白花蛇舌草对高糖诱导的人视网膜血管内皮细胞增殖、凋亡、活性氧水平的影响研究

Effect of Hedyotis diffusa willd on proliferation,apoptosis and ROS level of human retinal capillary endothelial cells induced by high glucose

目的探讨白花蛇舌草注射液(HDI)对高糖诱导的人视网膜血管内皮细胞(HRCECs)增殖、凋亡、氧化应激的影响及机制。方法2019年10月至2020年6月,体外培养HRCECs。将HRCECs分为对照组(5.5 mmol/L葡萄糖处理细胞)、高渗对照组(19.5 mmol/L甘露醇及5.5 mmol/L葡萄糖处理细胞)、高糖对照组(25 mmol/L葡萄糖处理细胞)、白花蛇舌草组(HDI组)(6.25 mL/L、12.5 mL/L、25 mL/L、50 mL/L HDI和25 mmol/L的葡萄糖处理细胞)。处理细胞48 h,通过CCK8法检测细胞活力;流式细胞术检测细胞周期及凋亡率;激光扫描共聚焦显微镜检测活性氧水平。蛋白质印迹法检测增殖细胞核抗原(PCNA)、细胞周期蛋白A1(CyclinA1)和活化胱天蛋白酶-3(cleaved caspase-3)蛋白表达。结果与对照组比较,高糖组细胞活性明显升高[(1.116±0.105)比(0.684±0.072)](P<0.05)。与高糖组比较,12.5 mL/L、25 mL/L和50 mL/L的白花蛇舌草组细胞活性明显降低[(0.847±0.076)、(0.639±0.058)、(0.421±0.042)比(1.116±0.105)](P<0.05)。选择50 mL/L的白花蛇舌草作为研究对象。与对照组比较,高糖组G_(0)/G期细胞百分比[(60.02±5.01)%比(54.72±4.31)%]、细胞凋亡率[(17.11±1.01)%比(3.32±0.47)%]、活性氧水平[(96.18±5.22)比(42.14±3.56)]及PCNA、CyclinA1和cleaved caspase-3表达均明显升高,G_(2)/M期和S期细胞百分比明显降低(P<0.05)。与高糖组比较,HDI组G_(0)/G期细胞百分比[(32.31±3.42)%比(60.02±5.01)%]、细胞凋亡率[(9.72±0.73)%比(17.11±1.01)%]、活性氧水平[(67.75±4.83)比(96.18±5.22)]及PCNA、CyclinA1和cleaved caspase-3表达均明显降低,G_(2)/M期和S期细胞百分比明显升高(P<0.05)。结论白花蛇舌草可降低高糖诱导的HRCECs增殖、凋亡及活性氧水平,阻滞细胞周期,机制可能与调节PCNA、CyclinA1和cleaved caspase-3表达有关。

Objective To investigate the effect and mechanism of Hedyotis diffusa injection(HDI)on the proliferation,apoptosis and oxidative stress of human retinal capillary endothelial cells(HRCECs)induced by high glucose.Methods From October 2019 to June 2020,HRCECs were cultured in vitro and assigned into control group(cells were treated with 5.5 mmol/L glucose),hypertonic control group(cells were treated with 19.5 mmol/L mannitol and 5.5 mmol/L glucose),high glucose control group(cells were treated with 25 mmol/L glucose),Hedyotis diffusa group(HDI group)(cells were treated with 6.25 mL/L,12.5 mL/L,25 mL/L,50 mL/L HDI and 25 mmol/L glucose).Cells were treated for 48 h,and then the cell viability was detected by CCK8 method;cell cycle and apoptosis rate were detected by flow cytometry;ROS level was detected by laser scanning confocal microscope.Western blotting was used to detect the ex‐pression of proliferating cell nuclear antigen(PCNA),cell cyclin A1(CyclinA1)and cleaved caspase-3.Results Compared with the control group,the cell activity in high glucose group increased significantly[(1.116±0.105)vs.(0.684±0.072)](P<0.05).Compared with the high glucose group,the cell activity in 12.5 mL/L,25 mL/L and 50 mL/L HDI group decreased significantly[(0.847±0.076),(0.639±0.058),(0.421±0.042)vs.(1.116±0.105)](P<0.05).50 mL/L Hedyotis diffusa was selected as the research object.Compared with the control group,the percentage of cells in G_(0)/G phase[(60.02±5.01)%vs.(54.72±4.31)%],apoptosis rate[(17.11±1.01)%vs.(3.32±0.47)%],ROS level[(96.18±5.22)vs.(42.14±3.56)]and the expression levels of PCNA,CyclinA1 and cleaved caspase-3 in high glucose group were significantly increased,while the percentages of cells in G_(2)/M phase and S phase were significantly decreased(P<0.05).Com‐pared with the high glucose group,the percentage of cells in G_(0)/G phase[(32.31±3.42)%vs.(60.02±5.01)%],apoptosis rate[(9.72±0.73)%vs.(17.11±1.01)%],ROS level[(67.75±4.83)vs.(96.18±5.22)]and the expression levels of PCNA,CyclinA1 and cleaved caspase 3 in HDI group decreased significantly,while the percentages of cells in G_(2)/M phase and S phase increased significantly(P<0.05).Conclusion Hedyotis diffusa willd can reduce the proliferation,apoptosis and ROS level of HRCECs induced by high glucose,and block cell cycle.The mechanism may be related to the regulation of the expression levels of PCNA,CyclinA1 and cleaved caspase 3.