Molecular hydrogen increases resilience to stress in mice

Abstract

The inability to successfully adapt to stress produces pathological changes that can lead to depression. Molecular hydrogen has anti-oxidative and anti-inflammatory activities and neuroprotective effects. However, the potential role of molecular hydrogen in stress-related disorders is still poorly understood. The present study aims to investigate the effects of hydrogen gas on resilience to stress in mice. The results showed that repeated inhalation of hydrogen-oxygen mixed gas [67%:33% (V/V)] significantly decreased both the acute and chronic stress-induced depressive- and anxiety-like behaviors of mice, assessed by tail suspension test (TST), forced swimming test (FST), novelty suppressed feeding (NSF) test, and open field test (OFT). ELISA analyses showed that inhalation of hydrogen-oxygen mixed gas blocked CMS-induced increase in the serum levels of corticosterone, adrenocorticotropic hormone, interleukin-6, and tumor necrosis factor-α in mice exposed to chronic mild stress. Finally, inhalation of hydrogen gas in adolescence significantly increased the resilience to acute stress in early adulthood, which illustrates the long-lasting effects of hydrogen on stress resilience in mice. This was likely mediated by inhibiting the hypothalamic-pituitary-adrenal axis and inflammatory responses to stress. These results warrant further exploration for developing molecular hydrogen as a novel strategy to prevent the occurrence of stress-related disorders.

Figure 1

Repeated inhalation of hydrogen gas enhanced resilience to acute stress in mice. (A) Experimental procedure. After a 5-day adaptation period, the mice were given daily administration of saline, fluoxetine (10 mg/kg, i.p.), or inhaled mixture gas of H₂/O₂ [67%/33% (v/v)] or O₂/N₂ [33%/67% (v/v)] for 1, 3 h daily for 14 days. Beginning on day 15, behavioural tests were conducted to assess the depressive- and anxiety-like behaviours. Inhalation of hydrogen gas significantly decreased the immobility time of mice in the TST (B) and the floating time in the FST (C), increased the time spent in the central zone (D) without affecting the crossing activities (E) in the OFT, and decreased the latency to feeding (F) without affecting the total feeding in homecages (G) during the NSF test. *P < 0.05, **P < 0.01 versus the saline-treated control group. n = 8–11 per group. TST, tail suspension test; FST, forced swimming test; OFT, open field test; NSF, novelty suppressed feeding test.

Figure 2

Repeated inhalation of hydrogen gas blocked the depressive- and anxiety-like behaviours in chronically stressed mice. (A) Experimental procedure. After a 5-day adaptation, mice were treated by chronic stress for 28 days. Beginning on day 14, mice inhaled mixture gas of H₂/O₂ [67%/33% (v/v)] or O₂/N₂ [33%/67% (v/v)] for 3 h daily or were injected with fluoxetine (10 mg/kg i.p.) daily 0.5 h before stress for 14 days. During day 28–30, behavioural tests were conducted to assess the depressive- and anxiety-like behaviours. Repeated inhalation of hydrogen gas significantly blocked the chronic stress-induced decrease in sucrose preference (B) without affecting the total intake (C) in the SPT, the increased latency to feeding (D) without affecting the total feeding in home cage (E) in the NSF test, and the decreased time spent in the central zone (F) without affecting the crossing activities (G) in the OFT. ^#P < 0.05 and ^##P < 0.01 versus the control group; *P < 0.05 and **P < 0.01 versus the saline-treated CMS group. n = 8–10 per group. CMS, chronic mild stress; SPT, sucrose preference test; NSF, novelty suppressed feeding test; OFT, open field test.

Figure 3

Repeated inhalation of hydrogen gas inhibited the chronic stress-induced increase in serum levels of CORT, ACTH, IL-6, and TNF-α. (A) Experimental procedure. After a 5-day adaptation, mice were treated with chronic stress procedure for 28 days. Beginning on day 14, mice were inhaled with mixture gas of H₂/O₂ [67%/33% (v/v)] or O₂/N₂ [33%/67% (v/v)] for 3 h daily 0.5 h before stress for 14 days. On day 29, mice were decapitated and serum samples were collected for ELISA analysis. Inhalation of hydrogen gas significantly blocked the increased serum levels of CORT (B), ACTH (C), IL-6 (D), and TNF-α (E) in chronically stressed mice. ^#P < 0.05 and ^##P < 0.01 versus the control group; *P < 0.05 and **P < 0.01 versus the saline-treated CMS group. n = 4–6 per group. CMS, chronic mild stress; CORT, corticosterone; ACTH, adrenocorticotropic hormone; IL-6, interlukin-6; TNF-α, tumour necrosis factor-α.

Figure 4

Repeated inhalation of hydrogen gas in adolescence increased resilience to acute stress in adulthood. (A) Experimental procedure. On the 28^th postnatal day (PND), the mice were inhaled mixture gas of H₂/O₂ [67%/33% (v/v)] or O₂/N₂ [33%/67% (v/v)] for 1, 3 h daily for 14 days. Behavioural tests were conducted to assess the acute stress-induced depressive- and anxiety-like behaviours. Inhalation of hydrogen gas significantly decreased the floating time in the FST (B) and the immobility time of mice in the TST (C), decreased the latency to feeding (D), but without affecting the total feeding in homecages (E) during the NSF test. *P < 0.05, **P < 0.01 versus the control group. n = 9–10 per group. TST, tail suspension test; FST, forced swimming test. NSF, novelty suppressed feeding test.