Tianjin State Key Laboratory of Modern Chinese Medicine, Tianjin University of Traditional Chinese Medicine, Tianjin, China; Research and Development Center of CM, Tianjin International Joint Academy of Biotechnology & Medicine, Tianjin, China.
Tianjin State Key Laboratory of Modern Chinese Medicine, Tianjin University of Traditional Chinese Medicine, Tianjin, China.
J Ethnopharmacol. 2019 Jun 12;237:9-19. doi: 10.1016/j.jep.2019.03.033. Epub 2019 Mar 14.
Shenmai injection (SMI) is a CFDA-approved and widely prescribed herbal medicine injection in China for treating cardiac dysfunction, especially myocardial ischemia and reperfusion (I/R) injury. However, despite of its known clinical efficacy, the cardioprotective mechanisms of SMI remain to be established.
The present study aimed to investigate the role of SMI on mitophagy and mitochondrial dynamics in cardiomyocytes with a hypoxia/reperfusion (H/R) injury setting.
H9c2 cardiomyocytes were subjected to 12 h of hypoxia followed by 2 h of reoxygenation to induce cellular injury. Multi-parameter imaging analysis was performed using Operetta High Content Imaging System to detect changes in mitochondrial function and morphological texture. The mPTP opening was directly assessed by analyzing mitochondrial calcein release in H9c2 and by Ca-induced swelling of isolated cardiac mitochondria. Mitochondrial respiration was measured by XF 24 analyzer of Seahorse Bioscience. RT-PCR and Western blotting analyses were used to detect mitophagy, mitochondrial fusion and fission biomarkers at the gene and protein levels.
Pretreatment of SMI significantly improved myocardial cell survival and protected against H/R-induced deterioration of mitochondrial structure and function, as evidenced by decreased mitochondrial mass and cytosolic Ca, increased mitochondrial membrane potential (ΔΨ) and mitochondrial morphology by SER Texture analysis, inhibited mPTP opening in H9c2 cells and isolated cardiac mitochondria, and alleviated severely impaired mitochondrial respiration. Mechanistically, SMI attenuated H/R injury by inducing mitophagy and then modulated mitochondrial dynamics as indicated by a significantly increased expression of LC3, Beclin 1, Parkin and Pink, and the inhibition of excessive mitochondria fission and increased mitochondrial fusion. Finally, the cardioprotective effect of SMI was confirmed in a LAD-induced cardiac dysfunction model in vivo.
We found that alleviation of H/R injury by pretreatment with SMI may be attributable to inducing mitophagy and modulating mitochondrial dynamics in cardiomyocytes, thereby providing a rationale for future clinical applications and potential mitoprotective therapy for MI/R injury.
参麦注射液（SMI）是中国食品药品监督管理局批准并广泛用于治疗心功能障碍，特别是心肌缺血再灌注（I / R）损伤的草药注射剂。然而，尽管其具有已知的临床疗效，但 SMI 的心脏保护机制仍有待确定。
本研究旨在探讨 SMI 在缺氧/复氧（H / R）损伤条件下对心肌细胞自噬和线粒体动力学的作用。
将 H9c2 心肌细胞置于 12 小时缺氧后再复氧 2 小时，以诱导细胞损伤。使用 Opera ta 高内涵成像系统进行多参数成像分析，以检测线粒体功能和形态纹理的变化。通过分析 H9c2 中线粒体钙黄绿素的释放以及通过 Ca 诱导的分离心脏线粒体肿胀，直接评估 mPTP 开放。使用 Seahorse Bioscience 的 XF 24 分析仪测量线粒体呼吸。通过 RT-PCR 和 Western blotting 分析在基因和蛋白水平上检测自噬、线粒体融合和裂变生物标志物。
SMI 预处理可显著提高心肌细胞存活率，并防止 H / R 诱导的线粒体结构和功能恶化，表现为线粒体质量和胞质 Ca 减少，线粒体膜电位（ΔΨ）和线粒体形态通过 SER 纹理分析增加，抑制 H9c2 细胞和分离的心脏线粒体中的 mPTP 开放，并缓解严重受损的线粒体呼吸。在机制上，SMI 通过诱导自噬来减轻 H / R 损伤，然后调节线粒体动力学，如 LC3、Beclin 1、Parkin 和 Pink 的表达显著增加，以及过度的线粒体分裂和增加的线粒体融合抑制。最后，在体内 LAD 诱导的心脏功能障碍模型中证实了 SMI 的心脏保护作用。
我们发现，预处理 SMI 减轻 H / R 损伤可能归因于诱导心肌细胞自噬和调节线粒体动力学，从而为 MI / R 损伤的未来临床应用和潜在的线粒体保护治疗提供了依据。
