Changes in the Subchondral Bone, Visfatin, and Cartilage Biomarkers after Pharmacological Treatment of Experimental Osteoarthritis with Metformin and Alendronate.

Subchondral bone that has intense communication with the articular cartilage might be a potential target for pharmacological treatment in the early stages of osteoarthritis (OA). Considering the emerging data about the role of adipokines in the pathogenesis of OA, the administration of drugs that influence their level is also intriguing. Metformin and alendronate were administered in mice with collagenase-induced OA (CIOA) as a monotherapy and in combination. Safranin O staining was used for the assessment of changes in subchondral bone and articular cartilage. Before and after treatment, serum levels of visfatin and biomarkers of cartilage turnover (CTX-II, MMP-13, and COMP) were assessed. In the current study, the combined administration of alendronate and metformin in mice with CIOA led to the protection against cartilage and subchondral bone damage. In mice with CIOA, metformin led to a decrease in visfatin level. In addition, treatment with metformin, alendronate, or their combination lowered the level of cartilage biomarkers (CTX-II and COMP), while the level of MMP-13 was not influenced. In conclusion, personalized combination treatment in OA according to clinical phenotype, especially in the early stages of the disease, might lead to the identification of a successful disease-modifying therapeutic protocol in OA.

Follicle stimulating hormone and estradiol alter immune response in osteoarthritic mice in an opposite manner.

Although a number of studies have shown that the occurrence and progression of osteoarthritis (OA) is related to endocrine system dysfunction, there is limited evidence about what roles sex hormones play. The aim of the present study was to examine the capacity of 17ß-estradiol (ED) and follicle stimulating hormone (FSH) to alter the differentiation of bone marrow (BM) cells in arthritic mice. The experiments were conducted in collagenase-induced osteoarthritis in mice. Cartilage degradation was observed by safranin and toluidine blue staining. Flow cytometry was used to define different BM and synovial cell populations. The influence of FSH and ED on osteoclastogenesis was studied in BM cultures and on the osteoblastogenesis in primary calvarial cultures. The levels of IL-8, TNF-α, FSH, and osteocalcin were estimated by ELISA. FSH increased cartilage degradation and serum osteocalcin levels, while ED abolished it and lowered serum osteocalcin. FSH elevated the percentage of monocytoid CD14+/RANK+ and B cell CD19+/RANK+ cells in contrast to ED which inhibited the accumulation of these osteogenic populations. Also, ED changed the percentage of CD105+/F4/80+ and CD11c+ cells in the synovium. FSH augmented and ED suppressed macrophage colony-stimulating factor (M-CSF) + receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast (OC) formation, and this correlated with a respective increase and decrease of IL-8 secretion. FSH did not influence osteoblast (OB) formation while ED enhanced this process in association with changes of TNF-α, IL-8, and osteocalcin production. ED reduced osteoclast generation in bone. The key outcome of the current study is that both hormones influenced BM cell differentiation, with FSH favoring osteoclast formation and ED favoring osteoblast accumulation.

Influence of estradiol treatment on bone marrow cell differentiation in collagenase-induced arthritis.

OBJECTIVE AND DESIGN: Estrogen is one of the important regulators of the balance between bone formation and bone resorption that can modulate the levels and activity of certain growth factors and cytokines. In this study, we investigated the effect of 17ß-estradiol (ED) on bone marrow (BM) cell differentiation in vivo and ex vivo in a mouse model of collagenase-induced osteoarthritis (CIOA). SUBJECT: ICR (CD-2) female mice were used in present experiments (total number = 75) and bone marrow cells were used for in vitro studies. TREATMENT: Mice were orally fed under different schemes with 17ß-estradiol at a dose of 2 µg or 4 µg for 30 days. METHODS: The effect of estradiol was estimated by histopathological, flow cytometry, and ELISA assays. Statistical differences were determined by one-way ANOVA. RESULTS: Estradiol treatment ameliorated cartilage destruction and osteophyte formation if started from day 0 of CIOA induction, attended with a decrease of uterine and ovarian weights. Long time treatment lowered the percentage of megakaryocyte/platelet (CD62P+) populations and osteoclast (RANK+) populations in BM. Cells obtained from estradiol-treated CIOA mice showed inhibited capacity to differentiate into RANK+ and mesenchymal cells under osteoclastogenic conditions in vitro. Estrogen decreased serum IL-6 levels. CONCLUSION: Results indicate a potential protective role for estrogen against the development of OA.

