京尼平苷对高糖诱导的人视网膜血管内皮细胞增殖及血管生成的影响及机制研究

Effect of geniposide on high glucose-induced proliferation and angiogenesis of human retinal vascular endothelial cells and its mechanism

目的研究京尼平苷(Gen)对高糖诱导的人视网膜血管内皮细胞(hRVECs)增殖、迁移及血管形成能力的影响,并探讨其作用机制。方法采用不同浓度(0、1、5、10、20、40、80 mg·L^(-1))Gen干预hRVECs 24 h,CCK-8检测Gen对hRVECs细胞增殖活性的影响。将hRVECs分为对照组,高糖(25 mmol·L^(-1))组,Gen低、中、高浓度(5、10、20 mg·L^(-1))组,贝伐珠单抗(BEV,250μg·L^(-1))组和Gen高浓度+BEV(250μg·L^(-1))组。CCK-8检测细胞增殖活性;划痕实验检测细胞迁移能力;体外成管实验检测细胞成管能力;Western blot检测细胞中血管内皮生长因子-A(VEGF-A)、可溶性VEGF受体-1(sFlt-1)、基质金属蛋白酶-2(MMP-2)和基质金属蛋白酶-9(MMP-9)等蛋白表达水平。结果与0 mg·L^(-1)Gen比较,1、5、10、20、40、80 mg·L^(-1)Gen对hRVECs增殖活性的影响差异均无统计学意义(均为P>0.05)。与对照组比较,高糖组hRVECs增殖活性和迁移能力均显著增强(均为P<0.05),细胞环形管状结构增多,VEGF-A、MMP-2和MMP-9蛋白表达水平均升高(均为P<0.05),sFlt-1蛋白表达水平降低(P<0.05)。与高糖组比较,Gen各浓度组和BEV组细胞增殖活性和迁移能力均减弱(均为P<0.05),细胞环状结构减少,VEGF-A、MMP-2和MMP-9蛋白表达水平均显著降低(均为P<0.05),sFlt-1蛋白表达水平均升高(P<0.05)。与Gen高浓度组比较,Gen高浓度+BEV组细胞增殖活性显著减弱(P<0.05),细胞环状结构减少,VEGF-A、MMP-2和MMP-9蛋白表达水平均显著降低(均为P<0.05),sFlt-1蛋白表达水平显著升高(P<0.05)。结论Gen可抑制高糖诱导的hRVECs增殖、迁移与血管形成,其作用机制可能与调控VEGF/sFlt-1轴平衡有关。

Objective To study the effect of geniposide(Gen)on the proliferation,migration and angiogenesis of human retinal vascular endothelial cells(hRVECs)induced by high glucose and explore its mechanism.Methods The hRVECs were intervened with different concentrations(0,1,5,10,20,40 and 80 mg·L^(-1))of Gen for 24 h,and Cell Counting Kit-8(CCK-8)was used to detect the effect of Gen on the proliferation activity of hRVECs.The hRVECs were divided into the control group,high glucose(25 mmol·L^(-1))group,low,middle and high Gen concentration(5,10 and 20 mg·L^(-1))groups,bevacizumab(BEV,250μg·L^(-1))group and high Gen concentration+BEV(250μg·L^(-1))group.Cell proliferation activity was detected by CCK-8.The cell migration ability was detected by scratch test.The tube formation ability of cells was detected by the in vitro tube formation assay.The protein expression levels of vascular endothelial growth factor-A(VEGF-A),soluble VEGF receptor-1(sFlt-1),matrix metalloproteinase 2(MMP-2)and matrix metalloproteinase 9(MMP-9)in cells were detected by Western blot.Results Compared with 0 mg·L^(-1)Gen,there was no statistically significant difference in the effect of Gen with concentrations of 1,5,10,20,40 and 80 mg·L^(-1)on the proliferation activity of hRVECs(all P>0.05).Compared with the control group,the proliferation activity and migration ability of hRVECs in the high glucose group were significantly enhanced(both P<0.05),the cell circular structure increased,the protein expression levels of VEGF-A,MMP-2 and MMP-9 significantly increased(all P<0.05),and the protein expression level of sFlt-1 significantly decreased(P<0.05).Compared with the high glucose group,the proliferative activity and migration ability of cells in all Gen concentration groups and BEV group significantly decreased(all P<0.05),the circular structure of cells was reduced,the protein expression levels of VEGF-A,MMP-2 and MMP-9 significantly decreased(all P<0.05),and the protein expression level of sFlt-1 significantly increased(P<0.05).Compared with the high Gen concentration group,the high Gen concentration+BEV group showed a significant decrease in cell proliferation activity(P<0.05),a decrease in cell circular structure,a significant decrease in VEGF-A,MMP-2 and MMP-9 protein expression levels(all P<0.05),and a significant increase in sFlt-1 protein expression level(P<0.05).Conclusion Gen can inhibit the high glucose-induced proliferation,migration and angiogenesis of hRVECs,and its mechanism may be related to the regulation of VEGF/sFlt-1 axis balance.