Treatment with Hydrogen-Rich Water Improves the Nociceptive and Anxio-Depressive-like Behaviors Associated with Chronic Inflammatory Pain in Mice

Abstract

Chronic inflammatory pain is manifested in many diseases. The potential use of molecular hydrogen (H₂) as a new therapy for neurological disorders has been demonstrated. Recent studies prove its analgesic properties in animals with neuropathic pain, but the possible antinociceptive, antidepressant, and/or anxiolytic actions of H₂ during persistent inflammatory pain have not been investigated. Therefore, using male mice with chronic inflammatory pain incited by the subplantar injection of complete Freud's adjuvant (CFA), we assessed the actions of hydrogen-rich water (HRW) systemically administered on: (1) the nociceptive responses and affective disorders associated and (2) the oxidative (4-hydroxy-2-nonenal; 4-HNE), inflammatory (phosphorylated-NF-kB inhibitor alpha; p-IKBα), and apoptotic (Bcl-2-like protein 4; BAX) changes provoked by CFA in the paws and amygdala. The role of the antioxidant system in the analgesia induced by HRW systemically and locally administered was also determined. Our results revealed that the intraperitoneal administration of HRW, besides reducing inflammatory pain, also inhibited the depressive- and anxiolytic-like behaviors associated and the over expression of 4-HNE, p-IKBα, and BAX in paws and amygdala. The contribution of the nuclear factor erythroid 2-related factor 2/heme oxygenase 1 and NAD(P)H: quinone oxidoreductase 1 pathway in the analgesic activities of HRW, systemically or locally administered, was also shown. These data revealed the analgesic, antidepressant, and anxiolytic actions of HRW. The protective, anti-inflammatory, and antioxidant qualities of this treatment during inflammatory pain were also demonstrated. Therefore, this study proposes the usage of HRW as a potential therapy for chronic inflammatory pain and linked comorbidities.

Keywords: allodynia; anxiety; apoptosis; depression; hyperalgesia; inflammatory pain; molecular hydrogen; oxidative stress.

Figure 1

Effects of the intraperitoneal injection of HRW or VEH on the mechanical allodynia and thermal hyperalgesia induced by paw inflammation. Effects of the repetitive administration of HRW or VEH, injected at 1T and 2T per day, over the decreased von Frey filaments strength (g) (A) and the withdrawal latency (s) (B) generated by CFA in the ipsilateral hind paws. For each test, * indicates significant changes vs. their respective SS-injected mice (SS + VEH, SS + HRW 1T, or SS + HRW 2T), + represents significant differences vs. CFA + VEH injected mice, and # denotes significant differences vs. CFA + HRW 1T (p < 0.05, on one-way ANOVA and Student–Newman–Keuls test). Data are presented as mean values ± SEM; n = 6 animals for group.

Figure 2

Effects of the subplantar administration of HRW or VEH on the mechanical allodynia and thermal hyperalgesia generated by paw inflammation. Effects of the subplantar administration of HRW or VEH, injected at 2T per day, over the decreased von Frey filaments strength (g) (A) and withdrawal latency (s) (B) provoked by CFA in the ipsilateral hind paws. For each test, * indicates significant variations vs. SS-injected mice treated with VEH (SS + VEH), and + represents significant differences vs. CFA-injected mice treated with VEH (CFA + VEH) (p < 0.05, one-way ANOVA and Student–Newman–Keuls test). Data are shown as mean values ± SEM; n = 6 animals for group.

Figure 3

Treatment with HRW inhibited the depressive-like behaviors linked with chronic inflammatory pain. Immobility times (s) in the TST (A) and FST (B) at day 16 after CFA injection in animals intraperitoneally treated with HRW or VEH, injected at 2T per day for two consecutive days, are represented. The effects of HRW and VEH in SS-injected mice are also shown. For each test evaluated, * denotes significant differences vs. SS-injected mice treated with VEH, # vs. SS-injected mice treated with HRW, and $ vs. CFA-injected mice treated with HRW (p < 0.05, one-way ANOVA and Student–Newman–Keuls test). Data are shown as mean values ± SEM; n = 8 animals for group.

