The Study Of Cinobufagin For Lymphocytes And Macrophages Immunomodulatory Activity

ObjectiveCinobufagin(CBG)is one of the main active ingredient in traditional Chinese medicine Toad,also known as Cinobufagin venom toad,an ether bond steroids have the efficacy,such as,local anesthesia,cardiac,boost and improve immunity,etc,widely distributed.To investigate the immunomodulatory effects of CBG,this paper established a mouse spleen lymphocytes,peritoneal macrophage model for the screening of target cells,in vitro studies CBG immunomodulatory activity,provide the experimental basis for the further development and utilization of CBG.MethodsIn this paper,use mouse spleen lymphocytes and peritoneal macrophages as a model,assay the effect of CBG,CBG with lipopolysaccharide(LPS)or concanavalin A(Con A)composited for lymphocyte proliferation by MTT;detected the effect of CBG for macrophages phagocytic function by MTT;detected the effect of CBG for spleen lymphocytes and macrophages secretion cytokine by ELISA;assay the effect of CBG for macrophages secretion NO by Griess;analyzed the effect of CBG for spleen lymphocyte membrane surface markers and cell cycle by flow cytometry.assay the effect of CBG,CBG and LPS or Con A composited on S.aureus infections lymphocyte proliferation;assay the effect of CBG for macrophage phagocytosis S.aureus function by MTT;detected the effect of CBG on S.aureus infections spleen lymphocytes secretion cytokine by ELISA.ResultsCBG monotherapy stimulated,CBG with LPS or Con A composited stimulation lymphocyte,both can improve lymphocyte stimulation index,but the lymphocyte stimulation index of complex drug were lower than the monotherapy group;CBG alone stimulation can induced the secretion of lymphocyte cytokines IL-2 and IL-10,improve the ratio of lymphocyte subsets CD4~+/CD8~+,improve the percentage of cell cycle S phase.CBG stimulate macrophages alone,can induced the secretion of macrophage NO,IL-6,TNF-a and GM-CSF,enhance the ability of macrophage phagocytic Neutral Red.when S.aureus infections lymphocyte,CBG alone stimulate,CBG with LPS or Con A composited stimulation can improve the stimulation index of lymphocyte;CBG alone stimulation can induced the secretion of lymphocyte cytokines IL-2 and IL-10;can enhance macrophage phagocytic S.aureus ability.ConclusionsIn a certain concentration range,CBG stimulate alone,CBG and LPS or Con A costimulatory which can promote lymphocyte proliferation,but CBG with LPS or Con A composite drug group can’t synergy.CBG alone stimulate lymphocytes,by increasing the ratio of lymphocyte subsets CD4~+/CD8~+,increasing the percentage that cells entering DNA synthesis,induced the secretion of Th1 and Th2 cytokines,and enhance the lymphocyte immune regulation.CBG stimulate macrophages alone,by increasing the macrophage phagocytic function,induced the secretion of NO,IL-6,TNF-α and GM-CSF,further improve the immunomodulatory effects of macrophages.Staphylococcus aureus infection lymphocytes and macrophages phagocytosis Staphylococcus aureus,CBG alone stimulate,CBG with LPS or Con A composited stimulation can improve lymphocyte proliferation;CBG alone stimulate can induced the secretion of Th1 and Th2 cytokines,enhance macrophage phagocytosis.further verification CBG can improve lymphocytes and macrophages immunomodulatory activity.