Epigallocatechin-3-gallate attenuates rotenone-induced oxidative stress in SH-SY5Y cells via regulating Nrf2/ARE signal pathway
Objective To explore the protective mechanism of epigallocatechin gallate(EGCG)on rotenone-induced oxidative stress of SH-SY5Y nerve cells.Methods SH-SH5Y cells were divided into control group(normal culture cells),model group(rotenone injury model)and experimental-L,-M,-H groups(10,20,40 μmol·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 20 μmol·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 were 0.26±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 si-experimental-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.
Epigallocatechin-3-gallaterotenoneoxidative stressnuclear factor erythroid-2p45-related factor 2/antioxidant response element pathway
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