Corrigendum: Exploiting curcumin synergy with natural products using quantitative analysis of dose-effect relationships in an experimental in vitro model of osteoarthritis.

[This corrects the article DOI: 10.3389/fphar.2019.01347.].

miR9 inhibits 6-mer HA-induced cytokine production and apoptosis in human chondrocytes by reducing NF-kB activation.

The present study aimed to investigate the expression of miR9 and its correlation with cytokines, proteolytic enzymes and apoptosis in an experimental model of 6-mer HA induced inflammation in human chondrocytes. Human articular chondrocytes, transfected with a miR-9 mimic and miR-9 inhibitor, were stimulated with 6-mer HA in presence/absence of a specific NF-kB inhibitor. 6-mer HA induced a significant increase of TLR-4, CD44, IL-8, IL-18, MMP-9, ADAMTS-5, BAX and BCL-2 mRNAs expression and the related proteins, as well as NF-kB activation, associated with a significant up regulation of miR-9. In chondrocytes transfected with the miR-9 mimic before 6-mer HA treatment we found a decrease of such inflammatory cytokines, metalloproteases and pro-apoptotic molecules, while we found them increased in chondrocytes transfected with the miR9 inhibitor before 6-mer HA stimulation. The activities of TLR-4 and CD44, up regulated by 6-mer HA, were not modified by miR9 mimic/inhibitor, while the NF-kB activation was significantly affected. We suggested that the up regulation of miR9, induced by 6-mer HA, could be a cellular attempt to limit cell damage during inflammation.

Endocan, a novel inflammatory marker, is upregulated in human chondrocytes stimulated with IL-1 beta.

Endocan is a circulating proteoglycan, involved in immunity, inflammation, and endothelial function. It has been recently suggested as a biomarker of inflammation, increased angiogenesis, and cancer. In vitro studies have shown that endocan expression could be upregulated by inflammatory cytokines and proangiogenic molecules. High endocan levels were also shown in arthritic joint tissues and particularly in sites characterized by severe inflammation. This study was performed to evaluate endocan expression in chondrocytes stimulated with IL-ß. mRNA and related protein production were measured for endocan, TNF-α, and IL-6. NF-kB activity was also evaluated. IL-1ß treatment induced a significant upregulation of both endocan and the inflammatory parameters as well as NF-kB activity. The treatment of chondrocytes with the specific NF-kB inhibitor before IL-1ß stimulation was able to reduce endocan and the inflammatory markers over-expression. The results of our study indicated that endocan is also expressed in human chondrocytes; furthermore, consistent with previous results in other cell types and tissues, IL-1ß-induced inflammatory response involves the expression of endocan through NF-kB activation. In this context, endocan seems to be an important inflammatory marker associated with the activation of NF-kB pathway.

miR146a up-regulation is involved in small HA oligosaccharides-induced pro-inflammatory response in human chondrocytes.

BACKGROUND: Small HA fragments are produced during cartilage degradation and their role seems to be preponderant during pathologies in which cartilage injury contribute to trigger and perpetuate the inflammatory mechanism. Several reports have increasingly shown that MicroRNAs (miRs), a small non-coding mRNAs are involved in the regulation of multiple biological processes, including cell proliferation and inflammation response in different pathologies, among them miR146a seems to be involved in inflammatory processes. METHODS: Starting by these evidences we investigated the levels of miR146a and its correlation with inflammatory mediators in an experimental model of 6-mer HA-induced inflammatory response in human cultured chondrocytes. RESULTS: Treatment of chondrocytes with 6-mer HA showed up-regulation in inflammation parameters such as TLR-4, and CD44 receptors activation, IL-6, IL-1ß and MMP-13 mRNA expression and proteins production, as well as NF-kB activation. We also revealed an up-regulation of miR146a. Transfection with a miR146a mimic or miR146a inhibitor produced the following effects: chondrocytes receiving miR146a mimic and then 6-mer HA significantly reduced inflammatory cytokines and MMP-13, while exposition of chondrocytes with miR146a inhibitor and then the 6-mer HA incremented the activity of damaging cytokines and MMP13. Expression of CD44 receptor was not affected by miR-146a treatments, while TLR-4 expression and NF-kB activation were modified. CONCLUSIONS: We concluded that up-regulation of miR146a occurred in 6-mer HA-induced inflammation response may reduce the inflammatory cascade by modulating TLR-4 and NF-kB activation. GENERAL SIGNIFICANCE: These results could be useful in develop new therapeutic strategies with the aim to reduce OA and RA incidence.

