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Small degradation fragments of hyaluronan (HA) may stimulate an inflammatory response in a variety of tissues at the injury site. HA oligosaccharides are endogenous ligands for the cluster determinant 44 (CD44) receptor as well as for toll-like receptor 4 (TLR-4). Previous data have shown that HA fragments may induce pro-inflammatory cytokine expression by interacting with both the CD44 receptor and TLR-4. CD44 and TLR-4 stimulation activates different inflammatory pathways that culminate with the activation of the transcriptional nuclear factor kappaB (NF-kappaB) which is responsible for the expression of inflammation mediators such as tumor necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6) and interleukin-1 beta (IL-1beta). The aim of this study was to investigate the inflammatory effects of very small HA oligosaccharides on both TLR-4 and CD44 involvement in normal human articular chondrocytes. Adding HA fragments to chondrocyte cultures up-regulated CD44 and TLR-4 expression, activated NF-kappaB translocation and increased the pro-inflammatory cytokines TNF-alpha, IL-6 and IL-1beta. The addition of a specific CD44 blocking antibody reduced CD44 and all inflammatory cytokine expression as well as protein production. However, cytokine expression remained significantly higher than in untreated chondrocytes. TLR-4 expression was not affected. The treatment with TLR-4 blocking antibody decreased TLR-4 and inflammatory cytokine expression, although cytokine expression was significantly higher than in control cells. CD44 expression was unaffected. The addition of both CD44 and TLR-4 blocking antibodies significantly reduced CD44, TLR-4 and inflammatory cytokine expression., (2010 Elsevier Inc. All rights reserved.)

Among Teleosts, Sparus aurata occupies a prominent place in the gastronomic and economic fields of the Mediterranean basin and other geographic districts. The knowledge of its molecular structures and functional features, such as hemoglobin, may be helpful to understand the adaptive biochemical mechanisms that allow this fish to live under extreme conditions, including fish farming. In Sparus aurata red blood cells two different alpha and one beta hemoglobin genes have been identified. The alpha1 gene codifies a putative protein of 144 amino acids, the alpha2 gene produces a protein of 143 amino acids, and the beta gene encodes a chain of 148 amino acids. Comparative analysis of various hemoglobins indicates that allosteric regulation can be modified by the substitution of one or a few key residues. The comparison of the percentage sequence differences for alpha and beta chains in fishes indicates that evolutionary relationships between different species may be helpful to understand the mechanisms of their differentiation from other vertebrates. Hemoglobin alpha and beta chains of about 50 teleostean temperate and Antarctic fishes were analyzed to build phylogenetic trees using different algorithms: the neighbor-joining method, the maximum likelihood approach, and the Bayesian inference computation. Sparus aurata alpha chains are positioned in a paraphyletic cluster, which includes the same subunit of Chrysophrys auratus and Seriola quinqueradiata, whereas the beta chain is on an homophyletic branch with that of Chrysophrys auratus. Therefore, the phylogenetic approach suggests that both Sparus aurata hemoglobin alpha genes are paralogues and may have derived from a duplication event.

