人参皂苷Rb3对高糖诱导的内皮细胞迁移和增殖及糖尿病小鼠视网膜功能的影响

Effect of Ginsenoside Rb3 on High Glucose-induced Migration and Proliferation of Endothelial Cells and the Retinal Function in Diabetic Mice

目的探究人参皂苷Rb3(Ginsenoside Rb3)对高糖诱导的恒河猴脉络膜-视网膜内皮细胞(RF/6A)迁移和增殖及db/db小鼠视网膜功能的影响及其作用机制。方法RF/6A细胞分为正常组(5.5 mmol/L D-葡萄糖)、高糖组(25 mmol/L D-葡萄糖)、高糖+Ginsenoside Rb3(100、150、200μg/mL)组。采用CCK-8法检测各组细胞活力,5-溴脱氧尿嘧啶核苷(5-Bromodeoxyuridinc,BrdU)染色评估细胞增殖能力以及Transwell法检测各组细胞迁移能力,Western blot技术检测各组RF/6A中YES相关蛋白1(yes-associated protein 1,YAP1)和磷酸化YES关联蛋白1(p-YAP1)的表达。db/db小鼠分为模型组和Ginsenoside Rb3组,给药4周后,运用光学相干断层扫描实验和视网膜电生理检查评价db/db小鼠视网膜形态和功能的变化,并采用免疫荧光染色法观察YAP1在视网膜中的表达。结果结果显示,与对照组比较,高糖组细胞活力和BrdU阳性细胞率显著增加(P<0.01),细胞迁移率显著提高(P<0.001);与高糖组相比,Ginsenoside Rb3降低细胞活力(150μg/mL,P<0.05)、阳性细胞率(200μg/mL,P<0.05;100μg/mL,P<0.01)和迁移细胞数量(P<0.05)。与正常组相比,高糖组YAP1和p-YAP1蛋白表达上调(P<0.01或P<0.05);与高糖组相比,Ginsenoside Rb3干预下调RF/6A中YAP1和p-YAP1(150μg/mL)的蛋白表达水平(P<0.05)。体内研究结果显示,Ginsenoside Rb3增加db/db小鼠的视网膜厚度,改善视网膜功能并抑制YAP1在视网膜中的表达(P<0.05)。结论Ginsenoside Rb3不仅抑制高糖诱导的RF/6A细胞增殖、迁移,亦能改善db/db小鼠视网膜的形态和功能,其药理机制可能与下调YAP1表达有关。

Objective To investigate the effect and mechanism of ginsenoside Rb3 on migration and proliferation of rhesus monkey choroidal-retinal endothelial cells(RF/6A cells)and db/db mice both induced by high glucose.Methods RF/6A cells were divided into a control group(5.5 mmol/L D-glucose),high glucose group(HG,25 mmol/L D-glucose),HG+Ginsenoside Rb3(100,150,and 200μg/mL)groups.The cell viability was detected by CCK-8 method.BrdU staining was used to evaluate cell proliferation,and Transwell method was used to detect cell migration.The protein expression of yesassociated protein 1(YAP1)and p-YAP1 was detected by Western blotting.The db/db mice were divided into the diabetes mellitus(DM)group and ginsenoside Rb3 group.Optical coherence tomography and electroretinogram were used to detect the effects of ginsenoside Rb3 on the retinal structure and function.The expression of YAP1 in the retinal tissues was detected by immunofluorescence.Results Compared with the control group,the cell viability and BrdU-positive cell rate were significantly increased(P<0.01),and cell migration was improved in the HG group(P<0.001).Compared with the HG group,ginsenoside Rb3 decreased the cell viability(150μg/mL,P<0.05),the BrdU-positive cell rate(200μg/mL,P<0.05;100μg/mL,P<0.01),and the number of migrated cells(P<0.05).Compared with the control group,the expression levels of YAP1 and p-YAP1 proteins in the HG group were upregulated(P<0.01 and P<0.05,respectively).Compared with the HG group,ginsenoside Rb3 downregulated the protein expression levels of YAP1(all P<0.05)and p-YAP1(150μg/mL)in RF/6A cells(P<0.05).In vivo experiment showed that ginsenoside Rb3 could increase the thickness of retina in the db/db mice,improve its function,and inhibit YAP1 protein expression(P<0.05).Conclusion Ginsenoside Rb3 not only inhibits HG-induced RF/6A cell proliferation,migration and improves the structure and function of retina in db/db mice,which may relate to the downregulation of YAP1 expression.