白藜芦醇对星形胶质细胞氧化损伤的保护作用

摘要

目的：探讨白藜芦醇( RES)对过氧化氢诱导星形胶质细胞氧化损伤的保护作用及其机制。方法星形胶质细胞传代培养,随机分为阴性对照组(以正常培养液培养),模型对照组(100μmol·L-1的过氧化氢作用12 h),RES小剂量组(20μmol·L-1 RES孵育24 h后,加入过氧化氢作用12 h)和RES大剂量组(40μmol·L-1 RES孵育24 h后,加入过氧化氢作用12 h),采用MTT比色法测定细胞活力,流式细胞仪检测细胞凋亡率,Hochest33258染色观察凋亡细胞形态,比色法检测凋亡相关因子caspses-3及caspase-9的表达。结果 MTT结果显示5,10,20,40μmol·L-1 RES孵育24 h后细胞活性分别为(100.46±3.17)％,(101.33±3.14)％,(101.33±1.30)％,(99.67±2.62)％,与阴性对照组(98.33±2.13)％比较,差异无统计学意义(P>0.05)。表明RES对星形胶质细胞活性无影响。用20及40μmol·L-1 RES处理后,星形胶质细胞在过氧化氢作用12 h后引起损伤,其活性分别提高到(54.67±4.11)％和(70.33±2.61)％,与模型对照组比较,差异有统计学意义(t=3.59,7.13,P<0.01)。 RES可以抑制过氧化氢诱导的细胞活性的下降,流式细胞计数结果显示用20,40μmol·L-1RES处理后,星形胶质细胞凋亡率分别下降到(35.51±3.56)％和(14.12±3.19)％,与模型对照组(46.31±4.16)％比较,差异有统计学意义(t=4.26,6.33 P<0.01)。 Hochest33258染色结果显示RES可以抑制过氧化氢诱导的细胞凋亡。此外,RES还可以减少过氧化氢所致星形胶质细胞内caspses-3及caspase-9的表达,并且伴随RES作用时间的延长其表达量呈下降趋势。结论 RES通过抑制caspses-3及caspase-9的表达有效抑制了过氧化氢对星形胶质细胞的损伤,从而为其用于治疗中枢神经疾病提供实验依据。%Objective To investigate the protective effect of resveratrol ( RES) against hydrogen peroxide ( H2 O2 )-induced oxidative injury to astrocytes and the related mechanism. Methods Subcultured astrocytes were randomly divided into four groups:negative control group ( treated with normal culture medium) , model control group ( treated with 100 μmol·L-1 H2 O2 for 12 h), resveratrol low dose group (treated with 20 μmol·L-1 RES for 24 h H2O2 for 12 h) and resveratrol high dose group ( treated with 40 μmol·L-1 RES for 24 h before H2 O2 for 12 h) . Cell viability was detected by MTT assay, apoptosis rate was detected by flow cytometry, apoptotic cell morphology was detected by hochest33258 staining, and the expression of apoptosis-related factors such as caspses-3 and caspase-9 were measured by colorimetric detection. Results MTT assay showed that after treatment with 5, 10, 20, and 40 μmol·L-1 RES for 24 h, cell viability was (100. 46±3. 17)%, (101. 33± 3.14)%, (101. 33±1. 30)%, and (99. 67±2. 62)%, respectively, and the difference was not statistically significant as compared with the negative control group [(98. 33±2. 13)%, P>0. 05]. RES showed no effect on astrocyte activity, after treatment with 20 and 40 μmol·L-1 RES, astrocyte activity was significantly elevated to (54. 67±4. 11)% and (70.33± 2. 61)% as compared with model control group (t=3. 59, 7. 13, P<0. 05), RES inhibited hydrogen peroxide-induced decrease in cell viability. Flow cytometry results showed that after treatment with 20, 40 μmol·L-1 RES, the apoptosis rate of astrocytes significantly decreased to (35.51±3. 56)% and (14. 12%±3. 19)% (t=4. 26, 6. 33, P<0. 01) as compared with model control group (46. 31±4. 16)%. Hochest 33258 staining showed that RES inhibited hydrogen peroxide-induced cell apoptosis, besides, the RES treatment also could reduce H2 O2-induced expression of caspses-3 and caspase-9 in astrocytesin a time-dependent manner. Conclusion RES can inhibit hydrogen peroxide-induced astrocytes apoptosis through inhibiting the expression of caspses-3 and caspase-9, which can provide experimental evidence for its treatment of central nervous disorders.