Research On The Molecular Mechanism Of Dihydroartemisinin Regulating The Splenic Immunity Based On Proteomics And Phosphorylation

Artemisinin is a novel sesquiterpene lactone extracted from the chrysanthemum plant Artemisia annua,which is a fast effective,low toxicity anti-malarial drug.Dihydroartemisinin(DHA)is a derivative of artemisinin,which has significant immunomodulatory effects in addition to its antimalarial effects.However,the potential mechanism of action remains unclear.In this study,DHA was used as an experimental drug to preliminarily study on the molecular mechanism of its immunomodulatory effect in mice spleen.In this study,the immune cell subsets of spleen of BALB/c mice treated with DHA were analyzed by flow cytometry.In view of the previous experiments,We found that phosphorylated proteins can lead to changes in protein activity and function,and then regulating cell proliferation.Then,the combined proteomic and phosphorylation modification conjoint analysis as an entry point for this project and validated the results of omics production,including Western blotting,validation of significant differential protein expression,immunohistochemistry,and Brd U staining for the detection of T cells.The results showed that the weight and volume of spleen were positively correlated with the prolongation of gavage time after DHA.Compared with the control group,DHA significantly induced the proportion of spleenic total CD8~+T cells to increase first and then decrease.DHA induced the percentage of total CD4~+T cells,neutrophils and cytotoxic T cells in spleen,and the increased proportion was positively correlated with prolonged gavage time after DHA.Brd U further confirmed that DHA significantly induced proliferation of CD8~+T,CD4~+T and CD25~+T cells in spleen.To further elucidate the molecular mechanism of DHA induced immune regulation,proteomics analysis was performed,which results showed that the cell cycle signaling pathway in DHA mice was significantly activated including Cyclin-dependent kinase 1(CDK1)(fold-change=2),CDK2(fold-change=2.676),Minichromosome Maintenance proteins 2(MCM2)(fold-change=2.916),Ki67(fold-change=532)and Proliferating cell nuclear antigen(PCNA)(fold-change=3.123).GO results showed that DHA significantly upregulated ribosome related signaling pathways,and KEGG results showed that DHA activated cell cycle signaling pathway.Analysis of DHA and control mice by phosphorylated proteomics showed that DHA treatment significantly activated key kinase activities in mitogen-activated protein kinase(MAPK)pathways.Finally,the results of phosphorylated proteomics were verified by the validation of phosphorylated antibodies.In order to further elucidate the molecular mechanism of MAPK signaling pathway in DHA mediated cell cycle activation,MAPK specific inhibitor SB203580 was used to inhibit MAPK signaling pathway.The results showed that the inhibitor could inhibit DHA-induced activation of cell cycle signaling pathway.In conclusion,this study suggests that DHA selectively induces the expansion of splenic T cell subsets through phosphorylation of key proteins(CDKs and MAPK)in the MAPK-AP-1 signaling pathway,thus regulating the immune response of splenic cells.The results of this research can provide a theoretical basis for further study of DHA mediated immune regulation mechanism.