枸杞多糖对H_2O_2诱导人晶状体上皮细胞氧化应激损伤的保护作用

Protective effects of lycium barbarum polysaccharides on human lens epithelial cells against H_2O_2-induced oxidative stress

目的探讨枸杞多糖(lycium barbarum polysaccharides,LBP)对H2O2诱导人晶状体上皮细胞(SRA01/04)氧化应激损伤的保护作用。方法将SRA01/04细胞传代培养24 h后,加入不同浓度(50 mg·L^(-1)、100 mg·L^(-1)、200 mg·L^(-1)、400 mg·L^(-1)、800 mg·L^(-1)和1600 mg·L^(-1))LBP预处理24 h,之后加入200μmol·L^(-1)H2O2继续培养24 h,用CCK-8检测LBP对H2O2诱导的SRA01/04细胞活力的影响并筛选出LBP最佳保护浓度用于后续实验。采用流式细胞仪检测SRA01/04细胞凋亡率和线粒体膜电位变化情况,Western blot检测各组凋亡相关蛋白Bcl-2及Bax的表达情况。结果 CCK-8检测结果显示:LBP浓度为200 mg·L^(-1)和400 mg·L^(-1)时,能够促进SRA01/04细胞增殖,细胞存活率分别为(121.10±5.56)%和(128.20±3.79)%,与对照组(99.98±4.73)%比较,差异均有统计学意义(均为P<0.05)。H2O2模型组SRA01/04细胞经氧化应激损伤后,细胞存活率(51.67±4.91)%明显降低,与对照组(99.67±2.52)%相比,差异有统计学意义(P<0.001)。用200 mg·L^(-1)和400mg·L^(-1)LBP预处理后,SRA01/04细胞存活率明显提高至(74.01±3.21)%及(84.67±4.33)%,与H2O2模型组比较差异均有统计学意义(均为P<0.01)。400 mg·L^(-1)LBP对RA01/04细胞氧化应激损伤保护作用最大。流式细胞仪检测结果显示:对照组细胞凋亡率为(5.1±1.2)%;H2O2模型组细胞凋亡率为(25.9±1.5)%;400 mg·L^(-1)LBP预处理后,细胞凋亡率降至(13.8±1.2)%,与H2O2模型组比较,差异有统计学意义(P<0.01)。线粒体膜电位检测结果显示:对照组阳性细胞数最多,线粒体膜电位高;H2O2模型组阳性细胞数最少,线粒体膜电位低;400 mg·L^(-1)LBP预处理后,阳性细胞数增多及线粒体膜电位升高,与H2O2模型组相比,差异有统计学意义(P<0.001)。Western blot检测显示:与对照组相比,H2O2模型组Bcl-2表达下降,Bax表达上升(P<0.01);经400 mg·L^(-1)LBP预处理后,Bcl-2表达上升,Bax表达下降,与H2O2模型组比较,差异有统计学意义(P<0.01)。结论 LBP对H2O2诱导人晶状体上皮细胞的氧化应激损伤具有保护作用,可抑制细胞凋亡。

Objective To investigate the protective effects of lycium barbarum polysaccharides（LBP） against H_2O_2-induced oxidative damage in human lens epithelial cells.Methods After 24 hours sub-culturing,SRA01 /04 cells were incubated with LBP of different concentrations（50 mg · L^-1,100 mg · L^-1 200 mg · L^-1,400 mg · L^-1,800 mg · L^-1 and1600 mg · L^-1） for 24 hours,and then H_2O_2（200 μmol · L^-1） was added to the culture medium.After 24-hour incubation,cell viability was measured using cell counting kit-8（CCK-8） assay.Protection concentration of LBP was selected by CCK-8 assay and used in subsequent experiment.Cell apoptosis and the loss of mitochondria membrane potential were detected by flow cytometric analyses.Expression levels of Bcl-2 and Bax proteins were measured by Western blot analysis.Results CCK-8 assay showed that LBP could promote SRA01/04 cell proliferation.After 24-hour incubation with LBP（200 mg · L^-1 and 400 mg ·L^-1）,survival rate of SRA01/04 cells were（121.10 ±5.56）%and（128.20 ±3.79）%,respectively,which were significantly higher than that of control（99.98 ± 4.73）%（all P〈0.05）.Survival rate of SRA01 /04 cells in oxidative damage group decreased significantly to（51.67 ±4.91）%after incubation with H_2O_2,there was significant difference compared with control group（99.67 ± 2.52）%（P〈0.001）.After incubation with 200 mg·L^-1 and 400 mg · L^-1 LBP,survival rate of SRA01 /04 cells increased to（74.01 ± 3.21）%and（84.67 ±4.33）%,which were significantly higher than that of oxidative damage group（P〈0.01）.According to FC M results,apoptotic rate of SRA01/04 cells in control group and H_2O_2 group was（5.1 ± 1.2%）%and（25.9 ± 1.5）%,after pre-cubation with 400 mg · L^-1 LBP,apoptotic rate of SRA01 /04 cells was reduced to（13.8 ± 1.2）%,which was significantly lower than that of H_2O_2 group（P〈0.01）.Detection of mitochondrial membrane potential showed that the control group had the most number of positive cells,H_2O_2 group had the least number of positive cells,the number of positive cells of LBP group had specifically significant different with H_2O_2 group（P〈0.001）.Western blot assay showed that the expression level of Bcl-2 were decreased,while the expression of Bax was increased in H_2O_2 group.After incubation with 400 mg · L^-1 LBP,the expression level of Bcl-2 were increased,while the expression of Bax was decreased,there was significant difference compared with H_2O_2 group（P〈0.01）.Conclusion The pre-administration of LBP can effectively protect SRA01/04 cells against H_2O_2-induced oxidative stress and inhibit cell apoptosis.