虫草素对高糖培养的恒河猴脉络膜-视网膜血管内皮细胞血管新生的影响

Anti-angiogenic mechanism of cordycepin on rhesus macaque choroid-retinal endothelial cell line cultured in high glucose condition

目的:观察虫草素对高糖培养的恒河猴脉络膜-视网膜血管内皮细胞(RF/6A)血管新生作用的影响,探讨其可能的作用机制。方法:培养RF/6A细胞,分为正常对照组(NC组)、高糖对照组(HG组)、高糖加不同浓度的虫草素组(HG+10μg/m L组、HG+50μg/m L组、HG+100μg/m L组)。采用CCK8法检测各组细胞的增殖活性;采用Transwell实验检测各组细胞的迁移;采用Matrigel检测各组管腔形成的情况;采用Western-blot检测各组细胞中VEGF和VEGFR2蛋白表达的情况。结果:与NC组相比,HG组RF/6A的增殖活性增加(P<0.05)。不同浓度的虫草素对RF/6A的增殖均有抑制作用,随着虫草素浓度增加,细胞活性下降。HG+10μg/m L组、HG+50μg/m L组、HG+100μg/m L组的增殖活性抑制率分别为(10.2±0.9)%、(23.4±1.5)%、(31.1±1.2)%,与HG组比较差异均有统计学意义(P<0.05)。NC组、HG组、HG+10μg/m L组、HG+50μg/m L组、HG+100μg/m L组细胞迁移个数分别为55.6±2.70、87.4±2.40、65.4±2.7、57.8±2.38、62.4±2.77个。与NC组相比,HG组迁移细胞数量增加(P<0.05);不同浓度虫草素组细胞迁移数量随着虫草素浓度的增加而减少,与HG组相比,差异具有统计学意义(P<0.05)。NC组、HG组、HG+10μg/m L组、HG+50μg/m L组、HG+100μg/m L组的细胞管腔形成个数分别为18.7±2.08、25.7±1.52、19.9±1.57、16.3±2.51、5.7±1.72个。与NC组相比,HG组细胞管腔形成个数增加(P<0.05);不同浓度虫草素组细胞管腔形成个数随着虫草素浓度的增加而减少,与HG组相比,差异具有统计学意义(P<0.05)。与NC组相比,HG组VEGF和VEGFR2蛋白表达的量增加(P<0.05);不同浓度虫草素组中细胞内VEGF和VEGFR2蛋白表达的量均低于高糖对照组(P<0.05)。结论:虫草素可能通过抑制高糖下VEGF和VEGFR2蛋白的表达,抑制RF/6A细胞增殖、迁移和管腔形成,进而抑制血管新生。

AIM： To investigate the angiogenesis effect and protective mechanism of cordycepin on rhesus macaque choroid- retinal endothelial （ RF/ 6A） cell line cultured in high glucose condition. METHODS： Cultured RF/ 6A cells were divided into normal control group, high glucose group and high glucose （HG） ＋ different concentration cordycepin groups （HG＋ 10μ g/ mL group, HG＋ 50μ g/ mL group, HG＋ 100μ g/mL group）. The cell proliferation was assessed using cholecystokinin octapeptide dye after treated for 48h. The cell migration was investigated by a Transwell assay. The tube formation was measured on Matrigel. Furthermore, the impact of cordycepin on high glucose - induced＆amp;nbsp;activation of VEGF and VEGF receptor 2 （VEGFR-2） was tested by Western blot analysis. RESULTS： Compared with normal control group, cell viability markedly increased in high glucose group （ P 〈0. 05）. Cordycepin inhibited RF/ 6A cell proliferation in a dose- dependent fashion： 10. 2 ± 0. 9%, 23. 4 ± 1. 5% and 31. 1±1. 2% inhibition as the concentrations of cordycepin were 10, 50 and 100μ g/ mL, respectively. The difference had statistically significant （P〈0. 05） compared with high glucose group. The number of cell migration were 55. 6±2. 70, 87. 4 ± 2. 40, 65. 4 ± 2. 7, 57. 8 ± 2. 38, 62. 4 ± 2. 77 in normal control group, high glucose group and HG＋10μ g/mL group, HG ＋ 50μ g/ mL group, HG ＋ 100μ g/ mL group respectively. Migration of RF/ 6A conspicuously increased in high glucose group （ P 〈 0. 05） compared with normal control group; while showing a gradually reducing trend with the increase of cordycepin dose and a statistically significant difference compared with high glucose group （P〈0. 05）. The number of tube formation were 18. 7±2. 08, 25. 7 ± 1. 52, 19. 9 ± 1. 57, 16. 3 ± 2. 51, 5. 67 ± 1. 72 in the abovementioned group. Similarly showing a gradually reducing trend with the increase of cordycepin dose and a statistically significant difference with high glucose group （P〈 0. 05）. In addition, the number of tube formation of RF/ 6A in high glucose group significant increased compared with normal control group （ P 〈 0. 05 ）. The expression of VEGF and VEGFR-2 dramaticlly increased in high glucose group vs normal control group, oppositely gradually decreased with the increase of cordycepin concentrations, and had a statistically significant difference vs high glucose group （P〈0. 05）. CONCLUSION： Cordycepin can suppress the proliferation, migration and tubu formation of RF/ 6A in high glucose condition, might via inhibiting expression of VEGF and VEGFR-2.