PDF(Pubmed)
Abstract：
BACKGROUND: Diabetic retinopathy (DR) is the primary cause of visual problems in patients with diabetes. The Heyingwuzi formulation (HYWZF) is effective against DR.
OBJECTIVE: To determine the HYWZF prevention mechanisms, especially those underlying mitophagy.
METHODS: Human retinal capillary endothelial cells (HRCECs) were treated with high glucose (hg), HYWZF serum, PX-478, or Mdivi-1 in vitro. Then, cell counting kit-8, transwell, and tube formation assays were used to evaluate HRCEC proliferation, invasion, and tube formation, respectively. Transmission electron microscopy was used to assess mitochondrial morphology, and Western blotting was used to determine the protein levels. Flow cytometry was used to assess cell apoptosis, reactive oxygen species (ROS) production, and mitochondrial membrane potential. Moreover, C57BL/6 mice were established in vivo using streptozotocin and treated with HYWZF for four weeks. Blood glucose levels and body weight were monitored continuously. Changes in retinal characteristics were evaluated using hematoxylin and eosin, tar violet, and periodic acid-Schiff staining. Protein levels in retinal tissues were determined via Western blotting, immunohistochemistry, and immunostaining.
RESULTS: HYWZF inhibited excessive ROS production, apoptosis, tube formation, and invasion in hg-induced HRCECs via mitochondrial autophagy in vitro. It increased the mRNA expression levels of BCL2-interacting protein 3 (BNIP3), FUN14 domain-containing 1, BNIP3-like (BNIP3L, also known as NIX), PARKIN, PTEN-induced kinase 1, and hypoxia-inducible factor (HIF)-1α. Moreover, it downregulated the protein levels of vascular endothelial cell growth factor and increased the light chain 3-II/I ratio. However, PX-478 and Mdivi-1 reversed these effects. Additionally, PX-478 and Mdivi-1 rescued the effects of HYWZF by decreasing oxidative stress and apoptosis and increasing mitophagy. HYWZF intervention improved the symptoms of diabetes, tissue damage, number of acellular capillaries, and oxidative stress in vivo. Furthermore, in vivo experiments confirmed the results of in vitro experiments.
CONCLUSIONS: HYWZF alleviated DR and associated damage by promoting mitophagy via the HIF-1α/BNIP3/NIX axis.
OBJECTIVE: To determine the HYWZF prevention mechanisms, especially those underlying mitophagy.
METHODS: Human retinal capillary endothelial cells (HRCECs) were treated with high glucose (hg), HYWZF serum, PX-478, or Mdivi-1 in vitro. Then, cell counting kit-8, transwell, and tube formation assays were used to evaluate HRCEC proliferation, invasion, and tube formation, respectively. Transmission electron microscopy was used to assess mitochondrial morphology, and Western blotting was used to determine the protein levels. Flow cytometry was used to assess cell apoptosis, reactive oxygen species (ROS) production, and mitochondrial membrane potential. Moreover, C57BL/6 mice were established in vivo using streptozotocin and treated with HYWZF for four weeks. Blood glucose levels and body weight were monitored continuously. Changes in retinal characteristics were evaluated using hematoxylin and eosin, tar violet, and periodic acid-Schiff staining. Protein levels in retinal tissues were determined via Western blotting, immunohistochemistry, and immunostaining.
RESULTS: HYWZF inhibited excessive ROS production, apoptosis, tube formation, and invasion in hg-induced HRCECs via mitochondrial autophagy in vitro. It increased the mRNA expression levels of BCL2-interacting protein 3 (BNIP3), FUN14 domain-containing 1, BNIP3-like (BNIP3L, also known as NIX), PARKIN, PTEN-induced kinase 1, and hypoxia-inducible factor (HIF)-1α. Moreover, it downregulated the protein levels of vascular endothelial cell growth factor and increased the light chain 3-II/I ratio. However, PX-478 and Mdivi-1 reversed these effects. Additionally, PX-478 and Mdivi-1 rescued the effects of HYWZF by decreasing oxidative stress and apoptosis and increasing mitophagy. HYWZF intervention improved the symptoms of diabetes, tissue damage, number of acellular capillaries, and oxidative stress in vivo. Furthermore, in vivo experiments confirmed the results of in vitro experiments.
