Ye Xiaojing, Xiang Feng, Hu Yiyi
Department of Pharmacy, The Second Affiliated Hospital of Wenzhou Medical University, Wenzhou, China.
Ann Transl Med. 2023 Feb 28;11(4):168. doi: 10.21037/atm-22-6591. Epub 2023 Feb 16.
This study aimed to investigate the effect and mechanism of gambogic acid (GA) on the apoptosis and inflammation of human retinal endothelial cells (HRECs) under high glucose conditions.
HRECs were cultured in a high glucose medium to simulate retinal endothelial cell injury induced by diabetic retinopathy. Flow cytometry was used to analyze the apoptosis level of HRECs. Cell viability was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Western blotting was applied to detect the intracellular apoptosis-related proteins and expression levels of NADPH oxidase 4 (NOX4), nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3), and interleukin (IL)-1β. Enzyme linked immunosorbent assay (ELISA) was utilized to detect the expression of IL-6, IL-8, IL-10, and tumor necrosis factor-α (TNF-α) in the cell supernatants. The messenger RNA (mRNA) levels of IL-6, IL-8, IL-10, and TNF-α were detected by reverse transcription-polymerase chain reaction (RT-qPCR).
We observed that high glucose induced the apoptosis and inflammation of HRECs. In addition, the high glucose environment promoted NOX/NLRP3 pathway activation. The activity of HRECs was not significantly affected by the presence of 20 μM or less of GA, and 15 μM of GA could restore the diminished activity of HRECs induced by high glucose. The apoptosis of HRECs cultured under high glucose conditions was significantly inhibited (P<0.05), the levels of IL-6, IL-8, and TNF-α in the cell supernatant were significantly decreased (P<0.05), and the levels of IL-10 were significantly increased (P<0.05). Meanwhile, the relative mRNA expression levels of IL-6, IL-8, and TNF-α in HRECs were significantly decreased (P<0.05), while those of IL-10 were significantly increased (P<0.05). The activity of the high glucose-induced NOX4/NLRP3 pathway in HRECs was significantly inhibited after treatment with 15 μM of GA (P<0.05). Following activation of the NOX4/NLRP3 pathway in HRECs, the apoptosis level was significantly increased (P<0.05), and the inflammatory response was aggravated (P<0.05). Inhibiting the activity of the intracellular NOX4/NLRP3 pathway markedly inhibited cell apoptosis and the inflammatory response (P<0.05).
GA can inhibit the apoptosis and inflammation of HRECs under high glucose conditions by inhibiting the activity of the NOX4/NLRP3 pathway. This has a significant inhibitory effect on diabetic retinopathy, which is worthy of further study.
本研究旨在探讨藤黄酸（GA）对高糖条件下人视网膜内皮细胞（HRECs）凋亡及炎症的影响及其机制。
将HRECs培养于高糖培养基中，以模拟糖尿病视网膜病变诱导的视网膜内皮细胞损伤。采用流式细胞术分析HRECs的凋亡水平。通过3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐（MTT）检测细胞活力。应用蛋白质免疫印迹法检测细胞内凋亡相关蛋白以及烟酰胺腺嘌呤二核苷酸磷酸氧化酶4（NOX4）、核苷酸结合寡聚化结构域样受体蛋白3（NLRP3）和白细胞介素（IL）-1β的表达水平。利用酶联免疫吸附测定（ELISA）检测细胞上清液中IL-6、IL-8、IL-10和肿瘤坏死因子-α（TNF-α）的表达。通过逆转录-聚合酶链反应（RT-qPCR）检测IL-6、IL-8、IL-10和TNF-α的信使核糖核酸（mRNA）水平。
我们观察到高糖诱导了HRECs的凋亡和炎症。此外，高糖环境促进了NOX/NLRP3途径的激活。20 μM及以下浓度的GA对HRECs的活性无显著影响，15 μM的GA可恢复高糖诱导的HRECs活性降低。高糖条件下培养的HRECs凋亡受到显著抑制（P<0.05），细胞上清液中IL-6、IL-8和TNF-α水平显著降低（P<0.05），IL-10水平显著升高（P<0.05）。同时，HRECs中IL-6、IL-8和TNF-α的相对mRNA表达水平显著降低（P<0.05），而IL-10的相对mRNA表达水平显著升高（P<0.05）。用15 μM的GA处理后，高糖诱导的HRECs中NOX4/NLRP3途径的活性显著受到抑制（P<0.05）。激活HRECs中的NOX4/NLRP3途径后，凋亡水平显著升高（P<0.05），炎症反应加剧（P<0.05）。抑制细胞内NOX4/NLRP3途径的活性可显著抑制细胞凋亡和炎症反应（P<0.05）。
GA可通过抑制NOX4/NLRP3途径的活性，抑制高糖条件下HRECs的凋亡和炎症。这对糖尿病视网膜病变具有显著的抑制作用，值得进一步研究。