Figure 4

Treatment with HRW inhibited the anxiety-like behaviors linked with chronic inflammatory pain. The effects of the intraperitoneal treatment with HRW or VEH in the EPM and OF tests, at 16 days after CFA injection, are represented. In the EPM test, the number of entries to the open arms (A), the percentage of time spent in the open arms (B), and the number of entries into the closed arms (C) are shown. In the OF test, the number of entries in the central area (D), the percentage of time spent in the central area (E), and the number of squares crossed (F) are represented. The effects of HRW and VEH in SS-injected mice are also shown. For each test evaluated, * denotes significant differences vs. SS-injected mice treated with VEH, # vs. SS-injected mice treated with HRW, and $ vs. CFA-injected mice treated with HRW (p < 0.05; one-way ANOVA and Student–Newman–Keuls test). Data are presented as mean values ± SEM; n = 8 animals for group.

Figure 5

Reversion of the analgesic effects of HRW produced by inhibitors of the Nrf2/HO-1-NQO1 signaling in animals with inflammatory pain. Effects of the intraperitoneal co-administration of HRW with a Nrf2 inhibitor (ML-385, 25 mg/kg) (A,B), an HO-1 inhibitor (SnPP, 10 mg/kg) (C,D), a NQO1 inhibitor (dicoumarol, 10 mg/kg) (E,F), or VEH in the mechanical allodynia (A,C,E) and thermal hyperalgesia (B,D,F) provoked by CFA in the ipsilateral paws of mice are shown. For each test, * denotes significant differences vs. their respective SS-injected mice, + denotes significant differences vs. CFA-injected mice treated with VEH plus VEH, and # denotes significant differences vs. CFA-injected mice treated with HRW plus VEH (p < 0.05; one-way ANOVA and Student–Newman–Keuls test). Data are presented as mean values ± SEM; n = 6 animals for group.

Figure 6

Reversion of the local analgesic actions of HRW produced by the Nrf2/HO-1-NQO1 signaling inhibitors during inflammatory pain. Effects of the subplantar co-administration of HRW with specific inhibitors of Nrf2 (ML-385, 650 μg), HO-1 (SnPP, 250 μg), and NQO1 (dicoumarol, 250 μg) or VEH on the mechanical allodynia (A) and thermal hyperalgesia (B) caused by CFA in the ipsilateral paws of mice are shown. For each test, ∞ denotes significant differences vs. other groups (p < 0.05; one-way ANOVA and Student–Newman–Keuls test). Data are presented as mean values ± SEM; n = 6 animals for group.

Figure 7

Effects of HRW on the expression of 4-HNE, BAX, and p-IKBα in the paw tissues of CFA-injected mice. The protein levels of 4-HNE (A), BAX (B), and p-IKBα (C) in the ipsilateral paws of CFA-injected mice treated with VEH or HRW are shown. SS-injected mice treated with VEH, used as controls, are also shown. Examples of blots for 4-HNE, BAX, and p-IKBα are displayed (D). 4-HNE and BAX are expressed relative to GAPDH levels, while p-IKBα is expressed relative to IKBα. In all graphics, * denotes significant differences vs. SS-injected mice plus VEH, and $ denotes significant differences vs. CFA-injected animals plus HRW (p < 0.05; one-way ANOVA and Student–Newman–Keuls test). Data are shown as the mean ± SEM; n = 3 samples.

Figure 8

Effects of treatment with HRW on the expression of 4-HNE, BAX, and p-IKBα in the amygdala of CFA-injected mice. The protein levels of 4-HNE (A), BAX (B), and p-IKBα (C) in the amygdala of CFA-injected mice treated with VEH or HRW are shown. SS-injected mice treated with VEH, used as controls, are also shown. Examples of blots for 4-HNE, BAX, and p-IKBα are displayed (D). 4-HNE and BAX are expressed relative to GAPDH levels, while p-IKBα is expressed relative to IKBα. In all graphics, * denotes significant differences vs. SS-injected mice plus VEH, and $ denotes significant differences vs. CFA-injected animals plus HRW (p < 0.05; one-way ANOVA and Student–Newman–Keuls test). Data are shown as the mean ± SEM; n = 3 samples.