CONCLUSIONS: HYWZF alleviated DR and associated damage by promoting mitophagy via the HIF-1α/BNIP3/NIX axis.
摘要：
背景：糖尿病视网膜病变（DR）是糖尿病患者视觉问题的主要原因。Heyingwuzi制剂（HYWZF）对DR有效。
目的：为了确定HYWZF预防机制，尤其是那些潜在的线粒体自噬。
方法：用高糖（hg）处理人视网膜毛细血管内皮细胞（HRCEC），HYWZF血清,PX-478或Mdivi-1体外。然后，细胞计数试剂盒-8,transwell,和试管形成试验用于评估HRCEC增殖，入侵,和管的形成，分别。透射电子显微镜用于评估线粒体形态，使用蛋白质印迹法测定蛋白质水平。流式细胞术用于评估细胞凋亡，活性氧（ROS）的产生，和线粒体膜电位.此外，使用链脲佐菌素在体内建立C57BL/6小鼠,并用HYWZF处理4周。连续监测血糖水平和体重。使用苏木精和曙红评估视网膜特征的变化，焦油紫罗兰,和高碘酸希夫染色。通过蛋白质印迹法测定视网膜组织中的蛋白质水平，免疫组织化学，和免疫染色。
结果：HYWZF抑制了过量的ROS产生，凋亡，管形成,并在体外通过线粒体自噬对hg诱导的HRCECs进行侵袭。它增加了BCL2相互作用蛋白3（BNIP3）的mRNA表达水平，含FUN14结构域的1，BNIP3样（BNIP3L，也称为NIX)，帕金,PTEN诱导的激酶1和缺氧诱导因子（HIF）-1α。此外，它下调了血管内皮细胞生长因子的蛋白质水平，并增加了轻链3-II/I的比例。然而,PX-478和Mdivi-1逆转了这些效应。此外,PX-478和Mdivi-1通过减少氧化应激和凋亡以及增加线粒体自噬来挽救HYWZF的作用。HYWZF干预改善了糖尿病的症状，组织损伤,无细胞毛细血管的数量，和体内氧化应激。此外，体内实验证实了体外实验的结果。
结论：HYWZF通过HIF-1α/BNIP3/NIX轴促进线粒体自噬减轻DR及相关损伤。
目的：为了确定HYWZF预防机制，尤其是那些潜在的线粒体自噬。
方法：用高糖（hg）处理人视网膜毛细血管内皮细胞（HRCEC），HYWZF血清,PX-478或Mdivi-1体外。然后，细胞计数试剂盒-8,transwell,和试管形成试验用于评估HRCEC增殖，入侵,和管的形成，分别。透射电子显微镜用于评估线粒体形态，使用蛋白质印迹法测定蛋白质水平。流式细胞术用于评估细胞凋亡，活性氧（ROS）的产生，和线粒体膜电位.此外，使用链脲佐菌素在体内建立C57BL/6小鼠,并用HYWZF处理4周。连续监测血糖水平和体重。使用苏木精和曙红评估视网膜特征的变化，焦油紫罗兰,和高碘酸希夫染色。通过蛋白质印迹法测定视网膜组织中的蛋白质水平，免疫组织化学，和免疫染色。
结果：HYWZF抑制了过量的ROS产生，凋亡，管形成,并在体外通过线粒体自噬对hg诱导的HRCECs进行侵袭。它增加了BCL2相互作用蛋白3（BNIP3）的mRNA表达水平，含FUN14结构域的1，BNIP3样（BNIP3L，也称为NIX)，帕金,PTEN诱导的激酶1和缺氧诱导因子（HIF）-1α。此外，它下调了血管内皮细胞生长因子的蛋白质水平，并增加了轻链3-II/I的比例。然而,PX-478和Mdivi-1逆转了这些效应。此外,PX-478和Mdivi-1通过减少氧化应激和凋亡以及增加线粒体自噬来挽救HYWZF的作用。HYWZF干预改善了糖尿病的症状，组织损伤,无细胞毛细血管的数量，和体内氧化应激。此外，体内实验证实了体外实验的结果。
结论：HYWZF通过HIF-1α/BNIP3/NIX轴促进线粒体自噬减轻DR及相关损伤。