Hyaluronan (HA) action depends upon its molecular size. Low molecular weight HA elicits pro-inflammatory responses by modulating the toll-like receptor-4 (TLR-4) or by activating the nuclear factor kappa B (NF-kB). In contrast, high molecular weight HA manifests an anti-inflammatory effect via CD receptors and by inhibiting NF-kB activation. Lipopolysaccharide (LPS) -mediated activation of TLR-4 complex induces the myeloid differentiation primary-response protein (MyD88) and the tumor necrosis factor receptor-associated factor-6 (TRAF-6), and ends with the liberation of NF-kB/Rel family members. The aim of this study was to investigate the influence of HA at different MWs (low, medium, high) on TLR-4 modulation in LPS-induced inflammatory response in mouse chondrocyte cultures. Messenger RNA and related protein levels were measured for TLR-4, MyD88, and TRAF-6 in both untreated and LPS-treated chondrocytes, with and without the addition of HA (two doses for each MW). NF-kB activation, TNF-alpha and IL-1beta levels, matrix metalloprotease-13 (MMP-13), and inducible nitric oxide synthase (iNOS) gene expression were also evaluated. LPS increased all the parameters studied as well as NF-kB activation. Low MW HA upregulated TLR-4 expression, increased MyD88 and TRAF-6 and the inflammation mediators in untreated chondrocytes, and it enhanced the LPS effect in LPS-treated cells. Medium and high MW HA exerted no activity in untreated cells and only the latter reduced the LPS effects. Specific TLR-4 blocking antibody was utilised to confirm TLR-4 as the target of HA action. These findings suggest that the regulatory effect exerted by HA (at any MW) on NF-kB activation may depend upon the interaction between HA and TLR-4 and HA may thereby modulate pro-inflammatory activity via its different state of aggregation., (2009 Elsevier Masson SAS. All rights reserved.)

Lipopolysaccharide (LPS)-mediated activation of toll-like receptor-4 (TLR-4) complex induces specific signaling pathways, such as the myeloid differentiation primary response protein-88 (MyD88) and the tumor necrosis factor receptor-associated factor-6 (TRAF-6), involving NF-kappaB activation. As previous data reported that hyaluronan (HA) and heparan sulfate (HS) may interact with TLR-4, the aim of this study was to investigate whether glycosaminoglycans (GAGs) may modulate the TLR-4 receptor in a model of LPS-induced inflammatory cytokines in mouse chondrocytes. LPS stimulation up-regulated all inflammation parameters. The GAG treatment produced various effects: HA reduced MyD88 and TRAF-6 levels and NF-kappaB activation at the higher dose only, and exerted a very low anti-inflammatory effect; chondroitin-4-sulfate (C4S) and chondroitin-6-sulfate significantly inhibited MyD88, TRAF-6 and NF-kappaB activation, the inflammation cytokines, and inducible nitric oxide synthase; HS, like C4S, significantly reduced MyD88, TRAF-6, NF-kappaB and inflammation. Specific TLR-4 blocking antibody confirmed that TLR-4 was the target of GAG action.

Background: Hyaluronan (HA) fragments elicit the expression of inflammatory mediators through a mechanism involving the CD44 receptor. This study investigated the effects of HA at different molecular weights on PMA-induced inflammation in mouse chondrocytes., Methods: mRNA and related protein levels were measured for CD44, PKCdelta, PKCepsilon, TNF-alpha, IL-1beta, MMP-13, and iNOS in chondrocytes, untreated or PMA treated, with and without the addition of HA. The level of NF-kB activation was also assayed., Results: CD44, PKCdelta, and PKCepsilon mRNA expression resulted higher than controls in chondrocytes treated with PMA. PMA also induced NF-kB up-regulation and increased TNF-alpha, IL-1beta, MMP-13, and iNOS expression. HA treatment produced different effects: low MW HA up-regulated CD44 expression, increased PKCdelta and PKCepsilon levels, and enhanced inflammation in untreated chondrocytes; while in PMA-treated cells it increased CD44, PKCdelta, PKCepsilon, NF-kB, TNF-alpha, IL-1beta, MMP-13, and iNOS expression and enhanced the effects of PMA; medium MW HA did not exert action; high MW HA had no effect on untreated chondrocytes; however, it reduced PKCdelta, PKCepsilon, NF-kB activation and inflammation in PMA-stimulated cells. Specific CD44 blocking antibody was utilised to confirm CD44 as the target of HA modulation., General Significance: These data suggest that HA via CD44 may modulate inflammation via its different molecular mass.

Acrylates (acrylic and methacrylic compounds) are commonly used in dentistry as restorative materials, and their use is progressively increasing because of the concern for mercury-related health problems (allergic and nonallergic) due to their metal equivalents. However, acrylates are not completely safe from an allergologic point of view. We report a case of multiple patch-test reactions to acrylates and methacrylates that occurred at various times between 24 hours and 7 days after test application.

