Fibrinogen-like Protein 2 Exacerbates Non-alcoholic Steatohepatitis Via Interaction With TLR4 Eliciting Inflammation In Macrophages And Inducing Hepatic Lipid Metabolism Disorder

?Backgrounds & Aims? Non-alcoholic liver disease(NAFLD)has become one of the most common chronic liver diseases in the world,which affects up to approximately 30% of the adult population.The disturbance of liver function caused by NAFLD is related to insulin resistance(IR)and metabolic stress.The NAFLD disease spectrum extends from non-alcoholic fat liver(NAFL)to non-alcoholic steatohepatitis(NASH),which can also progress to increasing stage of fibrosis and ultimately cirrhosis and cirrhosis-related complications such as hepatocellular carcinoma(HCC).Moreover,the high incidence of metabolic syndrome(Met S),coronary heart disease(CHD),type 2 diabetes mellitus(T2DM)and intestinal tumor is also parallel to NAFLD development.In the meanwhile,the population of patients with chronic hepatitis B virus(CHB)comorbid with NAFLD has been increasing in China for recent years,which seriously threatening public health.NASH is the most crucial stage in NAFLD progression.Compared with NAFL,the risk of NASH developing into end-stage of liver diseases such as ultimately cirrhosis and HCC is 30 to 50 times or even higher.Owing to its complicated pathogenesis,there is few effective therapeutic targets or predicable indicators for NASH.Therefore,it's particularly important to explore the pathogenesis of NASH and provide scientific evidence for therapeutic targets.Liver is not only the maximal parenchymatous and immune organ,but an important immune organ which regulates innate immunity through human body.Hepatic macrophages,which comprise liver-resident Kupffer cells(KCs)and monocyte derived macrophages(MDMs),are key components in liver innate immunity.Hepatic macrophages within the development of NASH can be activated and differentiated into M1 phenotype,releasing proinflammatory cytokines and reactive oxygen species(ROS)that can aggravate the lipid peroxidation and insulin resistance,then liver damage is further caught in a vicious circle.Thus,it is crucial to explore the cellular mechanisms in activation of hepatic macrophages during NASH development.It is critical to find the target molecules to regulate macrophages which have complex regulatory mechanisms.Fibrinogen-like protein 2(fgl2),also known as prothrombin enzyme,is an important member of the fibrinogen superfamily involved in the regulation of coagulation and immune regulation.In our previous study,we found that fgl2 was extensively expressed on activated macrophages,and increased expression in liver tissues of fulminant hepatitis mice model and patients with severe hepatitis B.Based on previous studies,this research used murine NASH model and in vitro primary macrophage to further explore the mechanism of fgl2 on macrophages combined with clinical samples,trying to provide new clues for the diagnosis and treatment of NASH.?Methods? 1.Patients with NASH were diagnosed by HE staining and NAFLD activity score(NAS),CD68 and fgl2 were labeled in liver sections.The expression of CD68+ macrophages and fgl2 in NAFL and NASH were observed by immunofluorescence,and the fgl2-positive CD68+ macrophages was further detected.We used methionine choline deficiency(MCD)diet and high fat diet(HFD)to establish murine NASH models,body weights were measured regularly,and expressions of F4/80 and fgl2 on hepatic macrophages were observed by immunofluorescence.2.HE staining and NAFLD score were used to evaluate pathological changes in wild type(WT)and fgl2 knockout(fgl2-/-)mice liver tissues.Alanine aminotransferase(ALT),aspartate transaminase(AST),lactate dehydrogenase(LDH)detected from serum were used to evaluate the liver function.The secretion of TNF-a,IL-6,IL-1?,MCP-1 and IL-18 and the activation of ROS in liver were also tested.3.The degree of liver steatosis was determined by oil red staining.Cholesterol(CHOL),triglyceride(TG)in liver and serum were detected.Bone marrow derived macrophages(BMDMs)stimulated by lipopolysaccharide(LPS)or free fatty acid(FFA)were considered NASH models in vitro.LPS-and FFA-conditioned medium(CM)were co-cultured with primary hepatocytes.Quantitative real-time PCR(q PCR)was used to detect gene expressions of sterol regulatory element binding protein 2(SREBP-2),fatty acid synthase(Fasn),peroxisome proliferator activated receptor a(PPARa)and carnitine palmitoyl transferase 1a(CPT1A)in liver tissues and primary hepatocytes.4.Western blot(WB)was used to detect phosphorylation levels of nuclear factor activated B cell k light chain enhancer(NF-kb)and p38 mitogen activated protein kinase(p38-MAPK)signaling pathways and activation of Nod-like receptor protein 3(NLRP3)inflammasome in liver tissues and BMDMs.5.Fgl2 in THP-1 cells was overexpressed by HBLV-h-Fgl2-GFP-PURO lentivirus,and tolllike receptor 4(TLR4)and its downstream molecules such as myeloid differentiation factor 88(My D88),TNF receptor associated factor 6(TRAF6)were detected by Western blot.Coimmunoprecipitation(Co IP)was used to test the combination of fgl2 and TLR4.?Results? 1.In NASH patients and murine models,the expression of fgl2 were upregulated,and the number of hepatic macrophages and fgl2-positve macrophages was also significantly increased.2.Fgl2 deficiency could alleviate levels of inflammation and steatosis in murine models of NASH.Compared with WT NASH mice,fgl2-/-NASH had lower NAFLD activity score.Indicators of liver functions such as ALT,AST and LDH were also alleviated.The activation of TNF-a,IL-6,IL-1?,MCP-1,IL-18 and ROS were decreased in fgl2-/-NASH mice and fgl2-/-BMDMs.3.In addition,genes involved in lipogenesis(Fasn,SREBP-2)were downregulated while those involved in lipolysis(PPARa,CPT1A)were upregulated both in liver tissues and primary hepatocytes co-cultured with BMDMs in fgl2-/-groups,which leading to lower lipid deposition.4.The activation of NF-k B,p38-MAPK and NLRP3 inflammasome were also downregulated when fgl2 was deficient with downregulated secretion of pro-inflammatory cytokines.5.Finally,we found that fgl2 could combine with TLR4 and activate the TLR4-My D88 dependent signaling pathway,which led to inflammatory and lipid metabolism disorder.?Conclusion? These data suggest that fgl2 aggravates progression of NASH by interacting with TLR4 on macrophages and activating TLR4-My D88 dependent signaling pathway,then activate NLRP3 inflammasome and downstream inflammatory signaling pathways including NF-k B and p38-MAPK,which consequently induce over-production of ROS and pro-inflammatory cytokines like TNF-a,IL-6,IL-1b,MCP-1 and IL-18.Hence genes involved in lipogenesis and lipolysis were interfered and led to hepatic lipid metabolism disorders.