The Inhibitory Effects of Slow-Releasing Hydrogen Sulfide Donors in the Mechanical Allodynia, Grip Strength Deficits, and Depressive-Like Behaviors Associated with Chronic Osteoarthritis Pain

Abstract

Osteoarthritis and its associated comorbidities are important clinical problems that have a negative impact on the quality of life, and its treatment remains unresolved. We investigated whether the systemic administration of slow-releasing hydrogen sulfide (H₂S) donors, allyl isothiocyanate (A-ITC) and phenyl isothiocyanate (P-ITC), alleviates chronic osteoarthritis pain and the associated emotional disorders. In C57BL/6 female mice with osteoarthritis pain induced by the intra-articular injection of monosodium iodoacetate, we evaluated the effects of repeated administration of A-ITC and P-ITC on the (i) mechanical allodynia and grip strength deficits; (ii) emotional conducts; and (iii) glial activity and expression of inducible nitric oxide synthase (NOS2), phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt), and antioxidant enzymes (heme oxygenase 1, NAD(P)H:quinone oxidoreductase-1, glutathione S-transferase mu 1 and alpha 1) in the hippocampus. The administration of A-ITC and P-ITC inhibited the mechanical allodynia, the grip strength deficits, and the depressive-like behaviors accompanying osteoarthritis. Both treatments inhibited microglial activation, normalized the upregulation of NOS2 and PI3K/p-Akt, and maintained high levels of antioxidant/detoxificant enzymes in the hippocampus. Data suggest that treatment with low doses of slow-releasing H₂S donors might be an interesting strategy for the treatment of nociception, functional disability, and emotional disorders associated with osteoarthritis pain.

Keywords: analgesia; anxiety; depression; grip strength; hydrogen sulfide donors; inflammation; microglia; osteoarthritis pain; oxidative stress.

Figure 1

Treatment with allyl isothiocyanate (A-ITC) reduces the mechanical allodynia and the grip strength deficits induced by the intra-articular injection of MIA. The development of (A) mechanical allodynia in the ipsilateral paw and (B) grip strength deficits in the hind paws of the MIA- or SS-injected mice treated with A-ITC or vehicle for 4 consecutive days are shown. The effects of A-ITC were evaluated at days 26, 27, and 29 after MIA or SS injection. For each test and time evaluated, * denotes significant differences vs. their respective SS-injected mice, and $ denotes significant differences vs. MIA-injected mice treated with A-ITC (p < 0.05; one-way ANOVA followed by the Student–Newman–Keuls test). The results are shown as the mean values ± SEM; n = 6 animals per experimental group.

Figure 2

Treatment with phenyl isothiocyanate (P-ITC) decreases the mechanical allodynia and the grip strength deficits induced by the intra-articular injection of MIA. The development of (A) the mechanical allodynia in the ipsilateral paw and (B) the grip strength deficits in the hind paws of the MIA- or SS-injected mice treated with P-ITC or vehicle for 10 consecutive days are presented. The effects of P-ITC were assessed at days 20, 22, 25, and 29 after MIA or SS injection. For each test and time evaluated, * denotes significant differences vs. their respective SS-injected mice, and $ denotes significant differences vs. MIA-injected mice treated with P-ITC (p < 0.05; one-way ANOVA followed by the Student–Newman–Keuls test). The results are shown as the mean values ± SEM; n = 6 animals per experimental group.

Figure 3

Treatment with A-ITC or P-ITC does not alter the anxiety-like behaviors associated with chronic osteoarthritis pain. The anxiety-like behaviors were evaluated on day 29 after MIA or SS injection and at 4 or 10 days of treatment with A-ITC or P-ITC in the elevated plus maze (EPM) and open field (OF) tests. In the EPM, (A) the number of entries into the open arms, (B) percentage of time spent in the open arms, and (C) the number of entries into the closed arms are shown. For OF, (D) the number of entries into the central area, (E) time spent in the central area (s), and (F) the number of squares crossed are shown. For each test evaluated, * denotes significant differences vs. their respective SS-injected mice (p < 0.05; one-way ANOVA followed by the Student-Newman-Keuls test). The results are presented as the mean ± SEM; n = 10 animals per experimental group.

Figure 4

Treatment with A-ITC or P-ITC decreases the depressive-like behaviors associated with chronic osteoarthritis pain. The immobility time (s) evaluated with the (A) tail suspension test (TST) and (B) forced swimming test (FST) at 29 days after MIA or SS injection in mice treated for 4 consecutive days with A-ITC or for 10 days with P-ITC is shown. For each test evaluated, * denotes significant differences vs. SS-injected mice treated with vehicle, and $ denotes significant differences vs. MIA-injected mice treated with a drug (p < 0.05; one-way ANOVA followed by the Student–Newman–Keuls test). The results are presented as the mean values ± SEM; n = 10 animals per experimental group.

