芪苈强心，一种复方草药配方，通过抑制 ROS/AMPK/mTOR 通路，减轻体外缺氧复氧诱导的细胞凋亡和自噬死亡。
Qili Qiangxin, a compound herbal medicine formula, alleviates hypoxia-reoxygenation-induced apoptotic and autophagic cell death via suppression of ROS/AMPK/mTOR pathway in vitro.
机构信息
Department of Cardiology, the First Affiliated Hospital of Jinan University, Jinan University, Guangzhou 510632, Guangdong Province, China.
College of Pharmacy, Jinan University, Guangzhou 510632, Guangdong Province, China.
出版信息
J Integr Med. 2022 Jul;20(4):365-375. doi: 10.1016/j.joim.2022.04.005. Epub 2022 Apr 27.
OBJECTIVE
Qili Qiangxin (QLQX), a compound herbal medicine formula, is used effectively to treat congestive heart failure in China. However, the molecular mechanisms of the cardioprotective effect are still unclear. This study explores the cardioprotective effect and mechanism of QLQX using the hypoxia-reoxygenation (H/R)-induced myocardial injury model.
METHODS
The main chemical constituents of QLQX were analyzed using high-performance liquid chromatography-evaporative light-scattering detection. The model of H/R-induced myocardial injury in H9c2 cells was developed to simulate myocardial ischemia-reperfusion injury. Apoptosis, autophagy, and generation of reactive oxygen species (ROS) were measured to assess the protective effect of QLQX. Proteins related to autophagy, apoptosis and signalling pathways were detected using Western blotting.
RESULTS
Apoptosis, autophagy and the excessive production of ROS induced by H/R were significantly reduced after treating the H9c2 cells with QLQX. QLQX treatment at concentrations of 50 and 250 μg/mL caused significant reduction in the levels of LC3II and p62 degradation (P < 0.05), and also suppressed the AMPK/mTOR signalling pathway. Furthermore, the AMPK inhibitor Compound C (at 0.5 μmol/L), and QLQX (250 μg/mL) significantly inhibited H/R-induced autophagy and apoptosis (P < 0.01), while AICAR (an AMPK activator, at 0.5 mmol/L) increased cardiomyocyte apoptosis and autophagy and abolished the anti-apoptotic effect of QLQX. Similar phenomena were also observed on the expressions of apoptotic and autophagic proteins, demonstrating that QLQX reduced the apoptosis and autophagy in the H/R-induced injury model via inhibiting the AMPK/mTOR pathway. Moreover, ROS scavenger, N-Acetyl-L-cysteine (NAC, at 2.5 mmol/L), significantly reduced H/R-triggered cell apoptosis and autophagy (P < 0.01). Meanwhile, NAC treatment down-regulated the ratio of phosphorylation of AMPK/AMPK (P < 0.01), which showed a similar effect to QLQX.
CONCLUSION
QLQX plays a cardioprotective role by alleviating apoptotic and autophagic cell death through inhibition of the ROS/AMPK/mTOR signalling pathway.
目的
芪苈强心（QLQX）是一种复方中药方剂，在中国被有效用于治疗充血性心力衰竭。然而，其心脏保护作用的分子机制仍不清楚。本研究采用缺氧复氧（H/R）诱导心肌损伤模型探讨 QLQX 的心脏保护作用及机制。
方法
采用高效液相色谱-蒸发光散射检测法分析 QLQX 的主要化学成分。建立 H9c2 细胞 H/R 诱导心肌损伤模型模拟心肌缺血再灌注损伤。通过检测细胞凋亡、自噬和活性氧（ROS）的产生来评估 QLQX 的保护作用。采用 Western blot 检测自噬、凋亡和信号通路相关蛋白。
结果
H/R 诱导的细胞凋亡、自噬和 ROS 过度产生，经 QLQX 处理后明显减少。浓度为 50 和 250 μg/mL 的 QLQX 可引起 LC3II 和 p62 降解的显著减少（P<0.05），并抑制 AMPK/mTOR 信号通路。此外，AMPK 抑制剂 Compound C（0.5 μmol/L）和 QLQX（250 μg/mL）均显著抑制 H/R 诱导的自噬和凋亡（P<0.01），而 AMPK 激活剂 AICAR（0.5 mmol/L）则增加心肌细胞凋亡和自噬，并消除 QLQX 的抗凋亡作用。凋亡和自噬蛋白的表达也出现类似现象，表明 QLQX 通过抑制 AMPK/mTOR 通路减少 H/R 诱导损伤模型中的细胞凋亡和自噬。此外，ROS 清除剂 N-乙酰-L-半胱氨酸（NAC，2.5 mmol/L）可显著减少 H/R 引起的细胞凋亡和自噬（P<0.01）。同时，NAC 处理下调 AMPK/AMPK 磷酸化的比值（P<0.01），这与 QLQX 作用相似。
结论
QLQX 通过抑制 ROS/AMPK/mTOR 信号通路减轻细胞凋亡和自噬，发挥心脏保护作用。
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