| Objectives:To investigate the effective monomers and their immunological mechanisms in triterpenoid of the alcohol extract from cimicifuga on acute liver injury induced by Concanavalin A(ConA).Mehtods:1.The triterpenoid of the alcohol extract from cimicifuga was separated into several monomers and latter were given to ConA-induced lethal model mice orally.The mice were 6-8 weeks age C57BL/6 male mice.The curative effects of the monomers were evaluated by observing the survival rate,analyzing serum transaminase levels and liver pathology with HE staining.The protective effects of monomers were evaluated by serum and liver cytokines secretion level.2.To discuss the effect of monomers on adaptive immunity:(1)The dynamic activations of splenic T lymphocytes of ConA-induced acute liver injury mice model was analyzed by detecting the expression of their surface markers and the intracellular IFN-y production with flow cytometry.(2)The CD4+ and CD8+T lymphocytes from mice spleen were cultured in vitro and activated with ConA or CD3/CD28 antibody respectively,monomers were given at the same time.IFN-gamma secretion in supernatant was detected by ELISA and CD4+,CD8+T lymphocytes surface markers expression were detected by flow cytometry.3.To discuss the effect of monomers on innate immunity:Mouse bone marrow-derived macrophages(BMDMs,innate immune cells)were cultured in vitro,and pretreated with the effective monomers 1 hour,then challenged with LPS 3 hours.The supernatant was collected and the cells were harvested.The cytokines TNF-alpha and IL-12 in the supernatant were detected by ELISA and the expression of inflammatory cytokines’ gene such as TNF-alpha,IL-6,IL-12a,IL-12b were detected by Real-time PCR.Results:1.Monomer 1 and monomer 2 of triterpenoid of the alcohol extract from cimicifuga could both improve the survival rate of ConA-induced liver lethal model.Monomer 1 improved the survival rate to 60%,while monomer 2 to 40%.Compared with model group,the serum ALT and AST levels of monomer 1 and monomer 2 group were both significantly decreased,so as to the serum inflammatory cytokines TNF-alpha,IL-12 secretion and at the same time,the liver pathological damage was reduced.2.(1)Monomer 1 and monomer 2 could afftect the dynamic activations of splenic T lymphocytes but did not affect IFN-gama production in ConA-induced acute liver injury murine model in vivo.(2)In vitro cell experiments,monomer 1 and monomer 2 could not affect CD4+,CD8+T lymphocyte activation and IFN-gama secretion induced by CD3/CD28 antibody or ConA respectively.3.Monomer 1 and monomer 2 could decrease BMDMs supernatant TNF-alpha secretion;monomer 1 could decrease TNF-alpha,IL-12 cytokines’ genes expression and other inflammatory cytokines’ genes expression of BMDMs cells activated by LPS.Conclusions:1.Monomer 1 and monomer 2 are effective monomers of triterpenoid of the alcohol extract from cimicifuga to cure ConA induced mice acute liver injury.2.Monomer 1 and monomer 2 does not inhibit the activation of lymphocytes directly.Macrophages are the target cells of monomer 1 and monomer.The protective effect of monomer 1 and monomer 2 maybe through inhibiting the expression and secretion of TNF-alpha,and inhibiting the expression of IL-12、IL-6 and other inflammatory cytokines’ genes to inhibit the activation of innate immune cells. |
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