The Effect And Regulatory Mechanism Of BRD4 In The Progression And Reversal Of Hepatic Fibrosis

Background:Hepatic fibrosis is an injury-repair response of the liver induced by various chronic stimuli including alcohol abuse,hyperfat diet,viral hepatitis,and autoimmune diseases.It is characterized by significant inflammatory response in the liver,major imbalance in the production and degradation of extracellular matrix（ECM）,continuous accumulation of ECM including collagen type I（COLⅠ）,and scarring,which disrupt the pathological structure and physiological function of the normal liver.Chronic irritation persists,resulting in irreversible progression to cirrhosis and hepatocellular carcinoma.Hepatic stellate cells（HSCs）are important members of hepatic non-parenchymal cells.Activated HSCs produce abundant collagen,which is the central point in the pathogenesis of hepatic fibrosis and becomes a routine cell in the study of the mechanism of hepatic fibrosis.BRD4 is a member of the Bromodomain and extraterminal domain（BET）family,which functions as a histone acetylation reader protein or contributes to the formation and induction of addition enhancers and super enhancers due to its unique bromodomain structure,and is involved in epigenetic modifications in the organism’s cells and plays an important role in transcription,replication and DNA It plays an important role in transcription,replication and DNA repair.It also binds non-histone proteins,DNA and RNA,which contribute to cell survival,tissue growth and various physiological processes.In addition,BRD4 has been associated with several diseases,including cancer,viral infections,inflammation,and neurological disorders.Purpose:This artical focuses on investigating the function and mechanism of BRD4 in models of hepatic fibrosis formation and reversal in vitro and in vivo,and confirming the function of BRD4 inhibition for hepatic fibrosis treatment and helping recovery.Methods and Results:We established a CCl₄-induced hepatic fibrosis model in mice and a reversal model for spontaneous recovery,and verified the success of the model by pathological slides,immunohistology and serological indexes,and then found that BRD4expression was elevated in the liver of fibrotic mice and slightly decreased in reversal mice by Western blot and q RT-PCR,which was consistent withα-SMA and COLⅠexpression changes were consistent.We established TGFβ-induced activation and lipogenic mixture（MDI）-induced inactivation state cells using LX2 cells to simulate the in vitro model of liver fibrosis formation and reversal,and examined the fibrosis indexes and BRD4 expression using immunofluorescence and Western blot,and the results were similar to those in vivo.Functionally,we treated TGFβ-activated LX2 cells in vitro with different concentrations of BET small molecule inhibitor JQ1 and i BET151,respectively,and constructed si RNA and overexpression plasmids to interfere with BRD4 expression in LX2 cells.The results showed that the BET small molecule inhibitor and si BRD4 significantly inhibited the expression of TGFβ-inducedα-SMA and COLⅠ,in contrast,the BRD4 overexpression with pc DNA3.1 vector promoted theα-SMA and COLⅠexpression in LX2 cells.In TGFβ-activated LX2 cells,the increased apoptosis rate of activated cells was detected by flow cytometry with si RNA inhibition of BRD4,and apoptosis-related proteins Bax/Bcl2 and Cleaved-Caspase3/Caspase3 were elevated as detected by Western blot.The CCK8 and flow cytometry showed that cell proliferation viability was diminished,and Western blot show that reduced expression of cycle-related proteins C-myc and Cyclin D1.In MDI-induced inactivated LX2 cells,after overexpression of BRD4 by pc DNA3.1 vector,flow cytometry,CCK8 and Western blot assays showed that apoptosis level was reduced and proliferation ability was increased in inactivated cells.In vivo,we treated CCl₄-induced fibrotic mice with adeno-associated virus AAV8-mediated BRD4 knockdown and verified the efficiency of BRD4 knockdown and the degree of fibrosis in mice using pathological sections,serological and molecular assays.In vivo,we used adeno-associated virus AAV8-mediated BRD4 knockdown to treat CCl₄-induced fibrosis mice,and verified the efficiency of BRD4 knockdown and the extent of fibrosis in mice using pathological sections,serological level and molecular level.The results showed that BRD4 knockdown significantly attenuated inflammation and fibrosis in the liver of mice,and the expression of fibrosis-related indexesα-SMA and COLⅠwas reduced.Mechanistically,we detected a significant decrease in PLK1 expression in activated LX2cells by Western blot and q RT-PCR after disrupting BRD4 expression with JQ1 and si RNA.We observed abundant H3K27ac signal at PLK1 transcription start site by UCSC Genome Browser database and elevated expression of H3K27ac in activated LX2 cells by Western blot.The expression of fibrosis-related indicators and PLK1 was decreased in activated LX2 cells by Western blot following inhibition of H3K27ac expression with the small molecule inhibitor C646.In activated LX2 cells,knockdown of histone acetyltransferase P300 which is upstream of H3K27ac resulted in inhibition of PLK1,α-SMA and COLⅠm RNA and protein expression.In activated LX2 cells,Ch IP and Co-IP assays demonstrated that si RNA-mediated P300 knockdown resulted in reduced enrichment of H3K27ac in the PLK1 promoter region and reduced interaction of H3K27ac with BRD4 compared to negative controls.Finally,after simultaneous knockdown of P300 and overexpression of BRD4,Western blot experiments detected that si P300 treatment in activated LX2 cells partially suppressed the elevated expression of PLK1,α-SMA and COLⅠgenerated by BRD4 overexpression.Conclusion:BRD4 exerts its epigenetic regulatory activity to modulate the P300/H3K27ac/PLK1 axis involved in hepatic stellate cell activation,proliferation,apoptosis and inactivation processes in hepatic fibrosis,providing new targets for the research and treatment of hepatic fibrosis.