EGCG通过Nrf2/ARE信号通路改善鱼藤酮诱导的SH-SY5Y细胞氧化损伤的研究

Epigallocatechin-3-gallate attenuates rotenone-induced oxidative stress in SH-SY5Y cells via regulating Nrf2/ARE signal pathway

目的研究表没食子儿茶素没食子酸酯(EGCG)对鱼藤酮诱导的SH-SY5Y细胞氧化损伤的作用并探讨可能的机制。方法将SH-SH5Y细胞分为对照组(正常培养细胞)、模型组(鱼藤酮诱导的氧化损伤模型)和低、中、高剂量实验组(分别给予10、20、40μmol·L^(-1)EGCG)。用小干扰RNA(siRNA)干扰细胞核转录因子红细胞系-2p45相关因子2(Nrf2)表达,将干扰后细胞分为si-对照组(细胞正常培养)、si-模型组(鱼藤酮诱导的氧化损伤模型)、si-低剂量实验组、si-中剂量实验组(分别给予10、20μmol·L^(-1)EGCG)。用细胞计数试剂盒-8(CCK-8)法检测细胞存活率;用乳酸脱氢酶(LDH)法检测细胞损伤情况;用还原型辅酶Ⅱ法检测总谷胱甘肽过氧化物酶(GPx)活性;用硫代巴比妥酸法检测丙二醛(MDA)活性;用荧光探针法检测活性氧(ROS)水平;用蛋白质印迹法检测Nrf2、酪氨酸羧化酶(TH)蛋白表达水平。结果对照组、模型组和低、中、高剂量实验组细胞存活率分别为(100.00±1.86)%、(45.33±4.25)%、(74.21±4.54)%、(80.30±3.62)%和(70.12±4.61)%;LDH水平分别为(100.00±3.67)、(222.56±11.46)、(125.57±20.52)、(129.60±6.29)和(149.51±11.99)U·L^(-1);ROS相对荧光强度分别为1.00±0.00、1.78±0.16、1.48±0.15、1.13±0.09和1.11±0.13;MDA活性分别为(2.11±0.34)、(6.39±0.55)、(4.84±0.36)、(3.96±0.46)和(4.43±0.43)nmol·mg^(-1);GPx活性分别为(668.80±32.79)、(533.03±57.72)、(718.30±73.78)、(735.03±87.25)和(797.98±66.76)mU·mg^(-1);TH蛋白相对表达水平分别为0.33±0.04、0.21±0.03、0.30±0.01、0.31±0.03和0.27±0.01;模型组上述指标与对照组比较,高剂量实验组上述指标与模型组比较,在统计学上差异均有统计学意义(P<0.05,P<0.01)。对照组、模型组和低、中、高剂量实验组Nrf2蛋白表达水平分别为0.26±0.07、0.28±0.02、0.39±0.05、0.54±0.09和0.53±0.02,低、中、高剂量实验组与模型组比较,在统计学上差异均有统计学意义(P<0.05,P<0.01)。si-对照组、si-模型组和si-低剂量实验组、si-中剂量实验组的细胞存活率分别为(100.00±5.73)%、(50.69±5.48)%、(61.95±8.15)%和(60.59±9.07)%;ROS相对荧光强度分别为1.00±0.05、1.48±0.10、1.30±0.15和1.28±0.22。si-模型组与si-对照组比较,在统计学上差异均有统计学意义(均P<0.05)。结论EGCG可能通过Nrf2/ARE信号通路对鱼藤酮所致SH-SY5Y细胞氧化损伤发挥保护作用。

Objective To explore the protective mechanism of epigallocatechin gallate(EGCG)on rotenone-induced oxidative stress of SH-SY5Y nerve cells.Methods SHSH5Y cells were divided into control group(normal culture cells),model group(rotenone injury model)and experimental-L,-M,-H groups(10,20,40μmol·L^(-1)EGCG,respectively).Using small interfering RNA(siRNA)to interfere with the expression of nuclear factor erythroid-2p45-related factor 2(Nrf2),the cells were divided into si-control group(normal cell culture),si-model group(rotenone injury model),si-experimental-L group and si-experimental-M group(10 and 20μmol·L^(-1)EGCG,respectively).Cell proliferation was measured by cell counting kit-8(CCK-8)method;cytotoxicity was detected by lactate dehydrogenase(LDH)method;activity of total glutathione peroxidase(GPx)was detected by NADPH method;activity of malondialdehyde(MDA)was detected by thiobarbituric acid method;reactive oxygen species(ROS)levels were detected by fluorescence probe;the expression levels of Nrf2 and tyrosine hydroxylase(TH)protein were detected by Western blotting.Results The cell viabilities of control group,model group and experimental-L,-M,-H groups were(100.00±1.86)%,(45.33±4.25)%,(74.21±4.54)%,(80.30±3.62)%and(70.12±4.61)%;the LDH release were(100.00±3.67),(222.56±11.46),(125.57±20.52),(129.60±6.29)and(149.51±11.99)U·L^(-1);the ROS relative fluorescence intensities were 1.00±0.00,1.78±0.16,1.48±0.15,1.13±0.09 and 1.11±0.13;the MDA levels were(2.11±0.34),(6.39±0.55),(4.84±0.36),(3.96±0.46)and(4.43±0.43)nmol·mg^(-1);the activities of GPx were(668.80±32.79),(533.03±57.72),(718.30±73.78),(735.03±87.25)and(797.98±66.76)mU·mg^(-1);the TH protein expression levels were 0.33±0.04,0.21±0.03,0.30±0.01,0.31±0.03 and 0.27±0.01,respectively.Among the above indicators,the differences between the model group and the control group,experimental-H group and the model group,were statistically significant respectively(P<0.05,P<0.01).Nrf2 protein expression levels in control group,model group and experimental-L,-M,-H groups were0.26±0.07,0.28±0.02,0.39±0.05,0.54±0.09 and 0.53±0.02,respectively,the differences between the experimental-L,-M,-H groups and the model group,were statistically significant respectively(P<0.05,P<0.01).The cell survival rates of si-control group,si-model group,si-experimental-L group and siexperimental-M group were(100.00±5.73)%,(50.69±5.48)%,(61.95±8.15)%and(60.59±9.07)%;the ROS relative fluorescence intensity were 1.00±0.05,1.48±0.10,1.30±0.15 and 1.28±0.22,respectively.The difference between si-model group and si-control group were statistically significant(all P<0.05).Conclusion EGCG may reduce the rotenone-induced oxidative stress in SH-SY5Y cells by activating the Nrf2/ARE signaling pathway.