桂枝芍药知母汤对类风湿关节炎Th17/Treg细胞失衡及JAK2/STAT3信号通路的影响

Influence of Guizhi Shaoyao Zhimu Decoction on Th17/Treg cell imbalance and JAK2/STAT3 signal pathway in rheumatoid arthritis

目的:探讨桂枝芍药知母汤对类风湿关节炎(RA)Th17/Treg细胞失衡及JAK2/STAT3信号通路的影响。方法:取6例RA患者外周血,用淋巴细胞分离液获取外周血单个核细胞(PBMC),进行CD_(4)^(+)T细胞磁珠分选纯化,应用桂枝芍药知母汤含药血清干预细胞,设中药低、中、高剂量组,应用CCK8法检测CD_(4)^(+)T细胞活力,流式细胞术检测CD_(4)^(+)T细胞中Th17、Treg细胞比例,RT-qPCR法检测各组细胞因子IL-17、IL-21、IL-22、TGF-β、IL-10 mRNA表达水平,Western blot检测p-JAK2、JAK2、p-STAT3、STAT3蛋白表达情况。结果:桂枝芍药知母汤含药血清低、中、高剂量对CD_(4)^(+)T细胞活力没有明显的影响。与空白组比较,桂枝芍药知母汤低、中、高剂量组均可显著抑制CD_(4)^(+)T细胞Th17比例、上调Treg比例、下调Th17/Treg比值(P<0.05,P<0.01),CD_(4)^(+)T细胞IL-17、IL-21、IL-22 mRNA表达水平显著降低(P<0.05,P<0.01),TGF-β、IL-10 mRNA表达水平显著升高(P<0.05,P<0.01),p-JAK2/JAK2和p-STAT3/STAT3表达水平显著下降(P<0.05,P<0.01)。结论:桂枝芍药知母汤能调控RA Th17/Treg平衡,纠正RA免疫紊乱状态,且其作用机制与JAK2/STAT3信号通路密切相关。

Objective:To investigate the influence of Guizhi Shaoyao Zhimu Decoction(GSZD)on Th17/Treg cell imbalance and JAK2/STAT3 signal pathway in rheumatoid arthritis(RA).Methods:The peripheral blood of 6 RA patients was collected,and the peripheral blood mononuclear cell(PBMC)was obtained with lymphocyte separation solution,and the CD_(4)^(+)T cells were sorted and purified by magnetic beads.The serum containing GSZD was used to intervene the cells,and low-,medium-and high-dose GSZD groups were set.The viability of CD_(4)^(+)T cells was detected by CCK8,and the proportion of Th17 and Treg cells in CD_(4)^(+)T cells was detected by flow cytometry.The mRNA expression of IL-17,IL-21,IL-22,TGF-βand IL-10 were detected by RT-qPCR.The expression of p-JAK2/JAK2 and p-STAT3/STAT3 were detected by Western blot.Results:GSZD contained low-,medium-and high-dose of serum on CD_(4)^(+)T cell viability had no obvious effect.Compared with the blank group,the low-,medium-and high-dose GSZD group could significantly inhibit the ratio of CD_(4)^(+)T cells Th17,up-regulate the ratio of Treg and down-regulated Th17/Treg ratio(P<0.05,P<0.01),the mRNA expression levels of IL-17,IL-21 and IL-22 in CD_(4)^(+)T cells were decreased,and the mRNA expression levels of TGF-βand IL-10 were increased(P<0.05,P<0.01).GSZD could significantly down-regulate the expression levels of CD_(4)^(+)T cells p-JAK2/JAK2 and p-STAT3/STAT3(P<0.05,P<0.01).Conclusion:GSZD regulate the balance of RA Th17/Treg and correct RA immune disorders,and its mechanism of action is closely related to the JAK2/STAT3 signal pathway.