Osteoarthritis Cartilage. 2014 Jul;22(7):1026-35.

Effect of hydrogen sulfide sources on inflammation and catabolic markers on interleukin 1b-stimulated human articular chondrocytes

E.F. Burguera1,2, A. Vela-Anero2,3, J. Magalhaes1,2, R. Meijide-Faílde3, F.J. Blanco1,2*

1CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Spain

2Grupo de Bioingieneria Tisular y Terapia Celular (GBTTC), Servicio de Reumatología, Instituto de Investigacion Biomédica (INIBIC), Complexo Hospitalario Universitario A Coruña (CHUAC), A Coruña, Spain

3Departmento de Medicina, INIBIC-Universidad de A Coruña, A Coruña, Spain

 

Abstract

Objective: Hydrogen sulfide (H2S), the third gassotransmiter together with NO and CO, is emerging as a regulator of inflammation. To test if it might offer therapeutic value in the treatment of osteoarthritis (OA) we evaluated the effects of two exogenous sources of H2S, NaSH and GYY4137, on inflammation and catabolic markers that characterize OA.

Method: Human chondrocytes were isolated from OA tissue. Cells were stimulated with a pro-inflammatory cytokine (interleukin-1β, IL1β, 5ng/ml) and the ability of the two H2S sources to ameliorate its effects on the cells was tested. Nitric oxide (NO) production was quantified through the Griess reaction. Protein levels of inducible NO synthase (NOS2) and matrix metalloproteinase 13 (MMP13) were visualized through immunocytochemistry. Relative mRNA expression was quantified with qRT-PCR. Prostaglandin-2 (PGE-2), interleukin 6 (IL6) and MMP13 levels were measured with specific EIAs. NFκB nuclear traslocation was visualized with immunofluorescence.

Results: Both H2S sources led to significant reductions in NO, PGE-2, IL6 and MMP13 released by the cells and at the protein level. This was achieved by downregulation of relevant genes involved in the synthesis routes of these molecules, namely NOS2, cyclooxigenase-2 (COX2), prostaglandin E synthase (PTGES), IL6 and MMP13. NFκB nuclear traslocation was also reduced.

Conclusion: NaSH and GYY4137 show anti-inflammatory and anti-catabolic properties when added to IL1β activated osteoarthritic chondrocytes. Supplementation with exogenous H2S sources can regulate the expression of relevant genes in osteoarthritis pathogenesis and progression, counteracting IL1β pro-inflammatory signals that lead to cartilage destruction in part by reducing NFκB activation.

Key words: Human articular chondrocytes; osteoarthritis; hydrogen sulfide; inflammation, metalloproteinase.

 

Supplementary

Osteoarthritis (OA) is a multifactorial, degenerative disease with a tremendously high prevalence in the developed world. OA can be initiated as a result of trauma (for instance, in high impact sports), congenital mechanical misalignments, a genetic predisposition, or through the progressive deleterious effects of a prevalent local inflammatory situation, whose etiology is not clearly understood. This inflammatory environment in the OA joint is the results of increased levels of inflammatory cytokines and other mediators that, by interacting with each other, perpetuate this unhealthy milieu. This collectively leads to an imbalance between the anabolic and the catabolic processes maintained by chondrocytes that inevitably results in the progressive loss of articular cartilage.

 

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Figure 1

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Figure 2

 

Interleukin 1β (IL1β) is the key cytokine that activates these harmful routes. High IL1β levels induce the synthesis of other proinflammatory cytokines such as IL6 and IL8, inflammatory soluble factors such as prostaglandin (PG) E2, molecules that subject chondrocytes to increased oxidative stress (chiefly, nitric oxide, NO and the anion superoxide, O2) and cartilage proteases (aggrecanases and matrix metalloproteinases, MMPs, 1, 3 and 13) (Figure 1) (1).

Hydrogen sulfide (H2S) is a gaseous molecule recognizable by its characteristic smell of rotten eggs. It is very toxic in high concentrations and it exerts its toxicity by inhibiting cytochrome C oxidase and reducing ATP production. However, it is also an endogenous mediator and gasotransmitter and it participates in many physiologic and pathological events (Figure 2). Recent literature provides ample evidence that H2S plays important roles in inflammation, although its role is not clearly understood yet. Exogenous administration of H2S has proven beneficial in a number of pathologies models, both in vitro and in animal models. Examples include endotoxic a shock rat model, RAW264.7 macrophages, murine microvascular endothelial cells, astrocytes and ischemic cardiomyocytes, to name a few (2).