Hyaluronan Fragmentation During Inflammatory Pathologies: A Signal that Empowers Tissue Damage.

The mechanisms that modulate the response to tissue injury are not fully understood. Abnormalities in the repair response are associated with a variety of chronic disease states characterized by inflammation, followed subsequently by excessive ECM deposition. As cell-matrix interactions are able to regulate cellular homeostasis, modification of ECM integrity appears to be an unspecific factor in promoting the onset and progression of inflammatory diseases. Evidence is emerging to show that endogenous ECM molecules supply signals to damage tissues and cells in order to promote further ECM degradation and inflammation progression. Several investigations have been confirmed that HA fragments of different molecular sizes exhibit different biological effects and responses. In fact, the increased deposition of HA into the ECM is a strong hallmark of inflammation processes. In the context of inflammatory pathologies, highly polymerized HA is broken down into small components, which are able to exacerbate the inflammatory response by inducing the release of various detrimental mediators such as reactive oxygen species, cytokines, chemokines and destructive enzymes and by facilitating the recruitment of leukocytes. However, strategies involving the modulation of the HA fragment with specific receptors on cell surface could represent different promising effects for therapeutic scope. This review will focus on the inflammation action of small HA fragments in recent years obtained by in vivo reports.

Exploiting Curcumin Synergy With Natural Products Using Quantitative Analysis of Dose-Effect Relationships in an Experimental In Vitro Model of Osteoarthritis.

Introduction: Drug combination is widely used to treat chronic inflammatory diseases. A similar strategy might be worth of interest to design plant-derived natural products to treat inflammatory conditions. Curcumin is a natural phenolic compound which shares anti-inflammatory activity with both flavocoxid, a flavonoid mixture of baicalin and catechin, and ß-caryophyllene, a bicyclic sesquiterpene. The aim of this study was to investigate the synergy potential of curcumin with both flavocoxid and ß-caryophyllene in human articular chondrocytes triggered with lipopolysaccharide (LPS), in an experimental in vitro model of osteoarthritis. Materials and Methods: Human articular chondrocytes were stimulated with LPS alone or in combination with different treatments. Total RNA was extracted 4 h after treatment to study interleukin 1ß (IL-1ß), NF-κB, and STAT3 mRNA expression. A drug combination study was designed choosing 5 doses to demonstrate a synergistic effect of compounds, according to Chou and Talalay method. A median-effect equation was applied and finally, the combination index (CI) was used to clarify the nature of the compounds interaction (synergistic versus additive versus antagonistic inhibitory effects); CI < 1, CI = 1, and CI > 1 indicated synergistic, additive, and antagonistic effects, respectively. Results: LPS prompted IL-1ß expression. Curcumin, flavocoxid and ß-caryophyllene suppressed IL-1ß expression with different IC50. A synergistic action for the reduction of the inflammatory phenotype in human chondrocytes was observed for the combination curcumin-flavocoxid with a percentage from 10% to 90%, and for the combination curcumin-ß-caryophyllene from 50% to 90%. IC50 doses of either flavocoxid, ß-caryophyllene and curcumin alone or in combination were safe and did not affect cell vitality. Moreover, the same IC50 doses reduced the transcription factors NF-κB and STAT3 mRNA expression and interestingly the effects of the combinations were greater than the natural products alone, thus suggesting that the site where the synergy takes place could be at the signal transduction level. Discussion: The results suggest that curcumin synergizes with either flavocoxid or ß-caryophyllene, exerting an anti-inflammatory activity and thus strongly suggesting the potential of a dual combination of these compounds for the management of osteoarthritis and unmasking a new feature of these natural products.

