基于STAT3信号通路调节Th17细胞增殖探讨温阳化浊通络方治疗系统性硬化病机制

Therapeutic mechanism of systemic sclerosis by Wenyang Huazhuo Tongluo Formula based on the regulation effect to human Th17 cell proliferation via STAT3 signaling pathway

目的:研究温阳化浊通络方对Th17细胞增殖的影响,探讨其对STAT3信号通路的调控作用,明确该方治疗系统性硬化病(SSc)的免疫调控机制。方法:收集SSc患者外周血,采用密度梯度离心法分离外周血单核细胞,人Th17分选试剂盒分选Th17细胞,同时制备温阳化浊通络方含药血清。荧光染料CFSE标记Th17细胞后,流式细胞术检测含药血清对Th17细胞增殖的影响。采用RT-qPCR和细胞免疫荧光技术检测含药血清对Th17细胞中JAK2、STAT3/p-STAT3 mRNA和蛋白表达的影响。结果:与空白组比较,各剂量含药血清组可剂量依赖性地抑制Th17细胞的增殖(P<0.01);阳性对照组对Th17细胞的增殖抑制作用介于含药血清低剂量和高剂量组之间(P<0.01,P<0.05);各含药血清组可剂量依赖性地降低JAK2和STAT3 mRNA和蛋白的表达,并可剂量依赖性地降低p-STAT3的表达(P<0.05,P<0.01);阳性对照组抑制JAK2和STAT3/p-STAT3表达的作用强于各含药血清组(P<0.01)。结论:温阳化浊通络方可抑制Th17细胞的增殖,其机制可能与抑制STAT3信号通路有关。

Objective: To investigate the effect of Wenyang Huazhuo Tongluo Formula(WYHZTL) on the proliferation of Th17 cells and explore its regulatory effect on STAT3 pathway, so that to clarify the immune regulation mechanism of WYHZTL in the treatment of systemic sclerosis(SSc). Methods: The peripheral blood was collected from the patients with SSc, the peripheral blood mononuclear cells were separated by density gradient centrifugation. The Th17 cells were sorted by stemcell Th17 kit. Meanwhile, the sera-containing WYHZTL was prepared. After Th17 cells were labeled with fluorescent dye CFSE, the effect of the sera-containing WYHZTL on the proliferation of Th17 cells was detected by flow cytometry. RT-qPCR and immunofluorescence techniques were used to detect the effect of the sera-containing WYHZTL on the expression of JAK2, STAT3/p-STAT3 mRNA and protein. Results: Compared with the blank sera group, the seracontaining WYHZTL could inhibit the proliferation of Th17 cells in a dose-dependent manner(P<0.01);the inhibitory effect the proliferation of Th17 cells in the positive control group was between low dose and high dose sera-containing WYHZTL(P<0.01, P<0.05). The sera-containing WYHZTL could decrease the expression of JAK2, STAT3 mRNA and protein in a dose-dependent manner, and inhibit the expression of p-STAT3(P<0.05, P<0.01);the inhibitory effect of the expression of JAK2 and STAT3/p-STAT3 in the positive control group was stronger than that of the sera-containing WYHZTL(P<0.01). Conclusion: WYHZTL can inhibit the proliferation of Th17 cells, and its mechanism may be related to the inhibition of STAT3 signaling pathway.