Alkaloids from the aerial parts of Leptopyrum fumarioides express immunomodulatory activity.

A new type aporphine-benzylisoquinoline alkaloid leptofumarine (1) was isolated from Leptopyrum fumarioides L. The structure of 1 was elucidated based on spectroscopic evidence. Leptofumarine together with the previously isolated leptopyrine (2) were evaluated for their effect on macrophages and bone marrow (BM) cells in vitro. Both alkaloids inhibited cytokine production by LPS-stimulated peritoneal macrophages. Leptopyrine suppressed osteoclast and osteoblast formation, while leptofumarine affected only osteoblastogenesis.

Functional complement activity is decisive for the development of chronic synovitis, osteophyte formation and processes of cell senescence in zymosan-induced arthritis.

Synovial inflammation plays a critical role in the symptoms and structural progression of arthritis which leads to irreversible damage of the adjacent cartilage and bone. Activation of complement system is strongly implicated as a factor in the pathogenesis of chronic synovitis in human rheumatoid arthritis (RA). In this study, we show that the depletion of functional complement activity at the time of the initiation of zymosan-induced arthritis, significantly reduced the expression of TGF-beta1/3, BMP2 and pSmad2 and decreased the number of Sudan Black B positive cells in the synovium. Also, the excessive synthesis of proteoglycans and glycosaminoglycans was diminished. The appearance of apoptotic and senescent cells among the adherent bone marrow cells cultivated in vitro was not observed in complement depleted mice. Therefore, the lack of functional complement prevented the development of chronic synovitis, osteophyte formation and the generation of pathologic senescent arthritic cells.

Distinct roles of TNF-related apoptosis-inducing ligand (TRAIL) in viral and bacterial infections: from pathogenesis to pathogen clearance.

INTRODUCTION: Apoptotic death of different cells observed during infection is thought to limit overwhelming inflammation in response to microbial challenge. However, the underlying apoptotic death mechanisms have not been well defined. Tumor necrosis factor (TNF) related apoptosis-inducing ligand (TRAIL) is a type II transmembrane protein belonging to the TNF superfamily, which is involved not only in tumor growth suppression but in infection control and also in the regulation of both innate and adaptive immune responses. FINDINGS: In this review, we have summarized data of recent studies on the influence of the TRAIL/TRAIL receptor (TRAIL-R) system on the development of viral and bacterial infections. TRAIL may have a dual function in the immune system being able to kill infected cells and also to participate in the pathogenesis of multiple infections. Moreover, many pathogens have evolved mechanisms to manipulate TRAIL signaling thus increasing pathogen replication. CONCLUSION: Present data highlight an essential role for the TRAIL/TRAIL-R system in the regulation and modulation of apoptosis and show that TRAIL has distinct roles in pathogenesis and pathogen elimination. Knowledge of the factors that determine whether TRAIL is helpful or harmful supposes its potential therapeutic implications that are only beginning to be explored.

Intravenous Immunoglobulin with Enhanced Polyspecificity Improves Survival in Experimental Sepsis and Aseptic Systemic Inflammatory Response Syndromes.

Sepsis is a major cause for death worldwide. Numerous interventional trials with agents neutralizing single proinflammatory mediators have failed to improve survival in sepsis and aseptic systemic inflammatory response syndromes. This failure could be explained by the widespread gene expression dysregulation known as "genomic storm" in these patients. A multifunctional polyspecific therapeutic agent might be needed to thwart the effects of this storm. Licensed pooled intravenous immunoglobulin preparations seemed to be a promising candidate, but they have also failed in their present form to prevent sepsis-related death. We report here the protective effect of a single dose of intravenous immunoglobulin preparations with additionally enhanced polyspecificity in three models of sepsis and aseptic systemic inflammation. The modification of the pooled immunoglobulin G molecules by exposure to ferrous ions resulted in their newly acquired ability to bind some proinflammatory molecules, complement components and endogenous "danger" signals. The improved survival in endotoxemia was associated with serum levels of proinflammatory cytokines, diminished complement consumption and normalization of the coagulation time. We suggest that intravenous immunoglobulin preparations with additionally enhanced polyspecificity have a clinical potential in sepsis and related systemic inflammatory syndromes.

Tyrosine kinase inhibitor tyrphostin AG490 reduces liver injury in LPS-induced shock.

