薯蓣皂苷对高糖诱导的视网膜色素上皮细胞损伤的保护作用  

Protective effect of diosgenin on retinal pigment epithelial cell injury induced by high glucose

作　　者：张新霞[1] 王小敏[1] 陆丽红[1] 王保君[1] 狄文玉[2] ZHANG Xinxia;WANG Xiaomin;LU Lihong;WANG Baojun;DI Wenyu(Department of Ophthalmology,the First Affiliated Hospital of Xinxiang Medical University,Xinxiang 453100,Henan Province,China;Department of Pathology,the First Affiliated Hospital of Xinxiang Medical University,Xinxiang 453100,Henan Province,China)

机构地区：[1]新乡医学院第一附属医院眼科,河南省新乡市453100 [2]新乡医学院第一附属医院病理科,河南省新乡市453100

出　　处：《眼科新进展》2022年第4期278-283,共6页Recent Advances in Ophthalmology

基　　金：河南省医学科技攻关计划项目(编号SBGJ2018055)。

摘　　要：目的探讨薯蓣皂苷(Dio)对高糖(HG)诱导的视网膜色素上皮(RPE)细胞损伤的保护作用,并分析其机制。方法用细胞计数试剂8(CCK-8)法筛选葡萄糖浓度和无细胞毒性的Dio剂量范围。将ARPE-19细胞分为对照组(5 mmol·L^(-1)葡萄糖处理48 h)、模型组(50 mmol·L^(-1)葡萄糖处理48 h)和低、中、高剂量Dio处理组(分别用50 mmol·L^(-1)葡萄糖联合0.5 mmol·L^(-1)、2.0 mmol·L^(-1)和8.0 mmol·L^(-1) Dio处理48 h)。CCK-8法检测细胞活性;FITC标记膜联蛋白-V/碘化丙啶(Annexin V-FITC/PI)染色检测细胞凋亡;荧光探针法检测活性氧(ROS)水平;试剂盒法检测丙二醛(MDA)水平、过氧化氢酶(CAT)和谷胱甘肽过氧化物酶(GSH-Px)活性;JC-1染色检测线粒体膜电位;Western blot法检测B细胞淋巴瘤/白血病-2(Bcl-2)、Bcl-2相关X蛋白(Bax)、cleaved Caspase-3和Yes相关蛋白(YAP)的表达水平;免疫荧光法观察YAP的表达。结果筛选的葡萄糖浓度为50 mmol·L^(-1),Dio的无细胞毒性的剂量为0.5 mmol·L^(-1)、2.0 mmol·L^(-1)、8.0 mmol·L^(-1)。与对照组比较,模型组细胞活性、线粒体膜电位、CAT和GSH-Px活性以及Bcl-2蛋白和YAP蛋白表达水平均明显降低(均为P<0.001),细胞凋亡水平、ROS和MDA水平以及cleaved Caspase-3蛋白和Bax蛋白表达水平均明显升高(均为P<0.001);与模型组比较,低、中、高剂量Dio处理组细胞活性、线粒体膜电位、CAT和GSH-Px活性以及Bcl-2蛋白和YAP蛋白表达水平均明显升高(均为P<0.05),细胞凋亡水平、ROS和MDA水平以及cleaved Caspase-3蛋白和Bax蛋白表达水平均明显降低,且均呈剂量依赖性(均为P<0.05)。结论Dio可通过激活YAP信号,降低氧化应激反应和抑制线粒体介导的细胞凋亡途径,进而减轻HG诱导的RPE细胞损伤。Objective To investigate the protective effect of diosgenin(Dio)on retinal pigment epithelial(RPE)cell injury induced by high glucose(HG),and to analyze its mechanism.Methods Glucose concentration and non-cytotoxic Dio dose range were screened by CCK-8 assay.ARPE-19 cells were divided into the control group(treated with 5 mmol·L^(-1) glucose for 48 h),model group(treated with 50 mmol·L^(-1) glucose for 48 h),and low-,medium-and high-dose Dio groups(treated with 50 mmol·L^(-1) glucose combined with 0.5 mmol·L^(-1),2.0 mmol·L^(-1) and 8.0 mmol·L^(-1) Dio,respectively for 48 h).Cell viability was detected by CCK-8 assay.Apoptosis was detected by Annexin V-FITC/PI staining.The levels of reactive oxygen species(ROS)were measured by fluorescence probe.The level of malondialdehyde(MDA)and the activity of catalase(CAT)and glutathione peroxidase(GSH-Px)were measured by assay kits.Mitochondrial membrane potential was tested by JC-1 staining.The expression levels of B-cell lymphoma/leukaemia-2(Bcl-2),Bcl-2-associated X protein(Bax),cleaved Caspase-3 and Yes-associated protein(YAP)were tested by Western blot.The expression of YAP was observed by immunofluorescence.Results The screened glucose concentration was 50 mmol·L^(-1),and the non-cytotoxic dose range of Dio was 0.5 mmol·L^(-1),2.0 mmol·L^(-1) and 8.0 mmol·L^(-1).Compared with the control group,cell viability,mitochondrial membrane potential,CAT and GSH-Px activity,Bcl-2 and YAP levels in the model group were significantly decreased(all P<0.001),while apoptosis,ROS and MDA levels,cleaved Caspase-3 and Bax levels were significantly increased(all P<0.001).Compared with the model group,cell viability,mitochondrial membrane potential,CAT and GSH-Px activity,Bcl-2 and YAP levels in low-,medium-and high-dose Dio groups were significantly increased,while apoptosis,ROS and MDA levels,cleaved Caspase-3 and Bax levels were significantly decreased,all in a dose-dependent manner(all P<0.05).Conclusion Dio can reduce HG-induced RPE cell injury by activating YAP signal,red

关 键 词：薯蓣皂苷 糖尿病视网膜病变 视网膜色素上皮细胞 高糖 氧化应激 细胞凋亡 

分 类 号：R966[医药卫生—药理学] R774.1[医药卫生—药学]