Dioscorea Saponin Regulates The Expression Of Th17/Treg In Aplastic Anemia Through The Hippo Pathway

Objective:Establish a cell model of aplastic anemia BMMSC+lymphocytes,observe the regulation of Treg/Th17cell number/proportion by Hippo pathway,provide corresponding animal and cell models for clinical treatment of aplastic anemia(AA),and explore the mechanism of aplastic anemia.Methods:(1)A cell model of aplastic BMMSC+lymphocytes was established to study the effect of Dioscorea saponin on Treg/Th17 cell number ratio and function after intervention.,Flow cytometry(FCM)was used to detect the percentage of Th17 and Treg cells in CD4~+T cells,Q-PCR was used to detect the content of STAT3mRNA and FOXP3 mRNA,and ELISA was used to detect IL-2,IL-6,IL-10 and IL-17 and other related indicators(2)The effects of Dioscorea saponin on Hippo pathway-related proteins in vitro,the expression of YAP,MST1,and LATS2 protein levels were detected by Westernblot,the expression of YAP mRNA,LATS2 mRNA,and MST1 mRNA were detected by QPCR method.Reaults:1.Effects of Dioscorea sapaion on the number/ratio and function of aplastic BMMSC+lymphocytes on T cells:Compared with the control group,the levels of IL-2,IL-6,IL-17 in the serum of model group BMMSC+lymphocytes co-cultured significantly increased(P<0.01),the levels of IL-10 and TGF-βdecreased..After the intervention of cyclosporine and dioscin,the content of IL-2,IL-6 and IL-17 was significantly reduced compared with the model group(P<0.01),and the content of IL-10 and TGF-βwas significantly compared with the model group were increased(P<0.01).Compared with the model group,the ratio of Th17 cells to CD4~+T cells in the dioscin group and cyclosporine group was significantly reduced(P<0.01),the ratio of Treg to CD4~+T cells was significantly increased,and the ratio of Th17/Treg cells was significantly reduced(P<0.01).Compared with the Dioscin group and the cyclosporine group,the ratio of Th17 cells to CD4~+T cells was significantly reduced(P<0.01),and the ratio of Treg cells to CD4~+T cells was significantly increased(P<0.01).2.Effect of Dioscorea saponin on Hippo pathway-related proteins in simulated bone marrow microenvironment(BMMSC+lymphocytes)in vitro:(1)Compared with the control group,the expression level of YAP mRNA in the model group decreased significantly(P<0.01);after the intervention of cyclosporine and dioscin,the expression levels of YAP and LATS2 mRNA decreased to varying degrees compared with the model group(P<0.01).Compared with the control group,the expression level of MST1 mRNA in the model group was significantly increased(P<0.01).After the intervention of cyclosporine and dioscin,the expression level of MST1 mRNA increased to varying degrees compared with the model group(P<0.01)(2)Compared with the control group,the protein expression levels of LATS2 and YAP in the model group increased(P<0.05),and the protein levels of MST1 in the model group decreased(P<0.05).After the model group was given cyclosporine and diosgenin,the expression levels of YAP and LATS2protein decreased(P<0.05),and the expression level of MST1 protein increased(P<0.05)Conclusion:Dioscorea saponin can improve the imbalance of Th17/Treg caused by aplastic BMMSC+lymphocytes in vitro,increased the number of Treg,and reduced the number of Th17,regulated the expression of MST1,YAP and LATS2,the key nodes of Hippo pathway,up-regulated the expression of MST1 protein and MST1mRNA and the expression of IL-10 and TGF-β,down-regulated the expression of LATS2mRNA,YAPmRNA and protein and the expression of IL-6,IL-17,increased the expression of MST1 and FOXP3 mRNA and reduced the expression of YAP,LATS2,STAT3mRNA,increased the number of Treg,reduced the amount of Th17,induced the differentiation of CD4~+Tcells into Treg cells,reduced the differentiation into Th17,and finally achieved the effect of restoring Th17/Treg balance.