Serglycin as part of IL-1ß induced inflammation in human chondrocytes.

Serglycin (SRGN) is an intracellular proteoglycan produced and secreted by several cell types. The increased expression of SRGN was associated with greater aggressiveness in cancer and inflammation. In this study, we demonstrated that SRGN is increased in human chondrocytes after IL-ß stimulation. Furthermore, we found that secreted SRGN was able to bind the CD44 receptor thus participating in the extension of the inflammatory response. Using SRGN knockdown cells we observed a significantly decrease in specific inflammatory markers and NF-kB activation. Similar results were observed by blocking the CD44 receptor. These data provide further evidences for a direct involvement of SRGN in the mechanisms regulating the non-infectious chondrocytes damage, and the consequent joint inflammation and cartilage destruction in arthritis.

Hyaluronan fragments produced during tissue injury: A signal amplifying the inflammatory response.

Inflammation is a complex mechanism that plays a key role during diseases. Dynamic features of the extracellular matrix (ECM), in particular, during phases of tissue inflammation, have long been appreciated, and a great deal of several investigations has focused on the effects of ECM derivatives on cell function. It has been well defined that during inflammatory and tissue injury, ECM components were degraded. ECM degradation direct consequence is the loss of cell homeostasis, while a further consequence is the generation of fragments from larger precursor molecules. These bio-functional ECM shred defined matrikines as capable of playing different actions, especially when they function as powerful initiators, able to prime the inflammatory mechanism. Non-sulphated glycosaminoglycan hyaluronan (HA) is the major component of the ECM that undergoes specific modulation during tissue damage and inflammation. HA fragments at very low molecular weight are produced as a result of HA depolymerization. Several evidence has considered the plausibility that HA breakdown products play a modulatory action in the sequential stages of inflammation, although the effective mechanism of these HA derivative compounds act is not completely defined. This review will focus on the pro-inflammatory effects of HA fragments in recent years obtained by in vitro investigations.

Serglycin is involved in inflammatory response in articular mouse chondrocytes.

Serglycin is expressed by a variety of cell types and mediates different functions in both normal and pathological conditions by interacting with different biological molecules, such as the CD44 receptor. Many studies suggest that serglycin has a crucial role in inflammatory response, but there are limited data on the functions of this proteoglycan in chondrocytes. In this study we investigated the effect of serglycin knockdown induced by a specific serglycin small interfering RNA (SRGN siRNA) in normal mouse chondrocytes stimulated with lipopolysaccharide (LPS). LPS administration in normal chondrocytes increased the expression of serglycin mRNA and related protein and the production of the pro-inflammatory mediators TNF-alpha, IL-1beta, IL-6, iNOS and MMP-9, through NF-kB activation. In addition, a marked increased expression of CD44 after LPS stimulation was observed. Notably, the CD44 expression and the inflammatory response were significantly reduced by SRGN siRNA treatment in LPS treated chondrocytes. Similar results were obtained in normal mouse chondrocytes exposed to LPS, using a specific blocking antibody against CD44. These results indicate that serglycin produced in LPS-induced inflammation in normal mouse chondrocytes is able to modulate inflammation by interacting with CD44 receptor, suggesting a possible key role in the cartilage inflammation.

The proteoglycan biglycan mediates inflammatory response by activating TLR-4 in human chondrocytes: Inhibition by specific siRNA and high polymerized Hyaluronan.

