Chaenomeles Fructus (CF), the Fruit of Chaenomeles sinensis Alleviates IL-1β Induced Cartilage Degradation in Rat Articular Chondrocytes

Osteoarthritis (OA) causes persistent pain, joint dysfunction, and physical disability. It is the most prevalent type of degenerative arthritis, affecting millions of people worldwide. OA is currently treated with a focus on pain relief, inflammation control, and artificial joint surgery. Hence, a therapeutic agent capable of preventing or delaying the progression of OA is needed. OA is strongly associated with the degeneration of the articular cartilage and changes in the ECM, which are primarily associated with a decrease in proteoglycan and collagen. In the progress of articular cartilage degradation, catabolic enzymes, such as matrix metalloproteinases (MMPs), are activated by IL-1β stimulation. Given the tight relationship between IL-1β and ECM (extra-cellular matrix) degradation, this study examined the effects of Chaenomeles Fructus (CF) on IL-1β-induced OA in rat chondrocytes. The CF treatment reduced IL-1β-induced MMP3/13 and ADAMTS-5 production at the mRNA and protein levels. Similarly, CF enhanced col2a and aggrecan accumulation and chondrocyte proliferation. CF inhibited NF-κB (nuclear factor kappa B) activation, nuclear translocation induced by IL-1β, reactive oxygen species (ROS) production, and ERK phosphorylation. CF demonstrated anti-OA and articular regeneration effects on rat chondrocytes, thus, suggesting that CF is a viable and fundamental therapeutic option for OA.     

Absolute Quantification of Selected Proteins in the Human Osteoarthritic Secretome

Osteoarthritis (OA) is characterized by a loss of extracellular matrix which is driven by catabolic cytokines. Proteomic analysis of the OA cartilage secretome enables the global study of secreted proteins. These are an important class of molecules with roles in numerous pathological mechanisms. Although cartilage studies have identified profiles of secreted proteins, quantitative proteomics techniques have been implemented that would enable further biological questions to be addressed. To overcome this limitation, we used the secretome from human OA cartilage explants stimulated with IL-1β and compared proteins released into the media using a label-free LC-MS/MS-based strategy. We employed QconCAT technology to quantify specific proteins using selected reaction monitoring. A total of 252 proteins were identified, nine were differentially expressed by IL-1 β stimulation. Selected protein candidates were quantified in absolute amounts using QconCAT. These findings confirmed a significant reduction in TIMP-1 in the secretome following IL-1β stimulation. Label-free and QconCAT analysis produced equivocal results indicating no effect of cytokine stimulation on aggrecan, cartilage oligomeric matrix protein, fibromodulin, matrix metalloproteinases 1 and 3 or plasminogen release. This study enabled comparative protein profiling and absolute quantification of proteins involved in molecular pathways pertinent to understanding the pathogenesis of OA.     

The Involvement of Mutual Inhibition of ERK and mTOR in PLCγ1-Mediated MMP-13 Expression in Human Osteoarthritis Chondrocytes

The issue of whether ERK activation determines matrix synthesis or degradation in osteoarthritis (OA) pathogenesis currently remains controversial. Our previous study shows that PLCγ1 and mTOR are involved in the matrix metabolism of OA cartilage. Investigating the interplays of PLCγ1, mTOR and ERK in matrix degradation of OA will facilitate future attempts to manipulate ERK in OA prevention and therapy. Here, cultured human normal chondrocytes and OA chondrocytes were treated with different inhibitors or transfected with expression vectors, respectively. The levels of ERK, p-ERK, PLCγ1, p-PLCγ1, mTOR, p-mTOR and MMP-13 were then evaluated by Western blotting analysis. The results manifested that the expression level of ERK in human OA chondrocytes was lower than that in human normal articular chondrocytes, and the up-regulation of ERK could promote matrix synthesis, including the decrease in MMP-13 level and the increase in Aggrecan level in human OA chondrocytes. Furthermore, the PLCγ1/ERK axis and a mutual inhibition of mTOR and ERK were observed in human OA chondrocytes. Interestingly, activated ERK had no inhibitory effect on MMP-13 expression in PLCγ1-transformed OA chondrocytes. Combined with our previous study, the non-effective state of ERK activation by PLCγ1 on MMP-13 may be partly attributed to the inhibition of the PLCγ1/mTOR axis on the PLCγ1/ERK axis. Therefore, the study indicates that the mutual inhibition of ERK and mTOR is involved in PLCγ1-mediated MMP-13 expression in human OA chondrocytes, with important implication for the understanding of OA pathogenesis as well as for its prevention and therapy.     