Sepsis remains a serious clinical problem despite continuous efforts to increase survival. Experimental animal models of sepsis are pointed to a great extent on blocking the activity of cytokines. A number of signal-transducing molecules are associated with the occurrence of excessive tissue inflammation. Through inhibition of tyrosine phosphorilation and thereby changing cell signaling, tyrosine kinase inhibitors can influence multiple inflammatory pathways. The purpose of the present investigation was to evaluate the effect of tyrosine kinase inhibitor tyrphostin AG490 in a mouse LPS-induced shock. Cytokine and chemokine blood levels were determined by ELISA assays. CD11b(+) Ly6C(+), CD3(+)CD69(+) and C5aR positive cell populations in the peritoneal exudate were detected by flow cytometry. The expression of iNOS and Signal Transducer and Activator of Transcription (STAT) in the liver were observed by immunohistochemistry. We found that tyrphostin AG490 inhibited Regulated upon activation normal T cell expressed and secreted (RANTES), IL-6 and IL-12 serum levels, decreased the number of CD11b(+)Ly6C(+) and CD3(+)CD69(+) subpopulations in the peritoneal exudate and prevented the decrease of cells expressing C5a receptor and TNF-alpha receptor. Tyrphostin ameliorated liver injury associated with suppressed iNOS, STAT3 and pSTAT3 expression. Our data suggest that tyrphostin AG490 diminished the degree of inflammation starting in peritoneal cavity and minimized liver dysfunction thus representing one approach for better outcome of sepsis conditions.

Kinase inhibitors with redox and anti-inflammatory activities.

The development of inflammatory immune response is related to an activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling. The intracellular molecules from this pathway are sensitive to the alterations in the microenvironment. The changes in cellular redox state, proliferation, gene expression pattern and genomic stability during inflammation induce the activation of non-canonical and atypical NK-κB signaling increasing the crosstalk with molecules involved in neddylation, cell cycle checkpoints regulation and DNA repair. This review article describes the reactive oxygen species (ROS)-sensitive kinases from the NF-κB pathway and presents the effects of their suppression by small kinase inhibitors. It illustrates that selective targeting of the redox sensor molecules from the inflammatory NK- κB cascades can influence cell survival and metabolism as well. We think that this issue is important when evaluating the drug efficacy in clinical studies and their side effects.

Acetylated derivative of glaucine inhibits joint inflammation in collagenase-induced arthritis.

CONTEXT: Osteoarthritis (OA) has become by far the most common joint disorder. A number of studies using OA animal models have explored the effects of agents that can modulate bone metabolism. OBJECTIVE: In the present study, we investigated the effect of acetylated derivative of plant alkaloid glaucine (ADG) on experimental OA in mice. MATERIALS AND METHODS: Arthritis was induced by two intraarticular (i.a.) injections of collaganase. Histopathological changes were observed through hematoxylin and eosine (H&E), safranin O and toluidine blue staining. Differentiation of bone marrow (BM) cells was evaluated by tartarate-resistant acid phosphatase (TRAP) assay. The expression of phospho-Janus kinase 2 (pJAK2) and phospho signal transducer and activator of transcription3 (pSTAT3) expression in the joints was determined by immunohistochemistry. RESULTS: We established that ADG significantly decreased cell infiltration (2.32 ± 0.14 versus 1.62 ± 0.13), cartilage loss (2.42 ± 0.12 versus 1.12 ± 0.10) and bone erosion (1.76 ± 0.13 versus 1.04 ± 0.14) in arthritic mice. It appeared that the substance inhibited in a dose-dependent manner osteoclast differentiation in vitro. ADG suppressed the expression of pJAK2 in the joint and partially affected the expression of pSTAT3. CONCLUSION: Present results suggest that ADG is a suitable candidate for further development as an anti-arthritic agent.

TLR2 elicits IL-17-mediated RANKL expression, IL-17, and OPG production in neutrophils from arthritic mice.

We investigated the ability of neutrophils to express receptor activator of nuclear factor kappa-B ligand (RANKL), to secrete osteoprotegerin (OPG), and to produce IL-17. Arthritis was induced by intra-articular injection of zymosan, a ligand for Toll-like receptor 2 (TLR2). Frequencies of neutrophils in bone marrow (BM), blood and synovial fluid (SF), receptor expression, and cytokine production were evaluated by flow cytometry. 1A8 antibody (1A8 Ab) was used to deplete neutrophils in zymosan-injected SCID mice. IL-17, RANKL, and OPG amounts in SF, serum, or cell cultures were determined by ELISA. The development of arthritis was associated with increased secretion of IL-17, RANKL, and OPG in serum and SF, elevated frequencies of Ly6G(+)CD11b(+) cells in BM, blood, and SF and upregulated RANKL expression. Both IL-17 and OPG were absent in serum and SF after neutrophil depletion; therefore we assume that they were released by neutrophils. In vitro blood Ly6G(+)CD11b(+) cells from arthritic mice produced spontaneously IL-17, IFN-γ, and OPG and expressed RANKL. This phenotype was sustained by IL-17. TLR2 engagement increased IL-17 and IFN-γ production, potentiated IL-17-mediated RANKL expression, and inhibited OPG secretion. We conclude that TLR2 regulates the destructive potential of neutrophils and its targeting might limit joint alterations in arthritis.