Introduction: There have been several cases reporting a significant increase in chondroitin sulphate plasma levels in patients with different types of disease, such as systemic lupus erythematosus, rheumatoid arthritis, and liver disease. At present, the precise role of chondroitin sulphate molecules in blood is unclear. Previous investigations have shown that the addition of purified human plasma glycosaminoglycans (GAGs), containing a high percentage of chondroitin-4-sulphate (C4S) was able to inhibit lipid peroxidation and to protect cells from reactive oxygen species damage, suggesting antioxidant activity. Starting from these reports, the aim of this study was to evaluate the effectiveness of GAG structures purified from normal human plasma in reducing inflammation using a model of lipopolysaccharide (LPS)-induced increase of pro-inflammatory cytokines in mouse articular chondrocyte cultures., Results: Chondrocyte stimulation with LPS (50 microg/ml) for 24 h enhanced gene expression of tumor necrosis factor alpha (TNF-alpha), interleukin-1 beta (IL-1beta), interleukin 6 (IL-6), interferon gamma (IFN-gamma), inducible nitric oxide synthase (iNOS) and increases in their related protein levels, as well as NF-kappaB activation, IkappaBalpha phosphorylation and apoptosis evaluated by the increase in caspase-3 expression and its related protein amount. LPS treatment also generated a high amount of nitric oxide (NO). The addition of different doses of purified human GAGs to LPS-stimulated chondrocytes reduced inflammatory cytokines and iNOS both at mRNA and protein levels, blocked NF-kappaB activation and cytoplasmic IkappaBalpha phosphorylation, limited cell death by inhibiting apoptosis, and reduced NO concentrations., Conclusions: These results further support the hypothesis that plasma GAGs may function as immunomodulators and their increased release and degradation could be a biological response acting to modulate inflammation during disease.

Activation of nuclear factor kappaB (NF-kappaB) and caspases may greatly amplify inflammation and cell damage in addition to that directly exerted by free radicals. Since reactive oxygen species (ROS) are involved in acute pancreatitis, we studied whether the administration of chondroitin-4-sulphate (C4S), in addition to its antioxidant activity, was able to modulate NF-kappaB and caspase activation in an experimental model of caerulein-induced acute pancreatitis in mice. Hyperstimulating doses of caerulein (50 microg/ kg), five injections per mouse given at hourly intervals produced the following: high serum lipase and amylase activity; lipid peroxidation, evaluated by 8-isoprostane concentrations; loss of antioxidant defenses such as glutathione reductase (GR) activity; NF-kappaB activation and loss of cytoplasmic IkappaBalpha protein; increases in tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), caspase-3, and caspase-7 gene expression and their related protein; accumulation and activation of neutrophils in the damaged tissue, evaluated by elastase (ELA) determination; and pancreatic injury, evaluated by histologic analysis. Pretreatment of mice with different doses of C4S, given 1 hr before caerulein injections and 1 and 2 hrs after the last caerulein injection, reduced lipid peroxidation, inhibited NF-kappaB translocation and cytoplasmic IkappaBalpha protein loss, decreased TNF-alpha, IL-6, and caspase gene expression and their related protein levels, limited endogenous antioxidant depletion, and reduced tissue neutrophils accumulation and tissue damage. Since molecules with antioxidant activity can block NF-kappaB and apoptosis activation, we suggest that C4S administration is able to block NF-kappaB and caspase activation by reducing the oxidative burst.

