风湿宁胶囊对活性氧介导的T细胞活化和滑膜凋亡的影响

Effect of Fengshining Capsule on Reactive Oxygen Species-mediated T Cell Activation and Apoptosis of Synovium

目的研究细胞内活性氧(reactive oxygen species,ROS)水平对胶原诱导性关节炎(collagen induced arthritis,CIA)大鼠T细胞活化和滑膜细胞凋亡的影响,探讨风湿宁胶囊(FSN)在类风湿关节炎(rheumatoid arthritis,RA)治疗中的作用机制。方法将60只大鼠随机分为正常对照组,CIA模型组,雷公藤多苷片组,FSN低、中、高剂量组[剂量分别为0.33、0.66、1.32 g/(kg·d)],每组10只。采用流式细胞术检测T淋巴细胞亚群水平,ELISA法检测干扰素-γ(interferon-γ,IFN-γ)、白细胞介素-4(interleukin-4,IL-4)含量,紫外分光光度法检测羟自由基水平,Western blot法检测滑膜天冬氨酸特异的半胱氨酸酶(cysteinyl aspartate specific proteinase,Caspase)-3、9蛋白表达。结果与模型组比较,FSN各给药组均可升高ROS水平(P<0.01),FSN中剂量组能够显著降低CD4+/CD8+水平(P<0.01),FSN各组的IFN-γ含量明显降低(P<0.01),FSN高剂量组的IL-4含量明显升高(P<0.01)。同时,FSN各给药组的Caspase-9、Caspase-3表达亦显著上升,与模型组比较差异有统计学意义(P<0.05);且FSN低、中剂量组Caspase-9蛋白平均灰度值显著高于雷公藤多苷组(P<0.05,P<0.01)。结论 FSN调节CIA大鼠免疫功能亢进及抑制滑膜细胞增殖的机制可能与上调体内ROS水平有关。

Objective To study the effect of intracellular reactive oxygen species （ROS） levels on T cell activation and apoptosis of synovial cells in collagen induced arthritis （CIA） rats, and to explore the mechanism of Fengshining Capsule （FSN） in the treatment of rheumatoid arthritis （RA）. Methods Sixty rats were randomly divided into the normal control group, the CIA model group, the Tripterygium Poly-glycoside Tablet （TPT） group, the low dose FSN group （at the daily dose of 0.33 g/kg）, the middle dose FSN group （at the daily dose of 0.66 g/kg）, and the high dose FSN group （at the daily dose of 1.32 g/kg）, 10 in each group. T lymphocyte subsets were detected by flow cytometry. The content of interfer- on-γ, （IFN-γ） and interleukin-4 （IL-4） in plasma of rats were detected by ELISA. Its expression of hydrox- yl radicals was detected by ultraviolet spectrophotometry. Caspase-3 and Caspase-9 protein expres- sions were measured by Western blot. Results Compared with the CIA model group, the levels of ROS were elevated in each dose FSN group （P 〈0.01 ）. The level of CD4＋/CD8 ＋was significantly reduced in the middle dose FSN group （P 〈0.01 ）. The content of IFN-γ, was obviously lowered in each dose FSN group （P 〈0.01）, while that of IL-4 was obviously elevated in the high dose FSN group （P 〈0.01 ）. Mean- while, the expression of Caspase-9 and Caspase-3 significantly increased in each dose FSN group （P 〈 0.05）. Besides, the average gray scale of Caspase-9 was significantly higher in the low and middle FSNgroups than in the TPT group （P 〈0.05,P 〈0.01 ）. Conclusion The mechanism of FSN for regulating the immune hyperfunction and inhibiting the proliferation of synovial cells in CIA rats might be associated with up-regulating in vivo ROS levels.