缺氧状态下SB431542对视网膜血管内皮细胞的作用

Effect of SB431542 on retinal vascular endothelial cells under hypoxia

目的观察并分析缺氧状态下SB431542对视网膜血管内皮细胞的作用。方法体内动物实验:健康C57BL/6J小鼠48只,随机分为正常组、氧诱导视网膜病变(OIR)组、OIR+二甲基亚砜(DMSO)组、OIR+SB431542组,每组各12只。小鼠17日龄时,作视网膜铺片观察新生血管情况;苏木精-伊红染色计数突破视网膜内界膜(ILM)的血管内皮细胞核数。体内细胞实验:人视网膜微血管内皮细胞(hRMEC)分为正常组、缺氧组、缺氧+DMSO组、缺氧+SB431542组。噻唑蓝(MTT)比色法检测细胞增殖情况;Matrigel体外三维成型法检测SB431542对hRMEC管腔形成的影响;细胞划痕实验检测hRMEC内细胞迁移情况;Seahorse XFe96细胞能量代谢分析仪测定细胞内糖酵解、糖酵解储备、糖酵解容量的细胞外酸化率(ECAR)。多组间比较采用单因素方差分析。结果体内动物实验:与正常组比较,OIR组新生血管增多(t=41.621,P<0.001);与OIR组比较,OIR+SB431542组突破ILM的血管内皮细胞核数明显减少,差异有统计学意义(F=36.183,P<0.001)。MTT比色法检测结果显示,与正常组、缺氧+SB431542组比较,缺氧组、缺氧+DMSO组细胞增殖显著增多,差异有统计学意义(F=39.316,P<0.01);缺氧+SB431542组细胞增殖较缺氧+DMSO组显著降低,差异有统计学意义(t=26.182,P<0.001)。正常组、缺氧组、缺氧+DMSO组、缺氧+SB431542组细胞完整管腔形成数、迁移细胞数比较,差异均有统计学意义(F=34.513、41.862,P<0.001、<0.01)。与缺氧+DMSO组比较,缺氧+SB431542组细胞完整管腔形成数、迁移细胞数明显下降,差异有统计学意义(t=44.723、31.178,P<0.001、<0.01)。细胞能量代谢测定结果显示,与缺氧+DMSO组比较,缺氧+SB431542组细胞内糖酵解、糖酵解储备的ECAR降低,糖酵解容量的ECAR增高,差异均有统计学意义(t=26.175、33.623、37.276,P<0.05)。结论SB431542可抑制缺氧诱导的hRMEC增殖、迁移及管腔形成能力,并降低细胞糖酵解水平。

Objective To investigate the effect of Nodal protein on retinal neovascularization under hypoxia.Methods In vivo animal experiment:48 healthy C57BL/6J mice were randomly divided into normal group,oxygen-induced retinopathy(OIR)group,OIR+dimethyl sulfoxide(DMSO)group and OIR+SB431542 group,with 12 mice in each group.Retinal neovascularization was observed in mice at 17 days of age by retina flat mount.Counts exceeded the number of vascular endothelial nuclei in the retinal inner boundary membrane(ILM)by hematoxylin eosin staining.In vivo cell experiment:human retinal microvascular endothelial cells(hRMEC)were divided into normal group,hypoxia group,hypoxia+DMSO group and hypoxia+SB431542 group.The cell proliferation was detected by thiazolyl blue colorimetry(MTT).The effect of SB431542 on hRMEC lumen formation was detected by Matrigel three-dimensional in vitro molding method.Cell migration in hRMEC was detected by cell scratch assay.The Seahorse XFe96 Cell Energy Metabolism analyzer measured extracellular acidification rate(ECAR)of intracellular glycolysis,glycolysis reserve,and glycolysis capacity.One-way analysis of variance was used to compare groups.Results In vivo animal experiment:compared with normal group,the neovascularization increased in OIR group(t=41.621,P<0.001).Compared with OIR group,the number of vascular endothelial nuclei breaking through ILM in OIR+SB431542 group was significantly reduced,and the difference was statistically significant(F=36.183,P<0.001).MTT test results showed that compared with normal group and hypoxia+SB431542 group,the cell proliferation of hypoxia group and hypoxia+DMSO group was significantly increased,and the difference was statistically significant(F=39.316,P<0.01).The cell proliferation of hypoxia+SB431542 group was significantly lower than that of hypoxia+DMSO group,and the difference was statistically significant(t=26.182,P<0.001).The number of intact lumen formation and migration cells in normal group,hypoxia group,hypoxia+DMSO group and hypoxia+SB431542 group were statistically significant(F=34.513,41.862;P<0.001,<0.01).Compared with the hypoxia+DMSO group,the number of intact lumen formation and migrating cells in the hypoxia+SB431542 group decreased significantly,and the differences were statistically significant(t=44.723,31.178;P<0.001,<0.01).The results of cell energy metabolism showed that compared with the hypoxia+DMSO group,the ECAR of intracellular glycolysis and glycolysis reserve in the hypoxia+SB431542 group was decreased,and the ECAR of glycolysis capacity was increased,with statistical significance(t=26.175,33.623,37.276;P<0.05).Conclusion SB431542 can inhibit the proliferation,migration and the ability to form lumens,reduce the level of glycolysis of hRMECs cells induced by hypoxia.