Objective: To investigate the effects of wogonoside on high glucose-induced dysfunction of human retinal microvascular endothelial cells (hRMECs) and streptozotocin (STZ)-induced diabetic retinopathy in rats and explore the underlying molecular mechanism.
Methods: HRMECs in routine culture were treated with 25 mmol/L mannitol or exposed to high glucose (30 mmol/L glucose) and treatment with 10, 20, 30, 40 μmol/L wogonoside. CCK-8 assay and Transwell assay were used to examine cell proliferation and migration, and the changes in tube formation and monolayer cell membrane permeability were tested. ROS, NO and GSH-ST kits were used to evaluate oxidative stress levels in the cells. The expressions of IL-1β and IL-6 in the cells were examined with qRT-PCR and ELISA, and the protein expressions of VEGF, HIF-1α and SIRT1 were detected using Western blotting. We also tested the effect of wogonoside on retinal injury and expressions of HIF-1α, ROS, VEGF, TNF-α, IL-1β, IL-6 and SIRT1 proteins in rat models of STZ-induced diabetic retinopathy.
Results: High glucose exposure caused abnormal proliferation and migration, promoted angiogenesis, increased membrane permeability (P < 0.05), and induced inflammation and oxidative stress in hRMECs (P < 0.05). Wogonoside treatment concentration-dependently inhibited high glucose-induced changes in hRMECs. High glucose exposure significantly lowered the expression of SIRT1 in hRMECs, which was partially reversed by wogonoside (30 μmol/L) treatment; interference of SIRT1 obviously attenuated the inhibitory effects of wogonoside against high glucose-induced changes in proliferation, migration, angiogenesis, membrane permeability, inflammation and oxidative stress in hRMECs. In rat models of STZ-induced diabetic retinopathy, wogonoside effectively suppressed retinal thickening (P < 0.05), alleviated STZ-induced retinal injury, and increased the expression of SIRT1 in the retinal tissues (P < 0.001).
Conclusion: Wogonoside alleviates retinal damage caused by diabetic retinopathy by up-regulating SIRT1 expression.
目的: 探究汉黄芩苷对高糖诱导的人视网膜微血管内皮细胞（hRMECs）功能障碍的作用和对链脲佐菌素（STZ）诱导的大鼠糖尿病视网膜的损伤的影响及潜在的分子机制。
方法: hRMECs常规培养，实验分为对照组（5 mmol/L葡萄糖）、渗透对照组（5 mmol/L葡萄糖+25 mmol/L甘露醇）、高糖组（30 mmol/L葡萄糖）、给药组（30 mmol/L葡萄糖+10、20、30、40 μmol/L汉黄芩苷）。通过CCK-8与Transwell实验分别检测细胞增殖和迁移能力，小管形成与单层细胞膜通透性实验分别检测细胞成管能力和细胞膜通透性，ROS、NO、GSH-ST试剂盒检测细胞氧化应激水平，qRT-PCR和ELISA试剂盒分别检测IL-1β、IL-6的表达水平和含量，Western blot检测VEGF、HIF-1α和SIRT1蛋白表达。选取40只SPF级雄性SD大鼠，随机分为对照组（Sham组）、模型组（STZ组）、汉黄芩苷组（Wog组）和汉黄芩苷治疗组（STZ+Wog组），10只/组。模型组和汉黄芩苷治疗组腹腔注射0.1 mol/L柠檬酸缓冲液（pH=4.5）溶解的STZ，剂量为60 mg/kg建立糖尿病大鼠模型，对照组与单独的汉黄芩苷组给予等剂量的柠檬酸缓冲液。糖尿病大鼠模型建立1周后给予汉黄芩苷治疗，对照组、模型组予等量生理盐水注射，连续6周。比较4组大鼠视网膜组织损伤、HIF-1-α、ROS、VEGF、TNF-α、IL-1β、IL-6水平以及sirt1蛋白表达情况。
结果: 与对照组相比，高糖诱导hRMECs异常增殖和迁移，提高了hRMECs小管形成能力及细胞膜通透性（P＜0.05），同时高糖诱导的hRMECs炎症水平和氧化应激水平上升（P＜0.05）。而与高糖组相比，汉黄芩苷可浓度依赖性抑制高糖诱导的hRMECs细胞增殖、迁移、小管形成及细胞膜通透性的增加（P＜0.05），此外汉黄芩苷可降低高糖诱导的hRMECs的炎症和氧化应激水平（P＜0.05）。SIRT1在高糖诱导的hRMECs中低表达，30 μmol/L剂量汉黄芩苷处理后高糖诱导的低SIRT1表达部分恢复（P＜0.01），干扰SIRT1表达可逆转汉黄芩苷对高糖诱导hRMECs异常增殖、迁移、小管形成、细胞膜通透性、炎症及氧化应激的抑制作用。动物实验显示，与对照组相比，STZ组视网膜组织厚度增加（P＜0.05），而汉黄芩苷治疗后能缓解糖尿病引起的大鼠视网膜增厚（P＜0.05）。同时STZ处理提高了VEGF、HIF- 1α、IL-1β、IL-6和ROS的水平（P＜0.001），汉黄芩苷的处理降低了STZ诱导的大鼠视网膜损伤且上调了SIRT1的表达（P＜0.001）。
结论: 汉黄芩苷通过上调SIRT1的表达缓解糖尿病视网膜病变引起的损伤。
Keywords: abnormal proliferation; diabetic retinopathy; inflammation; oxidative stress; wogonoside.