The Function And Mechanism Of BRD2 In Alcohol-induced Liver Injury

Background: Long-term excessive alcohol consumption is one of the most common causes of liver disease worldwide.Histopathologically,excessive alcohol abuse can lead to liver damage,from simple alcoholic fatty liver to severe hepatitis,liver fibrosis/cirrhosis and even hepatocellular carcinoma with high mortality.As reported,alcohol and its related metabolites induce the aggregation and activation of macrophages（inherent Kupffer cells and infiltrated macrophages）in the liver,and secrete inflammatory cytokines to further induce liver inflammation.Simultaneously,a variety of inflammation-related signaling pathways are also activated,and a large number of proinflammatory cytokines are released,further exacerbating liver damage in alcoholic liver disease（ALD）.In recent years,several potential mechanisms of macrophage regulation of inflammatory response have been reported,but there is still a lack of effective treatment for alcoholic liver injury.BRD2（Bromodomain-containing protein）is a member of BET Bromodomain and extra-terminal Domain protein family 2.Studies have shown that BRD2 is involved in regulation of a variety of inflammatory diseases,and knocking down BRD2 expression,the expression of pro-inflammatory factors in macrophages was reduced.Therefore,this study focuses on regulation and potential mechanism of BRD2 on macrophage inflammation in alcoholic liver injury,providing a new strategy for the prevention and treatment of alcoholic liver disease.Objective: To explore the regulation and potential mechanism of BRD2 on macrophage inflammation in alcoholic liver injury.Methods: In vivo,a total of 60 male C57 mice were randomly divided into 6 groups: pair-fed group,Eth-fed group,pair-fed+adenovirus empty vector group,Eth-fed+ adenovirus empty vector group,pair-fed+BRD2-silenced adenovirus group and Eth-fed +BRD2-silenced adenovirus group.Mice were firstly fed for a five-day adaptation period.The model period began on sixth day.Mice in Eth-fed group were fed with alcoholic liquid diet containing 5% alcohol,while mice in pair-fed group were fed with control liquid diet for 10 days.At 9:00 a.m.on the last day,mice in Eth-fed group were gavage with a concentration of 31.5% alcohol and at dose of 5 g/kg.Mice in pair-fed group were gavage with saline at the same dose and treated for 9 h.Liver tissues and serum were collected and primary liver macrophages were extracted.Serum was used for the detection of ALT、AST and inflammatory factors.Part of liver tissue was dehydrated and embedded in paraffin for HE staining,oil red staining,immunohistochemical staining.m RNA and protein were extracted from primary cells and liver tissue to detect the expression of BRD2 and proinflammatory factors.In vitro experiments,RAW264.7 cells were stimulated by alcohol（100 m M）for 12 h to establish inflammation model in vitro.Firstly,BRD2 protein expression was detected by Et OH stimulated at several time points.Then,BRD2 silenced by adenovirus or inhibitor JQ1,and BRD2 overexpression by p EX-3-BRD2.Western blot,ELISA and q RT-PCR methods were used to detect secretion of inflammatory factors and protein expression of NF-κB pathway.Result: In vivo,Results of histopathological staining and serological test showed that the liver structure of pair-fed group was intact,and only a small amount of lipid droplets accumulated.The liver damage of Eth-fed group was obvious,liver cells were damaged to varying degrees,and the nucleus disappeared,indicating that the mouse model of alcoholic liver injury was successfully established.F4/80 staining showed increased infiltration of macrophages in Eth-fed group,and increased secretion of inflammatory factors was detected by ELISA,indicating that alcohol induced activation of macrophages and inflammatory reaction occurred.BRD2 expression was significantly increased in primary hepatic macrophages and liver tissue by western blot assay.liver injury of mice was alleviated and secretion of inflammatory factors was also reduced after BRD2 gene silenced with adenovirus,suggesting that BRD2 gene silenced could improve alcoholic liver injury.In vitro,BRD2 expression was increased in RAW264.7 cells stimulated by 100 m M Et OH for 12 h.Subsequently,the release of proinflammatory factors was also decreased after silencing of BRD2.and proinflammatory factors expression was could not be increased after overexpressing BRD2.Further detection of protein expression of NF-κB pathway indicated that NF-κB signaling pathway was regulated by BRD2.Conclusion: BRD2 is involved in NF-κB signaling to regulate the inflammatory response of macrophages in alcoholic liver injury.