雀异黄酮对甲基苯丙胺诱导的神经细胞损伤保护作用及氧化应激机制研究

Study on protective effect of Genistein on methamphetamine-induced neuronal injury and mechanism of oxidative stress

目的 探究金雀异黄酮(GEN)对甲基苯丙胺(METH)诱导的神经细胞损伤的保护作用及机制。方法 体外培养鼠肾上腺嗜铬细胞瘤细胞系PC12细胞,分为空白对照组(PC12细胞不做特殊处理)、METH组(用终浓度为3 mmol/L METH溶液处理)、GEN低、中、高剂量组(用终浓度为1、10、100μmol/L GEN+3 mmol/L METH溶液处理)、阳性对照组(含终浓度为20μmol/L阿立哌唑溶液处理)。采用四唑盐比色法、Annexin-V FITC/PI双染法分别检测PC12细胞增殖、凋亡能力;氮蓝四唑光还原法检测超氧化物歧化酶(SOD)活性,采用硫代巴比妥酸法检测丙二醛(MDA)含量,铁离子还原法检测活性氧簇(ROS)水平;免疫印迹法检测凋亡相关蛋白[B淋巴细胞瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)]及核因子E2相关因子2/血红素加氧酶-1(Nrf2/HO-1)通路蛋白(Nrf2、HO-1)水平。结果 与空白对照组比较,METH组PC12细胞存活率、SOD活性及Bcl-2、Nrf2、HO-1蛋白水平降低(P<0.05),MDA含量、ROS水平及Bax蛋白水平升高(P<0.05);与METH组比较,GEN低、中、高剂量组及阳性对照组PC12细胞存活率、SOD活性及Bcl-2、Nrf2、HO-1蛋白水平升高(P<0.05),MDA含量、ROS水平及Bax蛋白水平降低(P<0.05),且均呈剂量依赖性;GEN高剂量组以上各指标水平与阳性对照组比较差异无统计学意义(P>0.05)。结论 GEN能促进METH诱导的PC12细胞增殖并抑制细胞凋亡,对神经细胞发挥保护作用,可能与促进Nrf2/HO-1通路活化抑制氧化应激损伤有关。

Objective To investigate the protective effect and mechanisms of Genistein(GEN)on methamphetamine induced neuronal injury.Methods In vitro culture of mouse pheochromocytoma cell line PC12,PC12 cells were divided into control group(no special treatment),METH group(treated with 3 mmol/L METH solution),low,middle and high GEN groups(treated with 1,10,100μmol/L GEN+3 mmol/L METH solution),positive control group(cells were treated with aripiprazole solution containing final concentration of 20μmol/L).The proliferation and apoptosis of PC12 cells were detected by tetrazole salt colorimetry and Annexin-V FITC/PI double staining;the activity of superoxide dismutase(SOD)was detected by blue tetrazole photoreduction assay;and the content of MDA was detected by barbituric acid;reactive oxygen species(ROS)levels were measured by iron ion reduction method.The levels of apoptosis related protein[B lymphocyte 2(Bcl-2),Bcl-2 related x protein(Bax)]and nuclear factor E2 related factor/Heme oxygenase 1(Nrf2/HO-1)pathway protein(Nrf2,HO-1)were detected by Western blotting.Results Compared with control group,the PC12 cell survival rate,SOD activity,Bcl-2,Nrf2,HO-1 protein level in METH group was decreased(P<0.05);MDA content,ROS level and Bax protein level was increased(P<0.05).Compared with METH group,the survival rate,SOD activity and Bcl-2,Nrf2,HO-1 protein level of PC12 cells in low,middle and high dose groups and positive control group were increased(P<0.05),while MDA content,ROS level and Bax protein level was decreased(P<0.05),which was dose dependent.There was no significant difference between the high dose group and the positive control group(P>0.05).Conclusion GEN can promote proliferation and inhibit apoptosis of PC12 cells induced by METH,which may be related to activation of Nrf2/HO-1 pathway and inhibition of oxidative stress injury.