP1.