富氢水对氧化应激细胞模型的保护作用及对PI3K/Akt信号通路的影响

Protective effect of hydrogen-rich water on oxidative stress cell model and the impact of the phosphatidylinositol 3 kinase/protein kinase B pathway

目的观察富氢水对小鼠星形胶质细胞氧化应激损伤的保护作用及对磷酸肌醇3激酶/蛋白激酶B(PI3K/Akt)信号通路的影响.方法体外培养小鼠星形胶质细胞,取对数生长期细胞进行实验.①实验一:取部分细胞,用1.25、2.50、5.00、10.00μmol/L过氧化氢(H2O2)作用细胞20 min,以确定H2O2诱导星形胶质细胞损伤所需的适宜浓度;用25、50、100、200μmol/L富氢水分别培养3、6、9、12 h,以确定富氢水预处理的最佳浓度及时间;用50μmol/L富氢水与PI3K/Akt信号通路抑制剂渥曼青霉素200 nmol/L或400 nmol/L共同培养,以确定渥曼青霉素最佳抑制浓度.采用四甲基偶氮唑盐(MTT)比色法检测星形胶质细胞存活率.②实验二:取部分细胞,按随机数字表法分为空白对照组、H2O2损伤组、富氢水预处理组(HW+H2O2组)、富氢水与渥曼青霉素共培养预处理组(HW+WM+H2O2组).采用反转录-聚合酶链反应(RT-PCR)检测PI3K、Akt的mRNA表达,用蛋白质免疫印迹试验(Western Blot)检测PI3K、Akt、磷酸化Akt(p-Akt)的蛋白表达.结果①实验一:以空白对照组细胞存活率为100%.细胞存活率随H2O2浓度的增加而逐渐降低,2.50μmol/L的H2O2作用20 min细胞存活率降至50%左右,故以此浓度建立细胞损伤模型.细胞存活率随富氢水预处理浓度升高、作用时间延长呈先升高后降低趋势,50μmol/L富氢水预处理9 h时细胞存活率最高,故以此建立富氢水预保护模型.200 nmol/L或400 nmol/L渥曼青霉素与富氢水共同培养后,细胞活性得到抑制,200 nmol/L的渥曼青霉素干预后细胞存活率与H2O2损伤组差异无统计学意义,故以此建立星形胶质细胞抑制模型.②实验二:与空白对照组比较,H2O2损伤组PI3K、Akt的mRNA表达及PI3K、Akt、p-Akt的蛋白表达均明显降低.与H2O2损伤组比较,HW+H2O2组PI3K、Akt的mRNA表达及PI3K、Akt、p-Akt的蛋白表达均明显升高〔PI3K mRNA(2^-ΔΔCT):0.843±0.019比0.631±0.038,Akt mRNA(2^-ΔΔCT):0.591±0.025比0.558±0.037,PI3K/β-actin:1.277±0.008比0.757±0.004,Akt/β-actin:1.308±0.015比0.682±0.006,p-Akt/β-actin:1.210±0.005比0.614±0.005,均P<0.05〕.HW+WM+H2O2组PI3K、Akt的mRNA表达分别为0.784±0.159和0.556±0.037,PI3K、Akt、p-Akt的蛋白表达分别为0.715±0.006、0.686±0.005和0.606±0.004,均明显低于HW+H2O2组(均P<0.05),而与H2O2损伤组比较差异无统计学意义(均P>0.05).结论富氢水通过激活PI3K/Akt信号通路介导小鼠星形胶质细胞而发挥抗氧化的生物学功能.

Objective To explore the protective effect of hydrogen-rich water on the oxidative stress injury of astrocytes in mice and its effect on phosphatidylinositol 3 kinase/protein kinase B (PI3K/Akt) signal pathway. Methods In vitro, mice astrocytes were cultured and the logarithmic growth period cells were taken for experiment.① Experiment one: some cells were acted by 1.25, 2.50, 5.00, 10.00 μmol/L hydrogen peroxide (H2O2) for 20 minutes to determine the appropriate concentration required for astrocyte damage induced by H2O2;cultivating 3, 6, 9, and 12 hours with hydrogen-rich water of 25, 50, 100, and 200 μmol/L, respectively, to determine the concentration and time of hydrogen-rich water pretreatment;the 50 μmol/L hydrogen-rich water was cultured together with PI3K/Akt signal pathway inhibitors wortmannin (WM) 200 nmol/L or 400 nmol/L to determine the best inhibition concentration of wortmannin. Astrocyte activity was detected by methyl thiazolyl tetrazolium (MTT) colorimetry.② Experiment two: some cells were divided into blank control group, H2O2 injury group, hydrogen-rich water pretreatment group (HW+H2O2 group), and co-culture of hydrogen-rich water and wortmannin pretreatment group (HW+WM+H2O2 group). The mRNA expressions of PI3K and Akt were detected by reverse transcription-polymerase chain reaction (RT-PCR);the protein expressions of PI3K, Akt and phosphorylated Akt (p-Akt) were detected by Western Blot. Results ① Experiment one: the survival rate of the blank control group was 100%. Cell activity gradually decreased with the increase of H2O2 concentration, and the survival rate of the H2O2 action 20 minutes cells of 2.50 μmol/L was reduced to about 50%, so a cell injury model was established at this concentration. With the increase of hydrogen-rich water pretreatment concentration, and the duration of action, the cell survival rate increased first and then decreased. The cell survival rate was highest when 50 μmol/L hydrogen-rich water was pretreated with 9 hours, so a hydrogen-rich water pre-protection model was established. After 200 nmol/L or 400 nmol/L wortmannin was cultured together with hydrogen-rich water, cell activity was inhibited, and the cell survival rate of 200 nmol/L wortmannin group was no significantly different compared with that of H2O2 injury group, so the astrocyte suppression model was established.② Experiment two: compared with the blank control group, the mRNA expressions of PI3K and Akt and the protein expressions of PI3K, Akt and p-Akt were significantly decreased in the H2O2 injury group. Compared with the H2O2 injury group, the PI3K, Akt mRNA expressions and PI3K, Akt, p-Akt protein expressions were significantly increased in the HW+H2O2 group [PI3K mRNA (2^-ΔΔCT): 0.843±0.019 vs. 0.631±0.038, Akt mRNA (2^-ΔΔCT): 0.591±0.025 vs. 0.558±0.037, PI3K/β-actin: 1.277±0.008 vs. 0.757±0.004, Akt/β-actin: 1.308±0.015 vs. 0.682±0.006, p-Akt/β-actin: 1.210±0.005 vs. 0.614±0.005, all P < 0.05]. The mRNA expressions of PI3K, Akt in the HW+WM+H2O2 group was 0.784±0.159 and 0.556±0.037, respectively, and the protein expressions of PI3K, Akt, p-Akt was 0.715±0.006, 0.686±0.005, and 0.606±0.004, respectively, both were significantly lower than those in HW+H2O2 group (all P < 0.05), and there was no significant difference with H2O2 injury group (all P >0.05). Conclusion Hydrogen-rich water activates the PI3K/Akt pathway, thereby mediates mice astrocytes to exert the biological function of antioxidant.