自噬在高糖条件下视网膜色素上皮细胞表达血管内皮生长因子促进RF/6A细胞血管生成中的作用

目的研究高糖条件下诱导的人视网膜色素上皮细胞株(ARPE-19)自噬对其表达血管内皮生长因子(vascular endothelial growth factor,VEGF),及恒河猴脉络膜视网膜血管内皮细胞(RF/6A)迁移和管腔形成的影响。方法根据不同干预将ARPE-19细胞随机分为对照组、高糖组及3-甲基腺嘌呤(3-MA)+高糖组。对照组细胞在无糖DMEM培养基中常规培养24 h,高糖组细胞在DMEM培养基中加入30 mmol·L^-1葡萄糖溶液处理24 h,3-MA+高糖组细胞先用10 mmol·L^-1 3-MA处理24 h,然后更换培养基再加入30 mmol·L^-1葡萄糖溶液处理24 h。Western blot法检测细胞自噬标志性蛋白微管相关蛋白1轻链3(microtubule-related protein 1 light chain 3,LC3)II型和I型的比值(LC3-II/LC3-I)、Beclin-1及细胞中自噬相关基因3(autophagy-related gene 3,Atg3)的蛋白表达。ELISA法检测ARPE-19细胞上清液中VEGF的蛋白表达。比较三组ARPE-19细胞LC3-II/LC3-I、Beclin-1、Atg3及VEGF的蛋白表达量。然后将以上三组ARPE-19细胞上清液分别加入RF/6A细胞培养基中,将RF/6A细胞也按以上方法分组,分别采用Transwell法及Matrigel胶法检测并比较三组RF/6A细胞迁移数及细胞管腔形成数。结果对照组、高糖组和3-MA+高糖组ARPE-19细胞中LC3-II/LC3-I比值分别为0.405±0.095、0.932±0.024和0.635±0.048;Beclin-1蛋白相对表达量分别为0.205±0.035、0.590±0.120和0.425±0.082;Atg3蛋白相对表达量分别为0.277±0.035、0.539±0.071和0.389±0.019。ARPE-19细胞上清液中VEGF的蛋白表达量三组分别为(44.03±9.08)ng·L^-1、(205.70±17.90)ng·L^-1和(112.52±21.06)ng·L^-1;RF/6A细胞迁移数三组分别为(125.60±6.35)个、(153.60±19.20)个和(67.40±7.95)个;细胞管腔形成数三组分别为(12.22±0.84)个、(18.44±1.68)个和(5.44±0.51)个;整体比较差异均有统计学意义(均为P<0.01)。与对照组相比,高糖组ARPE-19细胞的LC3-II/LC3-I比值、Beclin-1及Atg3蛋白相对表达量均明显增加(均为P<0.01),VEGF表达水平均明显升高(P<0.01),RF/6A细胞迁移数和管腔形成数均明显增加(均为P<0.01),3-MA+高糖组ARPE-19细胞中LC3-II/I比值、Beclin-1和Atg3蛋白相对表达量、VEGF表达量、RF/6A细胞迁移数和细胞管腔形成数均较高糖组明显降低(均为P<0.01)。结论高糖激活ARPE-19细胞自噬,进而上调VEGF的表达并促进RF/6A细胞迁移和管腔形成,自噬抑制剂3-MA可抑制ARPE-19细胞自噬,并下调VEGF的表达从而抑制RF/6A细胞的血管形成能力。

Autophagy promotes angiogenesis of RF/6A cells following upregulating the expression of vascular endothelial growth factor in retinal pigment epithelial cells under high glucose conditions

Objective　To study the effects of autophagy of human retinal pigment epithelial cell line (ARPE-19) induced by high glucose conditions on the expression of vascular endothelial growth factor (VEGF) as well as on the migration and tube formation of rhesus choroid-retinal endothelial cells (RF/6A).Methods　ARPE-19 cells were divided into the control group,high-glucose group and 3-methyladenine (3-MA)+high-glucose group.The cells in the control group were cultured in DMEM medium without glucose for 24 hours.The cells in the high-glucose group were cultured in DMEM medium adding 30 mmol·L^-1 glucose for 24 hours.The cells in the 3-MA+high-glucose group were pretreated with 10 mmol·L^-1 3-MA for 24 hours and then exposed to replaced medium adding 30 mmol·L^-1 glucose for 24 hours.The ratio of II and I form of microtubule-related protein 1 light chain 3 (LC3) (LC3-II/LC3-I),Beclin-1 and autophagy-related gene 3 (Atg3) protein expressions in the cells were detected by Western blot,and the expression of VEGF in the cell supernatants was examined by ELISA assay.The expressions of LC3-II/LC3-I,Beclin-1,Atg3 and VEGF among the three groups were compared.Then,the supernatant of ARPE-19 cells in the above three groups was respectively added into RF/6A cell culture medium and RF/6A cells were also grouped by the above method.The migration and tube formation of RF/6A cells were detected by transwell and matrigel assay,respectively.The number of migrated cells and tube formation of the RF/6A cells were compared.Results　In the control group,high-glucose group and 3-MA+high-glucose group,the LC3-II/LC3-I ratio in ARPE-19 cells was 0.405±0.095,0.932±0.024 and 0.635±0.048,respectively;the relative expression of Beclin-1 protein in the cells was 0.205±0.035,0.590±0.120 and 0.425±0.082,respectively;and the relative expression of Atg3 protein in the cells was 0.277±0.035,0.539±0.071 and 0.389±0.019,respectively.The protein expression of VEGF in the supernatant of ARPE-19 cells was (44.03±9.08)ng·L^-1,(205.70±17.90)ng·L^-1 and (112.52±21.06)ng·L^-1 for the three groups,respectively.The number of migrated RF/6A cells was 125.60±6.35,153.60±19.20 and 67.40±7.95;the number of tube formation was 12.22±0.84,18.44±1.68 and 5.44±0.51 for the three groups,respectively.There were significant differences among the groups (all P<0.01).Compared with the control group,the ratio of LC3-II/LC3-I and the expression levels of Beclin-1,Atg3 in ARPE-19 cells were significantly increased (all P<0.01) and the expression level of VEGF increased (P<0.01) in the high-glucose group.The number of migrated cells and tube formation of RF/6A cells were significantly increased in the high-glucose group (all P<0.01) compared with the control group.Compared with the high-glucose group,the ratio of LC3-II/LC3-I and the expression of Beclin-1,Atg3 and VEGF protein in ARPE-19 cells,the number of migrated cells and tube formation of RF/6A cells were significantly decreased in the 3-MA+high-glucose group (all P<0.01).Conclusion　High glucose conditions activate autophagy of ARPE-19 cells,which enhances the expression of VEGF and the migration and tube formation of RF/6A cells.Autophagy inhibitor 3-MA inhibits the angiogenesis abilities of RF/6A cells through inhibiting autophagy of ARPE-19 cells and the expression of VEGF.