当归对大鼠椎间盘髓核细胞自噬及氧化应激损伤的作用及机制研究

The autophagy of rat intervertebral disc nucleus pulposus cells and oxidative stress-induced injury in rat

目的 :探讨当归降低椎间盘髓核细胞自噬及氧化应激损伤的作用及其机制。方法 :从大鼠椎间盘中分离并培养SD大鼠原代髓核细胞,用不同浓度(0.1mg/ml、1mg/ml、5mg/ml)的当归注射液干预第3代细胞,噻唑蓝(methyl thiazolete trazolium,MTT)法检测适当的当归浓度。将原代髓核细胞随机分成3组:A组(对照组)、B组(H2O2组)和C组(当归+H2O2)组。A组用生理盐水处理24h,不进行H2O2刺激,B组用400μmol/L H2O2分别刺激0.5h、1.5h和3h,C组用适当浓度当归注射液预处理24h后,400μmol/L H2O2分别刺激0.5h、1.5h和3h。流式细胞术检测各组细胞不同时间的凋亡率,western blot检测凋亡相关蛋白细胞色素C、Bcl-2和Bax及自噬相关蛋白LC3-Ⅱ/Ⅰ、p62、p-AKT、p-m TOR和p-p70S6K的表达。结果:与其他浓度当归处理组比较,1mg/ml当归注射液可显著增加细胞存活(P<0.05)。与A组相比,B组细胞凋亡率随着时间的延长逐渐增加,细胞色素C的表达增加(P<0.05),Bcl-2/Bax的比例减小(P<0.05),LC3-Ⅱ/Ⅰ的比例增加(P<0.05),p62、p-AKT、p-m TOR和p-p70S6K的表达降低(P<0.05);与B组相比,C组中细胞凋亡率和细胞色素C、p62、p-AKT、p-m TOR和pp70S6K的表达均降低(P<0.05),Bcl-2/Bax和LC3-Ⅱ/Ⅰ的比例升高(P<0.05)。结论 :1mg/ml当归注射液能够抑制大鼠髓核细胞凋亡,这可能与线粒体凋亡通路被阻断相关;1mg/ml当归能促进其髓核细胞自噬,这可能与AKT/m TOR信号通路被抑制相关。

Objectives： To investigate the protective effect of angelica on intervertebral disc nucleus pulposus cells from oxidative stress-induced injury, and to explore its potential mechanism. Methods： Nucleus pulposus cells were isolated from rat lumbar discs and cultured into the third-passage to do the following experiments. The cells were incubated with different concentrations of angelica solution injection （0.1mg/ml, 1mg/ml, 5mg/ml）, and the optimum concentration of angelica was detected by MTT . After that, the cultured nucleus pulposus cells were randomly divided into group A, group B and group C. The cells in group B were stimulated with 400μmol/L H2O2 for 0.5h, 1.Sh and 3h. And the cells in group C were pretreated with proper concentration of angelica for 24h and then stimulated by 400μmol/L H2O2 for 0,Sh, 1.5h and 3h, respectively. The cells in control group were treated with saline for appropriate times. The apoptosis of nucleus pulposus cells was determined by flow cytometry. The expressions of cytochrome C, Bcl-2, Bax, LC3-Ⅱ/Ⅰ, p62, p- AKT, p-roTOR and p-p70S6K were analyzed by western blot. Results： Compared with other concentrations of angelica, 1mg/ml of angelica significantly increased the cell viability （P〈0.05）. Compared with the cells in group A, apoptosis, expression of cytochrome C and the ratio of LC3-Ⅱ/Ⅰ of the cells in group B were increased（P〈0.05）, while the ratio of Bcl-2/Bax and the expressions of p62, p-AKT, p-roTOR and p-p70S6K were decreased（P〈0.05）; compared with the ceils in group B, apoptosis and the expressions of cytochrome C, p62, p-AKT, p-roTOR and p-p7OS6K of the cells in group C were reduced（P〈O.05）, while the ratios of Bel- 2/Bax and LC3-Ⅱ/Ⅰ were raised in those of group C （P〈0.05）. Conclusions： 1mg/ml of angelica solution injection can decrease the apoptosis of nucleus pulposus cell, which may be related to suppression of the mitochondrial pathway. It also can enhance the autophagy of nucleus pulposus cell, which may be involved in the inhibition of AKT/mTOR signaling.