温经通络散调控Treg/Th17细胞平衡治疗类风湿关节炎效应机制研究

Study on Mechanism of Wenjing Tongluo Powder Regulating Treg/Th17 Cell Balance for Rheumatoid Arthritis

目的:观察温经通络散调控Treg/Th17细胞平衡治疗类风湿性关节炎的作用机制,为临床应用温经通络散治疗类风湿性关节炎提供科学依据。方法:构建牛Ⅱ型胶原诱导的DBA/1小鼠胶原关节炎模型(CIA),将造模成功小鼠分为CIA模型组(Model组)、甲氨蝶呤阳性组(MTX组)和温经通络散组(WJTLS组)。临床评分评价CIA小鼠关节炎严重程度;苏木精-伊红(HE)染色检测CIA小鼠关节炎病理损伤程度;酶联免疫吸附法检测CIA小鼠抗体水平和淋巴细胞培养上清炎症因子白细胞介素(IL)-10和IL-17水平;流式细胞术检测总CD4^(+)T细胞、Treg细胞(CD4^(+)Foxp3^(+)T细胞)、Th17细胞(CD4^(+)IL-17^(+)T细胞)、Th1(CD4^(+)IFN-γ^(+)T细胞)表达水平。结果:与Model组比较,MTX组和WJTLS组在给药后第10、12、14、16、18天的关节指数评分均降低(P<0.05)。与Model组比较,MTX组和WJTLS组小鼠滑膜组织增生、软骨和骨侵蚀显著减少,炎性细胞浸润程度显著减轻,关节面损伤程度显著缓解。病理评分结果显示,与Model组比较,MTX组和WJTLS组小鼠关节组织病理损伤评分均降低(P<0.05)。与Model组比较,WJTLS组和MTX组小鼠血清IgG3、IgG2a、IgG抗体水平降低,但差异无统计学意义(P>0.05)。与Model组比较,WJTLS组小鼠脾脏细胞培养上清中IL-10水平升高(P<0.05),IL-17水平降低(P<0.05)。流式细胞术检测结果显示,WJTLS干预能减少CIA模型小鼠脾脏CD4^(+)T细胞绝对细胞数(P<0.05),上调脾淋巴细胞CD4^(+)FoxP3^(+)T细胞表达(P=0.061),下调CD4^(+)IL-17^(+)T细胞比例和细胞总数(P<0.05),从而提高Treg/Th17比值(P<0.05),而WJTLS对CIA小鼠淋巴结淋巴细胞Treg和Th17表达无明显影响(P>0.05)。结论:温经通络散能够改善CIA模型小鼠的炎症评分和关节组织的病理损伤,减少滑膜组织增生和炎症细胞浸润,其作用机制与上调Treg细胞,抑制Th17细胞的表达,调控Treg/Th17细胞平衡有关。

Objective:To observe the effect mechanism of Wenjing Tongluo Powder regulating Treg/Th17 cell balance for rheumatoid arthritis,and provide scientific basis for the clinical application of Wenjing Tongluo Powder for rheumatoid arthritis.Methods:Constructed a bovine type II collagen induced DBA/1 mice of collagen-induced arthritis(CIA),and divided the successfully modeled mice into the CIA model group(Model group),the methotrexate positive group(MTX group),and the Wenjing Tongluo Powder group(WJTLS group).Clinical scoring evaluation was conducted on the severity of arthritis in mice;hematoxylin eosin(HE)staining was used to detect the pathological damage degree of arthritis in CIA mice;enzyme linked immunosorbent assay was used to detect antibody levels in CIA mice and levels of inflammatory cytokines IL-10 and IL-17 in lymphocyte culture supernatant;flow cytometry was used to detect the expression levels of total CD4^(+)T cells,Treg cells(CD4^(+)Foxp3^(+)T cells),Th17 cells(CD4^(+)IL-17^(+)T cells),and Th1 cells(CD4^(+)+IFN-γT cells).Results:Compared with those in the Model group,the joint index scores in the MTX group and WJTLS group were decreased on days 10,12,14,16,and 18 after administration(P<0.05).Compared with those in the Model group,the proliferation of synovial tissue,cartilage and bone erosion were significantly reduced,the degree of inflammatory cell infiltration was significantly relieved,and the degree of joint surface damage was significantly alleviated in mice in the MTX group and the WJTLS group.The pathological scoring results showed that when compared with those in the Model group,the joint tissue pathological injury scores in mice of the MTX group and the WJTLS group were reduced(P<0.05).Compared with those in the Model group,the serum IgG3,IgG2a,and IgG antibody levels of mice in the WJTLS and MTX groups were decreased,but the difference showed no significance(P>0.05).Compared with those in the Model group,the level of IL-10 in the supernatant of mice spleen cell culture in the WJTLS group was increased(P<0.05),and the level of IL-17 was decreased(P<0.05).The results of flow cytometry analysis showed that WJTLS intervention could reduce the absolute number of CD4^(+)T cells in the spleen of CIA model mice(P<0.05),upregulate the expression of CD4^(+)FoxP3^(+)T cells in spleen lymphocytes(P=0.061),downregulate the proportion and total number of CD4^(+)IL-17^(+)T cells(P<0.05),and thereby increasing the Treg/Th17 ratio(P<0.05);the expression of Treg and Th17 in lymph node lymphocytes of CIA mice in the WJTLS group showed no obvious effect(P>0.05).Conclusion:Wenjing Tongluo Powder can improve the inflammation score and pathological damage of joint tissue in rheumatoid arthritis models,reduce synovial tissue proliferation and infiltration of inflammatory cells.Its mechanism is related to upregulating Treg cells,inhibiting Th17 cell expression,and regulating the balance of Treg/Th17 cells.