Chen Jun, Li Li, Bai Xueyang, Xiao Lili, Shangguan Jiahong, Zhang Wenjing, Zhang Xiangqin, Wang Shen, Liu Gangqiong
Vasculocardiology Department, the First Affiliated Hospital of Zhengzhou University, Zhengzhou, China.
Front Pharmacol. 2021 Mar 8;12:620812. doi: 10.3389/fphar.2021.620812. eCollection 2021.
Endoplasmic reticulum (ER) stress is often closely linked to autophagy, hypoxia signaling, mitochondrial biogenesis and reactive oxygen species (ROS) responses. Understanding the interaction between ER stress, mitochondrial function and autophagy is of great importance to provide new mechanisms for the pathology, prevention and treatment of cardiovascular diseases. Our previous study has reported that Panax notoginseng saponins (PNS) protection against thapsigargin (TG)-induced ER stress response and associated cell apoptosis in cardiac myocytes is calcium dependent and mediated by ER Ca release through RyR. However, whether its protection upon ER stress and associated apoptosis is related to mitochondrial function and autophagy remains largely unknown. Here, we investigated the roles of PNS played in TG-induced mitochondrial function, ROS accumulation and autophagy. We also assessed its effects on Ca homeostasis, ER stress response and associated cell death in the presence of autophagy inhibition. PNS-pretreated primary cultured neonatal rat cardiomyocytes were stimulated with TG to induce ER stress response. Mitochondrial potential (Δm) was measured by JC-1. The general and mitochondrial ROS were measured by DCFH-DA and MitoSOX Red, respectively. Autophagy was evaluated by immunofluorescence of LC3, and immunoblots of LC3, p62, ATG7 and PINK1. In addition, mRFP-GFP-LC3 labeling was used to assess the autophagic influx. SiATG7 transfected H9c2 cells were generated to inhibit autophagy. Cytosolic and ER Ca dynamics were investigated by calcium imaging. RyR oxidation was tested by oxyblot. Cell viability was examined by TUNEL assay. ER stress response and cell apoptosis were detected by immunoblots of BiP, CHOP, Cleaved Caspase-3 and Caspase-12. The results demonstrated that firstly, PNS protects against TG-induced mitochondrial injury and ROS accumulation. Secondly, PNS enhances autophagy in TG-induced cardiac myocytes. Thirdly, inhibition of autophagy diminishes PNS prevention of TG-induced mitochondrial injury, ROS accumulation and disruption of Ca homeostasis. Last but not least, inhibition of autophagy abolishes PNS protection against TG-induced ER stress response and associated apoptosis. In summary, PNS protection against ER stress response and associated apoptosis is related to the regulation of mitochondrial injury and ROS overproduction via modulation of autophagy. These data provide new insights for molecular mechanisms of PNS as a potential preventive approach to the management of cardiovascular diseases.
内质网（ER）应激通常与自噬、缺氧信号传导、线粒体生物发生和活性氧（ROS）反应密切相关。了解内质网应激、线粒体功能和自噬之间的相互作用对于为心血管疾病的病理、预防和治疗提供新机制至关重要。我们之前的研究报道，三七总皂苷（PNS）对毒胡萝卜素（TG）诱导的心肌细胞内质网应激反应及相关细胞凋亡的保护作用是钙依赖性的，且由内质网通过兰尼碱受体（RyR）释放钙介导。然而，其对内质网应激及相关凋亡的保护作用是否与线粒体功能和自噬有关仍 largely 未知。在此，我们研究了 PNS 在 TG 诱导的线粒体功能、ROS 积累和自噬中所起的作用。我们还评估了在自噬抑制存在的情况下，其对钙稳态、内质网应激反应及相关细胞死亡的影响。用 TG 刺激经 PNS 预处理的原代培养新生大鼠心肌细胞以诱导内质网应激反应。用 JC - 1 测量线粒体膜电位（Δm）。分别用 2',7'-二氯二氢荧光素二乙酸酯（DCFH - DA）和 MitoSOX Red 测量总 ROS 和线粒体 ROS。通过 LC3 的免疫荧光以及 LC3、p62、自噬相关蛋白 7（ATG7）和线粒体自噬激活因子 1（PINK1）的免疫印迹评估自噬。此外，用 mRFP - GFP - LC3 标记评估自噬流。构建转染了 siATG7 的 H9c2 细胞以抑制自噬。通过钙成像研究细胞质和内质网钙动力学。通过氧化印迹法检测 RyR 氧化。通过 TUNEL 检测法检测细胞活力。通过 BiP、C/EBP 同源蛋白（CHOP）、裂解的半胱天冬酶 - 3 和半胱天冬酶 - 12 的免疫印迹检测内质网应激反应和细胞凋亡。结果表明，首先，PNS 可保护细胞免受 TG 诱导的线粒体损伤和 ROS 积累。其次，PNS 增强了 TG 诱导的心肌细胞中的自噬。第三，自噬抑制减弱了 PNS 对 TG 诱导的线粒体损伤、ROS 积累和钙稳态破坏的预防作用。最后但同样重要的是，自噬抑制消除了 PNS 对 TG 诱导的内质网应激反应及相关凋亡的保护作用。总之，PNS 对内质网应激反应及相关凋亡的保护作用与通过调节自噬来调控线粒体损伤和 ROS 过度产生有关。这些数据为 PNS 作为心血管疾病潜在预防方法的分子机制提供了新的见解。
