Junxiao Xi1, Yuezhao Rong1, Zifeng Zhao1, Yihai Huang1, Pu Wang1, Huiling Luan1, Yan Xing2, Siyuan Li1, Jun Liao3, Yue Dai1, Jingyu Liang1, Feihua Wu4. 1. School of Traditional Chinese Pharmacy, China Pharmaceutical University, Nanjing, 211198, China. 2. School of Science, China Pharmaceutical University, Nanjing, 211198, China. 3. School of Science, China Pharmaceutical University, Nanjing, 211198, China. Electronic address: liaojun@cpu.edu.cn. 4. School of Traditional Chinese Pharmacy, China Pharmaceutical University, Nanjing, 211198, China. Electronic address: fhwu2000@sina.com.
Abstract
ETHNOPHARMACOLOGICAL RELEVANCE: 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. AIM OF THE STUDY: 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. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSION: These results suggest that Scu may protect vascular endothelial cells against hyperglycemia-induced injury by up-regulating mitophagy via PINK1/Parkin signal pathway.
ETHNOPHARMACOLOGICAL RELEVANCE: 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. AIM OF THE STUDY: 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. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSION: These results suggest that Scu may protect vascular endothelial cells against hyperglycemia-induced injury by up-regulating mitophagy via PINK1/Parkin signal pathway.
Authors: M Sahyadri; Abhishek P R Nadiga; Seema Mehdi; K Mruthunjaya; Pawan G Nayak; Vipan K Parihar; S N Manjula Journal: 3 Biotech Date: 2022-08-18 Impact factor: 2.893