Ding Ke, Bao Qiying, He Jiaqi, Wang Jiahong, Wang Hui
Department of Pharmacy, The First Affiliated Hospital of Zhejiang Chinese Medical University, No. 54, Youdian Road, Hangzhou, 310006, Zhejiang, China.
Department of Pharmacy, Hangzhou Fuyang Hospital of TCM Orthopedics, Hangzhou, 311499, Zhejiang, China.
Open Med (Wars). 2025 Mar 17;20(1):20241059. doi: 10.1515/med-2024-1059. eCollection 2025.
Tetrahydropalmatine (THP) regulates mitochondrial function in vascular smooth muscle cells (VSMCs) to prevent or alleviate atherosclerosis (AS), with unclear specific mechanism.
AS models were constructed by oxidized low-density lipoprotein (ox-LDL)-treated VSMCs. Cell counting kit-8 for cell viability, wound scratch assay for cell migration, and flow cytometry for cell cycle, intracellular reactive oxygen species, and mitochondrial membrane potential (MMP) were performed. Malondialdehyde (MDA) and superoxide dismutase (SOD) levels by biochemical kits, oxygen consumption rate (OCR) by seahorse apparatus, apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay (TUNEL) staining, and apoptosis-related expression by western blot were detected. Ras homolog gene family A/Rho-associated protein kinase-1 (RhoA/ROCK1) levels were measured by western blot and ELISA. The RhoA agonist, U46619, was employed to validate mechanism of THP.
THP suppressed cell cycle progression and cell migration whereas alleviating cell viability and oxidative stress, as reduced MDA and enhanced SOD levels in ox-LDL-incubated VSMCs. THP protected mitochondrial function by higher MMP levels and OCR values. Additionally, THP decreased TUNEL-positive cells, Bax, Caspase-3, RhoA, ROCK1, and osteopontin expression, while increased Bcl-2 and smooth muscle myosin heavy chain levels. Furthermore, U46619 intervention antagonized effects of THP.
THP improved mitochondrial function in VSMCs of AS by inhibiting RhoA/ROCK1 signaling pathway.
四氢巴马汀（THP）可调节血管平滑肌细胞（VSMC）中的线粒体功能，以预防或减轻动脉粥样硬化（AS），但其具体机制尚不清楚。
通过氧化低密度脂蛋白（ox-LDL）处理的VSMC构建AS模型。采用细胞计数试剂盒-8检测细胞活力，划痕实验检测细胞迁移，流式细胞术检测细胞周期、细胞内活性氧种类和线粒体膜电位（MMP）。使用生化试剂盒检测丙二醛（MDA）和超氧化物歧化酶（SOD）水平，用海马仪器检测氧消耗率（OCR），用末端脱氧核苷酸转移酶介导的dUTP缺口末端标记法（TUNEL）染色检测细胞凋亡，用蛋白质免疫印迹法检测凋亡相关蛋白表达。通过蛋白质免疫印迹法和酶联免疫吸附测定法检测Ras同源基因家族A/ Rho相关蛋白激酶-1（RhoA/ROCK1）水平。使用RhoA激动剂U46619验证THP的作用机制。
THP抑制细胞周期进程和细胞迁移，同时提高细胞活力并减轻氧化应激，表现为降低ox-LDL处理的VSMC中的MDA水平并提高SOD水平。THP通过提高MMP水平和OCR值来保护线粒体功能。此外，THP减少TUNEL阳性细胞、Bax、Caspase-3、RhoA、ROCK1和骨桥蛋白的表达，同时增加Bcl-2和平滑肌肌球蛋白重链水平。此外，U46619干预可拮抗THP的作用。
THP通过抑制RhoA/ROCK1信号通路改善AS的VSMC中线粒体功能。