Adipose-Derived Extract Suppresses IL-1β-Induced Inflammatory Signaling Pathways in Human Chondrocytes and Ameliorates the Cartilage Destruction of Experimental Osteoarthritis in Rats

We investigated the effects of adipose-derived extract (AE) on cultured chondrocytes and in vivo cartilage destruction. AE was prepared from human adipose tissues using a nonenzymatic approach. Cultured human chondrocytes were stimulated with interleukin-1 beta (IL-1β) with or without different concentrations of AE. The effects of co-treatment with AE on intracellular signaling pathways and their downstream gene and protein expressions were examined using real-time PCR, Western blotting, and immunofluorescence staining. Rat AE prepared from inguinal adipose tissues was intra-articularly delivered to the knee joints of rats with experimental osteoarthritis (OA), and the effect of AE on cartilage destruction was evaluated histologically. In vitro, co-treatment with IL-1β combined with AE reduced activation of the p38 and ERK mitogen-activated protein kinase (MAPK) pathway and nuclear translocation of the p65 subunit of nuclear factor-kappa B (NF-κB), and subsequently downregulated the expressions of matrix metalloproteinase (MMP)-1, MMP-3, MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4, IL-6, and IL-8, whereas it markedly upregulated the expression of IL-1 receptor type 2 (IL-1R2) in chondrocytes. Intra-articular injection of homologous AE significantly ameliorated cartilage destruction six weeks postoperatively in the rat OA model. These results suggested that AE may exert a chondroprotective effect, at least in part, through modulation of the IL-1β-induced inflammatory signaling pathway by upregulation of IL-1R2 expression.     

Effects of Extracellular Vesicles from Blood-Derived Products on Osteoarthritic Chondrocytes within an Inflammation Model

Osteoarthritis (OA) is hallmarked by a progressive degradation of articular cartilage. One major driver of OA is inflammation, in which cytokines such as IL-6, TNF-α and IL-1β are secreted by activated chondrocytes, as well as synovial cells—including macrophages. Intra-articular injection of blood products—such as citrate-anticoagulated plasma (CPRP), hyperacute serum (hypACT), and extracellular vesicles (EVs) isolated from blood products—is gaining increasing importance in regenerative medicine for the treatment of OA. A co-culture system of primary OA chondrocytes and activated M1 macrophages was developed to model an OA joint in order to observe the effects of EVs in modulating the inflammatory environment. Primary OA chondrocytes were obtained from patients undergoing total knee replacement. Primary monocytes obtained from voluntary healthy donors and the monocytic cell line THP-1 were differentiated and activated into proinflammatory M1 macrophages. EVs were isolated by ultracentrifugation and characterized by nanoparticle tracking analysis and Western blot. Gene expression analysis of chondrocytes by RT-qPCR revealed increased type II collagen expression, while cytokine profiling via ELISA showed lower TNF-α and IL-1β levels associated with EV treatment. In conclusion, the inflammation model provides an accessible tool to investigate the effects of blood products and EVs in the inflammatory context of OA.     

The Regulatory Role of Signaling Crosstalk in Hypertrophy of MSCs and Human Articular Chondrocytes

Hypertrophic differentiation of chondrocytes is a main barrier in application of mesenchymal stem cells (MSCs) for cartilage repair. In addition, hypertrophy occurs occasionally in osteoarthritis (OA). Here we provide a comprehensive review on recent literature describing signal pathways in the hypertrophy of MSCs-derived in vitro differentiated chondrocytes and chondrocytes, with an emphasis on the crosstalk between these pathways. Insight into the exact regulation of hypertrophy by the signaling network is necessary for the efficient application of MSCs for articular cartilage repair and for developing novel strategies for curing OA. We focus on articles describing the role of the main signaling pathways in regulating chondrocyte hypertrophy-like changes. Most studies report hypertrophic differentiation in chondrogenesis of MSCs, in both human OA and experimental OA. Chondrocyte hypertrophy is not under the strict control of a single pathway but appears to be regulated by an intricately regulated network of multiple signaling pathways, such as WNT, Bone morphogenetic protein (BMP)/Transforming growth factor-β (TGFβ), Parathyroid hormone-related peptide (PTHrP), Indian hedgehog (IHH), Fibroblast growth factor (FGF), Insulin like growth factor (IGF) and Hypoxia-inducible factor (HIF). This comprehensive review describes how this intricate signaling network influences tissue-engineering applications of MSCs in articular cartilage (AC) repair, and improves understanding of the disease stages and cellular responses within an OA articular joint.     