Cartilage degeneration are hallmarks of wear, tear, mechanical and inflammatory damage of the joint cartilage. Tissue degradation as well as compromising the integrity and function of the organ, produces different intermediates, directly able to stimulate further inflammatory effect, therefore, amplifying the inflammation response. Biglycan is a soluble component of the extracellular matrix that is released during tissue injury. It has been reported that released biglycan is an endogenous ligand for TLR-2/4 in some cell type. We studied the role of biglycan in an experimental model of biglycan-induced inflammatory response in human chondrocytes and the effect of high polymerized HA on reducing its activity. Exposition of chondrocytes to LPS generated cell injury, including high levels of biglycan. Chondrocyte treatment with biglycan produces a high mRNA expression of several detrimental inflammation mediators such as IL-1ß, IL-6, MMP-13, and IL-17, as well as NF-kB and TLR-4 activation. Administration of high polymerized HA to chondrocytes exposed to biglycan was able to attenuate the inflammatory response by decreasing the expression of the inflammatory mediators. Involvement of the TLR-4 in the mediation of the biglycan action was confirmed using a specific silent agent (siRNA). Taken together, these data could be used to develop new anti-inflammatory approaches.

Hyaluronan in the experimental injury of the cartilage: biochemical action and protective effects.

INTRODUCTION: Our knowledge of extracellular matrix (ECM) structure and function has increased enormously over the last decade or so. There is evidence demonstrating that ECM provides signals affecting cell adhesion, shape, migration, proliferation, survival, and differentiation. ECM presents many domains that become active after proteolytic cleavage. These active ECM fragments are called matrikines which play different roles; in particular, they may act as potent inflammatory mediators during cartilage injury. FINDINGS: A major component of the ECM that undergoes dynamic regulation during cartilage damage and inflammation is the non-sulphated glycosaminoglycan (GAG) hyaluronan (HA). In this contest, HA is the most studied because of its different activity due to the different polymerization state. In vivo evidences have shown that low molecular weight HA exerts pro-inflammatory action, while high molecular weight HA possesses anti-inflammatory properties. Therefore, the beneficial HA effects on arthritis are not only limited to its viscosity and lubricant action on the joints, but it is especially due to a specific and effective anti-inflammatory activity. Several in vitro experimental investigations demonstrated that HA treatment may regulate different biochemical pathways involved during the cartilage damage. Emerging reports are suggesting that the ability to recognize receptors both for the HA degraded fragments, whether for the high-polymerized native HA involve interaction with integrins, toll-like receptors (TLRs), and the cluster determinant (CD44). The activation of these receptors induced by small HA fragments, via the nuclear factor kappa-light-chain enhancer of activated B cell (NF-kB) mediation, directly or other different pathways, produces the transcription of a large number of damaging intermediates that lead to cartilage erosion. CONCLUSIONS: This review briefly summarizes a number of findings of the recent studies focused on the protective effects of HA, at the different polymerization states, on experimental arthritis in vitro both in animal and human cultured chondrocytes.

Hyaluronan in experimental injured/inflamed cartilage: In vivo studies.

Joint disease is characterized by an imbalance between the synthesis and degradation of articular cartilage and subchondral bone accompanied by capsular fibrosis, osteophyte formation and varying degrees of inflammation of the synovial membrane. Many animal models have been developed to study arthritis and osteoarthritis that enable experimental conditions, diet and environmental risk factors to be carefully controlled. Animal-based studies have demonstrated the positive effects of exogenous HA on the preservation of joint cartilage in different models of arthritis and osteoarthritis. Although many promising effects of exogenous HA have been reported, there remains uncertainty as to its effectiveness in reversing cartilage injury and other manifestations of joint diseases because of difficulties in interpreting and unifying the results of these studies. A review of the literature of the last decade was conducted to report the results and to determine what we have learned from animal models in relation to joint inflammation induced by experimental models and HA treatment.

6-Mer Hyaluronan Oligosaccharides Modulate Neuroinflammation and α-Synuclein Expression in Neuron-Like SH-SY5Y Cells.

