芍药苷-6-O-苯磺酸酯通过调节JAK/STAT3/RORγ-t/IL-17A信号通路抑制人外周血Th17细胞的分化

Paeoniflorin-6′-O-benzene sulfonate inhibitsTh17 cell differentiation in human peripheral blood byregulating JAK/STAT3/RORγ-t/IL-17A signaling pathway

目的研究芍药苷-6′-O-苯磺酸酯(CP-25)对体外培养的人外周血单个核细胞(peripheral blood mononuclear cells,PBMCs)中JAK/STAT3/RORγ-t/IL-17A信号通路的调控及对Th17细胞分化的影响。方法从人外周血中分离出PBMCs,将细胞分为空白对照组、TNF-α(10μg·L-1)与包含佛波酯、离子霉素、布雷菲尔德菌素A的细胞刺激混合物(2 mL·L-1)联合刺激组、CP-25组(10μmol·L-1)、艾拉莫德组(80μmol·L-1)、托法替布组(10μmol·L-1),药物作用24 h后,收集细胞进行以下相关检测:CCK-8法检测CP-25对人PBMCs增殖功能的影响;流式细胞术检测p-STAT3、STAT3、RORγ-t和IL-17A在人PBMCs中CD4+Th细胞的表达情况;Western blot检测人PBMCs中p-JAK1、JAK1、p-JAK2、JAK2、p-JAK3、JAK3、p-STAT3、STAT3、RORγ-t、IL-17A蛋白表达情况。结果与空白对照组比较,CP-25能明显抑制刺激诱导的人PBMCs异常增殖,下调人PBMCs的p-JAK1、p-JAK2、p-JAK3、p-STAT3、STAT3、RORγ-t、IL-17A蛋白表达,降低CD4+Th细胞中p-STAT3、STAT3、RORγ-t的表达和CD4+IL-17A+Th17细胞的百分比。结论CP-25抑制人PBMCs中JAK/STAT3/RORγ-t/IL-17A信号通路,进一步抑制Th17细胞的分化。

Aim To study the effect of paeoniflorin-6′-O-benzene sulfonate(CP-25)on the regulation of the JAK/STAT3/RORγ-t/IL-17A signaling pathway in human peripheral blood mononuclear cells(PBMCs)cultured in vitro and the differentiation of Th17 cells.Methods PBMCs were isolated from human peripheral blood and divided into blank control group,TNF-α(10μg·L-1)and cell stimulation cocktail containing PMA,ionomycin,brefeldin A(2μL·mL-1)stimulated group,CP-25(10μmol·L-1),iguratimod(80μmol·L-1)and tofacitinib(10μmol·L-1).After drug stimulation for 24 h,human PBMCs were collected for the following tests:CCK-8 was used to test the effect of CP-25 on the proliferation function of human PBMCs.The expression of p-STAT3,STAT3,IL-17A and RORγ-t in human PBMCs CD4+Th cell were detected by flow cytometry.Western blot was used to detect the expression of p-JAK1,JAK1,p-JAK2,JAK2,p-JAK3,JAK3,p-STAT3,STAT3,RORγ-t and IL-17A protein in human PBMCs.Results Compared with blank control group,CP-25 significantly inhibited the abnormal proliferation of stimulated PBMCs,down-regulated the expression of p-JAK1,p-JAK2,p-JAK3,p-STAT3,STAT3,RORγ-t,IL-17A protein,and reduced the expression of p-STAT3,STAT3,RORγ-t in CD4+Th cells and the percentage of CD4+IL-17A+Th17 cells.Conclusions CP-25 inhibits the JAK/STAT3/RORγ-t/IL-17A signaling pathway in human PBMCs,further inhibiting Th17 cell differentiation.