Protective Effect of Resveratrol against IL-1β-Induced Inflammatory Response on Human Osteoarthritic Chondrocytes Partly via the TLR4/MyD88/NF-κB Signaling Pathway: An “in Vitro Study”

Resveratrol is a natural polyphenolic compound that prevents inflammation in chondrocytes and animal models of osteoarthritis (OA) via yet to be defined mechanisms. The purpose of this study was to determine whether the protective effect of resveratrol on IL-1β-induced human articular chondrocytes was associated with the TLR4/MyD88/NF-κB signaling pathway by incubating human articular chondrocytes (harvested from osteoarthritis patients) with IL-1β before treatment with resveratrol. Cell viability was evaluated using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and TNFα levels in culture supernatants were measured by ELISA(Enzymelinked immunosorbent assay). The levels of TLR4 and its downstream signaling targets (MyD88 and TRAF6) and IL-1β were assessed by measuring the levels of mRNA and protein expression by real-time RT-PCR and western blot analysis, respectively, in addition to assessing NF-κB activation. In addition, TLR4 siRNA was used to block TLR4 expression in chondrocytes further demonstrating that resveratrol prevented IL-1β-mediated inflammation by TLR4 inhibition. We found that resveratrol prevented IL-1β-induced reduction in cell viability. Stimulation of chondrocytes with IL-1β caused a significant up-regulation of TLR4 and its downstream targets MyD88 and TRAF6 resulting in NF-κB activation associated with the synthesis of IL-1β and TNFα. These IL-1β-induced inflammatory responses were all effectively reversed by resveratrol. Furthermore, activation of NF-κB in chondrocytes treated with TLR4 siRNA was significantly attenuated, but not abolished, and exposure to resveratrol further reduced NF-κB translocation. These data suggested that resveratrol prevented IL-1β-induced inflammation in human articular chondrocytes at least in part by inhibiting the TLR4/MyD88/NF-κB signaling pathway suggesting that resveratrol has the potential to be used as a nutritional supplement to counteract OA symptoms.     

Antiosteoarthritic Effect of Morroniside in Chondrocyte Inflammation and Destabilization of Medial Meniscus-Induced Mouse Model

Osteoarthritis (OA) is a common degenerative disease that results in joint inflammation as well as pain and stiffness. A previous study has reported that Cornus officinalis (CO) extract inhibits oxidant activities and oxidative stress in RAW 264.7 cells. In the present study, we isolated bioactive compound(s) by fractionating the CO extract to elucidate its antiosteoarthritic effects. A single bioactive component, morroniside, was identified as a potential candidate. The CO extract and morroniside exhibited antiosteoarthritic effects by downregulating factors associated with cartilage degradation, including cyclooxygenase-2 (Cox-2), matrix metalloproteinase 3 (Mmp-3), and matrix metalloproteinase 13 (Mmp-13), in interleukin-1 beta (IL-1β)-induced chondrocytes. Furthermore, morroniside prevented prostaglandin E2 (PGE2) and collagenase secretion in IL-1β-induced chondrocytes. In the destabilization of the medial meniscus (DMM)-induced mouse osteoarthritic model, morroniside administration attenuated cartilage destruction by decreasing expression of inflammatory mediators, such as Cox-2, Mmp3, and Mmp13, in the articular cartilage. Transverse microcomputed tomography analysis revealed that morroniside reduced DMM-induced sclerosis in the subchondral bone plate. These findings suggest that morroniside may be a potential protective bioactive compound against OA pathogenesis.     

Chondrocytes from Osteoarthritis Patients Adopt Distinct Phenotypes in Response to Central TH1/TH2/TH17 Cytokines

