Molecular hydrogen protects against oxidative stress-induced SH-SY5Y neuroblastoma cell death through the process of mitohormesis

Abstract

Inhalation of molecular hydrogen (H2) gas ameliorates oxidative stress-induced acute injuries in the brain. Consumption of water nearly saturated with H2 also prevents chronic neurodegenerative diseases including Parkinson's disease in animal and clinical studies. However, the molecular mechanisms underlying the remarkable effect of a small amount of H2 remain unclear. Here, we investigated the effect of H2 on mitochondria in cultured human neuroblastoma SH-SY5Y cells. H2 increased the mitochondrial membrane potential and the cellular ATP level, which were accompanied by a decrease in the reduced glutathione level and an increase in the superoxide level. Pretreatment with H2 suppressed H2O2-induced cell death, whereas post-treatment did not. Increases in the expression of anti-oxidative enzymes underlying the Nrf2 pathway in H2-treated cells indicated that mild stress caused by H2 induced increased resistance to exacerbated oxidative stress. We propose that H2 functions both as a radical scavenger and a mitohormetic effector against oxidative stress in cells.

Fig 1. Cell culture system under an atmosphere containing H₂ gas.

(A) Schematic representation of the culture system. 1. Pressure-reducing regulator. 2. Pressure gauge. 3. Flowmeter with flow control valve. 4. Three-way plug valve. 5. Multi-gas controller. 6. Bubbler bottle. 7. Gate valve. 8: Acrylamide box. 9. Culture dish. 10. H₂ electrode. 11. O₂ electrode. Note that, to avoid sudden ignition, H₂- and O₂-containing gases were mixed in a bubbler bottle with water. (B) H₂ and O₂ concentrations in culture medium were monitored with specific electrodes. Under H₂-mixed gas, the H₂ concentration was maintained at 390±40 μM. Under both H₂- and N₂-mixed gases, the O₂ concentration was maintained at 120±10 μM.

Fig 2. Protective effect of H₂ pretreatment against H₂O₂-induced cell death.

(A) For pretreatment with mixed gas, SH-SY5Y cells were incubated in culture medium containing either Glc or Gal under N₂- or H₂-mixed gas for 18 h. Immediately after the end of exposure to the mixed gas, the medium was replaced with fresh medium containing the indicated concentration of H₂O₂. Cells were further incubated in a conventional CO₂ incubator for 18 h. (B) For post-treatment with mixed gases, culture medium containing either Glc or Gal was replaced with fresh medium containing the indicated concentration of H₂O₂. Cells were further incubated under an appropriate mixed gas. After the final incubation, cell viability was estimated by a modified 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrasodium bromide viability assay (A, B). (C) The protective effects of pretreatment with mixed gas containing different concentrations of H₂ against 0.5 mM H₂O₂-induced cell death were examined. (D) The protective effects of pretreatment with N₂- or H₂-mixed gas for different durations against 0.5 mM H₂O₂-induced cell death were examined. Applying two-way ANOVA showed significant effects of mixed gas (P = 0.0017) and duration (P = 0.0315), however no interaction between them was observed (P = 0.2224). (E) After pretreatment with N₂- or H₂-mixed gas, culture medium was replaced with fresh medium, cells were incubated in a conventional CO₂ incubator for the indicated duration, and then H₂O₂ (final 0.5mM) was added. Cells were further incubated for 18 h. All cells used in C—E were cultured in medium containing Glc. After the final incubation, cell viability was estimated by a two-color fluorescence cell viability assay and expressed as a percentage compared with cells not treated with H₂O₂ (considered as 100%) (C—E). *P < 0.05, **P < 0.01, ***P < 0.001 versus treatment with N₂-mixed gas. ^#P < 0.05, ^##P < 0.01 versus an administration period of 0 h.

Fig 3. Enhancement of mitochondrial activities by H₂ treatment.

(A) H₂ treatment enhanced JC-1-indicated ΔΨ_m, which was expressed as the ratio of monomers to aggregates. (B) H₂ treatment enhanced the accumulation of ATP, which was expressed as a percentage compared with cells not treated with mixed gases (considered as 100%). SH-SY5Y cells were incubated in culture medium containing either Glc or Gal under N₂- or H₂-mixed gas for 18 h. **P < 0.01, ***P < 0.001 (A, B). (C) H₂ treatment enhanced the O₂ consumption rate in state 3. O₂ consumption was monitored with high-resolution respirometry. *P < 0.05. (D) The mtDNA copy number relative to nDNA in cells incubated under H₂-mixed gas was quantified by real-time PCR analysis, and expressed relative to those in cells incubated under N₂-mixed gas.

Fig 4. Induction of oxidative stress by H₂ treatment.

SH-SY5Y cells were incubated in culture medium containing either Glc or Gal under N₂- or H₂-mixed gas for 18 h. (A, B) H₂ treatment attenuated staining with MTR, which was expressed as a percentage compared with cells not treated with mixed gases (considered as 100%). The scale bar is 20 μm in (A). (C, D) H₂ treatment decreased the total glutathione and GSH levels. (E) H₂ treatment increased superoxide formation in cells cultured in Gal-containing medium, which was expressed as a percentage compared with cells not treated with mixed gases (considered as 100%). (F) H₂ treatment did not increase DCFDA-indicated ROS, which was expressed as a percentage compared with cells not treated with mixed gases (considered as 100%). *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 5. The H₂-induced anti-oxidative defense system.

(A) SH-SY5Y cells were stained with an anti-Nrf2 antibody. H₂ treatment induced translocation of Nrf2 into the nucleus (arrow heads). The scale bar is 100 μm. (B) Cells were incubated in culture medium containing either Glc or Gal under N₂- or H₂-mixed gas for 18 h. The transcript levels of genes involved in the anti-oxidative defense system, including CAT, GCLC, GPX1, GSR, HO-1 and SOD2, were quantified by real-time PCR analysis coupled with reverse transcription of total RNA, and expressed relative to those in cells incubated under N₂-mixed gas. (C) The expression levels of proteins involved in the anti-oxidative defense system, including γ-GCSc, HO-1, NQO1, Nrf2 and SOD2, were quantified by the intensity of representative immunoblots (n = 5 or 6, each), and expressed relative to those in cells incubated under N₂-mixed gas. *P < 0.05, **P < 0.01. ***P < 0.001.