Several studies have shown the degradation of the extracellular matrix at the site of neuroinflammation and increased release of degradation products of glycosaminoglycans. Among these, low molecular weight fragments of hyaluronan (HA) may play a key role in the events leading to neuroinflammation and/or neuronal degeneration. Small HA fragments are able to induce inflammation by stimulating both TLR-2 and TLR-4 as well as CD44 receptors. This stimulation culminates in the nuclear translocation of NF-kB that in turn induces the production of pro-inflammatory intermediates such as TNF-α and IL-1ß. The potential of HA fragments, as mediators of inflammation, it has been poorly investigated in neuron-like SH-SY5Y cells so the aim of this study was to investigate the neuroinflammatory effects of very small HA oligosaccharides, the involvement of TLR-2, TLR-4, and CD44 and the production of α-synuclein in such cells. The addition of HA fragments to cell cultures up-regulated TLR-2, TLR-4, and CD44 levels, induced NF-kB activity and increased both TNF-α and IL-ß as well as α-synuclein production. On blocking the activity of TLR-2, TLR-4, and CD44 the levels of inflammatory parameters and of α-synuclein were significantly reduced. Since several data have shown as α-synuclein, produced from neurons, is able to initiate ex novo or to maintain an existing neuroinflammatory response, which has been suggested as one of the principal components involved in neurodegenerative pathologies, as PD, we suggest that HA pathways should be given careful consideration when devising future anti-neuroinflammatory strategies to defend against the onset of neurodegenerative disorders. J. Cell. Biochem. 117: 2835-2843, 2016. © 2016 Wiley Periodicals, Inc.

Beta-arrestin 1 is involved in the catabolic response stimulated by hyaluronan degradation in mouse chondrocytes.

Beta-arrestin-1 (ß-arrestin-1) is an adaptor protein that functions in the termination of G-protein activation and seems to be involved in the mediation of the inflammatory response. Interleukin-1ß (IL-1ß) elicits the expression of inflammatory mediators through a mechanism involving hyaluronan (HA) degradation, thereby contributing to toll-like receptor 4 (TLR-4) and CD44 activation. Stimulation of both receptors induces nuclear factor kappaB (NF-kB) activation that, through transforming-growth-factor-activated-kinase-1 (TAK-1), in turn stimulates the inflammatory mediators of transcription. As ß-arrestin-1 seems to play an inflammatory role in arthritis, we have investigated the involvement of ß-arrestin-1 in a model of IL-1ß-induced inflammatory response in mouse chondrocytes. IL-1ß treatment significantly increases chondrocytes TLR-4, CD44, ß-arrestin-1, TAK-1, and serine/threonine kinase (AKT) mRNA expression and related protein levels. NF-kB is also markedly activated with consequent tumor-necrosis-factor-alpha, interleukin-6, and inducible-nitric-oxide-synthase up-regulation. Treatment of IL-1ß-stimulated chondrocytes with ß-arrestin-1 and/or AKT and/or TAK-1-specific inhibitors significantly reduces all parameters, although the inhibitory effect exerted by TAK-1-mediated pathways is more effective than that of ß-arrestin-1. ß-Arrestin-1-induced NF-kB activation is mediated by the AKT pathway as shown by IL-1ß-stimulated chondrocytes treated with AKT inhibitor. Finally, a specific HA-blocking peptide (Pep-1) has confirmed the inflammatory role of degraded HA as a mediator of the IL-1ß-induced activation of ß-arrestin-1.

Beta-arrestin-2 negatively modulates inflammation response in mouse chondrocytes induced by 4-mer hyaluronan oligosaccharide.