Septicaemia models using Streptococcus pneumoniae and Listeria monocytogenes: understanding the role of complement properdin.

Streptococcus pneumoniae and Listeria monocytogenes, pathogens which can cause severe infectious disease in human, were used to infect properdin-deficient and wildtype mice. The aim was to deduce a role for properdin, positive regulator of the alternative pathway of complement activation, by comparing and contrasting the immune response of the two genotypes in vivo. We show that properdin-deficient and wildtype mice mounted antipneumococcal serotype-specific IgM antibodies, which were protective. Properdin-deficient mice, however, had increased survival in the model of streptococcal pneumonia and sepsis. Low activity of the classical pathway of complement and modulation of FcγR2b expression appear to be pathogenically involved. In listeriosis, however, properdin-deficient mice had reduced survival and a dendritic cell population that was impaired in maturation and activity. In vitro analyses of splenocytes and bone marrow-derived myeloid cells support the view that the opposing outcomes of properdin-deficient and wildtype mice in these two infection models is likely to be due to a skewing of macrophage activity to an M2 phenotype in the properdin-deficient mice. The phenotypes observed thus appear to reflect the extent to which M2- or M1-polarised macrophages are involved in the immune responses to S. pneumoniae and L. monocytogenes. We conclude that properdin controls the strength of immune responses by affecting humoral as well as cellular phenotypes during acute bacterial infection and ensuing inflammation.

Tyrosine kinase inhibitor tyrphostin AG490 retards chronic joint inflammation in mice.

Tyrphostin AG490 is a Janus kinase (JAK) 2 inhibitor that is clinically used as an anticancer agent and is also effective in various models of inflammatory and autoimmune diseases. In this study, we examined the effects of tyrphostin AG490 on the development of collagenase-induced osteoarthritis (CIOA). Our results showed that tyrphostin-ameliorated cartilage and bone destructions. This effect was associated with decreased expression of signal transducers and activators of transcription 3 (STAT3), phosphorylated JAK2, Dickkopf homolog 1, and receptor activator of nuclear factor κB ligand (RANKL) in the joints of arthritic mice. Tyrphostin AG490 suppressed STAT3 phosphorylation and the expression of tumor necrosis factor-related apoptosis-inducing ligand and RANKL by synovial fluid cells. The drug inhibited RANKL-induced osteoclast differentiation in vitro. Molecules, such as tyrphostin AG490 that limit bone erosion and influence osteoclast generation, might have therapeutic utility in joint degenerative disorders.

The effect of interleukin 17 and Toll-like receptor 2 on CD11b expression and apoptosis of neutrophils in zymosaninduced arthritis and paw oedema.

We investigated the effect of interleukin (IL)-17 on Ly6G(+) cell apoptosis in zymosan-induced arthritis (ZIA) and oedema (ZIO). Zymosan injection at the ankle joint caused swelling and coincided with histological joint alterations and IL-17A expression in areas with cell infiltrates. Flow cytometry of blood demonstrated increased frequencies of Ly6G(+)CD11b(+) cells and their decreased apoptosis in ZIA. Annexin V(+) neutrophils had lower CD11b expression, unlike Annexin V(-) cells. Cell survival for 12 hours was affected neither by IL-17 nor by zymosan alone, while both stimuli diminished Annexin V(+) cell frequencies and up-regulated CD11b on Annexin V(-) cells. Interleukin 17 antagonised to the effects of zymosan in 24-hour cultures. The administration of IL-17 in ZIO increased paw thickness, enlarged the blood Ly6G(+) pool, elevated CD11b expression and decreased apoptosis. We suggest that altered neutrophil apoptosis in arthritis can be overcome by anti-IL-17 therapy combined with an inhibition of Toll-like receptor (TLR) 2 and CD11b signalling.

Harpagoside: from Kalahari Desert to pharmacy shelf.