Previous studies suggest that Transforming growth factor-1beta (TGF-1beta) administration in human fibroblasts exposed to oxidative stress is able to modulate hyaluronan synthases (HASs). HAS modulation in turn increases high molecular weight (Hyaluronan) HA concentration. Nuclear factor kB (NF-kB) is a response transcription factor involved in inflammation and acts by enabling the expression of certain detrimental molecules. Caspases are specific proteases responsible for regulating and programming cell death. HA at medium molecular weight together with chondroitin-4-sulphate proved to be effective on NF-kB and caspases. We investigated whether the protective effect afforded by the high molecular weight HA produced by TGF-1beta treatment has any effect on NF-kB and apoptosis activation in fibroblast cultures exposed to oxidative stress. Generation of free radicals gives rise to cell death, increases lipid peroxidation, activates NF-kB, reduces its cytoplasmic inhibitor IkBalpha, augments caspase-3 and caspase-7 gene expression and their relative protein activity, and depletes catalase (CAT) and glutathione peroxidase (GPx). Treatment of fibroblasts with TGF-1beta 12 h before inducing oxidative stress greatly increased HA levels, ameliorated cell survival, inhibited lipid peroxidation, blunted NF-kB translocation, normalized IkBalpha protein, reduced caspase gene expression and protein levels, and restored the endogenous antioxidants CAT and GPx. Since it was previously reported that antioxidants can work as inhibitors of NF-kB and apoptosis induction we can hypothesize that endogenous HA, by inhibiting lipid peroxidation, may block a step whereby free radical activity converges in the signal transduction pathway leading to NF-kB and caspase activation.

mRNA/cDNA gene expression of both small leucine-rich proteoglycans decorin and biglycan was evaluated by PCR real time in lymphocytes collected from patients with chronic lymphocytic leukaemia (CLL) at different stages of disease and from healthy controls. Lymphocytes obtained from healthy controls showed no or very low levels of mRNA expression of both decorin and biglycan. Biglycan expression was very low in CLL patients, values being close to those of controls. On the contrary, decorin mRNA was clearly expressed in patients with early B-cell CLL, while a low expression was found in advanced clinical stages. Furthermore, a significant higher decorin expression was found in patients with non-progressive CLL type in comparison with patients with aggressive type of the disease. Decorin expression resulted especially high in the low-progressive low-risk patients. The synthesis of decorin was also assessed by Western blot analysis. The peculiar occurrence of decorin in the non-aggressive type of CLL is consistent with its suggested anti-oncogenic role. Intracellular Bcl-2 level does not correlate with decorin mRNA transcription, suggesting that a Bcl-2 independent anti-cancer mechanism may occur. The measurement of galactosamine-containing proteoglycans concentration in plasma confirmed decorin expression results, with significant differences between CLL patients and controls. Significant changes were also seen between groups of patients of Rai stage 0 with recent diagnosis (less than 5 years, from analysis), (low amount of decorin) and less recent diagnosis (more than 5 years), (high amount of decorin).

Several reports have shown that a number of cytokines such as tumor necrosis-alpha (TNF-alpha), interferon-gamma (IFN-gamma), and interleukin-beta (IL-1beta) are capable to induce hyaluronan sinthases (HASs) mRNA expression in different cell culture types. The obvious consequence of this stimulation is a marked increment in hyaluronan (HA) production. It has been also reported that oxidative stress, by itself, may increase HA levels. The aim of this study was to evaluate how TNF-alpha, IFN-gamma,IL-1beta, and exposition to oxidative stress may modulate HAS activities in normal human skin fibroblasts. Moreover, the effects on HAS mRNA expression of the concomitant treatment with cytokines and oxidants, and the HA concentrations after treatments, were studied. TNF-alpha, IFN-gamma, and IL-1beta were added to normal or/and exposed to FeSO(4) plus ascorbate fibroblast cultures and HAS1, HAS2 and HAS3 mRNA content, by PCR-real time, was assayed 3,h later. HA levels were also evaluated after 24,h incubation. The treatment of fibroblasts with cytokines up-regulated HASs gene expression and increased HA production. IL-1beta induced HAS mRNA expression and HA production more efficiently than TNF-alpha and IFN-gamma. The exposition of the fibroblasts with the oxidant system markedly increased HAS activities while slightly HA production. The concomitant treatment of cells with the cytokines and the oxidant was able to further enhance, in a dose dependent way, with synergistic effect on HAS mRNA expression. On the contrary HA levels resulted unaffected by the concomitant treatment, and resemble those obtained with the exposure to FeSO(4) plus ascorbate only. This lack in HA production could be due to the deleterious action of free radicals on the HA synthesis.

