Mou You-Ling, Zhao Rui, Lyu Shi-Ying, Zhang Zi-Yi, Zhu Mei-Fei, Liu Qian
School of Pharmaceutical Sciences, Zhejiang Chinese Medical University, Hangzhou, China.
Department of Critical Care Medicine, the First Affiliated Hospital, Zhejiang Chinese Medical University, Hangzhou, China.
J Geriatr Cardiol. 2023 Jan 28;20(1):68-82. doi: 10.26599/1671-5411.2023.01.001.
Saffron ( L.) has been traditionally used as food, spice, and medicine. Crocetin (CRT), as main bioactive component of saffron, has accumulated pieces of beneficial evidence on myocardial ischemia/reperfusion (I/R) injury. However, the mechanisms are poorly explored. This study aims to investigate the effects of CRT on H9c2 cells under hypoxia/reoxygenation (H/R) and elucidated the possible underlying mechanism.
H/R attack was performed on H9c2 cells. Cell counting kit-8 was used to detect the cell viability. Cell samples and culture supernatants were evaluated via commercial kits to measure the superoxide dismutase (SOD) activity, malondialdehyde (MDA) content, and cellular adenosine triphosphate (ATP) content. Various fluorescent probes were used to detect cell apoptosis, intracellular and mitochondrial reactive oxygen species (ROS) content, mitochondrial morphology, mitochondrial membrane potential (MMP), and mitochondrial permeability transition pore (mPTP) opening. Proteins were evaluated via Western Blot.
H/R exposure severely reduced cell viability and increased LDH leakage. Peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) suppression and dynamin-related protein 1 (Drp1) activation were coincided with excessive mitochondrial fission, mitochondrial permeability transition pore (mPTP) opening and mitochondrial membrane potential (MMP) collapse in H9c2 cells treated with H/R. Mitochondria fragmentation under H/R injury induced ROS over-production, oxidative stress, and cell apoptosis. Notably, CRT treatment significantly prevented mitochondrial fission, mPTP opening, MMP loss, and cell apoptosis. Moreover, CRT sufficiently activated PGC-1α and inactivated Drp1. Interestingly, mitochondrial fission inhibition with mdivi-1 similarly suppressed mitochondrial dysfunction, oxidative stress and cell apoptosis. However, silencing PGC-1α with small interfering RNA (siRNA) abolished the beneficial effects of CRT on H9c2 cells under H/R injury, accompanied with increased Drp1 and p-Drp1 levels. Furthermore, over-expression of PGC-1α with adenovirus transfection replicated the beneficial effects of CRT on H9c2 cells.
Our study identified PGC-1α as a master regulator in H/R-injured H9c2 cells via Drp1-mediated mitochondrial fission. We also presented the evidence that PGC-1α might be a novel target against cardiomyocyte H/R injury. Our data revealed the role of CRT in regulating PGC-1α/Drp1/mitochondrial fission process in H9c2 cells under the burden of H/R attack, and we suggested that modulation of PGC-1α level may provide a therapeutic target for treating cardiac I/R injury.
藏红花传统上被用作食品、香料和药物。西红花酸（CRT）作为藏红花的主要生物活性成分，已积累了大量关于心肌缺血/再灌注（I/R）损伤的有益证据。然而，其机制尚未得到充分探索。本研究旨在探讨CRT对缺氧/复氧（H/R）条件下H9c2细胞的影响，并阐明可能的潜在机制。
对H9c2细胞进行H/R攻击。使用细胞计数试剂盒-8检测细胞活力。通过商业试剂盒评估细胞样本和培养上清液，以测量超氧化物歧化酶（SOD）活性、丙二醛（MDA）含量和细胞三磷酸腺苷（ATP）含量。使用各种荧光探针检测细胞凋亡、细胞内和线粒体活性氧（ROS）含量、线粒体形态、线粒体膜电位（MMP）和线粒体通透性转换孔（mPTP）开放情况。通过蛋白质免疫印迹法评估蛋白质。
H/R暴露严重降低细胞活力并增加乳酸脱氢酶（LDH）泄漏。在接受H/R处理的H9c2细胞中，过氧化物酶体增殖物激活受体γ共激活因子-1α（PGC-1α）抑制和动力相关蛋白1（Drp1）激活与过度的线粒体分裂、线粒体通透性转换孔（mPTP）开放和线粒体膜电位（MMP）崩溃同时发生。H/R损伤下的线粒体碎片化诱导ROS过度产生、氧化应激和细胞凋亡。值得注意的是，CRT处理显著预防了线粒体分裂、mPTP开放、MMP丧失和细胞凋亡。此外，CRT充分激活PGC-1α并使Drp1失活。有趣的是，用mdivi-1抑制线粒体分裂同样抑制了线粒体功能障碍、氧化应激和细胞凋亡。然而，用小干扰RNA（siRNA）沉默PGC-1α消除了CRT对H/R损伤下H9c2细胞的有益作用，同时伴有Drp1和磷酸化Drp1水平升高。此外，用腺病毒转染过表达PGC-1α复制了CRT对H9c2细胞的有益作用。
我们的研究确定PGC-1α是通过Drp1介导的线粒体分裂在H/R损伤的H9c2细胞中的主要调节因子。我们还提供了证据表明PGC-1α可能是对抗心肌细胞H/R损伤的新靶点。我们的数据揭示了CRT在H/R攻击负担下调节H9c2细胞中PGC-1α/Drp1/线粒体分裂过程中的作用，并且我们建议调节PGC-1α水平可能为治疗心脏I/R损伤提供一个治疗靶点。
