Abstract：
This study explored the effect of Tianma Gouteng Decoction on oxidative stress induced by angiotensin Ⅱ(AngⅡ) in vascular smooth muscle cell(VSMC) and its molecular mechanism. Primary rat VSMC were cultured using tissue block method, and VSMC were identified by α-actin immunofluorescence staining. AngⅡ at a concentration of 1×10~(-6) mol·L~(-1) was used as the stimulating factor, and Sprague Dawley(SD) rats were orally administered with Tianma Gouteng Decoction to prepare drug serum. Rat VSMC were divided into normal group, model group, Chinese medicine group, and inhibitor(3-methyladenine, 3-MA) group. Cell counting kit-8(CCK-8) assay was used to detect cell proliferation activity. Bromodeoxyuridine(BrdU) flow cytometry was used to detect cell cycle. Transwell assay was used to detect cell migration ability. Enzyme-linked immunosorbent assay(ELISA) was used to detect the activity of superoxide dismutase(SOD), catalase(CAT), and malondialdehyde(MDA) in VSMC. The intracellular reactive oxygen species(ROS) fluorescence intensity was detected using DCFH-DA fluorescent probe. Western blot was used to detect the expression of PTEN-induced putative kinase 1(PINK1), Parkin, p62, and microtubule-associated protein 1A/1B-light chain 3(LC3-Ⅱ) proteins in VSMC. The results showed that Tianma Gouteng Decoction-containing serum at a concentration of 8% could significantly inhibit VSMC growth after 48 hours of intervention. Compared with the normal group, the model group showed significantly increased cell proliferation activity and migration, significantly decreased levels of SOD and CAT, significantly increased levels of MDA, significantly enhanced ROS fluorescence intensity, significantly decreased expression of PINK1, Parkin, and LC3-Ⅱ proteins, and significantly increased expression of p62 protein. Compared with the model group, the Chinese medicine group showed significantly reduced cell proliferation activity and migration, significantly increased levels of SOD and CAT, significantly decreased levels of MDA, significantly weakened ROS fluorescence intensity, significantly increased expression of PINK1, Parkin, and LC3-Ⅱ proteins, and significantly decreased expression of p62 protein. Compared with the Chinese medicine group, the addition of the mitochondrial autophagy inhibitor 3-MA could block the intervention of Tianma Gouteng Decoction-containing serum on VSMC proliferation, migration, mitochondrial autophagy, and oxidative stress levels, with statistically significant differences. In summary, Tianma Gouteng Decoction has good antioxidant activity and can inhibit cell proliferation and migration. Its mechanism of action may be related to the activation of the mitochondrial autophagy PINK1/Parkin signaling pathway.
摘要：
本研究探讨天麻钩藤汤对血管紧张素Ⅱ(AngⅡ)诱导的血管平滑肌细胞(VSMC)氧化应激的影响及其分子机制。采用组织块法培养原代大鼠VSMC,通过α-肌动蛋白免疫荧光染色鉴定VSMC。使用浓度为1×10〜（-6）mol·L〜（-1）的AngⅡ作为刺激因子，用天麻钩藤汤口服SD大鼠制备药物血清。将大鼠VSMC分为正常组,模型组,中药集团,和抑制剂（3-甲基腺嘌呤，3-MA）组。细胞计数试剂盒-8(CCK-8)用于检测细胞增殖活性。使用溴脱氧尿苷(BrdU)流式细胞术检测细胞周期。用Transwell法检测细胞迁移能力。酶联免疫吸附试验(ELISA)检测超氧化物歧化酶(SOD)活性,过氧化氢酶(CAT),和VSMC中的丙二醛（MDA）。使用DCFH-DA荧光探针检测细胞内活性氧（ROS）的荧光强度。Westernblot用于检测PTEN诱导的推定激酶1（PINK1）的表达，Parkin,VSMC中p62和微管相关蛋白1A/1B-轻链3（LC3-Ⅱ）蛋白。结果表明,浓度为8%的天麻钩藤汤含药血清干预48h后可显著抑制VSMC生长。与正常组相比，模型组细胞增殖活性和迁移能力显著增强,显著降低SOD和CAT的水平，MDA水平显着增加，显著增强ROS荧光强度，PINK1、Parkin、和LC3-Ⅱ蛋白，p62蛋白表达明显增加。与模型组相比，中药组细胞增殖活性和迁移能力显著降低,显著增加SOD和CAT的水平，MDA水平显着降低，显著减弱ROS荧光强度，PINK1、Parkin、和LC3-Ⅱ蛋白，p62蛋白表达明显降低。与中药组相比，添加线粒体自噬抑制剂3-MA可以阻断天麻钩藤汤含药血清对VSMC增殖的干预，迁移,线粒体自噬，和氧化应激水平，具有统计学上的显著差异。总之,天麻钩藤汤具有良好的抗氧化活性,能抑制细胞增殖和迁移。其作用机制可能与线粒体自噬PINK1/Parkin信号通路的激活有关。
