Immunosuppressive Effect And Mechanism Of Phenethylchromone Compound GYF-21 In Agarwood

The immune system is an important system that protects against pathogenic bacteria and performs immune function.Immune system disfunction will lead to many diseases,including immunodeficiency diseases resulted from weakness of immune function and,autoimmune diseases,allergic reactions,and organ transplantation rejections resulted from hyperactivation of immune function.Immunosuppressant is used in the treatment of diseases originated from excessive immune reaction,including corticosteroids,microbial metabolites and small molecule inhibitors such as FTY-720,they have made curative effects in clinical treatment,but long-term use can lead to side effects such as high blood pressure,diabetes,and liver and kidney damage.In recent years,the researches focus on the immune suppression effects of Chinese medicine ingredient is becoming increasingly prominent due to its good therapeutic effect and little side effects,which providing new directions for finding new immunosuppressants.At the same time,questions of traditional Chinese medicine such as complex mechanisms and unclear drug targets cannot be ignored.So we should devoted to the immune function research of traditional Chinese medicine active monomers,especially some trace element.Although they are small in quantity but have great pharmacodynamic effects and clear target,suggesting great development and utilization prospects.Through the inhibitory effect screening of nitric oxide synthesis by macrophage,GYF-21,an epoxide 2-(2-Phenethyl)-chromone derivative which was isolated from Chinese agarwood ethyl acetate extract,showed obvious inhibitory effect on NO synthesis in macrophages stimulated by LPS,with EC50 of 2.1 ±0.02?M.In this research,the immunomodulation effects of GYF-21 were studied,including innate immunity and adaptive immunity.In the study of innate immune,GYF-21 could significantly suppress the mRNA transcription of NO synthase.RT-PCR and ELISA were used to detect the mRNA transcription and protein secretion of TNF-?,IL-6 and IL-1?,the results showed that GYF-21 had strong inhibitory effects on the expressions of these pro-inflammatory factors,and also had certain suppressive effects on the expressions of chemokine MCP-1 and MIP-1?.Using GM-CSF and IL-4 inducing mononuclear cells to obtain the primary dendritic cells,then flow cytometry results showed that GYF-21 significantly suppressed the upregulations of surface molecules CD80 and CD86 on dendritic cells induced by LPS,and inhibited the expressions and secretions of IL-12 and IL-23.Using the primary bone marrow cells which are rich in neutrophils as the research object,the flow cytometry results showed that GYF-21 significantly inhibited upregulation of adhesion molecule CD11b and downregulation of CD62L on the surface of neutrophil induced by inflammation conditioned medium.Furthermore,GYF-21 also significantly inhibited ROS production in neutrophil.In the mechanism study of innate immune regulating effects,we found that GYF-21 significantly suppressed the phosphorylations of I-?B and p65 subunit during the activation of NF-?B signaling pathway in BV2 cells induced by LPS.In addition,the phosphorylations of STAT1 and STAT3 were also strongly inhibited.In the adaptive immunity study,CCK-8 was used to detect the cytotoxic effects of GYF-21 on spleen cells,and the dosage was adjust to 0.5,1and 2?M.Magnetic bead separation method was used to obtain purified CD4+T cells,and the regulation of activation,proliferation and differentiation on CD4+T cells by GYF-21 were tested.The results showed that GYF-21 had no obvious inhibitory effects to upregulations of CD25 and CD69 on CD4+T cells which were induced by CD3e and CD28,but significantly inhibited CD4+T cells differentiate into Thl cells,Th2 cells and Th17 cells.In the study of CD8+T cells,we found that GYF-21 slightly inhibited the activation and proliferation of CD8+T cells,and moderately inhibited the release of IFN-y in CD8+T cells.In the study of B cells,we found that GYF-21 suppressed upregulations of the surface molecules CD80 and CD86 which were induced by LPS.Furthermore,GYF-21 also inhibited proliferation of B cells and differentiating into plasma cells and.However,in the activation and proliferation experiment of B cells stimulated by aIgM,aCD40 and mIL-4,differentiation experiment of B cells stimulated by aCD40 and mIL-4,GYF-21 showed no significant inhibitory effects.In conclusion,GYF-21 can significantly inhibit the activation of innate immune cells such as microglial cells,dendritic cells and neutrophils.It also has significant inhibition effects to the expressions and secretions of cytokine IL-6,IL-1? and IL-23,which play important roles in Th17 cells differentiation.Furthermore,GYF-21 has remarkable and selective inhibitory effects on the differentiation of CD4+T cells into Th1 cells,Th2 cells and Th17 cells.At last,GYF-21 can also inhibit proliferation and IFN-y release of the CD8+T cells.So GYF-21 exerts excellent inhibitory effects on the innate and adaptive immunity and has huge potential to be developed into a new type of small molecule immune inhibitor.