Beta-arrestin-2 is an adaptor protein that terminates G protein activation and seems to be involved in the modulation of the inflammatory response. Small hyaluronan (HA) fragments, such as 4-mer HA oligosaccharides, are known to interact with the toll-like receptor-4 (TLR-4) with consequent activation of the nuclear factor kappaB (NF-kB) that in turn stimulates the inflammation response. NF-kB activation is mediated by different pathways, in particular by the transforming growth factor-activated kinase-1 (TAK-1). Conversely, increased levels of protein kinase A (PKA), induced by cyclic adenosine monophosphate (cAMP), seem to inhibit NF-kB activation. We studied the involvement and role of beta-arrestin-2 in mouse chondrocytes stimulated with 4-mer HA fragments. The exposure of chondrocytes to 4-mer HA produced a significant up-regulation in TLR-4, cAMP, beta-arrestin-2, TAK-1, protein 38 mitogen-activated protein kinase (p38MAPK), and PKA, both in terms of mRNA expression and of the related protein levels. NF-kB was significantly activated, thereby producing the transcription of pro-inflammatory mediators, including tumor necrosis factor alpha, interleukin-6, and interleukin-17. The treatment of 4-mer HA-stimulated chondrocytes with antibodies against beta-arrestin-2 and/or a specific PKA inhibitor, significantly increased the inflammatory response, while the treatment with a specific p38MAPK inhibitor significantly reduced the inflammatory response. Interestingly, the anti-inflammatory action exerted by beta-arrestin-2 appeared to be mediated in part through the direct inhibition of p38MAPK, preventing NF-kB activation, and in part through cAMP and PKA activation primed by G protein signaling, which exerted an inhibitory effect on NF-kB. Taken together, these results could be useful for future anti-inflammatory strategies.

Inhibition of small HA fragment activity and stimulation of A2A adenosine receptor pathway limit apoptosis and reduce cartilage damage in experimental arthritis.

Recent studies have found that the inactivation of small hyaluronan (HA) fragments originating from native HA during inflammation reduced the inflammatory response in models of experimental arthritis. The stimulation of adenosine receptors A2A reduced inflammation by inhibiting NF-kB activation. The combination of both treatments was significantly more effective than either of the individual treatments. The aim of this study was to further investigate the effects of a combined treatment using the HA inhibitor Pep-1 and a selective A2AR agonist (CV-1808) on the structure and ultrastructure of the articular cartilage and on apoptosis in a model of collagen-induced arthritis (CIA) in mice. Arthritic mice were treated with Pep-1 and/or CV-1808 intraperitoneally daily for 20 days. At day 35, the hind limbs were processed for light microscopy (hematoxylin/eosin and Safranin-O-Fast Green) and for transmission and scanning electron microscopy. CIA increased IL-6, caspase-3 and caspase-7 mRNA expression and the related protein levels in arthritic articular cartilage, and significantly increased concentrations of Bcl-2-associated X protein (Bax), while B cell-lymphoma-2 protein (Bcl-2) was markedly reduced. The combined Pep-1/CV-1808 treatment significantly reduced CIA injury, particularly at the highest doses, demonstrated by the presence of Safranin-O-positive cartilage, with a smooth surface and normal chondrocytes in the superficial, intermediate and deep zones. Morphological data and histological scoring were strongly supported by the reduction in inflammation and apoptotic markers. The results further support the role of HA degradation and A2A receptors in arthritis.

Inhibition of the hyaluronan oligosaccharides inflammatory response: reduction of adenosine 2A receptor activation by EPAC and PKA.

The aim of this study was to investigate the involvement of exchange proteins directly activated by cyclic adenosine (ADO) monophosphate (EPAC) in 4-mer hyaluronan (HA) oligosaccharide-induced inflammatory response in mouse normal synovial fibroblasts (NSF). Treatment of NSF with 4-mer HA increased Toll-like receptor-4, TNF-alpha and IL-1beta mRNA expression and of the related proteins, as well as nuclear factor kappaB (NF-kB) activation. Addition to NSF, previously stimulated with 4-mer HA oligosaccharides, of ADO significantly reduced NF-kB activation, TNF-alpha and IL-1beta expression. The pre-treatment of NSF with cyclic ADO monophosphate and/or PKA and/or EPAC-specific inhibitors significantly inhibited the anti-inflammatory effect exerted by ADO. In particular, the EPAC inhibitor reduced the ADO effect to a major extent than the PKA inhibitor. These results mean that both PKA and EPAC pathways are involved in ADO-induced NF-kB inhibition although EPAC seems to be more involved than PKA.

