Fgl2 Regulates NLRP3 Inflammasome Of Hepatic Macrophage In Experimental Viral Fulminant Hepatitis

Background and objective: Severe hepatitis is a serious disease that threatens human health with a large number of liver cell necrosis as the main pathological characteristics.Severe hepatitis can cause liver failure and endanger the lives of patients.It is one of the main causes of death in patients with liver disease.There are many causes of severe hepatitis,mainly caused by hepatitis viruses in Asia,including hepatitis a,b,c,e virus,and most importantly,hepatitis b virus.In western countries,it is mainly caused by drug poisoning and chronic alcoholic liver damage.Severe hepatitis is critically ill with rapid progress,many complications which are difficult to treat,and the mortality rate is very high(40-60%)clinically.Therefore,the study of its pathogenesis is very necessary and urgent.The pathogenesis of severe hepatitis is complex,from cell injury and dysfunction to apoptosis and necrosis.The study of its pathogenesis is still a hot topic in medical research.More and more studies suggest that innate immunity plays an important role in the pathogenesis of severe hepatitis.Natural killer cells(NK cells),Kupffer cells(KCs)and other innate immune cells and various cytokines jointly participate in the formation of a complex and huge network system,in which IL-1? and inflammasomes(NLRP3,ASC,caspase-1)play an important role.It has been found that in mice with fulminant hepatitis caused by MHV-3,the NLRP3 inflammasome is activated,producing IL-1? in large amounts.In combination with TNF-a,IL-1? can promote the high expression of Fgl2 in liver KCs through the NF-k B pathway,leading to massive necrosis of liver cells.Fgl2 can play a regulatory role through the MAPK pathway.The purpose of this study is to investigate whether liver macrophage Fgl2 can regulate the expression of NLRP3 inflammasome,and thus play an important role in the pathogenesis of fulminant hepatitis in mice induced by MHV-3.Method: In the pathogenesis of MHV-3-induced fulminant hepatitis in mice,the regulation of Fgl2 expression in mice,namely fgl2 gene knockout mice and normal mice,was used to study the regulation of Fgl2 on NLRP3 inflammasome of liver macrophages.1.Fgl2 gene knockout mice and wild-type mice were intraperitoneally injected with mouse hepatitis virus MHV-3 to establish a fulminant hepatitis model.After the mice were infected with MHV-3 virus,liver tissues were collected at different time points for HE staining to detect the pathological damage of liver in mice.At the same time,serum ALT and AST levels were detected to assess liver inflammation.The expression levels of IL-1? in liver tissues of fgl2 knockout mice and wild-type mice were detected by immunohistochemistry.The expression levels of cytokine IL-1? in serum and liver homogenates of mice were detected by ELISA.And the expression of IL-1? in liver tissues was detected by Western blot and RT-PCR.2.Fgl2 gene knockout mice and wild-type mice were intraperitoneally injected with mouse hepatitis virus MHV-3 to establish a fulminant hepatitis model.After the mice were infected with MHV-3 virus,hepatic macrophages were extracted by hepatic portal vein perfusion /digestion and density gradient centrifugation at different time points.The percentages of NLRP3,ASC and IL-1? in KCs(CD11b-F4/80hi)and monocyte derived macrophages(Mo MFs CD11bhiF4/80int)in fgl2 knockout mice and wild-type mice at different time points after MHV-3 infection were observed by flow cytometry.3.In vitro,macrophage colony stimulating factor was used to induce marrow derived macrophages in mice with fgl2 gene knockout and wild-type mice,and then LPS was used to make the targeted differentiation into M1.The expressions of NLRP3,ASC,pro-caspase-1,caspase-1,pro-IL-1? and IL-1? in the cells were detected by RT-PCR and Western blot respectively.Result: 1.After fgl2 gene knockout mice and wild-type mice were infected with MHV-3 respectively,the liver tissue pathological damage of fgl2 gene knockout mice was significantly less than that of wild-type mice at different time points,and the serum transaminase level was lower than that of wild-type mice.Immunohistochemistry showed that the expression of IL-1? in liver tissue of fgl2 knockout mice was significantly lower than that of WT mice.2.The expression levels of IL-? in serum and liver homogenate of fgl2 knockout mice were lower than those of wild-type mice,and the expression levels of genes of NLRP3 and ASC in liver tissue were lower than those of wild-type mice,and the expression level of IL-1? protein was lower than that of wild-type mice.3.At different time points after MHV-3 infection,the expression of NLRP3,ASC and IL-1? in fgl2 knockout mouse liver macrophages were lower than those in wild-type mouse liver macrophages.4.After LPS stimulation,BMDM of fgl2 knockout mice showed lower expressionof ASC,pro-caspase-1,caspase-1,pro IL-1?,and IL-1? than that of WT mice at the RNA level.At the protein level,the expression of pro-IL-1? was lower in fgl2 knockout mice stimulated by LPS than that of WT mice BMDM,and the activation of pro-caspase-1 in fgl2 knockout mice BMDM was lower than that of WT mice.Conclusion: In mice with fulminant hepatitis,Fgl2 can regulate the activation of NLRP3 inflammasome in liver macrophages.After fgl2 gene knockout,the activation of inflammasome in mouse liver macrophages was weakened,and liver tissue damage was also significantly reduced.Those indicate that both fgl2 and NLRP3 are important targets in the pathogenesis of fulminant hepatitis,and through the control of these two targets,they can be used as a potential treatment for severe hepatitis.