Regulation Of Yin Chen Si Ling Granule On The Polarization Of Liver Macrophages In Mice With Acute Liver Failure Based On JAK-STAT Pathway

Objective: To investigate the mechanism of Yin Chen Si Ling Granule on the polarization of liver macrophages in mice with acute liver failure based on JAK-STAT pathway.Methods: Through the establishment of ALF mice model,we studied the polarization trend of liver macrophages and the expression of JAK-STAT related pathway proteins in mice after the intervention of Yin Chen Si Ling Granule,and explored the regulatory mechanism of Yin Chen Si Ling Granule on the polarization of liver macrophages in mice with acute liver failure mediated by JAK-STAT pathway.First,60 male C57BL/6 mice were randomly numbered and divided into 6 groups according to the normal group,model group,control group(0.22mg/kg dexamethasone),low dose group of Yin Chen(2.25g/kg),medium dose group of Yin Chen(4.5g/kg)and high dose group of Yin Chen(9g/kg).Next,Five days before the establishment of the model.Normal group and model group were gavaged same amount of normal saline in advance,twice a day;Dexamethasone was administered to the control group with dose of 0.22 mg/kg in advance,twice a day;TCM were given to in advance in the low dose group,medium dose group and high dose group of Yin Chen twice a day with dose of 2.25g/kg,4.5g/kg and 9g/kg.The ALF mice model was established: the model group was injected intraperitoneally with D-Gal N/LPS(500mg/Kg,20μg/kg)to form a membrane at one time,and model could be established after 8 hours.Observe the 24-hour survival rate of mice in each group.After 24 hours of intervention,the liver tissue and blood samples of the surviving mice were collected in each group(n=3).Liver function ALT and AST in mice serum were detected by liver function kit.HE staining was used to observe the pathological changes of liver tissue.Detection of TNF-α in tissue fluid and serum by ELISA-α,TGF-β expressions of cytokines.Immunofluorescence assay was used to detect the expressions of INOS gene and Arg-1 gene,the surface markers of macrophages M1 and M2.Western blot was used to detect the expressions of JAK2 and STAT3 proteins on the related JAK-STAT pathway.Results: 1.The results of liver function in mice showed that after 24 h,ALT and AST in each test group were improved compared with those in the model group.Within Yin Chen Si Ling group: ALT and AST decreased in a concentration dependent manner with increasing concentrations in TCM,among which Yin Chen high-dose group had the most obvious effect on improving liver function in mice(P<0.05).2.Observation of pathological morphology of mice liver: diffuse necrosis of hepatocytes,infiltration of neutrophils and lymphocytes in some areas,disorder of Hepatic lobule morphology,blurred structure of hepatocytes,and severe liver injury were observed in liver tissue of model group;In the high dose group of Yin Chen,the inflammatory cell infiltration in liver tissue decreased,the structure of Hepatic lobule was less damaged,the structure was relatively complete,and the degree of damage was smaller.3.ELISA: Between the Yin Chen Si Ling Granule groups,the concentration of TNF-α is negatively correlated with the concentration of TCM,and in the high dose group,the concentration of TNF-αwas significantly lower(P<0.05);4.The expressions of M1 and M2 in liver macrophages was detected by immunofluorescence: Between the Yin Chen Si Ling Granule groups,the expression of INOS gene in liver macrophages of mice decreased in a concentration-dependent manner with the increase of the concentration of TCM,and the expression of Arg-1 gene increased with the increase of the concentration of TCM,in which the high dose group of Yin Chen was the most obvious,with statistical significance(P<0.05);5.Western Blot detected the expressions of JAK2 and STAT3 proteins on the relevant JAK-STAT pathway: Among TCM groups,JAK2 and STAT3 proteins expression increased in the high dosage group of Yin Chen with statistical significance(P <0.05).Conclusions: 1.Yin Chen Si Ling Granule can improve the liver function,inflammation in vivo and pathological damage of liver tissue in mice with acute liver failure;2.Yin Chen Si Ling granule can Inhibit polarization towards M1 type and TNF-α Secretion,attenuates inflammatory injury in mice with D-galactosamine/lipopolysaccharide induced acute liver failure.3.The mechanism by which Yin Chen Si Ling granule regulate on D-galactosamine/lipopolysaccharide induced injury in mice with acute liver failure may be that upregulating the expressions of JAK2,STAT3 proteins in the JAK-STAT pathway,promoting macrophage polarization toward M2 type,restoring the balance of M1/M2 associated.