双乌宣痹颗粒对类风湿关节炎滑膜成纤维细胞凋亡及PI3K/Akt信号通路的影响

Effects of Shuangwuxuanbi granule on the apoptosis of rheumatoid arthritis fibroblast-like synoviocytes and PI3K/Akt signaling pathway

目的观察双乌宣痹颗粒对类风湿关节炎(RA)滑膜成纤维细胞(FLS)凋亡及磷脂酰肌醇3激酶(PI3K)/蛋白激酶B(Akt)信号通路的影响,探讨其作用机制。方法胶原酶消化法分离RA患者膝关节FLS,传代培养并鉴定后,取4～7代细胞用于实验。RA-FLS分别用高[17.28g/(kg.d)]、中[8.64g/(kg.d)]、低[4.32g/(kg.d)]剂量双乌宣痹颗粒含药血清培养24h,即高、中、低剂量双乌宣痹组;另设阳性药物对照[来氟米特,1.04mg/(kg.d)]组和阴性对照(生理盐水)组。采用流式细胞仪检测细胞凋亡情况,Western blotting检测PI3K、蛋白激酶B(Akt)、磷酸化蛋白激酶B(p-Akt)、B淋巴细胞瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)的表达水平。结果高、中、低剂量双乌宣痹含药血清及来氟米特含药血清均能明显促进FLS的凋亡,下调PI3K、p-Akt、Bcl-2蛋白的表达,上调Bax蛋白表达,与阴性对照组比较差异有统计学意义(P<0.05);与来氟米特组比较,高、中双乌宣痹组PI3K、p-Akt、Bcl-2蛋白的表达明显下调,Bax蛋白表达明显上调,差异有统计学意义(P<0.05)。结论双乌宣痹含药血清能促进RA-FLS凋亡,下调Bcl-2、PI3K、p-Akt蛋白的表达,这可能是其治疗RA的机制之一。

Objective To observe the effect of serum containing Shuangwuxuanbi granule drug on the apoptosis of fibroblast-like synoviocytes（FLS） from rheumatoid arthritis（RA） patients and PI3 K/Akt signal pathway, and to explore its mechanism of treating RA. Methods FLS were extracted from the knee synovial membrane of the RA patient and isolated. Subsequent to subculture and identification, the cells at passages 4 to 7 were chosen for the experiment. The cells were incubated in Dulbecco＇s modified eagle medium（DMEM） supplemented with different concentrations of Shuangwuxuanbi granule containing serum [high dose： 17.28 g/（kg.d）, middle dose： 8.64 g/（kg.d） and low dose：（4.32 g/（kg.d）], leflunomide containing serum [1.04 mg/（kg.d）] and saline containing serum. After 24 hours, the cell apoptosis was detected by flow cytometry. The levels of Bax, Bcl-2, PI3 k, Akt, and phosphorylation Akt protein were detected by Western blotting. Results Shuangwuxuanbi granule containing serum and leflunomide containing serum obviously promoted the apoptosis of RA-FLS, decreased the proteins expression of PI3 K, p-Akt, Bcl-2 and increased the protein expression of Bax when compared with the control group（saline group）, the difference was statistically significant（P〈0.05）. In comparison with the leflunomide group, the expression of PI3 K, p-Akt and Bcl-2 proteins decreased, and the protein expression of the Bax increased in the high and middle dose groups of Shuangwuxuanbi granule, the difference was statistically significant（P〈0.05）. Conclusion The Shuangwuxuanbi granule containing serum promoted the apoptosis of RA-FLS, and down-regulated the proteins expression of Bcl-2, PI3 K, p-Akt, which may be one of the mechanisms for its efficacy in treating RA.