紫草素对高糖诱导的血管内皮细胞凋亡和氧化应激的影响

目的:探讨紫草素(shikonin)对高浓度葡萄糖诱导的血管内皮细胞凋亡和氧化应激水平的影响及其可能的作用机制。方法:体外培养的大鼠胸主动脉内皮细胞随机分为5组:正常对照组(培养基中葡萄糖浓度为5.5 mmol/L)、高糖组(培养基中葡萄糖浓度为33 mmol/L)、高糖+低浓度紫草素组(培养基中葡萄糖浓度为33mmol/L,紫草素浓度为0.1μmol/L)、高糖+中浓度紫草素组(培养基中葡萄糖浓度为33 mmol/L,紫草素浓度为1μmol/L)和高糖+高浓度紫草素组(培养基中葡萄糖浓度为33 mmol/L,紫草素浓度为10μmol/L)。各组细胞经相应处理后,CCK-8法检测细胞活力,流式细胞术检测细胞的凋亡率;此外,检测细胞中丙二醛(malondialdehyde,MDA)、活性氧簇(reactive oxygen species,ROS)、超氧化物歧化酶(superoxide dismutase,SOD)及谷胱甘肽过氧化物酶(glutathione peroxidase,GSH-Px)的水平,以反映细胞的氧化应激状态;Western blot检测Nrf2/HO-1信号通路的活性。结果:相较于高糖组,紫草素处理可呈剂量依赖性地逆转高糖所致的内皮细胞活力降低及凋亡率增加。与正常对照组相比,高浓度葡萄糖可升高内皮细胞中MDA和ROS的含量,同时降低SOD和GSH-Px的活性;相较于高糖组,给予紫草素干预后,细胞内MDA和ROS的含量降低,SOD和GSH-Px的活性升高。此外,高糖可致内皮细胞中cleaved caspase-3、HO-1及核内Nrf2蛋白表达的增加;与高糖组相比,给予紫草素干预后细胞中cleaved caspase-3、HO-1和核内Nrf2的表达部分下降。结论:紫草素可显著改善高糖所致的血管内皮细胞凋亡,其作用机制可能与激活Nrf2/HO-1信号通路并降低细胞的氧化应激水平有关。

Effect of shikonin on high glucose-induced apoptosis and oxidative stress in vascular endothelial cells

AIM:To investigate the effect of shikonin on the apoptosis and oxidative stress induced by high concentration of glucose in vascular endothelial cells. METHODS:Rat thoracic aortic endothelial cells were randomly divided into 5 groups:normal control group (with glucose at concentration of 5.5 mmol/L in cell culture medium), high glucose group (with glucose at concentration of 33 mmol/L in cell culture medium), high glucose+low shikonin group (with glucose at concentration of 33 mmol/L and shikonin at concentration of 0.1 μmol/L in cell culture medium), high glucose+medium shikonin group (with glucose at concentration of 33 mmol/L and shikonin at concentration of 1 μmol/L in cell culture medium), and high glucose+high shikonin group (with glucose at concentration of 33 mmol/L and shikonin at concentration of 10 μmol/L in cell culture medium). After treatments, the cell viability was measured by CCK-8 assay and cell apoptotic rate was analyzed by flow cytometry. In addition, the status of oxidative stress was evaluated by determining the levels of malondialdehyde (MDA), reactive oxygen species (ROS), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px). The activation of Nrf2/HO-1 signaling pathway was determined by Western blot. RESULTS:Compared with high glucose group, shikonin reversed high glucose-induced decrease in cell viability and increase in apoptosis in a concentration-dependent manner. High concentration of glucose induced high levels of MDA and ROS, while decreased the levels of SOD and GSH-Px. However, after treatment with shikonin, the contents of MDA and ROS were decreased, while the activities of SOD and GSH-Px were increased as compared with high glucose group. Furthermore, the high concentration of glucose up-regulated the protein levels of cleaved caspase-3, HO-1 and Nrf2 (nuclear). Compared with high glucose group, the protein levels of cleaved caspase-3, HO-1 and Nrf2 (nuclear) were partly decreased after treatment with shikonin. CONCLUSION:Shikonin alleviates high glucose-induced vascular endothelial cell apoptosis. Its mechanism may be related to activation of Nrf2/HO-1 signaling pathway and down-regulation of oxidative stress in vascular endothelial cells.