Imbalance between matrix metalloproteinases (MMPs) and tissue inhibitor of matrix metalloproteinases (TIMPs) is an important control point in tissue remodelling. Several findings have reported a marked MMP/TIMP imbalance in a variety of in vitro models in which oxidative stress was induced. Since previous studies showed that commercial hyaluronan and chondroitin-4-sulphate are able to limit lipid peroxidation during oxidative stress, we investigated the antioxidant capacity of purified human plasma chondroitin-4-sulfate in reducing MMP and TIMP imbalance in a model of ROS-induced oxidative injury in fibroblast cultures. Purified human plasma chondroitin-4-sulfate was added to the fibroblast cultures exposed to FeSO4 plus ascorbate. We assayed cell death, MMP and TIMP mRNA expression and protein activities, DNA damage, membrane lipid peroxidation, and aconitase depletion. FeSO4 plus ascorbate produced severe death of cells and increased MMP-1, MMP-2 and MMP-9 expression and protein activities. It also caused DNA strand breaks, enhanced lipid peroxidation and decreased aconitase. TIMP-1 and TIMP-2 protein levels and mRNA expression remain unaltered. Purified human plasma C4S, at three different doses, restored the MMP/TIMP homeostasis, increased cell survival, reduced DNA damage, inhibited lipid peroxidation and limited impairment of aconitase. These results further support the hypothesis that these biomolecules possess antioxidant activity and by reducing ROS production C4S may limit cell injury produced by MMP/TIMP imbalance.

The main role of superoxide dismutases (SODs) is to eliminate reactive oxygen species in cells and tissues. Extracellular SOD (EC-SOD/SOD3) is a major superoxide scavenger and it is located on cell surfaces and primarily in extracellular matrix, and binds heparan sulfates by its carboxyterminal portion. Human EC-SOD gene is located on chromosome 4 and comprises three exons and two introns. The SOD3 coding sequence is entirely located within exon 3 and has missense polymorphisms. The Arg213Gly mutation affects the function of the carboxyterminus and correlates with several diseases. In this work, we explored genetic variants within EC-SOD gene of subjects living in southern Italy. Four new variations were detected: one was silent mutation, while three were missense variations that give rise to amino acid substitutions at position 131 (F>C), 160 (V>L) and 202 (R>L) in the mature product. The Arg213Gly variant was not found. The missense mutations in the DNA of assayed 2400 chromosomes had frequencies of 5.34% for the F131C variation, 0.25% for the V160L variation and 0.84% for the R202L variation. The effect of these alterations on the metabolic activity and diseases remains to be further explained.

A number of studies in vivo and in vitro showed that high levels of glycosaminoglycans (GAGs) are found as a consequence of free radical damage. The GAG over production may represent an endogenous mechanism capable to limit oxidative damage. Based on these hypotheses, the aim of this study was to evaluate the antioxidant property of GAGs of human origin in fibroblast cultures. Purified human plasma GAGs were added to the fibroblast cultures in which oxidative stress was induced by the oxidizing system employing iron (Fe2+) plus ascorbate. We assessed cell death, lactate dehydrogenase activity, membrane lipid peroxidation, DNA damage, protein oxidation, hydroxyl radical (OH*) generation and endogenous antioxidant depletion. The exposure of fibroblasts to FeSO4 produced cell death and increased OH* production. It also caused DNA strand breaks and protein oxidation as shown by the DNA fragment analysis and protein carbonyl content, respectively. In addition, FeSO4 enhanced lactate dehydrogenase activity and lipid peroxidation while decreased antioxidant defences. Purified human GAGs, at three different doses, reduced cell death, limited DNA fragmentation and protein oxidation, decreased OH* generation and lactate dehydrogenase activity, inhibited lipid peroxidation and improved endogenous antioxidant defences. These results further support the hypothesis that these molecules may function as antioxidants.

