The Study On The Effect And Mechanism Of BMP9 On The Development Of Nonalcoholic Steatohepatitis

Objective: To investigate the role of bone morphogenetic protein 9(BMP9)in the development of nonalcoholic steatohepatitis(NASH)and explore the molecular mechanism.Methods: 1.Adeno-associated viral(AAV)vectors containing the BMP9 transgene(AAVBMP9)or a null transgene(AAV-null)were injected into the tail vein of mice,and the NASH model induced by methionine and choline deficiency(MCD)diet or control diet(CD)for 4 weeks was established.The clodronate liposomes(Clod.Lipo)or the control liposomes(CT.Lipo)were administered 1 day before and 8,15 and 22 days after the diet intervention to deplete hepatic macrophages.The mice were divided into six groups with 6 mice in each group as follows: AAV-null mice fed a CD diet with control liposomes(AAVnull+CD+CT.Lipo);AAV-BMP9 mice fed a CD diet with control liposomes(AAVBMP9+CD+CT.Lipo);AAV-null mice fed a MCD diet with control liposomes(AAVnull+MCD+CT.Lipo);AAV-BMP9 mice fed a MCD diet with control liposomes(AAVBMP9+MCD+CT.Lipo);AAV-null mice fed a MCD diet with clodronate liposomes(AAVnull+MCD+Clod.Lipo);AAV-BMP9 mice fed a MCD diet with clodronate liposomes(AAV-BMP9+MCD+Clod.Lipo).After 4 weeks of dietary intervention,all the mice were sacrificed,serum and liver tissues were collected.Serum transaminase ALT,AST and liver triglyceride(TG)were detected.Liver tissue was stained with hematoxylin-eosin(HE),oil red O,and Sirius red staining to evaluate the degree of liver injury.Immunohistochemical staining,RT-PCR,Western Blot were used to detect liver inflammation and fibrosis,and Western Blot were used to detect the expression of nuclear factor κB(NF-κB)signaling pathway-related proteins.2.Mouse macrophage cell line RAW264.7 was cultured in vitro.Sh-smad1 plasmids were transfected into RAW264.7 cells to silence Smad1(RAWsh Smad1).RAW264.7 cells were pretreated with recombinant human BMP9 protein(rh-BMP9,100 ng/ml)for 2 h and then treated with lipopolysaccharide(LPS,100 ng/ml)for 24 h.RT-PCR was used to detect the BMP9 signaling pathway molecular and the level of M1 type polarization markers.RAW264.7 or RAW-sh Smad1 cells were pretreated with NF-κB inhibitor(BAY11-7085,5 μM)for 2 h,then rh-BMP9 was administered for 16 h.RT-PCR,Western Blot and immunofluorescent staining were used to detect inflammation and the expression of NF-κB signaling pathway-related proteins.Results:(1)The expression of BMP9 in NASH liver tissue induced by MCD diet was upregulated.(2)Under CD diet,BMP9 overexpression had no significant effect on the liver of mice;In MCD diet-induced NASH mice,overexpression of BMP9 increased the level of AST and ALT,increased the quantity of the liver M1 type macrophages(i NOS-stained positive cells),and aggravated hepatic steatosis and inflammatory infiltration and fibrosis.(3)In MCD diet-induced NASH mice,Overexpression of BMP9 increased the m RNA levels of F4/80,i NOS,CD86,IL6,IL1β,TNFα,MCP-1,COX2 and IL12β and IL1β protein expression,increased the profibrotic factors TGFβ1,COL-1 m RNA expression,and the protein levels of TLR4,P-IκBα,PNFκB were also upregulated;After the depletion of hepatic macrophages,the effect of BMP9 on increasing liver inflammation was abolished,there was no significant difference between AAV-BMP9 and AAV-null group.2.(1)In RAW264.7 cells,after 24 h LPS intervention,the m RNA levels of BMP9 and ALK1 were increased,and LPS-mediated M1 type polarization was increased by rh-BMP9, resulting in further enhanced expression of CD86,i NOS,TNFα,MCP-1 and COX2.(2)In RAW264.7 and RAW-sh Smad1 cells,rh-BMP9 increased the m RNA levels of IL1β,TNFα,i NOS,MCP-1,COX2 and NF-κB nuclear translocation,and these effects were inhibited by NF-κB inhibitor.Conclusion:BMP9 promoted MCD diet-induced NASH through NF-κB dependent M1 type polarization of hepatic macrophages.BMP9 may play a proinflammatory role in NASH,providing potential evidence for treatment of NASH.