The Role Of Fgl2 In Macrophage Polarization In Severe Hepatitis

ObjectiveSevere hepatitis can be induced by diverse causes,in our country mainly caused by viral hepatitis infection,due to the lack of specific and effective clinical therapeutic targets and interventions,the vast majority of patients with poor prognosis.Pathological morphology and immunology research shows that microcirculation obstacle and cell apoptosis in the early stage is vital for disease progression of severe hepatitis.Our institute early found that in MHV-3 induced fulminant hepatitis model,kupffer cell polarized to M1,and high expression of fgl2 molecule on the M1,after fgl2 knockout,mouse liver inflammation relieve,M1 polarization decreased.On the basis of early work,this study use of vitro cell model to further study of fgl2 impact on macrophage polarization and its relationship with severe hepatitis disease progression.In order to further clarify the immunological pathogenesis of severe hepatitis,provides new targets for the treatment of severe hepatitis.Methods1.Stimulate THP-1 cells by PMA and LPS,then use FCM detect cell surface molecular CD80 and fgl2,use Elisa detect inflammation factors(TNF-??IL-6?IL-1?)level in the supernatant,use Real-time PCR detect fgl2 and M1-realted gene expression(i NOS?TNF-??IL-6?IL-1?).2.Stimulate by 3% starch broth,then collect peritoneal macrophage from Balb/c J wild type mice and fgl2-/-mice,join in MHV-3 to simulate fulminant hepatitis antigen stimulation in mice.3.FCM detect the change of macrophage polarization and fgl2 after MHV-3 infection.4.CBA detect the inflammatory factors(TNF-??IL-6?IL-1??MCP-1?IL-10?IL-12)level in the supernatant of MHV-3 infected macrophage cells(WT and fgl2-/-).5.Realtime PCR detect the change of fgl2 gene expression and M1-realted(i NOS?TNF-??IL-6?IL-1?)?M2-realted(CD206?Arg1?TGF-?)gene expression.Results 1.Stimulated with PMA and LPS,THP-1 cells polarized to M1 accompanied by elevated fgl2 expression.Experimental group compared with control group,FCM found that CD80+(M1)ratio increased significantly(79.67±2.11% VS 32.43±2.11%,P<0.001),and fgl2+/CD80+ ratio also increased(70.8±5.26% VS 36.83±3.47%,P<0.001);Elisa found in the supernatant,inflammation factors(TNF-??IL-6?IL-1?)secretion increased;Real-time PCR found that fgl2 and M1-realted(i NOS?TNF-??IL-6?IL-1?)gene expression increased.2.Wild type Balb/c J mice peritoneal macrophages after MHV-3 stimulated,prefer to M1 polarization and associated with elevated fgl2 expression.Experimental group compared with control group,FCM found that i NOS+(M1)ratio increased significantly,and fgl2+/i NOS+ ratio also increased;CBA found in the supernatant,inflammation factors TNF-??IL-6?MCP-1 secretion increased,but IL-1??IL-12?IL-10 have no difference;Real-time PCR found that fgl2 and M1-realted(i NOS?TNF-??IL-6?IL-1?)gene expression increased,M2-realted(CD206?TGF-?)gene expression decreased.3.Peritoneal macrophages from WT and fgl2-/-,after MHV-3 stimulate,both polarized to M1,but fgl2-/-M1 polarization ratio decrease.Under the condition of the same pathogen stimulus,fgl2-/-mice compared with wild type mice,FCM found i NOS+(M1)ratio decreased;CBA found inflammation factors(TNF-??IL-6?MCP-1)secretion decreased;Real-time PCR found that M1-realted(i NOS ? TNF-? ? IL-6 ? IL-1?)gene expression decreased,M2-realted(CD206?TGF-?)gene expression increased.Conclusion 1.Fgl2 can promote to the M1 polarization,inhibit M2 polarization;2.Fgl2 closely related to the M1 polarization,but it is not the must exist molecule for macrophage polarization,may be combined with other molecules.