紫花牡荆素对BEAS-2B细胞氧化应激损伤的作用研究

Study on the effect of casticin on oxidative stress injury in BEAS-2B cells

目的探讨紫花牡荆素对BEAS-2B细胞氧化应激的影响及可能作用机制。方法用H_(2)O_(2)诱导人正常支气管上皮(BEAS-2B)细胞构建氧化应激模型。将细胞分为空白组(正常培养细胞)、模型组(氧化应激模型)、紫花牡荆素组(10μmol·L^(-1)紫花牡荆素预处理后造模)、ML385组(20μmol·L^(-1)ML385预处理后造模)、联合组(10μmol·L^(-1)紫花牡荆素+20μmol·L^(-1)ML385预处理后造模)和阳性对照组(40μg·L^(-1)维生素C预处理后造模)。用流式细胞术检测细胞中活性氧(ROS)水平;用细胞计数-8(CCK-8)法检测细胞的增殖能力;用生化实验检测细胞中超氧化物歧化酶(SOD)和丙二醛(MDA)的水平;用蛋白质印迹法检测细胞中Kelch样环氧氯丙烷相关蛋白-1(Keap1)、核因子E2相关因子2(Nrf2)和血红素氧合酶1(HO-1)的相对表达水平。结果空白组、模型组、紫花牡荆素组、ML385组、联合组和阳性对照组ROS水平分别为(3.85±0.17)%,(29.80±1.15)%,(20.57±1.21)%,(31.55±1.04)%,(21.81±0.39)%和(21.23±0.67)%;这6组的细胞增殖率分别为(100.00±1.60)%,(67.95±1.10)%,(77.31±1.34)%,(66.48±0.51)%,(74.74±0.61)%和(79.57±1.32)%;这6组SOD水平分别为(75.44±1.34),(27.32±1.96),(42.48±0.59),(38.62±1.49),(56.93±0.68)和(58.29±0.92)U·mg^(-1);这6组MDA水平分别为(6.15±0.46),(31.51±0.60),(24.65±0.62),(22.96±0.19),(16.34±0.53)和(14.57±0.75)nmol·mg^(-1);这6组Keap1的相对表达水平分别为0.27±0.02,0.66±0.01,0.44±0.03,0.67±0.05,0.54±0.03和0.65±0.06;这6组Nrf2相对表达水平分别为0.55±0.03,0.32±0.02,0.47±0.03,0.33±0.04,0.39±0.03和0.32±0.02;这6组HO-1相对表达水平分别为0.49±0.03,0.30±0.01,0.41±0.02,0.30±0.02,0.36±0.02和0.29±0.03。紫花牡荆素组、联合组与模型组比较,细胞增殖能力、SOD、Nrf2蛋白和HO-1蛋白的相对表达水平均明显增加,MDA、ROS和Keap1蛋白的相对表达水平均明显降低,差异均有统计学意义(均P<0.05)。结论紫花牡荆素能够缓解H_(2)O_(2)诱导的BEAS-2B细胞的氧化应激损伤,增加细胞的增殖能力,其作用机制可能与Keap1-Nrf2/ARE信号通路有关。

Objective To investigate the effects and possible mechanisms of casticin on oxidative stress in BEAS-2B cells.Methods Oxidative stress model in human normal bronchial epithelial(BEAS-2B)cells was induced with H_(2)O_(2).Cells were divided into blank group(normal cultured cells),model group(oxidative stress model),casticin group(10μmol·L^(-1)zymosan pretreated and modeled),ML385 group(20μmol·L^(-1)ML385 pretreated and modeled),combined group(10μmol·L^(-1)casticin+20μmol·L^(-1)ML385 pretreated and modeled)and positive control group(40μg·L^(-1)vitamin C pretreated and modeled).Reactive oxygen species(ROS)levels in cells was detected by flow cytometry;the proliferative capacity of each group of cells was detected by the cell counting-8(CCK-8)method;the levels of superoxide dismutase(SOD)and malondialdehyde(MDA)in each group of cells were measured by biochemical assays;the relative expression levels of Kelch-like ECH-associated protein 1(Keap1),nuclear factor erythroid-2 related factor(Nrf2)and heme oxygenase 1(HO-1)in the cells were measured by Western Blot.Results The ROS levels of the blank group,model group,casticin group,ML385 group,combined group and positive control group were 3.85±0.17,29.80±1.15,20.57±1.21,31.55±1.04,21.81±0.39 and21.23±0.67,respectively;cell proliferation capacities in these six groups were(100.00±1.60)%,(67.95±1.10)%,(77.31±1.34)%,(66.48±0.51)%,(74.74±0.61)%and(79.57±1.32)%,respectively;SOD levels in these six groups were(75.44±1.34),(27.32±1.96),(42.48±0.59),(38.62±1.49),(56.93±0.68)and(58.29±0.92)U·mg^(-1),respectively;MDA levels in these six groups were(6.15±0.46),(31.51±0.60),(24.65±0.62),(22.96±0.19),(16.34±0.53)and(14.57±0.75)nmol·mg^(-1),respectively;Keap1 levels in these six groups were 0.27±0.02,0.66±0.01,0.44±0.03,0.67±0.05,0.54±0.03 and 0.65±0.06,respectively;Nrf2 levels in these six groups were 0.55±0.03,0.32±0.02,0.47±0.03,0.33±0.04,0.39±0.03 and 0.32±0.02,respectively;HO-1 levels in these six groups were 0.49±0.03,0.30±0.01,0.41±0.02,0.30±0.02,0.36±0.02 and 0.29±0.03,respectively.The casticin group and combine group showed statistically significant increases in cell proliferation capacity,SOD levels,and relative expression of Nrf2,HO-1,and Nrf2,and statistically significant decreases in MDA levels,ROS levels,and Keap1 relative expression compared with those in model group(all P<0.05).Conclusion Casticin can alleviate H_(2)O_(2)-induced oxidative stress damage in BEAS-2B cells and increase cell proliferation,and its mechanism of action may be related to the Keap1-Nrf2/ARE signaling pathway.