The Mechanism Of BRD2 In Activation Rat Hepatic Stellate Cells

Objective BRD2 can regulate gene transcription.The researches showed that PPAR-γ,TGF-β/Samd and NF-κB may be regulated by BRD2.These signaling are highly related with liver fibrosis,so that BRD2 may be an important target for treatment of liver fibrosis.The purpose of this study is to investigate the possibility of BRD2 involvement in the process of hepatic stellate cell activation and may provide new ideas and experimental basis for the treatment of hepatic fibrosis.Methods(1)Constructing eukaryotic expression plasmid pcDNA3.1(+)-BRD2 and overexpressing BRD2 protein in culturing HSC-T6 cells by transient transfection.(2)Screening BRD2-specific siRNA to knock down the BRD2 expression in cultruring HSC-T6 cells.(3)Western blot assay type I collagen expression with over-expression or knocking down of BRD2.(4)Reporter gene for analyzing the PPAR-γ,TGF-β,NF-κB response element reporter genes expression with knocking down BRD2 expression.(5)Western blot assay PPAR-γ,Smad7,NF-κB(p65)expression and Smad3,NF-κB(p65)phosphorylation when BRD2 overexpression or knocked down.(6)Reporter gene for analyzing the PPAR-γ,TGF-β,NF-κB response element reporter genes expression after treatment of culturing HSC-T6 cells with BRD2 peptide mimic(BIP mimic).Results(1)The eukaryotic expression plasmid pcDNA3.1(+)-BRD2 was successfully constructed,BRD2-specific siRNA siRNA was obtened and were confirmed by detecting BRD2 expression in rat HSC cells.(2)Over-expression of BRD2 up-regulated type I collagen by western blotting assay and it is speculated that BRD2 may be involved in the activation of hepatic stellate cells.(3)Reporter gene analysis showed that knocked down BRD2 expression has enhanced PPAR-γ promoter transcription,and suppressed TGF-β,NF-κB response element reporter gene expression.(4)Over-expression of BRD2down-regulated PPAR-γ expression,up-regulated Smad7,NF-κB(p65)expression,and promoted Smad3,NF-κB(p65)phosphorylation by detecting with western blotting assay.(6)Reporter gene analysis showed that BRD2 peptide mimic(BIP mimic)has enhanced PPAR-γ promoter gene transcription,and suppressed TGF-β,NF-κB response element reporter gene expression.Conclusion The results here suggested that BRD2 promoted the activation of hepatic stellate cells in vitro by directly or indirectly inhibiting PPAR-γ and promoting TGF-β and NF-κB pathways.BRD2 may serve as a potential therapeutic target for liver fibrosis.