白藜芦醇对高浓度葡萄糖诱导人晶状体上皮细胞氧化损伤的拮抗作用及其可能机制探讨

Resveratrol alleviates oxidative injury from high glucose-induced human lens epithelial cells and its possible mechanism

目的 探讨白藜芦醇对高糖诱导的人晶状体上皮细胞（LEC）氧化损伤的拮抗作用及其可能的机制.方法 实验研究.使用不同浓度（5.5、15.0、25.0、35.0、45.0 mmol/L）葡萄糖培养LEC;25.0 mmol/L葡萄糖培养LEC不同时间（0、6、12、24、48、72 h）并建立（5.0、15.0、25.0、35.0 mg/L）白藜芦醇干预模型.连接素V-碘化丙锭双染色流式细胞法测定检测细胞凋亡,流式细胞术检测细胞中活性氧（ROS）含量,免疫印迹法检测凋亡蛋白Bcl-2、Bax、iNOS、NF-κB、IκB、锰超氧化物歧化酶（MnSOD）的表达,试剂盒分析氧化损伤标记物MDA的含量变化.组间差异比较采用单因素方差分析.结果 高糖诱导SRA01/04细胞凋亡表现为浓度和时间的依赖性,随着葡萄糖浓度的升高和作用时间的延长,抗凋亡蛋白Bcl-2表达降低,促凋亡蛋白Bax表达增多.葡萄糖诱导的细胞内ROS和丙二醛（MDA）含量变化也具有明显的浓度和时间依赖性;与5.5 mmol/L组相比,高糖培养ROS产生量显著增加[15.0 mmol/L（F=14.06,P=0.035）,25.0 mmol/L（F=17.46,P=0.000）,35.0 mmol/L（F=16.58,P=0.001）、45.0 mmol/L（F=12.88,P=0.000）],差异均有统计学意义;与5.5 mmol/L培养相比,25.0 mmol/L高糖培养6h（F=6.778,P=0.014）、12 h（F =6.551,P=0.001）、24 h（F=7.327,P=0.001）,48 h（F=10.84,P =0.000）、72 h（F=13.36,P=0.000）的LEC中ROS产生量显著增加,差异均有统计学意义;与5.5 mmol/L组相比,高糖培养MDA含量显著增加15.0 mmol/L（F=1.177,P=0.035）,25.0 mmol/L（F=1.704,P=0.000）,35.0 mmol/L（F=2.412,P =0.001）、45.0 mmol/L（F=2.347,P=0.000）,差异均有统计学意义;与5.5 mmol/L培养相比,25 mmol/L高糖培养6 h（F=1.704,P=0.014）、12 h（F=5.676,P=0.001）、24 h（F=3.325,P=0.001）,48 h（F =6.669,P=0.000）、72 h（F=3.011,P=0.000）的LEC中MDA含量显著增加,差异均有统计学意义.25.0 mmol/L高糖培养中加入5.0、15.0、25.0、35.0 mg/L白藜芦醇后细胞的凋亡减少,促凋亡蛋白Bax表达降低,抗凋亡蛋白Bcl-2表达增多,细胞中的ROS（与基础浓度5.5 mmol/L相比,F值分别为14.76,7.018,13.96,4.733,1.921,P值分别为0.000,0.000,0.003,0.086,0.100）、MDA（与基础浓度5.5 mmol/L相比,F值分别为2.454,1.108,1.630,1.563,2.250,P值分别为0.000,0.001,0.026,0.068,0.183）的表达量均减少;且MnSOD的表达量显著增多,iNOS、NF-κB的活性被抑制.结论 白藜芦醇对高糖刺激LEC氧化损伤具有保护作用,推测其作用机制可能是通过抑制NF-κB的转录活性,从而抑制iNOS介导的细胞氧化损伤.

Objective To investegate the effects and its mechanism of resveratrol against human lens epithelial cells （LEC） apoptosis mediated by high glucose-induced oxidative injury.Methods An experimental study.LEC were cultured in different concentrations （5.5,15.0,25.0,35.0,45.0 mmol/L） of glucose medium or 25.0 mmol/L glucose medium at different time （0,6,12,24,48,72 h）,and established an interventional models of （5.0,15.0,25.0,35.0 mg/L） resveratrol.Av-FITC-PI （annexin V-fluorescein isothiocyanate-propidium iodium） was used to detect apoptosis.The amount of ROS was calculated by flow cytometry.The expression of apoptosis of the protein Bcl-2 and Bax,iNOS,NF-κB,IκB and MnSOD were showed by Western blotting and the amount of oxidative damage marker MDA was explored by Spectrometers and Analytical Photometers.Differences between the two groups were evaluated by One-way ANOVA.Results The apoptosis of LEC induced by high glucose was time-and dose-dependent obviously.As the glucose concentration increased and duration prolonged,the expression of anti-apoptotic protein Bcl-2 was decreased and pro-apoptotic protein Bax was increased.Intracellular ROS and MDA induced by high glucose were increased significantly with dose-and time-dependenc.Compared with 5.5 mmol/L group,ROS generation increased significantly in the concentration of 15.0 mmol/L （F =14.06,P =0.035）,25.0 mmol/L （F =17.46,P =0.000）,35.0 mmol/L （F =16.58,P =0.001）,45.0 mmol/L （F =12.88,P =0.000） and were statistically significant.Compared with 5.5 mmol/L glucose cultured group,ROS generation increased significantly at 6 h （F =6.778,P =0.014）,12 h （F =6.551,P=0.001）,24h （F=7.327,P =0.001）,48 h （F=10.84,P =0.000）,72 h （F=13.36,P =0.000） in LEC cultured group by 25.0 mmol/L glucose and were statistically significant.Compared with 5.5 mmol/L group,the content of MDA were significantly increased in 15.0 mmol/L （F =1.177,P =0.035）,25.0 mmol/L （F=1.704,P =0.000）,35.0 mmol/L （F =2.412,P =0.001） and 45.0 mmol/L （F =2.347,P =0.000） glucose medium and were statistically significant.Compared with 5.5 mmol/Lcultured group,the content of MDA were significantly increased at 6 h （F =1.704,P =0.014）,12 h （F =5.676,P=0.001）,24h （F=3.325,P=0.001）,48 h （F=6.669,P=0.000）,72 h （F=3.011,P =0.000） in LEC cultured group by 25.0 mmol/L high glucose and were statistically significant.When resveratrol （5.0,15.0,25.0,35.0 mg/L） was added to 25.0 mmol/L glucose medium,respectively,the apoptotic cells were decreased,the expression of pro-apoptotic protein Bax was decreased and anti-apoptotic protein Bcl-2 was increased.Intracellular ROS （compared with the basic concentration of 5.5 mmol/L,F values were 14.76,7.018,13.96,4.733,1.921,P values were 0.000,0.000,0.003,0.086,0.100respectively） and MDA （compared with the basic concentration of 5.5 mmol/L,F values were 2.454,1.108,1.630,1.563,2.250,P values were 0.000,0.001,0.026,0.068,0.183 respectively） were decreased.MnSOD expression was increased,iNOS and NF-κB activation were inhibited.Conclusion Resveratrol could alleviates oxidative injury from high glucose-induced LEC,and inhibited of iNOS-mediated oxidative damage through inhibiting the activities of NF-κB could be the mechanism of this effect.