College of Basic Medicine &Public Health, Zhejiang Chinese Medical University, Hangzhou, Zhejiang 310053, China; College of Life Science, Zhejiang Chinese Medical University, Hangzhou, Zhejiang 310053, China.
College of Basic Medicine &Public Health, Zhejiang Chinese Medical University, Hangzhou, Zhejiang 310053, China.
Phytomedicine. 2021 Mar;83:153480. doi: 10.1016/j.phymed.2021.153480. Epub 2021 Jan 23.
Autophagy plays an important role in cellular homeostasis. Oxidative stress stimulated endothelial excessive autophagy has been proposed as a major risk factor for cardiovascular diseases (CVD). Danhong injection (DHI), the most prescribed traditional Chinese medicine for the treatment of CVD, has been shown to elicit vascular protective effects. However, its underlying mechanisms remain poorly defined. This study aimed to uncover the protective effects of DHI and its main bioactive components on autophagy injury of human umbilical vein endothelial cells (HUVECs) induced by HO and reveal the possible mechanisms.
HUVECs were treated with different concentrations of DHI or its components, after exposed to HO. The protective effects of DHI and its components in HO-induced HUVECs were examined via a cytotoxicity assay and western blot. Apoptosis was evaluated with flow cytometry. Autophagy flux was assessed by transmission electron microscopy and LC3 plasmid transfection. Besides, the role miR-19a and SIRT1 in DHI and components-mediated anti-autophagy responses were validated with inhibitors transfection.
Our results showed that DHI and its components do have different effects on different aspects. In terms of HUVECs survival rate, Salvianolic acid B (Sal B) and danshensu (DSS) performed better than DHI, Hydroxysafflor yellow A (HSYA) and Tanshinone IIA (DST-IIA). As for the proliferation effect on HUVECs, only Sal B has the most obvious performance as same as 3MA. Besides, DHI and its components are sensitive and superior in regulating and balancing ROS concentration. Among the GSH/GSSG indicators, DSS and HSYA performed better. In terms of SOD content and apoptotic rate, the SOD level showed the opposite trend compared with HO group. For the expression of LC3, Beclin-1 and P62, DHI and its components all had significant effects. When miR-19a or SIRT1 was inhibited, Sal B (0.5 μg/ml) can not decrease autophagy-related protein effectively.
DHI and its components all had anti-autophagy effects. And Sal B (0.5 μg/ml) inhibited HUVECs autophagy via miR-19a/SIRT1 pathway.
自噬在细胞稳态中发挥重要作用。氧化应激刺激内皮细胞过度自噬已被认为是心血管疾病 (CVD) 的主要危险因素。丹红注射液 (DHI) 是治疗 CVD 最常用的中药，已显示出血管保护作用。然而，其潜在机制仍不清楚。本研究旨在揭示 DHI 及其主要生物活性成分对 HO 诱导的人脐静脉内皮细胞 (HUVEC) 自噬损伤的保护作用，并揭示可能的机制。
用不同浓度的 DHI 或其成分处理 HUVECs，然后用 HO 处理。通过细胞毒性测定和 Western blot 检测 DHI 及其成分对 HO 诱导的 HUVECs 的保护作用。用流式细胞术评估细胞凋亡。通过透射电镜和 LC3 质粒转染评估自噬通量。此外，用抑制剂转染验证 miR-19a 和 SIRT1 在 DHI 和成分介导的抗自噬反应中的作用。
结果表明，DHI 及其成分对不同方面的影响不同。就 HUVECs 存活率而言，丹参酸 B (Sal B) 和丹参素 (DSS) 的表现优于 DHI，羟基红花黄色 A (HSYA) 和丹参酮 IIA (DST-IIA)。至于对 HUVECs 的增殖作用，只有 Sal B 与 3MA 一样具有最明显的作用。此外，DHI 及其成分在调节和平衡 ROS 浓度方面具有敏感性和优越性。在 GSH/GSSG 指标方面，DSS 和 HSYA 表现较好。就 SOD 含量和凋亡率而言，与 HO 组相比，SOD 水平呈现相反的趋势。对于 LC3、Beclin-1 和 P62 的表达，DHI 和其成分均有显著影响。当抑制 miR-19a 或 SIRT1 时，Sal B (0.5μg/ml) 不能有效降低自噬相关蛋白的表达。
DHI 及其成分均具有抗自噬作用。Sal B (0.5μg/ml) 通过 miR-19a/SIRT1 通路抑制 HUVECs 自噬。