Chronic low-grade inflammation plays a central role in the pathogenesis of osteoarthritis (OA), and several pro- and anti-inflammatory cytokines have been implicated to mediate and regulate this process. Out of these cytokines, particularly IFNγ, IL-1β, IL-4 and IL-17 are associated with different phenotypes of T helper (T_H) cells and macrophages, both examples of cells known for great phenotypic and functional heterogeneity. Chondrocytes also display various phenotypic changes during the course of arthritis. We set out to study the hypothesis of whether chondrocytes might adopt polarized phenotypes analogous to T_H cells and macrophages. We studied the effects of IFNγ, IL-1β, IL-4 and IL-17 on gene expression in OA chondrocytes with RNA-Seq. Chondrocytes were harvested from the cartilage of OA patients undergoing knee replacement surgery and then cultured with or without the cytokines for 24 h. Total RNA was isolated and sequenced, and GO (Gene Ontology) functional analysis was performed. We also separately investigated genes linked to OA in recent genome wide expression analysis (GWEA) studies. The expression of more than 2800 genes was significantly altered in chondrocytes treated with IL-1β [in the C(IL-1β) phenotype] with a fold change (FC) > 2.5 in either direction. These included a large number of genes associated with inflammation, cartilage degradation and attenuation of metabolic signaling. The profile of genes differentially affected by IFNγ (the C(IFNγ) phenotype) was relatively distinct from that of the C(IL-1β) phenotype and included several genes associated with antigen processing and presentation. The IL-17-induced C(IL-17) phenotype was characterized by the induction of a more limited set of proinflammatory factors compared to C(IL-1β) cells. The C(IL-4) phenotype induced by IL-4 displayed a differential expression of a rather small set of genes compared with control, primarily those associated with TGFβ signaling and the regulation of inflammation. In conclusion, our results show that OA chondrocytes can adopt diverse phenotypes partly analogously to T_H cells and macrophages. This phenotypic plasticity may play a role in the pathogenesis of arthritis and open new therapeutic avenues for the development of disease-modifying treatments for (osteo)arthritis.     

The DNA Repair Enzyme Apurinic/Apyrimidinic Endonuclease (Apex Nuclease) 2 Has the Potential to Protect against Down-Regulation of Chondrocyte Activity in Osteoarthritis

Apurinic/apyrimidinic endonuclease 2 (Apex 2) plays a critical role in DNA repair caused by oxidative damage in a variety of human somatic cells. We speculated that chondrocyte Apex 2 may protect against the catabolic process of articular cartilage in osteoarthritis (OA). Higher levels of Apex 2 expression were histologically observed in severely compared with mildly degenerated OA cartilage from STR/OrtCrlj mice, an experimental model which spontaneously develops OA. The immunopositivity of Apex 2 was significantly correlated with the degree of cartilage degeneration. Moreover, the OA-related catabolic factor interleukin-1β induced the expression of Apex 2 in chondrocytes, while Apex 2 silencing using small interfering RNA reduced chondrocyte activity in vitro. The expression of Apex 2 in chondrocytes therefore appears to be associated with the degeneration of articular cartilage and could be induced by an OA-related catabolic factor to protect against the catabolic process of articular cartilage. Our findings suggest that Apex 2 may have the potential to prevent the catabolic stress-mediated down-regulation of chondrocyte activity in OA.     

Intra-Articular Administration of Cramp into Mouse Knee Joint Exacerbates Experimental Osteoarthritis Progression

Osteoarthritis (OA) is the most common type of arthritis and is associated with wear and tear, aging, and inflammation. Previous studies revealed that several antimicrobial peptides are up-regulated in the knee synovium of patients with OA or rheumatoid arthritis. Here, we investigated the functional effects of cathelicidin-related antimicrobial peptide (Cramp) on OA pathogenesis. We found that Cramp is highly induced by IL-1β via the NF-κB signaling pathway in mouse primary chondrocytes. Elevated Cramp was also detected in the cartilage and synovium of mice suffering from OA cartilage destruction. The treatment of chondrocytes with Cramp stimulated the expression of catabolic factors, and the knockdown of Cramp by small interfering RNA reduced chondrocyte catabolism mediated by IL-1β. Moreover, intra-articular injection of Cramp into mouse knee joints at a low dose accelerated traumatic OA progression. At high doses, Cramp affected meniscal ossification and tears, leading to cartilage degeneration. These findings demonstrate that Cramp is associated with OA pathophysiology.     

Obeticholic Acid Derivative,T-2054 Suppresses Osteoarthritis via Inhibiting NF-κB-Signaling Pathway

