Mechanism Of NHWD-870,a Novel BET Inhibitor,in Improving Cholestatic Liver Injury

Background and objectives:Cholestatic liver injury(CLI)is a disease characterized by elevated bile acids due to various causes,which triggers an inflammatory response and results in serious liver damage.The current mechanism remains elusive.Bromodomain 4(BRD4)is one of the members of the bromodomain and extra-terminal domain(BET)family,and plays a pro-inflammatory role in diseases such as sepsis and rheumatoid arthritis.As reported,BRD4 promoted pathophysiological process of CLI,and the BET inhibitor JQ-1 improved CLI by inhibiting BRD4.However,the molecular mechanism of BRD4 in CLI has not been fully elucidated.NHWD-870,a novel type of BET inhibitor,presents stronger effect and better safety compared with traditional inhibitors such as JQ1,but its effect in CLI is not clear.This study aims to further clarify the pro-inflammatory role of BRD4 in CLI,and explore the therapeutic effect and mechanism of NHWD-870 in CLI.We hope to provide a novel strategy for the treatment of CLI.Methods:(1)To detect the expression levels of BET family members.Real-time polymerase chain reaction(RT-q PCR)was used to detect the expression of BET family in CLI patients and mouse models,as well as primary mouse hepatocytes(PMH).Immunohistochemistry(IHC)was used to detect the expression of BRD4 in CLI patients and mouse models.Spearman correlation analysis was used to explore the correlation between the expression of BRD4 in CLI patients and the liver injury.(2)To explore the therapeutic effect of NHWD-870 in CLI mouse.BDL and DDC mouse models were constructed respectively,and the mice were intraperitoneally injected with NHWD-870 on the second day after BDL or DDC diet.The mice were sacrificed on the 10 th day after BDL or the 14 th day after DDC diet,and the liver tissue and serum were collected.Hematoxylin and eosin(HE)staining and serum liver function tests were carried out to evaluate liver necrosis.In mouse liver tissue,IHC was applied to detect the expression of CD45,F4/80,and MPO,and RT-q PCR was used to detect the expression of inflammatory factors such as IL-6 and IL-1β.Masson’s Trichrome and Sirius Red were applied to detect collagen deposition.IHC was used to detect the expression of CK19,and RT-q PCR was performed to detect liver fibrosis indicators such as TGFβ1 and Timp1.RT-q PCR was applied to evaluate the expression of bile acid transporters and synthetases such as cholesterol7a-hydroxylase(CYP7a1)and bile salt export pump(BSEP).(3)To clarify the main biological processes of CLI affected by NHWD-870.RNA-Sequencing(RNA-Seq)was performed on the BDL liver tissue to clarify the main biological process of NHWD-870.RT-q PCR was used to detect the expression of chemokines and pro-inflammatory factors such as CXCL10 and CXCL2 in mouse liver tissue and PMH.Co-culture of BRD4-overexpressing L02 cell line(L02-OE)with THP1 cell line/human primary T cells was carried out to detect the chemotaxis.Lactate dehydrogenase(LDH)activity was detected in the supernatant of the co-culture system.(4)To explore the downstream molecule affected by NHWD-870 in CLI.We analysed the RNA-Seq to predict the downstream molecule of BRD4 and verified it via enzyme-linked immunosorbent assay(ELISA),RT-q PCR and IHC.We also used neutralizing antibody of the targeted molecule to interfere L02-OE+THP1/T cell co-culture system,and detected THP1/T cell chemotaxis.Chromatin Immunoprecipitation Sequencing(Ch IP-Seq)was used to explore the regulatory relationship between BRD4 and downstream molecules in mouse liver tissue.(5)To explore the potential signaling pathways of NHWD-870 in CLI.Co-Immunoprecipitation(Co-IP)and Ch IP-Seq were used to explore the regulatory relationship between BRD4 and TEA domain family member 1(TEAD1),and the JASPAR software was used to predict whether TEAD1 bound to the downstream molecular promoter region.Results:(1)BRD4 was significantly increased in CLI patients and mouse models,as well as in PMH.The expression of BRD4 in liver tissue of CLI patients was positively correlated with liver injury.(2)After being treated with NHWD-870 in CLI mouse,the liver necrosis and inflammatory infiltration,collagen deposition,and serum liver function were improved,but the bile acid transporters and synthetases were not significantly changed.(3)RNA-Seq analysis suggested that NHWD-870 was mainly involved in CLI by inhibiting inflammation-related signaling pathways such as leukocyte migration and leukocyte adhesion.RT-q PCR found that the expression of pro-inflammatory factors and chemokines such as CXCL10 and CXCL2 in CLI was increased,and was decreased after NHWD-870 intervention.The number of chemotactic inflammatory cells in the L02-OE group was increased in the co-culture experiment of L02-OE+THP1/T cells,and was decreased after NHWD-870 intervention.The activity of LDH in the supernatant was increased,and was inhibited after NHWD-870 intervention.(4)RNA-Seq analysis suggested that Secreted Phosphoprotein1(SPP1)might be the downstream molecule of BRD4.RT-q PCR and ELISA detected the expression of SPP1 were elevated in CLI liver tissue and PMH,and were decreased and NHWD-870 intervention.After the SPP1 neutralizing antibody intervened in the co-culture system of L02-OE and THP1/T cells,the chemotactic number of inflammatory cells decreased.Ch IP-Seq data analysis found that there was no direct binding signal between BRD4 and the promoter region of SPP1.(5)Co-IP and Ch IP-Seq showed that BRD4 might directly bind to TEAD1 and regulated its expression,and JASPAR software predicted that TEAD1 might bind to the promoter region of SPP1 and regulate its expression.Conclusions:(1)The expression of BRD4 is significantly increased in CLI and may promote the pathophysiological process of CLI.(2)BET inhibitor NHWD-870 improves CLI by inhibiting hepatic BRD4.(3)NHWD-870 improves CLI mainly by inhibiting hepatic BRD4,and reducing the chemotaxis of inflammatory cells in CLI.(4)NHWD-870 mainly regulates SPP1 indirectly by inhibiting hepatic BRD4.(5)NHWD-870 may participate in CLI by inhibiting the BRD4-TEAD1-SPP1 signaling axis.