School of Traditional Chinese Pharmacy, China Pharmaceutical University, Nanjing, 211198, China.
School of Science, China Pharmaceutical University, Nanjing, 211198, China.
J Ethnopharmacol. 2021 May 10;271:113855. doi: 10.1016/j.jep.2021.113855. Epub 2021 Jan 22.
Scutellarin (Scu) is one of the main active ingredients of Erigeron breviscapus (Vant.) Hand.-Mazz which has been used to treat cardiovascular disease including vascular dysfunction caused by diabetes. Scu also has a protective effect on vascular endothelial cells against hyperglycemia. However, molecular mechanisms underlying this effect are not clear.
This aim of this study was to investigate the effect of Scu on human umbilical vein endothelial cells (HUVECs) injury induced by high glucose (HG), especially the regulation of PTEN-induced kinase 1 (PINK1)/Parkin-mediated mitophagy.
HUVECs were exposed to HG to induce vascular endothelial cells injury in vitro. Cell viability was assessed by MTT assay. The extent of cell apoptosis was measured by Hoechst staining and flow cytometry. Mitophagy was assayed by fluorescent immunostaining, transmission electron microscope and immunoblot. Besides, virtual docking was conducted to validate the interaction of PINK1 protein and Scu.
We found that Scu significantly increased cell viability in HG-treated HUVECs. Scu reduces the expression of Bcl-2, Bax and cytochrome C (Cyt.c) to inhibit apoptosis through a mitochondria-dependent pathway. Meanwhile, Scu improved the overload of reactive oxygen species (ROS), superoxide dismutase (SOD) activity and SOD2 protein expression, and reversed the collapse of mitochondrial membrane potential. Besides, Scu increased autophagic flux, improved the expression of microtubule-associated protein 1 light chain 3 Ⅱ (LC3 II), Beclin 1 and autophagy-related gene 5 (Atg 5) and decreased the expression of Sequestosome1/P62 in HG-treated HUVECs. Furthermore, Scu improved the expressions of PINK1, Parkin, and Mitofusin2, which revealed the enhancement of mitophagy. Moreover, the beneficial effects of Scu on HG-induced low expression of Parkin, overproduction of ROS, and over expressions of P62, Cyt.c and Cleaved caspase-3 were weakened by PINK1 gene knockdown. Molecular docking suggested good interaction of Scu and PINK1 protein.
These results suggest that Scu may protect vascular endothelial cells against hyperglycemia-induced injury by up-regulating mitophagy via PINK1/Parkin signal pathway.
野黄芩苷（Scu）是灯盏细辛（Vant。）Hand.-Mazz 的主要活性成分之一，已用于治疗包括糖尿病引起的血管功能障碍在内的心血管疾病。Scu 对高血糖引起的血管内皮细胞也有保护作用。然而，其作用的分子机制尚不清楚。
本研究旨在探讨 Scu 对高糖（HG）诱导的人脐静脉内皮细胞（HUVEC）损伤的作用，特别是对 PTEN 诱导的激酶 1（PINK1）/Parkin 介导的线粒体自噬的调节。
体外采用 HG 诱导 HUVEC 损伤，MTT 法检测细胞活力。Hoechst 染色和流式细胞术检测细胞凋亡程度。荧光免疫染色、透射电镜和免疫印迹法检测线粒体自噬。此外，还进行了虚拟对接，以验证 PINK1 蛋白与 Scu 的相互作用。
我们发现 Scu 可显著增加 HG 处理的 HUVEC 中的细胞活力。Scu 通过线粒体依赖性途径降低 Bcl-2、Bax 和细胞色素 C（Cyt.c）的表达，从而抑制细胞凋亡。同时，Scu 改善了活性氧（ROS）、超氧化物歧化酶（SOD）活性和 SOD2 蛋白表达的过载，并逆转了线粒体膜电位的崩溃。此外，Scu 增加了自噬通量，提高了微管相关蛋白 1 轻链 3 Ⅱ（LC3 II）、Beclin 1 和自噬相关基因 5（Atg 5）的表达，并降低了 HG 处理的 HUVEC 中 Sequestosome1/P62 的表达。此外，Scu 改善了 PINK1、Parkin 和 Mitofusin2 的表达，表明线粒体自噬增强。此外，通过 PINK1 基因敲低减弱了 Scu 对 HG 诱导的 Parkin 低表达、ROS 过度产生以及 P62、Cyt.c 和 Cleaved caspase-3 过度表达的有益作用。分子对接表明 Scu 与 PINK1 蛋白具有良好的相互作用。
这些结果表明，Scu 可能通过 PINK1/Parkin 信号通路上调线粒体自噬来保护血管内皮细胞免受高血糖诱导的损伤。
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