Effect And Molecular Mechanisms Of Kinsenoside Against Autoimmune Hepatitis

Objective: To explore the regulation and mechanism of kinsenoside(KD) on Con A-induced AIH.Methods:(1) The ConA-induced AIH model was established. 42 male C57BL/6J mice were randomly divided into 7 groups: Control; Model(Con A); Silymarin(Silymarin 50 mg/kg); KD High-dose(KD 30 mg/kg); KD Middle-dose(KD 20 mg/kg); KD Low-dose(KD 10 mg/kg); KD/NS(KD 30 mg/kg),( n=6). Each group of mice orally administered continuously for 3 days. Autoimmune hepatitis model was induced at 3 day by intravenous injection of Con A(20 mg/kg). 12 hours after the last administration, the mice were sacrificed. The hepatoprotective ability of KD were estimated by measuring serum aminotransferase which indicate the liver function(AST and ALT) and SOD, MDA, NO, IL-12, IFN-? levels in serum, liver histology(HE). Flow cytometry measured phenotypic and function of Liver and spleen CD8+T cells, Treg cells and DCs derived from murine bone-marrow. Comprehensively evaluation KD hepatoprotective effect.(2) In Semi-vitro and vitro experiments: The splenocytes were isolated from mice and cultured in RPMI-1640 medium supplemented with KD 10 ?g/ml, 5 ?g/ml, 2.5 ?g/ml. DCs cultured in RPMI-1640 medium with GM-CSF stimulation treated with KD 10 ?g/ml, 5 ?g/ml, 2.5 ?g/ml. To investigate KD affect T cell proliferation and DCs mature function. Also detected KD on cells proliferation of RAW264.7 cells, HSC-T6 cells.(3) Real-time quantitative polynucleotide chain reaction, Western blot detected JAK2/STAT3/SOCS3 signaling pathway in autoimmune hepatitis mice induced by Con A as well as the therapeutic effect of KD in AIH.Results:(1) KD high, medium and low dose could significantly decrease serum aminotransferase of Con A induced autoimmune hepatitis model mice(P <0.01), ELISA method detection showed serum MDA, NO, IL- 12, IFN-? concentrations significantly reduced and SOD concentration significantly increased. Liver histology(HE) showed KD could reduce the infiltration of inflammatory cells, improving liver damage. KD suppressed DCs phagocytosis and antigen-presenting ability to inhibit T lymphocyte proliferation and reduced the CD8+T cell differentiation and killing function, regulated the expression of Treg cells to play an impotent role in immune tolerance.(2) In Semi-vitro and vitro experiments, each dose group of KD could reduce the proliferation of T lymphocytes and can suppress DCs maturation and function. Proliferation of RAW264.7 cells, HSC-T6 cells was significantly inhibited.(3) Real-time quantitative polynucleotide chain reaction, Western blot results showed that, KD upregulate the expression of SOCS3, inhibit the activation of JAK2 / STAT3 signaling pathway.Conclusions:(1) KD showed a good therapeutic effect on Con A induced autoimmune hepatitis mice. KD could significantly improve liver function, upregulated of the concentration SOD, reduced the concentration of MDA, decreased the level of NO, IL-12, IFN-?, reduced oxidative stress and improved hepatic inflammation reaction.(2) KD can inhibited the proliferation of T lymphocytes and reduced DCs maturation, decrease immune reaction in liver. KD inhibited the proliferation of RAW264.7 cells and HSC-T6 cells, showed anti-inflammatory and anti-fibrotic effect.(3) KD possess immunosuppression function through inhibition of the expression of the JAK2/STAT3/SOCS3 signaling pathway in liver and spleen.