利金方降低大鼠Th17细胞/CD4^(+)T细胞比值减轻香烟烟雾提取物引起的炎症反应

Liking Prescription reduces cigarette smoke extract-induced inflammatory response via inhibiting JAK2/STAT3/ROR-γt pathway to reduce Th17/CD4^(+)T cell ratio in rats

目的探讨利金方对香烟提取物诱导的CD4^(+)T细胞向Th17细胞分化及对炎症的影响及作用机制。方法分离和培养CD4^(+)T细胞,分为空白对照组、香烟烟雾提取物(CSE)组、(0.25、0.5、1.0)mg/mL利金方处理组、Janus激酶2(JAK2)抑制剂AG-490组、信号转导子和转录激活子3(STAT3)抑制剂TG-101348组。利金方处理组和抑制剂组都是先给予相应处理后再进行CSE刺激。流式细胞术检测各组CD4^(+)T细胞、Th17细胞比例,ELISA检测CD4^(+)T细胞培养基中肿瘤坏死因子α(TNF-α)、白细胞介素17A(IL-17A)水平;实时定量PCR检测CD4^(+)T细胞JAK2、STAT3、维甲酸相关孤核受体γt(ROR-γt)的mRNA水平,Western blot法检测CD4^(+)T细胞JAK2、磷酸化的JAK2(p-JAK2)、STAT3、磷酸化的STAT3(p-STAT3)、ROR-γt的蛋白水平。结果CSE处理增加Th17细胞/CD4^(+)T细胞比值,培养基中TNF-α、IL-17A含量;ROR-γt的mRNA和蛋白表达增加,JAK2、STAT3磷酸化增加。利金方处理后降低Th17细胞/CD4^(+)T细胞比值,减少TNF-α、IL-17A分泌,抑制ROR-γt的mRNA和蛋白表达以及JAK2、STAT3的磷酸化。结论利金方通过抑制JAK2-STAT3-ROR-γt通路降低Th17细胞/CD4^(+)T细胞比值减轻CSE诱导的炎症反应。

Objective To investigate the effects of Liking Prescription on cigarette smoke extract(CSE)-induced differentiation into Th17 cells from CD4^(+)T cells and inflammation,and to study the possible mechanism.Methods CD4^(+)T cells isolated and cultured were divided into control group,CSE group,(0.25,0.5,1.0)mg/mL Liking Prescription treatment group,JAK2 inhibitor AG-490 group,STAT3 inhibitor TG-1013480 group.The Liking Prescription treatment groups and the inhibitor groups were given corresponding treatments before CSE stimulation.The proportions of CD4^(+)T cells and Th17 cells in each group were detected by flow cytometry.TNF-α and IL-17A in CD4^(+)T cell culture medium were analyzed by ELISA.The expression of JAK2,STAT3 and retinoic acid-related orphan nuclear receptorγt(ROR-γt)mRNA in CD4^(+)T cells was measured by real-time quantitative PCR.The expression of JAK2,phosphorylated JAK2(p-JAK2),STAT3,phosphorylated STAT3(p-STAT3)and ROR-γt protein in CD4^(+)T cells were analyzed by Western blotting.Results CSE treatment increased the ratio of Th17 cells/CD4^(+)T cells and the content of TNF-αand IL-17A in the culture medium;the expression of ROR-γt mRNA and protein increased;the phosphorylation of JAK2 and STAT3 increased.After treatment with Liking Prescription,the ratio of Th17 cells/CD4^(+)T cells was reduced;the secretion of TNF-αand IL-17A dropped;the expression of ROR-γt mRNA and protein and the phosphorylation of JAK2 and STAT3 were inhibited.ConclusionLiking Prescription can reduce CSE-induced inflammatory response via inhibiting JAK2/STAT3/ROR-γt pathway to reduce the ratio of Th17 cells/CD4^(+)T cells.