Study On Immunosuppressive Activity And Mechanism Of Sphingolipids In Cordyceps Sinensis

ObjectiveCordyceps Sinensis（CS）is a traditional Chinese medicine in my country.Studies have reported that CS has various pharmacological effects such as relieving fatigue,anti-aging,anti-cancer,hyperlipidemia,immune regulation,post-severe asthma,protecting liver and kidney functions.In 780 AD,in“Tibetan Materia Medica”,it was recorded that CS has the effect of moistening the lungs and invigorating the kidneys.In recent years,studies have found that CS has a significant inhibitory effect on immune rejection after organ transplantation,but the specific mechanism of action is still unclear.Through activity tracking,we found that sphingolipid compounds in CS are the main components that exert immunosuppressive effects.These compounds not only exist in mycelium of CS but also in wild CS,after analyzing by Thermo Q Exactive-high-resolution mass spectrometry.This thesis aims to establish a method for preparing these sphingolipid chemical components from the fermentation broth of CS mycelium,and further to study its immunosuppressive activity.MethodsSphingolipids were prepared by silica gel chromatography and reversed-phase chromatography.Its components were identified and quantified by Quadrupole-Orbitrap UHPLC-MS/MS.The PBMC were prepared from human blood and the splenic lymphocyte,B cells,and T cells were prepared from mouse spleen.The inhibitory effect of Sphingolipids on cell viability was evaluated by CCK-8 assay.The PBMC apoptosis and the ratio of CD4⁺T cells and CD8⁺T cells was quantified by flow cytometry analysis.The expression of IL-2,IL-10,and TNF-αin PBMC were detected by the ELISA kits.ResultsFraction of sphingolipids prepared from the mycelia of Cordyceps sinensis,and sphingolipids accounted for 84.83%of the Fr-SPLs.15 SPLs were identified from the Fr-SPLs.Fr-SPLs were applied to human peripheral blood mononuclear cell（PBMC）,and the inhibitory activity of the cells was detected by the CCK-8 method.The results showed that the inhibitory activity of Fr-SPLs on PBMC was dose-dependent,with an IC₅₀ value of 9.82μg/m L.The results of flow cytometry analysis of Fr-SPLs on PBMC cell apoptosis showed that Fr-SPLs promoted PBMC cell apoptosis in a dose-dependent manner,and the early apoptosis rate was particularly obvious.Moreover,the inhibitory activity of Fr-SPLs on mouse splenocytes and mouse B lymphocytes was dose-dependent,with an IC₅₀ values of 8.79μg/m L for mouse splenocytes and an IC₅₀ values of 7.34μg/m L for mouse B lymphocytes.Fr-SPLs reduced the ratio of CD4⁺T cells to CD8⁺T cells in the spleen of mice.Furthermore,we also checked the activity of Fr-SPLs by ELISA kits found that the Fr-SPLs could reduce the expression of IL-2,IL-10and TNF-αin PBMC cells after PHA or LPS inducing.ConclusionThe Fr-SPLs shows an immunosuppressive activity and this study will be useful for preparing immunosuppressive components from CS and its mycelia for hyperimmune disease.