白藜芦醇对过氧化氢诱导人晶状体上皮细胞氧化损伤的保护作用

Protective effect of resveratrol against H_2O_2-induced oxidative damage in human lens epithelial cells

目的探讨白藜芦醇(resveratrol,RES)对过氧化氢(H2O2)诱导人晶状体上皮细胞(human lens epithelial cells,HLEC)氧化损伤的保护作用。方法 HLEC传代培养24h后,分别加入不同浓度(5μmol·L-1、10μmol·L-1、20μmol·L-1、40μmol·L-1)RES预处理12h后,加入100μmol·L-1H2O2继续孵育24h,倒置相差显微镜观察细胞形态改变,MTT比色法检测RES对H2O2诱导的HLEC活力的影响,流式细胞仪检测HLEC细胞凋亡率,比色法检测凋亡相关因子caspses-3及caspase-9的表达。结果氧化损伤可以诱导HLEC形态改变,RES处理后,细胞形态逐渐得到改善。MTT结果显示RES对HLEC活性无抑制作用,RES(5μmol·L-1、10μmol·L-1、20μmol·L-1、40μmol·L-1)孵育24h后细胞存活率分别为(101.30±4.49)%、(100.31±3.53)%、(101.71±3.33)%、(99.30±3.00)%,与对照组(99.67±2.67)%比较,差异均无统计学意义(均为P>0.05);模型组HLEC经氧化损伤处理后,细胞存活率(34.33±3.71)%明显下降,用20μmol·L-1及40μmol·L-1RES处理后,HLEC存活率分别提高到(57.33±5.61)%和(72.67±6.98)%,与模型组比较差异均有统计学意义(均为P<0.05)。流式细胞计数结果显示:对照组HLEC凋亡率为(1.99±0.17)%,经H2O2处理后,模型组HLEC凋亡率为(51.73±4.97)%,20μmol·L-1、40μmol·L-1RES处理后,HLEC凋亡率分别为(34.43±3.67)%、(26.55±2.07)%,与模型组比较,差异均有统计学意义(均为P<0.05)。此外,RES还可以减少H2O2所致HLEC内caspses-3及caspase-9的表达。结论 RES可以明显抑制HLEC凋亡,其抑制凋亡的作用可能是其防止和延缓白内障发生发展的细胞学基础,从而为寻求有效的防治白内障药物提供可靠的实验依据。

Objective To investigate the protective effect of resveratrol（RES） a gainst H： 02-induced oxidative damage in human lens epithelial cells（HLEC）. Methods After 24-hour subculturing, HLEC were incubated with RES of different concentrations （5 -1mol. L -1,10 -1mol. L--1,20 -μmol. L -1,40 -1mol. L--1） for 12 hours , then H2 02 （ 100 -1mol -1 L--1 ） was added to the culture medium. After 24-hour incubation,morpho logical change of HLEC was detected by inverted phase contrast microscope;Influence of RES on viability of cells processed with H2O2 was detected by MTT assay;Apoptosis rate was determined by flow cytometry（FCM） ;Expressions of apoptosis-related factors of caspses-3 and caspase-9 were detected by colorimetric detection. Results Morphological change of HLEC can be induced by oxidative damage, but that could gradually be improved by RES. MTP assay result showed that RES had no inhibitive effect on HLEC viability. After 24-hour incubation with RES（5 -1mol -1 L -1, l0 txmol -1 L--1 ,20 -1mol -1 L--1 ,40 -1mol -1 L--1 ） ,survival rate of HLEC were（ 101.30 ±4.49）% , （ 100.31 ± 3.53 ）% , （101.71 ±3.33）% and （99.30 ± 3.00）% ,respectively,between any one of which and that of control group was no significant difference （ all P 〉 0.05 ）. Survival rate of HLEC in oxidative damage group decreased significantly to（34.33 ± 3.71 ）% after oxidative damage processing, after incubation with RES （ 20μmol -1 L - 1 and 40 -μmol -1 L - 1 ）, sur vival rate of HLEC increased to （ 57.33 ±5.61 ） % and （ 72.67 ± 6. 98 ） % , respectively, which were significantly higher than that of oxidative damage group （ both P 〈 0.05 ）. According to FCM results, apoptosis rate of HLEC in control group was （1. 99 ± 0. 17）% ,after H202 processing that increased to（51.73 ±4. 97 ）%. After incubation with RES（20 txmol -1 L--1 ,40 txmol -1 L t ） ,apoptosis rate of HLEC was reduced to（34.43 ± 3.67）% and（ 26.55 ±2.07 ）% , which were significantly higher than that of oxidative damage group（ both P 〈 0.05 ）. Moreover, RES could inhibit the expression of caspses-3 and caspase-9 induced by H2 02. Conclusion RES has a apparent inhibitive effect on HLEC apoptosis, which might be the cytological basis of preventing and delaying occur- Fhis could provide reliable experimental basis of seeking efficacious medicine to cataract.