Effect of ganoderic acid A on a mouse model of concanavalin A-induced acute immune liver injury and its mechanism
Objective To investigate the therapeutic effect of ganoderic acid A(GA-A)on a mouse model of concanavalin A(ConA)-induced autoimmune hepatitis(AIH).Methods A total of 35 mice were randomly divided into control group(NC group),model group(ConA group),and low-,middle-,and high-dose GA-A treatment groups(GA-A-L,GA-A-M,and GA-A-H groups,respectively),with 7 mice in each group.ConA was injected via the caudal vein of mice to establish a classic mouse model of AIH,and different doses of GA-A were administered via intraperitoneal injection 1 hour later for treatment.Proteomic techniques were used to investigate the protective mechanism of GA-A on hepatocytes,and HL-60 cells were differentiated into dHL-60 neutrophils by all-trans retinoic acid in vitro to validate the mechanism of action of GA-A.Related indicators were measured,including inflammatory markers(the activities of serum alanine aminotransferase[ALT]and aspartate aminotransferase[AST],HE staining,and inflammation-related genes),apoptosis markers(TUNEL staining),neutrophils,and neutrophil extracellular trap(NET)markers(myeloperoxidase[MPO],citrullinated histone H3[CitH3],Ly6G,and free double-stranded DNA[dsDNA]),and p38 phosphorylation markers.The independent samples t-test was used for comparison of continuous data between two groups;a one-way analysis of variance was used for comparison between multiple groups,and the least significant difference t-test was used for further comparison between two groups.Results Compared with the NC group,the ConA group had significant increases in the serum levels of ALT and AST(both P<0.001),and compared with the ConA group,GA-A treatment significantly reduced the levels of ALT and AST(both P<0.01).HE staining showed that the mice in the ConA group had significant liver necrosis,while GA-A treatment significantly reduced the area of liver necrosis and the number of TUNEL-positive cells(both P<0.05).Compared with the ConA group,the GA-A group had significant reductions in the expression levels of the inflammatory factors interleukin-6,tumor necrosis factor-α,and interferon gamma in serum and liver tissue(all P<0.05).The proteomic analysis showed that GA-A alleviated ConA-induced acute immune liver injury by inhibiting the release of NET and the p38 MAPK pathway.Immunofluorescent staining of mouse liver tissue showed that compared with the ConA group,the GA-A group had significant reductions in the number of MPO-positive neutrophils and the number of cells with positive Ly6G and CitH3(all P<0.01).Western Blot and dsDNA testing showed that GA-A significantly inhibited the levels of the NET markers dsDNA and CitH3 and the level of p38 phosphorylation in liver tissue and dHL-60 cells(all P<0.05).Conclusion GA-A alleviates liver inflammatory response and hepatocyte death by inhibiting the p38 MAPK pathway and the release of NET,thereby alleviating ConA-induced acute immune liver injury.This study provides a theoretical basis for the use of GA-A to treat immune liver injury by regulating neutrophil function.
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