Role Of Macrophage XBP1 In Regulating Non-alcoholic Steatohepatitis Progression

Background and Aims:The incidence of non-alcoholic steatohepatitis（NASH）is increasing,and if not treated,it will eventually lead to liver cirrhosis and liver cancer.Studies have shown that hepatic macrophages play an important role in the progression of NASH.However,the mechanism of hepatic macrophages affecting the progression of NASH remains to be further explored.X-box binding protein-1（XBP1）plays a key role in regulating the mammalian unfolded protein response or ER stress response.During ER stress,the endribonucliase IRE1αinduces splicing of XBP1 mRNA to produce mature mRNA encoding the active transcription factor XBP1s.Mice lacking XBP1 gene show different effects in different diseases,such as reducing pain degree in pain models,increasing T-cell infiltration into tumors,helping to enhance antitumor immunity,delaying malignant tumor progression,and improving overall survival.In addition,XBP1 also plays an important role in regulating embryonic liver development.Therefore,XBP1disorder plays different roles in the development of different diseases.Currently,the role of XBP1 in the regulation of liver inflammation,fibrosis and lipid metabolism disorders in NASH remains unclear.This study aims to explore the role and mechanism of macrophage XBP1 in non-alcoholic steatohepatitis,so as to provide a new target and theoretical basis for clinical intervention.Methods:In this study,we first analyzed the expression level of XBP1 in the specimens of patients diagnosed as NASH by liver biopsy,and the tissue samples of patients with hepatic hemangioma who underwent partial hepatectomy were used as the control group.Our study constructed macrophage Xbp1 myeloid specific knockout mice.The NASH model was established by feeding mice with high-fat diet for 26 weeks and methionine-and choline-deficient diet for 6 weeks,which were divided into normal diet 26w NC group,high fat diet 26w HFD group,normal diet 6w NC group,methionine/choline-deficiency diet 6w MCD group（n=6/group）.Our study further detected the changes of liver function（ALT,AST,ect）,liver lipid deposition（TG）and liver fibrosis（α-SMA,Collagen I,TIMP1）in myeloid specific Xbp1 knockout mice with NASH models.Macrophages were isolated from wild-type floxed mice and myeloid specific Xbp1 knockout mice and co-cultured with hepatocytes and hepatic stellate cells.The effects of Xbp1 knockout on lipid deposition of hepatocyte and hepatic stellate cell activation were detected by oil red staining andα-SMA immunofluorescence.High-throughput sequencing was performed to detect the specific mechanism of macrophage Xbp1 knockout affecting NASH progression.qRT-PCR,western blotting,immunohistochemistry and immunofluorescence staining were used to explore the specific mechanism of XBP1 affecting the progression of NASH.To further determine the mechanism of macrophage Xbp1 knockout affecting liver inflammation and fibrosis,adeno-associated virus overexpressing downstream key molecular were injected into Xbp1 myeloid specific NASH mice in our study.At the same time,myeloid specific knockout mice of downstream signaling molecule were constructed to further verify the specific effect of XBP1 on the progression of NASH.In vivo intervention with specific drugs of XBP1 activation was used to further verify the effect of XBP1 activation on NASH progression.Results:In the liver samples of NASH patients,the expression of XBP1 was significantly up-regulated.Myeloid specific macrophage Xbp1 knockdown inhibited the development of steatohepatitis and fibrosis in mice fed with high-fat diet or methionine-and choline-deficient diet.Macrophage-specific Xbp1 knockout mice promoted the transformation of macrophages into anti-inflammatory M2 phenotype,and inhibited the expression of Tnfa,Il6,Il1b and Cxcl10 proinflammatory genes,all P value were less than0.05.Macrophage Xbp1 depletion ameliorates steatohepatitis by inhibiting NLRP3expression and proinflammatory cytokine secretion,thus mediating macrophage toward M2 anti-inflammatory polarization.Steatohepatitis was less severe in macrophage-specific Nlrp3 knockout mice than in wild-type Nlrp3^FL/FLmice.Oil red staining and the immunofluorescence ofα-SMA showed that macrophage Xbp1deficiency inhibited the lipid deposition and inflammation of hepatocytes and inhibited the activation of hepatic stellate cells,when macrophages were co-cultured with hepatocytes and hepatic stellate cells in vitro.Further investigation revealed that Xbp1deletion in macrophages inhibited inflammatory cytokines secretion by macrophage via inhibiting NLRP3 expression,thereby inhibiting hepatocyte lipid deposition and inflammation.In vitro viral overexpression of Nlrp3 promoted lipid deposition in hepatocytes.In vivo viral overexpression of Nlrp3 reversed the ameliorative effect of macrophage Xbp1 knockout on NASH.Macrophage XBP1 promotes lnc RNA NEAT1expression of hepatic stellate cells by promoting TGF-β1 secretion,inhibits miR139-5p,and promotes hepatic stellate cell activation byβ-catenin/SOX9/TGF-β1 signaling,eventually promoted NASH fibrosis progression in mice.Pharmacological inhibition of XBP1 expression could alleviate the progression of NASH.Conclusion:In conclusion,macrophage XBP1 promotes the development of NASH including inflammation and fibrosis through NLRP3 and TGF-β1 signaling.XBP1 inhibition can improve NASH.XBP1 is therefore a potential target for the treatment of NASH.