穿心莲内酯对体外大鼠髓核细胞生物行为的影响△

Effects of andrographolide on biological behavior of rat nucleus pulposus cells in vitro

摘要:

[目的] 探讨穿心莲内酯 (andrographolide, Andro) 对体外大鼠髓核细胞 (nucleus pulposus cells, NPCs) 生物行为的影响及其机制。[方法]利用叔丁基过氧化氢(tert-buttyl hydroerioxide, TBHP)处理 NPCs 构建凋亡模型，不同浓度的 Andro 分别处理正常 NPCs 和 TBHP 诱导的 NPCs，采用 CCK8 检测毒性以筛选 Andro 的最佳作用浓度和时间。将细胞分为空白对照 (blank control, BC) 组、TBHP (T) 组、TBHP+Andro (TA) 组、TBHP+Andro+PI3K inhibitor (LY294002)(TA-Pih) 组和 TBHP+PI3K inhibitor (T-Pih) 组，并给予相应体外处理。流式细胞术检测细胞凋亡，ELISA 检测炎性细胞因子水平，Western blot 检测凋亡标志物蛋白的表达水平。[结果]Andro 的最佳作用浓度和时间为 18 μmol/L 和 24 h。与 BC 组相比，T 组细胞凋亡率、TNF-α、IL-1β、IL-6 水平、Bax 蛋白水平显著升高 (P＜0.05)，Bcl-2 和 p-Akt 蛋白水平显著降低 (P＜0.05)；与 T 组相比，TA 组细胞凋亡率、TNF-α、IL-1β、IL-6 水平、Bax 蛋白水平显著降低(P＜0.05)，Bcl-2 和 p-Akt 蛋白水平显著升高(P＜ 0.05)，T-Pih 组的趋势则相反；与 T-Pih 组相比，TA-Pih 组细胞凋亡率、TNF-α、IL-1β、IL-6 水平、Bax 蛋白水平显著降低 (P＜0.05)，Bcl-2 和 p-Akt 蛋白水平显著升高(P＜0.05)。[结论]Andro 能抑制细胞凋亡，降低炎性细胞因子的水平，其机制可能与 Andro 调控 PI3K/Akt 通路有关。

Abstract:

[Objective] To explore the effect of andrographolide (Andro) on the biological behavior of rat nucleus pulposus cells (NPCs) in vitro and its mechanism. [Methods] NPCs were treated with tert-buttyl hydroerioxide (TBHP) to construct apoptosis model. The normal NPCs and TBHP-induced NPCs were treated with different concentrations of Andro, CCK8 assay for toxicity was conducted to select the op- timal concentration and time of Andro action. The cells were divided into blank control (BC) group, TBHP (T) group, TBHP+Andro (TA) group, TBHP+Andro+PI3K inhibitor (LY294002) (TA-Pih) group and TBHP+PI3K inhibitor (T-Pih) group, and were given corresponding treatment in vitro. The cell apoptosis was detected by flow cytometry, the levels of inflammatory cytokines were detected by ELISA, and the protein expression levels of apoptotic markers were detected by Western blot. [Results] The optimum concentration and reaction time of An- dro were 18μmol/L and 24 h respectively. Compared with the BC group, the apoptosis rate, TNF-α, IL-1β, IL-6 levels and Bax protein lev- el in the T group were significantly increased (P＜0.05) , while the protein levels of Bcl-2 and p-Akt were significantly decreased (P＜0.05) . Compared with the T group, the apoptosis rate, TNF-α, IL-1β, IL-6 levels and Bax protein level in the TA group were significantly de- creased (P＜0.05) , while the protein levels of Bcl-2 and p-Akt were significantly increased (P＜0.05) , while which as a trend was opposite in the T-Pih group. Compared with the T-Pih group, the apoptosis rate, TNF-α, IL-1β, IL-6 levels and Bax protein level in the TA-Pih group were significantly decreased (P＜0.05) , while the protein levels of Bcl-2 and p-Akt were significantly increased (P＜0.05) . [Conclusion] Andro does inhibit cell apoptosis and reduce the level of inflammatory cytokines, which may be related to the regulation of PI3K/Akt pathway.