1. Introduction
Osteoarthritis (OA) is the most common articular chronic disease. Despite this high incidence in the population, especially in the elderly, its etiology has not been completely elucidated yet. Likewise, there is no cure for this pathology and its current treatment is limited and mainly focused on alleviating pain, attenuating its progression, and maintaining articular function [
1].
Although OA was classically considered to be mainly a consequence of the “wear and tear” of the articular cartilage, we now know that it is a more complex pathology that involves both mechanical and biological processes occurring not only in the cartilage but in the entire joint [
1]. In this regard, the increased production of pro-inflammatory mediators in the cartilage and synovial tissue contribute to cartilage destruction that in turn amplifies joint inflammation, creating a vicious circle that contributes to OA development [
2,
3,
4]. Chondrocytes from OA cartilage actively produce pro-inflammatory cytokines, prostaglandins (PGs), or metalloproteinases (MMPs), among others. PGs are key factors in inflammatory processes that are generated by cycloxygenase and PG synthase enzymes after metabolizing arachidonic acid. The expression of inducible isoform of cyclooxygenase, COX-2, is elicited by inflammatory stimuli and oxidative stress [
5,
6]. This enzyme is responsible for the synthesis of PGE
2, one of the most important pro-inflammatory prostaglandins in the OA [
7]. Additionally, the reactive oxygen species (ROS) production is upregulated in OA, causing oxidative stress. As a result, the byproduct of oxidative damage to proteins, lipids, or DNA could strongly promote the activation of pathological pathways in the joint, including inflammatory and destructive processes [
8,
9]. Thus, oxidative stress may participate in the pathogenesis of this disease [
5]. For instance, the excessive production of nitric oxide (NO) by inducible NO synthase (iNOS) is considered a catabolic event responsible for perpetuating OA pathogenesis by mediating the expression of proinflammatory cytokines, inhibiting the synthesis of the extracellular matrix proteins collagen and proteoglycan [
10]. Besides this, NO has been demonstrated to modulate COX-2 expression and prostaglandin production [
11] as well as the activation of MMPs in the cartilage [
12]. MMPs are well characterized enzymes involved in the degradation of the extracellular matrix (ECM) of the cartilage [
13,
14]. Among them, MMP-13 (collagenase 3) is an enzyme constitutively expressed in the chondrocyte that degrades collagenous ECM [
15]. However, its aberrant expression and activity elicited by pro-inflammatory mediators and oxidative stress has been associated with diseases such as OA [
15,
16,
17].
In relation to OA treatment, disease-modifying drugs have yet to be identified, and the current consensus guidelines recommends the use of a combination of conservative measures, including physical therapy, analgesia, and surgical interventions such as arthroplasty [
18]. In the pursuit of an effective and alternative pharmacological treatment for this disease, hydrogen sulfide (H
2S), an endogenous gas with recognized physiological activities, has emerged as a putative molecule showing a protective effect on different pathological pathways activated in OA cartilage [
19,
20,
21,
22]. These actions could be due largely to the role of H
2S in cellular redox homeostasis. This gas shows antioxidant properties, as it quenches reactive oxygen species (ROS) and reactive nitrogen species (RNS) or increases the expression of antioxidant enzymes by activating the transcription factor nuclear factor erythroid-derived 2-like 2 (Nrf-2), among other mechanisms [
23]. Nonetheless, the biosynthesis of this gas is reduced in OA cartilage [
24]. Likewise, our group previously detected that the exogenous administration of H
2S reduced the pro-inflammatory production induced by IL-1β in cultured human chondrocytes, as well as protecting against the degradation of the matrix in ex vivo experiments in OA cartilage explants [
19,
20]. Additionally, other authors observed in vitro similar actions of the gas in the chondrocyte, such as attenuating inflammatory signaling and exerting chondroprotective effects [
21,
22,
25]. By in vivo experimental approaches, different studies have also detected the beneficial effect of the administration of H
2S donors on rheumatic disease progression and pain [
26,
27]. Interestingly, we have recently shown that balneotherapy in sulfur-rich water attenuates the destruction of cartilage and pain levels in an OA model in rats by the surgical destabilization of the joint [
28].
Overall, a number of findings suggest the effect of H
2S-releasing molecules in alleviating symptoms and attenuating progression in different pathologies [
29]. However, scarce studies have directly evaluated in vivo the impact of exogeneous H
2S administration on the signaling pathways involved in the pathogenesis of disorders such as OA. To better understand the potential role of H
2S as a therapeutic candidate in the treatment of rheumatic diseases, we analyzed in this study the effect of the intraarticular injection of GYY-4137, a slow-releasing H
2S donor [
30], in a surgically induced model of OA in rats.
