The Mechanism Of β-arrestin 2 Aggravating Non-alcoholic Steatohepatitis Via The Metabolic Reprogramming Of Macrophages

Background and purpose: Nonalcoholic fatty liver disease（NAFLD）,the most common diffuse liver disease,is characterized by accumulation of lipids in hepatocytes in the absence of chronic or excessive alcohol consumption,including from simple fat Degeneration（Nonalcoholic fatty liver,NAFL）to non-alcoholic steatohepatitis（NASH）,liver cirrhosis and even liver cancer.The rising incidence of NAFLD will create a huge economic burden,accompanied by an increasing number of patients with cirrhosis and end-stage liver disease requiring liver transplantation,and the incidence of hepatocellular carcinoma（HCC）is increasing year by year.The pathogenesis of NAFLD and NASH has been extensively studied,especially immunology has become one of the focuses of research.Innate immunity plays a central role in disease progression.Macrophages are key players in the innate immune system,and hepatic macrophages include liver-resident Kupffer cells（KCs）and recruited macrophages.In NAFLD,fatty acids,cholesterol and its metabolites accumulated in the liver,as well as molecules related to hepatocyte injury,can activate hepatic macrophages and make macrophages polarized.Activated inflammatory macrophages can also activate hepatic stellate cells and promote the development of liver fibrosis in NAFLD.However,the specific mechanisms involved in regulating hepatic macrophage activation in NAFLD remain to be further elucidated.Beta-arrestin 2（Arrb2）is a multifunctional adaptor protein that participates in the desensitization and internalization of G-protein-coupled receptors（GPCRs）on different cell surfaces,as well as intracellular kinases and phosphatase signal transduction.Arrb2 has been reported to be involved in various intracellular metabolisms,such as adipocytes,pancreatic β cells,etc.The database suggests that Arrb2 is generally highly expressed in human bone marrow,monocytes,and macrophages,while its expression is lower in hepatocytes.This study aimed to investigate the roles and mechanisms of hepatocyte Arrb2 and macrophage Arrb2 in the pathogenesis of NAFLD.Methods: 1.This study used C57BL/6J-Arrb2 knockout mice（Arrb2 KO mice）and C57BL/6J wild-type mice（WT mice）,using quantitative real-time PCR（q PCR）and Western blotting（western blot,WB）to verify the knockout efficiency.2.Arrb2 KO and WT mice were fed with high-fat diet（HFD）for 24 weeks or methionine-choline deficient（MCD）for 8 weeks to construct NAFLD model.3.The levels of alanine aminotransferase（ALT）,aspartate aminotransferase（AST）and triglyceride（TG）in serum were detected by an automatic biochemical analyzer.4.Hematoxylin and eosin（H&E）staining was used to evaluate the degree of liver tissue pathological damage.5.Oil red O staining was used to observe and compare lipid deposition in liver tissue.6.The degree of liver fibrosis was assessed by Sirius red staining,Masson staining andα-smooth muscle actin（α-SMA）immunohistochemical（IHC）staining.7.The IHC staining of MPO and F4/80 were used to observe the infiltration of neutrophils and macrophages in liver tissue.8.The NAFLD mouse model was established after HFD feeding for 24 weeks or MCD feeding for 8 weeks.Liver primary hepatocytes and primary macrophages were isolated by liver perfusion,and the expression changes of Arrb2 in hepatocytes and macrophages were detected by qPCR and WB.9.After depleting Arrb2 in mouse hepatocytes by tail vein injection of Adeno-associated virus 8（AAV8-si Arrb2）,the role of Arrb2 in hepatocytes in NAFLD was investigated by feeding with MCD for 8 weeks.10.In vitro Palmitic acid（PA）& Oleic acid（OA）stimulated primary hepatocytes to construct an in vitro model of NAFLD to study Arrb2 in hepatocytes.11.Detection of NASH by immunofluorescence co-staining of ARRB2 and CD68 Changes in Arrb2 expression in patient macrophages.12.The expression of Arrb2 in HFD mouse liver macrophages was detected by immunofluorescence co-staining of Arrb2 and F4/80.13.The specific type and polarization of hepatic macrophages were analyzed by in situ perfusion and flow cytometry.14.The m RNA levels of inflammatory factors and chemokines in the liver were detected by q PCR.15.The recipient mice were irradiated with a single dose of 11 Gy to construct recipients,and the bone marrow of donor mice was isolated and injected into recipient mice via tail vein to construct bone marrow transplanted mice to study the role of myeloid Arrb2 in NAFLD.16.To study the mechanism of action of Arrb2 in liver macrophages by RNA-sequencing（RNA-seq）and gene set enrichment analysis（GSEA）.17.The levels of mitochondrial oxidative phosphorylation（OXPHOS）and glycolysis were detected by extracellular acidification rate（ECAR）and oxygen consumption rate（OCR）of energy metabolism.18.Measure mitochondrial ROS by Mito SOX^TM,respectively.19.The M1-type polarization of bone marrow macrophages was detected by i NOS immunofluorescence staining.20.The human peripheral blood monocytes isolation kit was used to isolate peripheral blood monocytes from healthy people,NAFLD and HCC patients,and then the expression of ARRB2 in monocytes was detected by q PCR.Results:The expression of Arrb2 was significantly increased in hepatic macrophages of NAFLD mice,while it was unchanged in hepatocytes.The systemic loss of Arrb2 could alleviated liver steatosis,inflammation and fibrosis,and this reduction was mainly mediated by the loss of Arrb2 in liver macrophages,and hepatocyte Arrb2 did not play a key role in NAFLD.Moreover,the above-mentioned consistent experimental phenomenon was also obtained in in vitro experiments.Deletion of Arrb2 in inflammatory macrophages significantly reduced M1-type polarization of macrophages.Likewise,depletion of Arrb2 in macrophages inhibited LPS & IFN-γ-induced M1-type polarization in vitro.Changes in the production of effectors by macrophages after Arrb2 deletion were mainly a decrease in IL-1β secretion and an increase in IL-10 release.Arrb2 in NAFLD affectd the energy metabolism of hepatic macrophages,resulting in enhanced intracellular glycolysis and decreased OXPHOS levels.This process was mainly mediated by the inhibition of immune suppressor gene 1（IRG1）by Arrb2.Arrb2 in inflammatory macrophages could interacted with IRG1 to inhibit its expression,thereby increasing SDH activity,promoting the release of mitochondrial ROS.Increased mitochondrial ROS release led to increased Hif-1α expression,increased IL-1β release,and decreased IL-10 secretion.In addition,the expression of ARRB2 in peripheral blood monocytes of NAFLD population was significantly higher than that of healthy population,and was correlated with the severity of fatty liver.In clinical HCC disease,the expression of ARRB2,which has the potential to promote M1-type polarization,was significantly reduced in tumor-associated macrophages（TAMs）compared with macrophages in adjacent tissues.Moreover,the expression level of ARRB2 in peripheral blood monocytes of HCC patients was also lower than that of healthy people.Conclusions: In NAFLD/NASH,Arrb2 promotes hepatic steatosis,inflammation and fibrosis by promoting metabolic reprogramming of hepatic macrophages.The expression of Arrb2 in peripheral blood monocytes of clinical NAFLD patients is expected to be a marker of disease progression in NAFLD.Studying the macrophage Arrb2 signaling pathway may provide new opportunities for developing new treatments for NAFLD and HCC.