Department of Cardiovascular, Guang'anmen Hospital, China Academy of Chinese Medical Sciences, Beijing, China.
Department of Cardiovascular, Guang'anmen Hospital, China Academy of Chinese Medical Sciences, Beijing, China.
J Ethnopharmacol. 2024 Jan 10;318(Pt B):117006. doi: 10.1016/j.jep.2023.117006. Epub 2023 Aug 5.
β-tubulin is a skeletal protein of sinoatrial node cells (SANCs) that maintains the physiological structure of SANCs and inhibits calcium overload. Tongyang Huoxue decoction (TYHX) is widely used to treat sick sinus syndrome (SSS) owing to its effects on calcium channels regulation and SANCs protection.
This study focuses on the mechanism of TYHX in improving the hypoxia/reoxygenation (H/R)-induced disequilibrium of calcium homeostasis in SANCs via regulating β-tubulin.
Real-Time PCR (RT-PCR) and Western Blot were adopted to detect the mRNA and protein expression levels of calcium channel regulatory molecules. Laser confocal method was employed to examine β-tubulin structure and fluorescence expression levels in SANCs, as well as calcium wave and calcium release levels.
It was found that the fluorescence expression level decreased and the β-tubulin structure of SANCs was damaged after H/R treatment. The mRNA and protein expression levels of SERCA2a/CaV1.3/NCX and β-tubulin decreased, while the mRNA and protein expression of RyR2 increased. The results of calcium wave and calcium transient experiments showed that the fluorescence expression level of Ca increased and calcium overload occurred in SANCs. After treatment with TYHX, the mRNA and protein expression levels of SERCA2a/CaV1.3/NCX and β-tubulin increased, while the mRNA and protein expression levels of RyR2 decreased and the cell structure was restored. Interestingly, the regulation of TYHX on calcium homeostasis was further enhanced after Ad-β-tubulin treatment and counteracted after siRNA-β-tubulin treatment. These results suggest that TYHX could maintain calcium homeostasis via regulating β-tubulin, thus protecting against H/R-induced SANCs injury, which may be a new target for SSS treatment.
β-微管蛋白是窦房结细胞（SANCs）的骨架蛋白，它维持着 SANCs 的生理结构并抑制钙超载。通阳活血汤（TYHX）由于其对钙通道调节和 SANCs 保护的作用，被广泛用于治疗病态窦房结综合征（SSS）。
本研究旨在通过调节β-微管蛋白，研究 TYHX 改善 SANCs 缺氧/复氧（H/R）诱导的钙稳态失衡的机制。
采用实时 PCR（RT-PCR）和 Western blot 检测钙通道调节分子的 mRNA 和蛋白表达水平。采用激光共聚焦法检测 SANCs 中β-微管蛋白结构和荧光表达水平，以及钙波和钙释放水平。
发现 H/R 处理后 SANCs 的荧光表达水平降低，β-微管蛋白结构受损。SERCA2a/CaV1.3/NCX 和 β-微管蛋白的 mRNA 和蛋白表达水平降低，而 RyR2 的 mRNA 和蛋白表达水平升高。钙波和钙瞬变实验结果表明，SANCs 中 Ca 的荧光表达水平增加，发生钙超载。TYHX 处理后，SERCA2a/CaV1.3/NCX 和 β-微管蛋白的 mRNA 和蛋白表达水平增加，而 RyR2 的 mRNA 和蛋白表达水平降低，细胞结构得到恢复。有趣的是，Ad-β-微管蛋白处理后 TYHX 对钙稳态的调节作用进一步增强，而 siRNA-β-微管蛋白处理后则拮抗了这种作用。这些结果表明，TYHX 可以通过调节β-微管蛋白来维持钙稳态，从而防止 H/R 诱导的 SANCs 损伤，这可能是 SSS 治疗的一个新靶点。