3. Discussion
OA is a multifactor chronic disease that causes pain and disability as, during its development, the entire joint organ is affected. There is no cure for OA, and treatment mainly involves drug intervention strategies in order to relieve pain and symptoms. Nonetheless, available structure-modifying agents show limited efficacy and, in some cases, adverse effects [
31,
32]. In the pursuit of an effective and alternative pharmacological treatment for this disease, H
2S has emerged as a putative therapeutic candidate, showing a beneficial effect in OA [
33]. However, the precise mechanisms of action of H
2S have not been completely clarified yet. In this study, we described for the first time to our knowledge the effect of the intraarticular administration of GYY-4137, a slow-releasing H
2S donor, in an in vivo experimental model of OA. Our results suggest a protective effect of the exogenous induction of H
2S on joint pain and motor dysfunction, as well as cartilage destruction, likely as a result of the activation of antioxidant responses and the inhibition of catabolic signaling pathways in the joint.
A growing number of findings suggest that impaired H
2S biosynthesis in the joint might be a contributing factor to OA [
24,
34]. We recently observed that OA cartilage shows reduced levels of the mitochondrial enzyme 3-Mercaptopyruvate sulfurtransferase (3-MPST) that could be responsible for the diminished H
2S levels in this tissue [
24]. Additionally, Nasi et al. (2020) described that the oxidative stress decreases the 3-MPST expression in the chondrocyte and in turn may reinforce the impairment of H
2S biosynthesis in the OA joint [
34]. Thus, in the current study the increment in oxidative stress after the surgical destabilization of the joint could be underlying a reduction in the H
2S levels. Interestingly, the intraarticular administration of a H
2S donor reduced oxidative damage, suggesting its protective impact on the maintenance of redox and H
2S homeostasis in the joint. However, future experiments should elucidate whether GYY-4137 modulates H
2S levels in the joint by inducing gas release [
35,
36], but also by modulating the expression of H
2S-synthesizing enzymes, such as cystathionine γ-lyase (CSE), cystathionine β-synthetase (CBS), and MPST. In this sense, the effect of H
2S donors on H
2S-synthesizing enzymes is still unclear, as, for instance, different studies show contradictory results [
37,
38,
39].
One of the main goals in OA management is focused on the reduction in pain and stiffness, and subsequently maintaining or improving physical functioning. In the current study, our results seem to indicate that the intraarticular administration of H
2S reduces pain induced by the surgical destabilization of the joint, as well as recovering the joint flexibility and attenuating the impaired motor function detected by the Rotarod performance test to a higher extent than saline. This is despite the fact that the intraarticular injection of saline has also shown some beneficial effects, as it is now considered to be effective at alleviating nociceptive pain and in turn is not a true placebo in OA [
40]. Accordingly, Batalle et al. (2019) observed that the systemic administration of slow-releasing H
2S donors reduces mechanical allodynia and the grip strength deficits induced by the intraarticular injection of monosodium iodoacetate (MIA) in mice [
27]. Lucarini et al. (2018) also demonstrated the antinociceptive effects of different compounds exhibiting slow H
2S-release properties in an osteoarthritic pain model [
41]. Similarly, we previously detected that balneotherapy with sulfurous-rich water was more effective against surgically induced OA, in terms of reduction in articular pain and improvement of physical response, than a bath in tap water [
28].
The etiology of pain and disability in OA is complex and multifactorial. Pathological changes in the joint capsule and periarticular ligaments are a likely source [
42,
43]. Thus, one of the main mechanisms that characterizes this pathology and contributes to pain and stiffness is cartilage loss. We observed that intraarticular GYY-4137 administration protected cartilage against deterioration induced by the OA model. Previous studies support these findings [
26,
28,
34]. For instance, the intra-articular injection of sodium hydrosulfide (NaSH), a fast-releasing H
2S donor, slowed the development of degenerative changes in the articular cartilage in a model of gonarthrosis [
26]. Likewise, cartilage damage as well as proteoglycan loss were exacerbated in 3-MST KO joints compared to WT joints in an experimental OA in mice [
34]. By ex vivo and in vitro experimental approaches, a great number of studies also indicate the activation of H
2S signaling as a protective pathway against cartilage break down. Hence, we have previously observed that H
2S donors, including GYY-4137, reduced the activation of catabolic pathways in the cartilage, such as the expression of MMP-3 and-13 and the loss of cartilage extracellular matrix components [
20]. Similarly, other studies have detected that NaSH inhibited the MMP-13 expression induced by inflammatory stimuli in chondrocytes [
21,
22]. Interestingly, we observed in the current study that H
2S donor also attenuated the increment in MMP-13 levels in the cartilage elicited by the surgical destabilization of the joint. Overall, these findings further suggest the inhibition of this catabolic enzyme as an underlying mechanism responsible for the protective effect of H
2S on cartilage destruction, although more studies will be necessary to elucidate this pathway.
