丹参酮ⅡA对缺氧状态下视网膜色素上皮细胞增生的抑制作用及其机制  

Inhibitory effect of Tanshinone ⅡA on the proliferation of human retinal pigment epithelial cells in hypoxia and its mechanism

作　　者：雷祥 栗占荣[1] 范珂[1] Lei Xiang;Li Zhanrong;Fan Ke(Department of Ophtalmology, Henan Provincial People's Hospital, Henan Provincial Eye Hospital, Henan Provincial Ophthalmology Institute, Zhengzhou 450003, China)

机构地区：[1]河南省人民医院眼科河南省立眼科医院河南省眼科研究所,郑州450003

出　　处：《中华实验眼科杂志》2019年第5期342-347,共6页Chinese Journal Of Experimental Ophthalmology

基　　金：国家自然科学基金项目(U1304811).

摘　　要：目的探讨丹参酮ⅡA在缺氧条件下对人视网膜色素上皮(RPE)细胞增生的抑制作用及相关信号通路的影响。方法体外培养ARPE-19细胞,细胞分为空白对照组、缺氧对照组、不同质量浓度丹参酮ⅡA干预组和缺氧诱导因子1α(HIF-1α)抑制剂组。利用氯化钴(CoCl2)建立缺氧环境,MTT法检测各组细胞加药培养24、48和72 h后的增生抑制率,流式细胞仪检测各组细胞的凋亡率及细胞周期分布情况,real-time PCR及Western blot法检测各组HIF-1α和血管内皮生长因子(VEGF)mRNA及蛋白表达情况。结果MTT法检测结果显示,丹参酮ⅡA可呈时间和质量浓度依赖性地抑制细胞增生,1、5、10 mg/L丹参酮ⅡA组细胞增生抑制率依次升高,两两比较差异均有统计学意义(均P<0.05)。流式细胞术检测结果显示,1、5和10 mg/L丹参酮ⅡA组细胞凋亡率依次升高,两两比较差异均有统计学意义(均P<0.05);缺氧对照组、1 mg/L丹参酮ⅡA组、5 mg/L丹参酮ⅡA组、10 mg/L丹参酮ⅡA组G0/G1期细胞比例依次升高,S期细胞比例依次降低,两两比较差异均有统计学意义(均P<0.05)。RT-PCR和Western blot检测结果显示,空白对照组、缺氧对照组、1 mg/L丹参酮ⅡA组、5 mg/L丹参酮ⅡA组、10 mg/L丹参酮ⅡA组和HIF-1α抑制剂组中VEGF mRNA、HIF-1α蛋白和VEGF蛋白相对表达量的总体比较,差异均有统计学意义(均P<0.05),其中缺氧对照组、1 mg/L丹参酮ⅡA组、5 mg/L丹参酮ⅡA组、10 mg/L丹参酮ⅡA组VEGF mRNA、HIF-1α蛋白、VEGF蛋白相对表达量依次降低,两两比较差异均有统计学意义(均P<0.05)。10 mg/L丹参酮ⅡA组与HIF-1α抑制剂组各检测指标比较,差异均无统计学意义(均P>0.05)。结论丹参酮ⅡA在缺氧条件下可以抑制RPE细胞增生,将细胞阻滞在G0/G1期,促进细胞凋亡,可能与其对HIF-1α/VEGF信号通路的抑制作用有关。Objective To investigate the effect of Tanshinone ⅡA on the proliferation and the signaling pathway of human retinal pigment epithelial (RPE) cells in hypoxia. Methods CoCl2 (150 μmol/L) was used to simulate hypoxic condition and the ARPE-19 cells cultured in vitro were divided into blank control group, hypoxia control group, Tanshinone ⅡA group and hypoxia-inducible factor-1α(HIF-1α) inhibitor group.The different doses of Tanshinone ⅡA were used to treat ARPE-19 for 24, 48 and 72 hours, respectively.The inhibitory rate of cell proliferation of different groups were detected by MTT after 24, 48 and 72 hours of administration cultured, and the apoptosis rate and the cell cycle distribution of cells in the hypoxia were analyzed by flow cytometry.Real-time PCR and Western blot were used to detect the expressions of mRNA and protein of HIF-1α and vascular endothelial growth factor (VEGF). Results MTT assay showed that Tanshinone ⅡA could inhibit the proliferation of ARPE-19 cells in a dose- and time-dependent manner, and the proliferation inhibitory rate gradually increased in the 1, 5 and 10 mg/L Tanshinone ⅡA groups, with significant differences between any two groups (all at P<0.05). Flow cytometry showed that the apoptosis rate of ARPE-19 in 1, 5 and 10 mg/L Tanshinone ⅡA groups gradually increased with the elevation of Tanshinone ⅡA dosage, with significant differences between any two groups (all at P<0.05). The cell proportion in the G0/G1 phase gradually increased, while the cell proportion in the S phase gradually decreased along with the elevation of Tanshinone ⅡA concentration, significant differences were obtained among the hypoxia control group, 1, 5 and 10 mg/L Tanshinone ⅡA groups (all at P<0.05). RT-PCR and Western blot showed that the relative expression of VEGF mRNA, HIF-1α and VEGF protein in the the blank control group, hypoxia control group, 1, 5 and 10 mg/L Tanshinone ⅡA groups and HIF-1α inhibitor group were significantly different (all at P<0.05). The expression of VEG

关 键 词：丹参酮ⅡA 视网膜色素上皮细胞 缺氧诱导因子1Α 血管内皮生长因子 增生 

分 类 号：R774.1[医药卫生—眼科]