Lu Ruiting, Liu Yadi, Wang Difen
Department of Critical Care Medicine, the Affiliated Hospital of Guizhou Medical University, Guiyang 550004, Guizhou, China. Corresponding author: Wang Difen, Email:
Zhonghua Wei Zhong Bing Ji Jiu Yi Xue. 2019 Jun;31(6):762-767. doi: 10.3760/cma.j.issn.2095-4352.2019.06.020.
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.
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 (HO) for 20 minutes to determine the appropriate concentration required for astrocyte damage induced by HO; 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, HO injury group, hydrogen-rich water pretreatment group (HW+HO group), and co-culture of hydrogen-rich water and wortmannin pretreatment group (HW+WM+HO 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.
(1) Experiment one: the survival rate of the blank control group was 100%. Cell activity gradually decreased with the increase of HO concentration, and the survival rate of the HO 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 HO 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 HO injury group. Compared with the HO injury group, the PI3K, Akt mRNA expressions and PI3K, Akt, p-Akt protein expressions were significantly increased in the HW+HO group [PI3K mRNA (2): 0.843±0.019 vs. 0.631±0.038, Akt mRNA (2): 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+HO 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+HO group (all P < 0.05), and there was no significant difference with HO injury group (all P > 0.05).
Hydrogen-rich water activates the PI3K/Akt pathway, thereby mediates mice astrocytes to exert the biological function of antioxidant.
探讨富氢水对小鼠星形胶质细胞氧化应激损伤的保护作用及其对磷脂酰肌醇3激酶/蛋白激酶B（PI3K/Akt）信号通路的影响。
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：部分细胞分别用1.25、2.50、5.00、10.00 μmol/L过氧化氢（HO）作用20分钟，以确定HO诱导星形胶质细胞损伤所需的适宜浓度；分别用25、50、100、200 μmol/L富氢水培养3、6、9、12小时，以确定富氢水预处理的浓度和时间；将50 μmol/L富氢水与PI3K/Akt信号通路抑制剂渥曼青霉素（WM）200 nmol/L或400 nmol/L共同培养，以确定渥曼青霉素的最佳抑制浓度。采用甲基噻唑基四氮唑（MTT）比色法检测星形胶质细胞活性。（2）实验二：部分细胞分为空白对照组、HO损伤组、富氢水预处理组（HW+HO组）、富氢水与渥曼青霉素共同预处理组（HW+WM+HO组）。采用逆转录-聚合酶链反应（RT-PCR）检测PI3K和Akt的mRNA表达；采用蛋白质免疫印迹法检测PI3K、Akt及磷酸化Akt（p-Akt）的蛋白表达。
（1）实验一：空白对照组细胞存活率为100%。随着HO浓度增加，细胞活性逐渐降低，2.50 μmol/L HO作用20分钟时细胞存活率降至约50%，故以此浓度建立细胞损伤模型。随着富氢水预处理浓度增加及作用时间延长，细胞存活率先升高后降低。50 μmol/L富氢水预处理9小时时细胞存活率最高，故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，细胞活性受到抑制，200 nmol/L渥曼青霉素组细胞存活率与HO损伤组相比差异无统计学意义，故建立星形胶质细胞抑制模型。（2）实验二：与空白对照组相比，HO损伤组PI3K和Akt的mRNA表达及PI3K、Akt和p-Akt的蛋白表达均显著降低。与HO损伤组相比，HW+HO组PI3K、Akt mRNA表达及PI3K、Akt、p-Akt蛋白表达均显著升高[PI3K mRNA（2）：0.843±0.019比0.631±0.038，Akt mRNA（2）：0.591±0.025比0.558±0.037，PI3K/β-肌动蛋白：1.277±0.008比0.757±0.004，Akt/β-肌动蛋白：1.308±0.015比0.682±0.006，p-Akt/β-肌动蛋白：1.210±0.005比0.614±0.005，均P<0.05]。HW+WM+HO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，均显著低于HW+HO组（均P<0.05），与HO损伤组相比差异无统计学意义（均P>0.05）。
富氢水激活PI3K/Akt通路，从而介导小鼠星形胶质细胞发挥抗氧化生物学功能。
