Vitamin C Has A Moderating Effect On Treg/Th17 Balance

Objective Different concentrations of vitamin C or prednisone were used to treat EL4 cells,and the effects of vitamin C or prednisone on the proliferation of EL4 cells were observed,and the best concentration for EL4 cells proliferation was selected for subsequent experiments.Using cytokines TGF-β1,IL-6,IL-23 induced EL4 cells to have a Treg/Th17 differentiation to the Th17 offset,and the intervention of vitamin C or prednisone was performed before and after differentiation,to observe if the regulatory effects of vitamin C on the Treg/Th17 imbalance are similar to prednisone.Methods Different concentration(10mM,8mM,6mM,4mM,2mM,1mM,0.5mM and0.2mM)of vitamin C was treated with EL4 cells,respectively.Then cells survival rate was detected by 24 h,48h,72 h with CCK-8.Different concentration(100uM,10 uM,1 uM,0.1 uM,0.01 uM,0.001 uM,0.0001 uM,0.0001 uM and0.00001 uM)of prednisone was treated with EL4 cells,respectively.Then cells survival rate was detected by 24 h,48h,72 h with CCK-8.Differentiation before intervention:Vitamin C of 0.2mM and prednisone of 1 uM were respectively used to culture EL4 cells for 24 h.The cytokines(TGF-β1 5ng/ml,IL-620ng/ml,IL-23 10ng/ml)were added to the culture,and then cultured for 48 h.RT-PCR was used to detect the expression of RORγt mRNA and Foxp3 mRNA,and ELISA was used to detect the secretion of IL-17 A and IL-10.Differentiation after intervention:The differentiation of EL4 cells was stimulated by cytokine TGF-β1 5ng/ml,IL-6 20ng/ml and IL-23 10ng/ml for 48 h,to have the Treg/Th17 imbalance shifted to Th17.Then individually cultured with addition of 0.2 mM vitamin C or 1 uM prednisone for 24 h.RT-PCR was used to detect the expression of RORγt mRNA and Foxp3 mRNA,and ELISA was used to detect the secretion of IL-17 A and IL-10.Results When the concentration of vitamin C was 0.2 mM,the EL4 cells proliferation was unsignificantly affected,and the cell proliferation does not change with the increase of the action time.It was found that when the concentration of vitamin C was great than 0.5 mM,the cell proliferation of EL4 cells was significantly inhibit,and the inhibitory effect increased with the increasing of the time of action.EL4 cells viability was lower in prednisone concentration at 100 μM,compared with prednisone in lower concentration.In different time,prednisone did not have any effect of the EL4 cells proliferation.Before the differentiation,0.2mm of vitamin C could increase the expression of RORγt mRNA and Foxp3 mRNA in EL4 cells,and decreased the secretion of IL-10,which was similar to the prednisone of 1 uM.After the differentiation,the 0.2 mM of vitamin C would increased the expression of RORγt mRNA and decreased the secretion of IL-10,which was similar to the pre-differentiation of vitamin C.Conclusion Vitamin C inhibits the proliferation of EL4 cells,and the inhibitory effect is related to the concentration and time.While prednisone have no significantinhibitory effect on EL4 cells proliferation.When vitamin C exists,it can interfere with the result of Treg/Th17 differentiation,which is similar to prednisone.When the Treg/Th17 is out of balance to the Th17,vitamin C has a regulation effect to it.