摘要
目的观察黄芪多糖(astragalus polysaccharide,APS)对过氧化氢诱导的人视网膜色素上皮细胞(retinal pigment epithelium,RPE)氧化损伤的保护作用。方法培养的人RPE细胞系ARPE-19分为正常对照组、过氧化氢损伤组和不同浓度(1000 mg·L-1、500mg·L-1)APS干预组,正常对照组不作任何处理,过氧化氢损伤组加入200μmol·L-1过氧化氢,APS干预组加入不同浓度(1000 mg·L-1、500 mg·L-1)APS后加入200μmol·L-1过氧化氢。用倒置荧光显微镜观察各组细胞形态变化;MTT检测各组细胞活性;DAPI染色观察各组细胞核形态学变化;实时细胞电子分析系统(real-time cell electronic sensing system,RT-CES)实时记录细胞生长状态;应用caspase-3活性检测试剂盒测定各组细胞中caspase-3的活性。结果过氧化氢损伤组细胞皱缩,悬浮细胞增多,APS干预后细胞状态改善。MTT检测结果显示:1000 mg·L-1、500 mg·L-1APS孵育24 h后细胞存活率分别为(99.86±3.64)%和(101.03±3.52)%,与正常对照组(100%)相比,差异均无统计学意义(均为P>0.05),过氧化氢损伤组细胞存活率为(56.49±2.30)%,与正常对照组相比显著降低(P<0.01),1000 mg·L-1、500 mg·L-1APS干预后,细胞存活率升高到(88.14±2.97)%和(80.75±3.13)%,与过氧化氢损伤组相比差异均有显著统计学意义(均为P<0.01)。DAPI染色显示正常细胞核均匀淡染,过氧化氢损伤组出现强荧光点和致密的细胞核,APS处理后凋亡程度减轻。RT-CES显示,APS处理可以减少过氧化氢造成的细胞损伤。Caspase-3检测发现过氧化氢造成细胞caspase-3水平升高,APS处理后caspase-3水平下降。结论 APS可以抑制过氧化氢诱导的人RPE细胞系ARPE-19的凋亡,这为APS的临床应用提供了一定的实验基础。
Objective To observe the protective effects of astragalus polysaccharide （APS） on human retinal pigment epithelium ceils （ARPE-19） against H2O2-induced oxidative damage. Methods ARPE-19 cells were cultured in DMEM/F12 medium, and were divided into 4 groups randomly,including normal control group ,H2O2 injury group and H2O2 ＋ 1000 mg·L^-1 APS group and H2O2 ＋ 500 mg ·L^-1 APS group. Normal control group received no treatment, H2O2 injury group was treated with 200 μmol ·L^-1 H2O2 ,H2O2 ＋ 1000 mg ·L^-1 APS group and H2O2 ＋ 500 mg·L^-1 APS group were treated with different concentrations of APS （1000 mg ·L^-1 ,500 mg ·L^-1） and then treated with 200 μmol·L^-1 H2O2. The cellular morphology was observed by inverted fluorescence microscopy, the cell viability was measured using MTT assay and apoptosis was evaluated using DAPI staining, the cell proliferation was dynamically monitored using the real-time cell electronic sensing system （ RT-CES）, and the caspase-3 activity was evaluated using the caspase-3 cellular activity assay kit. Results Cells disposed in H2O2 were sparse and shrinkage, but it could be improved by the adding of APS. MTT assay determination showed that the cell viability treated with APS （ 1000 mg·L^-1 ,500 mg·L^-1 ） for 24 hours were （99.85 ±3.64）% and （ 101.03 ±3.52）% ,respectively, which was not significantly different with normal control group （ 100% ） （ all P 〉 0.05 ）. However,the cell viability in H2O2 injury group was （55.49 ±2.30）% ,which was lower than normal control group（P 〈0.01 ） ,and the cell viability in H2O2 ＋ 1000 mg·L^-1 APS group and H2O2 ＋ 500 mg·L^-1 APS group were （88. 14 ±2.97）% and （80. 75 ±3.13）% ,respectively,which were higher than H2O2 injury group （all P 〈0.01 ）. DAPI staining revealed that characteristic apoptotic changes, such as convoluted nuclei with cavitations and strong fluorescent spots appeared in ARPE-19 cells in H2O2 injury group,these apoptotic changes were alleviated in H2O2 ＋ 1000 mg ·L^-1 APS group and H2O2 ＋ 500 mg·L^-1 APS group. RT-CES result demonstrated that cell growth in H2O2 injury group was suppressed, while cells in other groups grew well. Meanwhile, the caspase-3 activity was inhibited by APS in H2O2 ＋ 1000 mg ·L^-1 APS group and H2O2 ＋ 500 mg·L^-1 APS group compared with that in H2O2 injury group. Conclusion APS can exert protective effects on ARPE-19 cells against H2O2 -induced oxidative stress. These results may provide some experimental basis for the possible application of astragalus polysaccharide in clinical practice.
出处
《眼科新进展》
CAS
北大核心
2015年第1期18-21,共4页
Recent Advances in Ophthalmology
基金
山东省自然科学基金资助(编号:ZR2009CM135)
山东省中医药科技发展计划(编号:2011-130)~~
关键词
黄芪多糖
视网膜色素上皮细胞
氧化损伤
凋亡
astragalus polysaccharide
retinal pigment epithelium
oxidative damage
apoptosis
