Application Study Of Taraxasterol In Reducing Fulminant Hepatitis Induced By D-GalN/LPS

Objective: Taraxasterol(TAR)was used to regulate SOCS3/JAK2/STAT3 and JNK signaling pathways to find a new method for the treatment of D-galactosamine/lipopolysaccharide(D-Gal N/LPS)-induced fulminant hepatitis(FH).Methods: In vivo experiment,70 mice were randomly divided into 7groups: normal Control group,D-Gal N/LPS group,D-Gal N/LPS+Silymarin group(120 mg/kg),D-Gal N/LPS+TAR group(2.5 mg/kg,5 mg/kg,10 mg/kg)and TAR10 group(10 mg/kg),with 10 mice in each group.All mice were given intragastric administration at a volume of 10 m L/kg for 15 d.2 h after the last administration,the Control group and TAR10 group was intraperitoneally injected with saline.Mice in the other groups were intraperitoneally injected with D-Gal N(500mg/kg)and LPS(10 μg/kg)to establish FH models.After 6 hours of fasting,the eyeballs were removed to collect blood and the livers of mice were collected.Biochemical methods were used to detect the changes of liver function indexes in serum and the activity of oxidative stress related factors in liver tissue of mice.HE and immunohistochemical staining were used to find the pathological damage changes of liver in mice.q RT-PCR and ELISA were used to detect the inflammatory reaction in liver tissue of mice,The expressions of SOCS3/JAK2/STAT3,JNK signaling pathway and apoptosis-related proteins were detected by q RT-PCR and Western blot.In vitro experiment,RAW264.7 cells were treated with LPS and TAR.MTT assay was used to screen the dose of TAR and detect the toxicity of TAR on RAW264.7 cells.Immunofluorescence assay was used to detect the protein distribution of p-STAT3 and p-JNK in RAW264.7 cells.The m RNA expressions of SOCS3,p-STAT3,p-JNK and p-JAK2 were detected by q RT-PCR.The apoptosis of RAW264.7 cells was observed by Hoechst33258 staining and the apoptosis was quantitatively analyzed by annexin V-FITC/PI double staining.The expressions of SOCS3/JAK2/STAT3,JNK signaling pathway and apoptosis-related proteins were detected by Western blot.Results: In vivo experiment,the liver function of mice in the D-Gal N/LPS group was severely damaged.The liver was significantly blackened and the HE staining section showed obvious necrosis of hepatocytes.These results indicated that the FH model of mice was successfully constructed.After TAR intervention,compared with D-Gal N/LPS group,the liver function index in mice serum was significantly reduced.The liver was restored to ruddy and the necrosis part in the section was significantly improved.Compared with D-Gal N/LPS group,after TAR administration,the content of oxidative stress factor MDA in liver tissue of mice was decreased,and the activities of MDA and GSH Px were increased.The results of ELISA and q RT-PCR showed that with the influence of TAR,the level of inflammatory factor TNF-α,IL-6 and IL-1 β was reduced.The results of q RT-PCR also showed that TAR could significantly reduce p-STAT3,p-JAK2 and p-JNK induced by D-Gal N/LPS and increase the m RNA expression of SOCS3.Immunohistochemical staining showed that there were positive staining regions of p-JNK and p-STAT3 in D-Gal N/LPS group.While with TAR intervention,the positive staining regions of p-JNK and p-STAT3 were decreased.Western blot showed that with TAR intervention,the expression of SOCS3/JAK2/STAT3,JNK signaling pathway and apoptosis related proteins in the liver of mice was significantly reduced.In vitro experiment,LPS was used to induce the inflammatory microenvironment of RAW264.7 cells.Hoechst33258 staining and annexin V-FITC/PI double staining showed that the apoptosis rate of RAW264.7 cells were decreased significantly after TAR intervention.Immunofluorescence detection showed that TAR could down-regulate the over-expression of p-JNK and p-STAT3 proteins induced by LPS in RAW264.7 cells.q RT-PCR and Western blot also confirmed that TAR could reduce the expression of SOCS3/JAK2/STAT3,JNK signaling pathway and apoptosis-related proteins.Conclusion: TAR can alleviate D-Gal N/LPS-induced fulminant hepatitis,and its hepatoprotective mechanism may be related to the inhibition of SOCS3/JAK2/STAT3 and JNK signaling pathways and apoptosis.