Harpagoside is an iridoid glycoside that was first isolated from Harpagophytum procumbens (devil's claw, Pedaliaceae), a medicinal plant in which it is the major constituent of the iridoid pool. Both the pure compound and devil's claw extracts have potent anti-rheumatic, anti-inflammatory and analgesic effects. According to the European Pharmacopoeia commercial devil's claw products should contain at least 1.2% harpagoside. However, the compound has also been isolated from several other plant species and in vitro plant culture systems. Recent advances in knowledge of harpagoside distribution, biosynthesis/accumulation and pharmacology are summarized in this review. We also discuss the possible synergism and/or antagonism between major constituents in harpagoside-containing phytopharmaceutical products. Finally, future perspectives for its potential application are highlighted.

Abrogated RANKL expression in properdin-deficient mice is associated with better outcome from collagen-antibody-induced arthritis.

INTRODUCTION: Properdin amplifies the alternative pathway of complement activation. In the present study, we evaluated its role in the development of collagen antibody-induced arthritis (CAIA). METHODS: Arthritis was induced by intraperitoneal injection of a collagen antibody cocktail into properdin-deficient (KO) and wild-type (WT) C57BL/6 mice. Symptoms of disease were evaluated daily. The degree of joint damage was assessed histologically and with immunostaining for bone-resorption markers. Phenotypes of cell populations, their receptor expression, and intracellular cytokine production were determined with flow cytometry. Osteoclast differentiation of bone marrow (BM) precursors was evaluated by staining for tartrate-resistant acid phosphatase (TRAP). RESULTS: Properdin-deficient mice developed less severe CAIA than did WT mice. They showed significantly improved clinical scores and downregulated expression of bone-resorption markers in the joints at day 10 of disease. The frequencies of Ly6G⁺CD11b⁺ cells were fewer in BM, blood, and synovial fluid (SF) of KO than of WT CAIA mice. The receptor activator of nuclear factor κB ligand (RANKL) was downregulated on arthritic KO neutrophils from BM and the periphery. Decreased C5a amounts in KO SF contributed to lower frequencies of CD5aR⁺-bearing neutrophils. In blood, surface C5aR was detected on KO Ly6G⁺ cells as a result of low receptor engagement. Circulating CD4⁺ T cells had an altered ability to produce interleukin (IL)-17 and interferon (IFN)-γ and to express RANKL. In KO CAIA mice, decreased frequencies of CD4⁺ T cells in the spleen were related to low CD86 expression on Ly6GhighCD11b⁺ cells. Arthritic KO T cells spontaneously secreted IFN-γ but not IL-17 and IL-6, and responded to restimulation with less-vigorous cytokine production in comparison to WT cells. Fewer TRAP-positive mature osteoclasts were found in KO BM cell cultures. CONCLUSIONS: Our data show that the active involvement of properdin in arthritis is related to an increased proinflammatory cytokine production and RANKL expression on immune cells and to a stimulation of the RANKL-dependent osteoclast differentiation.

Pro-inflammatory action of Candida albicans DNA in zymosan-induced arthritis.

OBJECTIVE: This study was designed to examine the potential ability of Candida albicans DNA to influence joint inflammation in a mouse model of zymosan-induced arthritis (ZIA) relating to Toll-like receptor-9 (TLR9) expression and cytokine production in different compartments. METHODS: To induce ZIA, mice were injected in the ankle joint with 180 µg zymosan. TLR9 expression in synovial extracts, peritoneal macrophages, splenocytes and popliteal lymph node cells was analyzed by flow cytometry. The levels of interferon (IFN)-γ, interleukin (IL)-6 and IL-10 in synovial fluid and sera were measured by ELISA. The expression of TLR9 in the joints was determined by immunohistochemistry. RESULTS: A single intraperitoneal injection of C. albicans DNA did not elevate TLR9 expression and cytokine levels in the joints. It increased TLR9 expression by peritoneal macrophages isolated from healthy and arthritic mice and elevated the IFN-γ level in circulation. In-vitro stimulation with DNA enhanced IL-6, IFN-γ and IL-10 production by different cells isolated from mice with ZIA. CONCLUSION: These results suggest that small quantities of C. albicans DNA can provoke a pro-inflammatory systemic response rather than locally in the joint.

Antiinflammatory properties of extracts and compounds isolated from Verbascum xanthophoeniceum Griseb.