MiRNome expression is deregulated in the peripheral lymphoid compartment of multiple myeloma.

MicroRNAs (miRNAs) are short non-coding RNAs involved in the regulation of gene expression. Selected groups of miRNAs are differentially expressed in various types of cancers. Alterations in miRNAs gene expression have been shown in cells from the B-cell malignancy, multiple myeloma (MM). However, although MM is a disease of plasma cells, abnormalities have been detected in the peripheral blood of the patients. The goal of our study was to analyse the entire miRNome in peripheral lymphocytes of MM patients using reverse transcription quantitative polymerase chain reaction. Using in silica analysis, we also evaluated some of the most interesting and significant pathways. Analysis revealed that MM samples had a distinct miRNA profile compared to the controls. This resulted in the identification of 203 miRNAs, 85 of which were over-expressed and 118 under-expressed. Of these, 184 possessed validated or highly predicted mRNA targets. We identified 12 354 mRNA targets of the transcriptome: 36·4% of the related proteins are involved in death processes while the 21% are required for growth and cell proliferation. We have demonstrated that miRNAs are differentially expressed in the peripheral blood of MM patients compared to controls, affecting some pathways involved in the anti-apoptotic process, cell proliferation and maybe anti-angiogenesis.

The SOD mimic MnTM-2-PyP(5+) reduces hyaluronan degradation-induced inflammation in mouse articular chondrocytes stimulated with Fe (II) plus ascorbate.

In pathological conditions, oxidative burst generates hyaluronan (HA) fragmentation with a consequent increase in the number of small HA oligosaccharides. These fragments are able to stimulate an inflammatory response in different cell types by activating the CD44 and the toll-like receptors 4 (TLR-4) and 2 (TLR-2). The stimulation of CD44 and TLRs in turn activates the NF-kB which induces the production of several pro-inflammatory mediators that amplify and perpetuate inflammation. We aimed to study the antioxidant effect of the SOD mimic, synthetic manganese porphyrin, Mn(III) 5,10,15,20-tetrakis(N-methylpyridinium-2-yl)porphyrin (MnTM-2-PyP(5+)) on preventing HA degradation in mouse articular chondrocytes stimulated with Fe (II) plus ascorbate. Fe (II) plus ascorbate stimulation induced oxidative burst confirmed by high levels of hydroxyl radical/peroxynitrite production, increased lipid peroxidation and HA degradation. HA fragments highly induced mRNA expression and the related protein production of CD44, TLR-4 and TLR-2, NF-kB activation and significantly up-regulated the inflammatory cytokines, tumor necrosis factor alpha (TNF-alpha), interleukin-1beta (IL-1beta), and other pro-inflammatory mediators, i.e. matrix metalloprotease 13 (MMP-13) and inducible nitric oxide synthase (iNOS). Treatment of cells with MnTM-2-PyP(5+)was able to attenuate oxidative burst, HA degradation and NF-kB activation, and markedly decreased mRNA expression of CD44, and TLRs and the related protein synthesis, as well as the levels of up-regulated inflammatory mediators. Adding a specific HA-blocking peptide (PEP-1) to cells significantly reduced all the inflammatory parameters up-regulated by Fe (II) plus ascorbate, and increased MnTM-2-PyP(5+) activity. These findings suggest that HA degradation plays a key role in the initial inflammatory response of cartilage and antioxidants and could be a useful tool to prevent the propagation of this mechanism.