[1]司俊康,郭俊国,郭大东,等.黄芪多糖对过氧化氢诱导人视网膜色素上皮细胞氧化损伤的保护作用[J].眼科新进展,2015,35(1):018-21.[doi:10.13389/j.cnki.rao.2015.0005]
　SI Jun-Kang,GUO Jun-Guo,GUO Da-Dong,et al.Protective effects of astragalus polysaccharide on human retinal pigment epithelium cells against H202-induced oxidative damage[J].Recent Advances in Ophthalmology,2015,35(1):018-21.[doi:10.13389/j.cnki.rao.2015.0005]
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黄芪多糖对过氧化氢诱导人视网膜色素上皮细胞氧化损伤的保护作用
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《眼科新进展》[ISSN:1003-5141/CN:41-1105/R]

卷:
35卷
期数:
2015年1期
页码:
018-21
栏目:
实验研究
出版日期:
2015-01-05

文章信息/Info

Title:
Protective effects of astragalus polysaccharide on human retinal pigment epithelium cells against H202-induced oxidative damage
作者:
司俊康; 郭俊国; 郭大东; 唐凯; 杜宇翔; 王兴荣
２５０００２　山东省济南市，山东中医药大学
Author(s):
SI Jun-Kang ;  GUO Jun-Guo;  GUO Da-Dong ;  TANG Kai;  DU Yu-Xiang ;  WANG Xing-Rong
Shandong University of Traditional Chinese Medicine , Jinan 250002 . Shandong Province , China
关键词:
黄芪多糖; 视网膜色素上皮细胞; 氧化损伤; 凋亡
Keywords:
astragalus polysaccharide ;  retinal pigment epithelium ;  oxidative damage ;  apoptosis
DOI:
10.13389/j.cnki.rao.2015.0005
文献标志码:
A
摘要:
目的　观察黄芪多糖（ａｓｔｒａｇａｌｕｓｐｏｌｙｓａｃｃｈａｒｉｄｅ，ＡＰＳ）对过氧化氢诱导的人视网膜色素上皮细胞（ｒｅｔｉｎａｌｐｉｇｍｅｎｔｅｐｉｔｈｅｌｉｕｍ，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（１０００ｍｇ?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（１０００ｍｇ?Ｌ－１、５００ｍｇ?Ｌ－１）ＡＰＳ后加入２００μｍｏｌＬ－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ｔｉｍｅｃｅｌｌｅｌｅｃｔｒｏｎｉｃｓｅｎｓｉｎｇｓｙｓｔｅｍ，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：１０００ｍｇ?Ｌ－１、５００ｍｇ?Ｌ－１ＡＰＳ孵育２４ｈ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），１０００ｍｇ?Ｌ－１、５００ｍｇ?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。
Abstract:
Objective To observe the protective effects of astragalus polysaccharide ( APS) on human retinal pigment epithelium cells ( ARPE-19) against H202-induced oxidative damage. Methods ARPE-19 cells were cultured in DMEM/F12 medium . and were divided int0 4 groups randomly ,including normal control group , H202 injury group and H202 + 1000 mg . L-l APS group and H202 + 500 mg . L-l APS group. Normal control group received no treatment,H202 injury group was treated with 200 Vmol . L-1 H202 ,H202 + 1000 mg . L-l APS group and H202 + 500 mg . L-l APS group were treated with different concentrations of APS ( 1000 mg . 1’1 ,500 mg . L’l ) and then treated with 200 ymol . L -1 H2 02. 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 electroruc sensing system ( RT-CES) , and the caspase-3 activity was evaluated using the caspase-3 cellular activity assay kit. Results Cells disposed in H202 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") for 24 hours were ( 99. 86 + 3. 64 ) % and ( 101. 03 + 3. 52 ) % , respectively, which was not sigruficantly different with normal control group ( 100% ) ( all P > 0. 05 ) . However , the cell viability in H2 02 injury group was ( 56. 49 + 2. 30) % , which was lower than normal control group ( P < 0. 01 ) , and the cell viability in H2 02 + 1000 mg . L - l APS group and H202 + 500 mg ’ L-1 APS group were ( 88. 14 + 2. 97)% and (80. 75 + 3. 13 ) % .respectively, which were higher than H202 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 H202 injury group , these apoptotic changes were alleviated in H202 + 1000 mg . L -l APS group and H202 + 500 mg . L -1 APS group. RT-CES result demonstrated that cell growth in H202 injury group was suppressed, while cells in other groups grew well. Meanwhile, the caspase-3 activity was inhibited by APS in H202 + 1000 mg . L -1 APS group and H202 + 500 mg . L-l APS group compared with that in H202 injury group. Conclusion APS can exert protective effects on ARPE-19 cells against H202-induced oxidative stress. These results may provide some experimental basis for the possible application of astragalus polysaccharide in clinical practice.

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备注/Memo

备注/Memo:
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）
更新日期/Last Update: 2015-01-04