Article Publish Status: FREE
Abstract Title:

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

Abstract Source:

Zhonghua Wei Zhong Bing Ji Jiu Yi Xue. 2019 Jun ;31(6):762-767. PMID: 31315738

Abstract Author(s):

Ruiting Lu, Yadi Liu, Difen Wang

Article Affiliation:

Ruiting Lu


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, 10.00μmol/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 200μmol/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, HOinjury group, hydrogen-rich water pretreatment group (HW+HOgroup), and co-culture of hydrogen-rich water and wortmannin pretreatment group (HW+WM+HOgroup). 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 HOconcentration, and the survival rate of the HOaction 20 minutes cells of 2.50μmol/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 HOinjury 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 HOinjury group. Compared with the HOinjury group, the PI3K, Akt mRNA expressions and PI3K, Akt, p-Akt protein expressions were significantly increased in the HW+HOgroup [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+HOgroup 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+HOgroup (all P<0.05), and there was no significant difference with HOinjury group (all P>0.05).

CONCLUSIONS: Hydrogen-rich water activates the PI3K/Akt pathway, thereby mediates mice astrocytes to exert the biological function of antioxidant.

Study Type : In Vitro Study

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