Figure 5

The administration of XE-991 reverses the inhibition of the mechanical allodynia and grip strength deficits of A-ITC and P-ITC during chronic osteoarthritis pain. The mechanical allodynia in the ipsilateral paw (A) and grip strength deficits in the hind paws (B) of the MIA-injected mice treated with A-ITC or P-ITC during 4 or 10 days alone and combined with the selective Kv7 potassium channel blocker XE-991 are shown. The effects of XE-991 administered alone are also represented. For each test evaluated, * denotes significant differences vs. saline-saline-injected mice treated with vehicle and + denotes significant differences vs. MIA saline-injected mice treated with a drug (p < 0.05; one-way ANOVA followed by the Student–Newman–Keuls test). The results are shown as the mean values ± SEM; n = 6 animals per experimental group.

Figure 6

The administration of XE-991 reverses the antidepressant effects of A-ITC and P-ITC in mice with chronic osteoarthritis pain. The immobility time (s) evaluated with the (A) TST and (B) FST in mice treated for 4 or 10 consecutive days with A-ITC or P-ITC alone and combined with the selective Kv7 potassium channel blocker XE-991 is shown. The effects of XE-991 administered alone are also represented. For each test evaluated, * denotes significant differences vs. saline–saline-injected mice treated with vehicle and + denotes significant differences vs. MIA–saline-injected mice treated with a drug (p < 0.05; one-way ANOVA followed by the Student–Newman–Keuls test). The results are shown as the mean values ± SEM; n = 6–8 animals per experimental group.

Figure 7

A-ITC and P-ITC treatments inhibit the overexpression of CD11b/c and NOS2 in the hippocampus of mice with osteoarthritis pain. The relative protein levels of (A) CD11b/c, (B) GFAP, and (C) NOS2 in the hippocampus of MIA-injected mice treated with A-ITC, P-ITC, or vehicle are presented. The SS-injected mice treated with vehicle were used as controls. (D) Representative blots for CD11b/c (160 kDa), GFAP (50 kDa), NOS2 (110 kDa), and GAPDH (37 kDa). All proteins are expressed relative to GAPDH levels. In all panels, * denotes significant differences vs. SS-injected mice treated with vehicle, # vs. MIA-injected mice treated with A-ITC and $ vs. MIA-injected mice treated with P-ITC (p < 0.05; one-way ANOVA followed by the Student–Newman–Keuls test). The results are presented as the mean ± SEM; n = 4 samples per experimental group.

Figure 8

A-ITC and P-ITC treatments inhibit the overexpression of phosphatidylinositol 3-kinase (PI3K) and protein kinase B (p-Akt) in the hippocampus of mice with osteoarthritis pain. The relative protein levels of (A) PI3K and (B) p-Akt/Akt in the hippocampus of MIA-injected mice treated with A-ITC, P-ITC, or vehicle are presented. The SS-injected mice treated with vehicle were used as controls. (C) Representative blots for PI3K (140 kDa), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (37 kDa), p-Akt (60 kDa), and Akt (60 kDa). PI3K is expressed relative to GAPDH levels and p-Akt relative to total Akt. In all panels, * denotes significant differences vs. SS-injected mice treated with vehicle, # vs. MIA-injected mice treated with A-ITC and $ vs. MIA-injected mice treated with P-ITC (p < 0.05; one-way ANOVA and Student–Newman–Keuls test). Results are presented as the mean ± SEM; n = 4 samples per experimental group.

Figure 9

A-ITC and/or P-ITC treatments maintain the overexpression of heme oxygenase 1 (HO-1), quinone oxidoreductase-1 (NQO1), glutathione S-transferase mu 1 (GSTM1), and/or glutathione S-transferase alpha 1 (GSTA1) in the hippocampus of mice with osteoarthritis pain. The relative protein levels of (A) HO-1, (B) NQO1, (C) GSTM1, and (D) GSTA1 in the hippocampus of MIA-injected mice treated with A-ITC, P-ITC, or vehicle are presented. The SS-injected mice treated with vehicle were used as controls. (E) Representative blots for HO-1 (32 kDa), NQO1 (28 kDa), GSTM1 (26 kDa), GSTA1 (25 kDa), and GAPDH (37 kDa). All proteins are expressed relative to GAPDH levels. In all panels, * denotes significant differences vs. SS-injected mice treated with vehicle, # vs. MIA-injected mice treated with A-ITC and $ vs. MIA-injected mice treated with P-ITC (p < 0.05; one-way ANOVA and Student–Newman–Keuls test). The results are presented as the mean ± SEM; n = 4 samples per experimental group.