Conversely, the intraarticular administration of H
2S failed to show any modulation in the inflammation of the synovial tissue in our model. This is despite the fact that previous in vitro and in vivo studies have indicated that the induction of H
2S biosynthesis attenuates the expression of pro-inflammatory mediators as well as pathological responses in the synovium [
44,
45,
46]. However, most of these studies evaluated the early response to H
2S administration, when an acute inflammatory reaction has taken place and, subsequently, the modulation of these responses is more easily detectable. Different studies have described that, in the model of the destabilization of the medial meniscus, an acute inflammatory response occurs very early after joint injury, which is sustained in the first two weeks and dropped at a lower level at the later phases, becoming a chronic low-grade inflammation [
47,
48] that mimics OA progression [
1,
3,
49]. In agreement, we performed the histological analysis of synovial tissue at a later stage of the postramautic OA, so that all joints under surgical destabilization only showed a low grade of synovitis according to the grading system of Krenn [
50], and likely differences between experimental conditions in terms of synovitis could be still indistinguishable between a phase of persistent chronic inflammation or in resolution. Future studies are warranted to further address this issue.
Different authors have observed in vitro that H
2S presents antioxidant and anti-inflammatory effects on articular cells differently activated [
21,
22,
44]. Here, we observed that the administration of H
2S donor downregulated the levels of MMP-13, COX-2, and iNOS in the cartilage. Additionally, although the modulation of circulating pro-inflammatory makers in the joint was not evaluated in our study, we also detected a reduction in the gene expression of COX-2 and TNF-α at a systemic level under H
2S treatment. In agreement, we have previously described that GYY-4137 decreased the expression of pro-inflammatory markers in chondrocytes by an in vitro experimental approach [
19]. Besides this, other authors have also detected the capacity of H
2S to reduce the levels of inflammatory mediators in the joint in in vivo studies [
27] [
44,
46]. iNOS and COX-2 are enzymes responsible for the synthesis of NO and PGE
2, respectively, which are widely known to be key mediators that contribute to the pain and pathogenesis of OA [
6,
51]. Tumor necrosis factor-α (TNF-α) is considered a pivotal cytokine in joint inflammation and hypernociception and initiates catabolic responses in the chondrocyte, including the generation of ROS and the activation of iNOS and COX-2 expression [
10,
52,
53,
54]. Interestingly, ATB-346, a novel H
2S-releasing naproxen, more efficiently reduces TNF-α release in a rat model of arthritis [
55], and GYY-4137 inhibits the expression of effectors of the TNF pathway [
56]. Thus, the inhibition of the production of these mediators has been associated with the chondroprotective effect of H
2S [
27,
55,
56], and subsequently it could be responsible for the lesser OA severity and pain observed in our model under GYY-4137 treatment.
The effects of H
2S are likely mediated through its capacity to inhibit nuclear factor-κB (NF-κB) signaling and promote Nrf-2 transcriptional activity [
57]. Nrf-2 is a master regulator of antioxidant, anti-inflammatory, and other cytoprotective mechanisms. It regulates the expression of genes that code for phase II detoxification enzymes, such as NADPH:quinone oxidoreductase (NQO1) and heme oxygenase-1 (HO-1), which lead to amelioration of oxidative stress in cellular and animal models [
58]. In our study, the intraarticular induction of H
2S biosynthesis attenuated oxidative stress, reducing the presence in the cartilage of the markers of oxidative damage 4-HNE and 8-oxo-dG. Likewise, we observed that animals under H
2S treatment showed an upregulated expression of Nrf-2, NQO1 and HO-1, suggesting that the activation of this signaling pathway could be involved in the antioxidant effect of H
2S. These findings are in accordance with the fact that the downregulation of Nrf-2/HO-1 signaling increases OA severity, whereas its induction elicits protective actions in this pathology [
59,
60]. Nonetheless, we and other authors observed an upregulation of Nrf-2 levels in OA joint, likely as an insufficient response against the oxidative stress generated during this articular disorder [
61,
62]. Additionally, a recent study described that the expression of Nrf-2-regulated detoxicant enzymes, such as HO-1 and NQO1, is significantly increased in MIA-injected mice and that treatment with slow H
2S-release donors maintains these high levels [
27]. Wu et al. (2016) observed that S-propargyl-cysteine, an endogenous inductor of H
2S synthesis, activates Nrf-2 signaling in adjuvant-induced arthritis rats and inhibits inflammatory response, ameliorating the severity of the arthritis model [
46]. Hence, a growing number of evidences suggest that the Nrf-2 activation induced by H
2S is also responsible for its anti-inflammatory effects [
46,
63,
64]. In our study, we observed for the first time that the administration of a slow-releasing H
2S donor in the joint induces the expression of Nrf-2 in the cartilage and that this event could contribute to the attenuation of the expression of pro-inflammatory markers detected in our model and subsequently to its pathogenesis. Nonetheless, future experiments should be encouraged to further enlighten us about the involvement of Nrf-2 in H
2S actions in the joint.