Some recent studies in our group, now featured in this site, have focused on investigating the effects of the exogenous administration of H2S, through two well known synthetic molecules, to diseased chondrocytes (i.e. cells isolated from OA cartilage) stimulated with interleukin 1β (IL-1β). Tissue biopsies were donated, after the corresponding informed consent, by patients of the Traumatology and Rheumatology Services of the A Coruña University Hospital (CHUAC), A Coruña, Spain. The Rheumatology Research Unit, directed by Francisco J. Blanco, PhD, MD is a world-known referent in the study of osteoarthritis. Its goal is to improve the quality of life of osteoarthritis patients by counterattacking the disease from several flanks. This includes, the search for new biomarkers to improve diagnosis (Proteomics group), understanding genetic predisposition and the different OA phenotypes (Genomics group), improving our knowledge of the disease’s etiology and progression (Cartilage Biology group), and lastly, finding new therapeutic options, including the development of tissue engineering approaches to regenerate hyaline cartilage (Cellular Therapy group).

 

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Figure 3

The present study was conducted by researchers in this latter group. As expected, stimulation of articular chondrocytes with IL-1β induced the production of NO, PGE2, IL-6, MMP3 and MMP13, all measured through appropriate biochemical, immunoenzymatic and immunocytochemical assays. These were, in turn, the result of the overexpression of their respective synthesis genes, i.e. NOS2, COX-2 and PTGES, IL-6 and MMP13, respectively, measured with qRT-PCR (Figure 3). Our study proved that co-administration of either of the two H2S compounds resulted in a dose-dependent reduction in these deleterious markers in the chondrocytes, both at the mRNA and protein levels. Of particular relevance is the arrest of MMP13 mRNA induction and protein reduction. This collagenase has been singled out in a recently review (3) as the key gene upon which many of the signalling pathways (i.e. JNK or p38 MAPK) and transcription factors (such as NFκB, HIF2α, Ihh, or Runx2) involved at the different stages of OA converge. Most of these regulatory factors directly or indirectly, impact on MMP13 transcription and activity. This is of significance because, there is an agreement that cartilage degradation by aggrecanases is a reversible process, but once MMPs are induced, cartilage extracellular matrix destruction becomes irreversible. Therefore, our study demonstrates for the first time that exogenous supplementation with H2S can counteract the induction of important markers in OA and indicates that OA cartilage, and therefore OA patients, might benefit from it administration (Figure 4).

 

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Figure 4

In general, whether H2S is a cytoprotective or cytotoxic compound is still a matter of controversy. It seems to depend on the specific pathology. In a number of diseases, including diabetes, hypertension, chronic obstructive pulmonary disease and even neurodegenerative diseases such as Alzheimer’s, patients seem to have decreased blood levels of H2S. Our group has evidence that this might be the case with osteoarthritis and that H2S biosynthesis in the joint might be impaired (unpublished results). As a result, articular chondrocytes might have a deficit of this molecule that normally exerts local anti-inflammatory and anti-oxidant effects. This would explain why exogenous administration of H2S has beneficial effects in these cells. Our group is conducting further experiments in this line.

Lastly, H2S supplementation using synthetic chemicals is not the only option to supply H2S to cells. There are a few studies on the effects of the administration of sulfurous minero-medicinal spring waters on oxidation, inflammation or pain markers in arthritides patients undergoing bath and/or hydropinic regimes. Further, the new Osteoarthritis Research Society International (OARSI) recommendations for the non-surgical management of OA include balneotherapy with sulfurous spring waters for the treatment in multi-joint OA patients with co-morbidities, pointing out, however, that although the quality of evidence is fair, further randomized controlled trials are required (4).

In this line, our laboratory is starting studies on the effects of sulfurous waters, from our local springs, on OA animal models as a logical next step after our in vitro experiments, and before moving to clinical human trials.

 

References

  1. Kapoor M, Martel-Pelletier J, Lajeunesse D, Pelletier JP, Fahmi H. Role of proinflammatory cytokines in the pathophysiology of osteoarthritis. Nature Reviews Rheumatology 2011;7: 33-42
  2. Whiteman M, Winyard PG. Hydrogen sulfide and inflammation: the good, the bad, the ugly and the promising. Expert Rev Clin Pharmacol 2011;4: 13-32
  3. Goldring MB, Otero M, Plumb DA, Dragomir C, Favero M, El Hachem K, et al. Roles of inflammatory and anabolic cytokines in cartilage metabolism: signals and multiple effectors converge upon Mmp-13 regulation in osteoarthritis. Eur Cell Mater 2011;21:202e20.
  4. T.E. McAlindon, R.R. Bannuru, M.C. Sullivan, N.K. Arden, F. Berenbaum, S.M. Bierma-Zeinstra, G.A. Hawker, Y. Henrotin, D.J. Hunter, H. Kawaguchi, K. Kwoh, S. Lohmander, F. Rannou, E.M. Roos, M. Underwood. OARSI guidelines for the non-surgical management of knee osteoarthritis. Osteoarthritis and Cartilage 22 (2014) 363e388.
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