Verbascum xanthophoeniceum Griseb. is an endemic plant of the Balkan region, a representative of the genus Verbascum used in traditional medicine for respiratory and inflammatory disorders. The objective of this study was to evaluate in vivo and in vitro the antiinflammatory action of crude extract, different fractions and pure compounds obtained from V. xanthophoeniceum Griseb. Bioactive metabolites were isolated by the use of low-pressure chromatographic separation. Crude methanol extract (CME) was applied in a model of paw oedema and different fractions and substances were tested in vitro for their effect on NO and cytokine production by peritoneal macrophages, and on the COX-1 and COX-2 expression. The CME exerted inhibition on cobra venom factor (CVF)-induced oedema in mice, in correlation with reduced alternative pathway (AP) complement activity. A highly suppressive effect was expressed by nigroside VI on IL-6 and NO production and by forsythoside B on TNF-α production. Leucosceptoside B lowered NO release and COX-1 expression in macrophages. Verbascum xanthophoeniceum could serve as a promising source of active compounds with antiinflammatory action, particularly in complement-mediated disorders.

Involvement of soluble receptor activator of nuclear factor-κB ligand (sRANKL) in collagenase-induced murine osteoarthritis and human osteoarthritis.

Joint destruction and excessive bone formation are associated with high expression of soluble receptor activator of nuclear factor-κB ligand (sRANKL). This study was undertaken to investigate the role of sRANKL in collagenase-induced osteoarthritis (CIOA) in mice and in patients with osteoarthritis (OA). The initial phase of CIOA was associated with severe proteoglycan depletion, decreased collagen density, and up-regulation of bone morphogenetic protein (BMP)-2. At the late stage of CIOA, bone remodeling was related with increased BMP2 and RANKL expression in the joints, high sRANKL, and decreased number of activated neutrophils in synovium. CIOA mice showed elevated plasma level of sRANKL but low RANKL expression on blood neutrophils. The percentage of RANKL-positive blood neutrophils was higher in patients with OA than in healthy individuals. Our data indicate that increased local and systemic levels of soluble RANKL might be indicative for OA disorders in mouse and human.

Bone resorption and remodeling in murine collagenase-induced osteoarthritis after administration of glucosamine.

INTRODUCTION: Glucosamine is an amino-monosaccharide and precursor of glycosaminoglycans, major components of joint cartilage. Glucosamine has been clinically introduced for the treatment of osteoarthritis but the data about its protective role in disease are insufficient. The goal of this study was to investigate the effect of long term administration of glucosamine on bone resorption and remodeling. METHODS: The effect of glucosamine on bone resorption and remodeling was studied in a model of collagenase-induced osteoarthritis (CIOA). The levels of macrophage-inflammatory protein (MIP)-1α, protein regulated upon activation, normal T-cell expressed, and secreted (RANTES), soluble receptor activator of nuclear factor kappa-B ligand (RANKL), tumor necrosis factor (TNF)-α, and interleukin (IL)-6, 4 and 10 in synovial fluid were measured by enzyme-linked immunosorbent assay (ELISA). Cell populations in synovial extracts and the expression of RANKL, of receptors for TNF-α (TNF-αR) and interferon γ (IFN-γR) on clusters of differentiation (CD) three positive T cells were analyzed by flow cytometry. Transforming growth factor (TGF)-ß3, bone morphogenetic protein (BMP)-2, phosphorylated protein mothers against decapentaplegic homolog 2 (pSMAD-2), RANKL and Dickkopf-1 protein (DKK-1) positive staining in CIOA joints were determined by immunohistochemistry. RESULTS: The administration of glucosamine hydrochloride in CIOA mice inhibited loss of glycosaminoglycans (GAGs) and proteoglycans (PGs) in cartilage, bone erosion and osteophyte formation. It decreased the levels of soluble RANKL and IL-6 and induced IL-10 increase in the CIOA joint fluids. Glucosamine limited the number of CD11b positive Ly6G neutrophils and RANKL positive CD3 T cells in the joint extracts. It suppressed bone resorption via down-regulation of RANKL expression and affected bone remodeling in CIOA by decreasing BMP-2, TGF-ß3 and pSMAD-2 expression and up-regulating DKK-1 joint levels. CONCLUSIONS: Our data suggest that glucosamine hydrochloride inhibits bone resorption through down-regulation of RANKL expression in the joints, via reduction of the number of RANKL positive CD3 T cells and the level of sRANKL in the joints extracts. These effects of glucosamine appear to be critical for the progression of CIOA and result in limited bone remodeling of the joints.