Hepatic fibrosis involves the interplay of many factors including reactive oxygen species. Recent reports described antioxidant properties of glycosaminoglycans (GAGs). Since several findings have shown that hyaluronic acid (HYA) and chondroitin-4-sulphate (C4S) may act as antioxidant molecules, the aim of this research was to evaluate the antioxidant effects of HYA and C4S treatment in a rat model of liver fibrosis. The effect on tissue inhibitors of metalloproteinases (TIMPs) was also studied. Liver fibrosis was induced in rats by eight intraperitoneal injections of CCl4, twice a week for 6 weeks. HYA or C4S alone (25 mg/kg) or HYA and C4S in combination (12.5 + 12.5 mg/kg) were administered daily by the same route during the 6 weeks. At the end of the 6-week treatment period (24 h after the last dose of GAGs), the following parameters were evaluated: (1) serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities, as index of hepatic cell disruption; (2) hepatic conjugated dienes (CD), as index of lipid peroxidation; (3) hepatic TIMPs activity and expression; (4) hepatic superoxide dismutase (SOD) and glutathione peroxidase (GPx) activity, as index of endogenous defences; (5) hepatic hydroxyproline, as index of collagen deposition. CCl4-induced liver fibrosis enhanced lipid peroxidation and TIMPs activation, increased ALT and AST, depleted antioxidants SOD and GPx, and caused collagen deposition in liver tissue. Treatment with GAGs, especially when in combination, successfully reduced ALT and AST rise, lipid peroxidation by evaluating conjugated dienes, TIMPs activation and mRNA expression, partially restored SOD and GPx activities, and limited collagen deposition in the hepatic tissue. The data obtained showed that these molecules were able to limit hepatic injury induced by chronic CCl4 intoxication and especially limited liver fibrosis. They also confirm that HYA and C4S may exert antioxidant mechanism, while reduction of TIMPs expression suggests that GAGs may influence MMPs and TIMPs imbalance in liver fibrosis.

PON gene family includes at least three members termed PON1, PON2 and PON3, and it is mapped on human chromosome 7q21-q22. PON1 and PON3 gene products are constituents of high density lipoprotein (HDL) and have many enzymatic properties and antioxidant activity. PONs are proposed to participate in the prevention of low density lipoprotein (LDL) oxidation. PON1 and PON2 genes have missense polymorphisms, but, to date, no missense variants are reported in PON3 gene. In this work we explored the existence of genetic variants within the PON3 coding sequences. Five point mutations were identified by direct sequencing of genomic DNA derived from 250 randomly selected DNA samples of 1143 blood donors living in southern Italy. Three were silent mutations, while two were missense mutations that give rise to amino acid substitutions at positions 311 (S>T) and 324 (G>D). The missense variations in the DNA of the 1143 samples had frequencies of 0.22% (5 out of 2286 alleles) for the S311T mutation, and 0.57% (13 out of 2286 alleles) for the G324D mutation. The effect of these variants on the metabolic activity of paraoxonase 3 remains to be further evaluated.

