背景:TYHX通阳活血汤在临床应用已近40年。TYHX的成分是黄芪(黄芪),红参(红参),地黄(地黄),普通山药根状茎(山药)和决明子树皮(肉桂)。我们先前的实验证实TYHX可以保护窦房结细胞。然而,其作用机制尚不完全清楚。
目的:本研究旨在研究TYHX对低氧应激下窦房结细胞损伤的保护作用,并阐明其保护机制。
方法:通过RNA测序分析和网络药理学分析,我们发现线粒体相关基因在缺氧模拟SNC前后存在显著差异,解决了TYHX的主要监管机制。通过TYHX对SNC的干预,激光共聚焦等一系列检测方法,荧光共定位,线粒体膜电位和RT-PCR。通过体外实验验证了TYHX对窦房结细胞β-微管蛋白的调节作用。TYHX及其活性成分槲皮素通过线粒体自噬维持线粒体稳态和保护窦房结细胞的作用机制,线粒体融合/裂变和线粒体生物合成得到证实。
结果:通过RNA测序分析,我们发现在缺氧建立SNC模型前后线粒体相关基因存在显著差异。通过药理实验,我们发现TYHX可以抑制Drp1向线粒体的迁移,抑制过度的线粒体裂变,激活线粒体自噬并增加线粒体膜电位。这些保护作用主要由β-微管蛋白介导。此外,TYHX中的活性成分槲皮素可以通过SIRT1抑制线粒体过度裂变,维持线粒体能量代谢,保护SNCs。我们的结果表明,通过维持β-微管蛋白和激活SIRT1来保护线粒体功能是TYHX减轻SNCs缺氧应激损伤的主要机制。TYHX和槲皮素对线粒体质量监测的调节作用也是必要的。我们的发现提供了经验证据,支持使用TYHX作为病态窦房结综合征的靶向治疗。
结论:我们的数据表明TYHX在低氧应激下对窦房结细胞损伤具有保护作用,这可能与线粒体质量监测(MQS)的调节和抑制线粒体稳态介导的细胞凋亡有关。
BACKGROUND: TYHX-Tongyang Huoxue decoction has been used clinically for nearly 40 years. The ingredients of TYHX are Radix Astragali (Huangqi), Red Ginseng (Hongshen), Rehmannia Glutinosa (Dihuang), Common Yam Rhizome (Shanyao) and Cassia-bark-tree Bark (Rougui). Our previous experiments confirmed that TYHX can protect sinoatrial node cells. However, its mechanism of action is not completely understood yet.
OBJECTIVE: The present study aimed to determine the protective effects of TYHX against Sinus node cell injury under hypoxic stress and elucidate the underlying mechanisms of protection.
METHODS: Through RNA sequencing analysis and network pharmacology analysis, we found significant differences in mitochondrial-related genes before and after hypoxia-mimicking SNC, resolved the main regulatory mechanism of TYHX. Through the intervention of TYHX on SNC, a series of detection methods such as laser confocal, fluorescence co-localization, mitochondrial membrane potential and RT-PCR. The regulatory effect of TYHX on β-tubulin in sinoatrial node cells was verified by in vitro experiments. The mechanism of action of TYHX and its active ingredient quercetin to maintain mitochondrial homeostasis and protect sinoatrial node cells through mitophagy, mitochondrial fusion/fission and mitochondrial biosynthesis was confirmed.
RESULTS: Through RNA sequencing analysis, we found that there were significant differences in mitochondrial related genes before and after SNC was modeled by hypoxia. Through pharmacological experiments, we showed that TYHX could inhibit the migration of Drp1 to mitochondria, inhibit excessive mitochondrial fission, activate mitophagy and increase the mitochondrial membrane potential. These protective effects were mainly mediated by β-tubulin. Furthermore, the active component quercetin in TYHX could inhibit excessive mitochondrial fission through SIRT1, maintain mitochondrial energy metabolism and protect SNCs. Our results showed that protection of mitochondrial function through the maintenance of β-tubulin and activation of SIRT1 is the main mechanism by which TYHX alleviates hypoxic stress injury in SNCs. The regulatory effects of TYHX and quercetin on mitochondrial quality surveillance are also necessary. Our findings provide empirical evidence supporting the use of TYHX as a targeted treatment for sick sinus syndrome.
CONCLUSIONS: Our data indicate that TYHX exerts protective effects against sinus node cell injury under hypoxic stress, which may be associated with the regulation of mitochondrial quality surveillance (MQS) and inhibition of mitochondrial homeostasis-mediated apoptosis.