Oxidative stress is involved in the pathogenesis of chemically mediated liver injury. Since glycosaminoglycans possess antioxidant activity, the aim of this work was to assess the protective effects of hyaluronic acid and chondroitin-4-sulphate treatment in a model of carbon tetrachloride-induced liver injury. Liver damage was induced in male rats by an intraperitoneal injection of carbon tetrachloride (1 ml/kg in vegetal oil). Serum alanine aminotransferase and aspartate aminotransferase, hepatic malondialdehyde, plasma TNF-alpha, hepatic reduced glutathione and catalase, and myeloperoxidase, an index of polymorphonuclear infiltration in the jeopardised hepatic tissue, were evaluated 24 h after carbon tetrachloride administration. Carbon tetrachloride produced a marked increase in serum alanine aminotransferase and aspartate aminotransferase activities, primed lipid peroxidation, enhanced plasma TNF-alpha levels, induced a severe depletion of reduced glutathione and catalase, and promoted neutrophil accumulation. Intraperitoneal treatment of rats with hyaluronic acid (25 mg/kg) or chondroitin-4-sulphate (25 mg/kg) failed to exert any effect in the considered parameter, while the combination treatment with both glycosaminoglycans (12,5 + 12,5 mg/kg) decreased the serum levels of alanine aminotransferase and aspartate aminotransferase, inhibited lipid peroxidation by reducing hepatic malondialdehyde, reduced plasma TNF-alpha, restored the endogenous antioxidants, and finally decreased myeloperoxidase activity. These results suggest that hyaluronic acid and chondroitin-4-sulphate possess a different antioxidant mechanism and consequently the combined administration of both glycosaminoglycans exerts a synergistic effect with respect to the single treatment.

Acid glycosaminoglycans (GAGs) antioxidant activity was assessed in a fibroblast culture system by evaluating reduction of oxidative system-induced damage. Three different methods to induce oxidative stress in human skin fibroblast cultures were used. In the first protocol cells were treated with CuSO4 plus ascorbate. In the second experiment fibroblasts were exposed to FeSO4 plus ascorbate. In the third system H2O2 was utilised. The exposition of fibroblasts to each one of the three oxidant systems caused inhibition of cell growth and cell death, increase of lipid peroxidation evaluated by the analysis of malondialdehyde (MDA), decrease of reduced glutathione (GSH) and superoxide dismutase (SOD) levels, and rise of lactate dehydrogenase activity (LDH). The treatment with commercial GAGs at different doses showed beneficial effects in all oxidative models. Hyaluronic acid (HA) and chondroitin-4-sulphate (C4S) exhibited the highest protection. However, the cells exposed to CuSO4 plus ascorbate and FeSO4 plus ascorbate were better protected by GAGs compared to those exposed to H2O2. These outcomes confirm the antioxidant properties of GAGs and further support the hypothesis that these molecules may function as metal chelators.

To evaluate the antioxidant activity of the glycosaminoglycans hyaluronic acid (HYA) and chondroitin-4-sulphate (C4S), we used a rat model of collagen-induced arthritis (CIA). Arthritis was induced in Lewis rats by multiple intradermal injections of 250 microl of emulsion containing bovine type II collagen in complete Freund's adjuvant at the base of the tail and into three to five other sites on the back. Rats were challenged again with the same antigen preparation 7 days later. Disease developed about 11 days after the second immunization. The effects of treatment in the rats were monitored by biochemical parameters and by macroscopic and histological evaluations in blood, synovial tissue and articular cartilage. Arthritis produced the following symptoms: severe periarticular erythema, edema and inflammation in the hindpaws; membrane peroxidation in the cartilage of the joints; endogenous antioxidant wasting; high tumour necrosis factor-alpha (TNF-alpha) plasma levels; and synovial neutrophil accumulation. Treatment with HYA and C4S, starting at the onset of arthritis for 10 days, limited the erosive action of the disease in the articular joints of knee and paw, reduced lipid peroxidation, restored the endogenous antioxidants reduced glutathione (GSH) and superoxide dismutase, decreased plasma TNF-alpha levels, and limited synovial neutrophil infiltration. These data confirm that erosive destruction of the joint cartilage in CIA is due at least in part to free radicals released by activated neutrophils and produced by other biochemical pathways. The beneficial effects obtained with the treatment suggest that HYA and C4S could be considered natural endogenous macromolecules to limit erosive damage in CIA or as a useful tool with which to study the involvement of free radicals in rheumatoid arthritis.