Osteoarthritis (OA), a degenerative joint disorder, has been reported as the most common cause of disability worldwide. The production of inflammatory cytokines is the main factor in OA. Previous studies have been reported that obeticholic acid (OCA) and OCA derivatives inhibited the release of proinflammatory cytokines in acute liver failure, but they have not been studied in the progression of OA. In our study, we screened our small synthetic library of OCA derivatives and found T-2054 had anti-inflammatory properties. Meanwhile, the proliferation of RAW 264.7 cells and ATDC5 cells were not affected by T-2054. T-2054 treatment significantly relieved the release of NO, as well as mRNA and protein expression levels of inflammatory cytokines (IL-6, IL-8 and TNF-α) in LPS-induced RAW 264.7 cells. Moreover, T-2054 promoted extracellular matrix (ECM) synthesis in TNF-α-treated ATDC5 chondrocytes. Moreover, T-2054 could relieve the infiltration of inflammatory cells and degeneration of the cartilage matrix and decrease the levels of serum IL-6, IL-8 and TNF-α in DMM-induced C57BL/6 mice models. At the same time, T-2054 showed no obvious toxicity to mice. Mechanistically, T-2054 decreased the extent of p-p65 expression in LPS-induced RAW 264.7 cells and TNF-α-treated ATDC5 chondrocytes. In summary, we showed for the first time that T-2054 effectively reduced the release of inflammatory mediators, as well as promoted extracellular matrix (ECM) synthesis via the NF-κB-signaling pathway. Our findings support the potential use of T-2054 as an effective therapeutic agent for the treatment of OA.     

eNAMPT Is Localised to Areas of Cartilage Damage in Patients with Hip Osteoarthritis and Promotes Cartilage Catabolism and Inflammation

Obesity increases the risk of hip osteoarthritis (OA). Recent studies have shown that adipokine extracellular nicotinamide phosphoribosyltransferase (eNAMPT or visfatin) induces the production of IL-6 and matrix metalloproteases (MMPs) in chondrocytes, suggesting it may promote articular cartilage degradation. However, neither the functional effects of extracellular visfatin on human articular cartilage tissue, nor its expression in the joint of hip OA patients of varying BMI, have been reported. Hip OA joint tissues were collected from patients undergoing joint replacement surgery. Cartilage explants were stimulated with recombinant human visfatin. Pro-inflammatory cytokines and MMPs were measured by ELISA and Luminex. Localisation of visfatin expression in cartilage tissue was determined by immunohistochemistry. Cartilage matrix degradation was determined by quantifying proteoglycan release. Expression of visfatin was elevated in the synovial tissue of hip OA patients who were obese, and was co-localised with MMP-13 in areas of cartilage damage. Visfatin promoted the degradation of hip OA cartilage proteoglycan and induced the production of pro-inflammatory cytokines (IL-6, MCP-1, CCL20, and CCL4) and MMPs. The elevated expression of visfatin in the obese hip OA joint, and its functional effects on hip cartilage tissue, suggests it plays a central role in the loss of cartilage integrity in obese patients with hip OA.     

The Role of Synovial Membrane in the Development of a Potential In Vitro Model of Osteoarthritis

There is a lack of in vitro models able to plausibly represent the inflammation microenvironment of knee osteoarthritis (OA). We analyzed the molecules released from OA tissues (synovial membrane, cartilage, infrapatellar fat pad) and investigated whether the stimulation of human synovial fibroblasts (SFs), with synthetic cytokines (IL-1β and TNF-α or IFN-γ) or conditioned media (CM) from OA tissues, influence the SFs’ response, in the sense of pro-inflammatory cytokines, chemokines, growth factors, and degradative enzymes modulation. Human SFs were obtained from OA synovial membranes. SFs and their CM were analyzed for biomarkers, proliferation rate, protein profile and gene expression, before and after stimulation. Real-time PCR and multiplex assays quantified OA-related gene expression and biomolecule production. Unlike other activators, CM from OA synovial membrane (CM-SM), significantly up-regulated all genes of interest (IL-6, IL-8, MMP-1, MMP-3, RANTES, MCP-1, TSG-6, YKL-40) in SFs. Multiplex immunoassay analysis showed that levels of OA-related cytokines (IL-6, IL-8, MCP 1, IL-1Ra), chemokine (RANTES) and growth factor (VEGF), produced by CM-SM stimulated SFs, increased significantly compared to non-stimulated SFs. Molecules released from the SM from OA patients induces OA-like changes in vitro, in specific OA synovial populations (SFs). These findings promote the use and establish a compelling in vitro model that simulates the versatility and complexity of the OA disease. This model, in the future, will allow us to study new cell therapies or test drugs by reducing or avoiding animal models.     

Inhibition of Inducible Nitric Oxide Synthase Prevents IL-1β-Induced Mitochondrial Dysfunction in Human Chondrocytes

Interleukin (IL)-1β is an important pro-inflammatory cytokine in the progression of osteoarthritis (OA), which impairs mitochondrial function and induces the production of nitric oxide (NO) in chondrocytes. The aim was to investigate if blockade of NO production prevents IL-1β-induced mitochondrial dysfunction in chondrocytes and whether cAMP and AMP-activated protein kinase (AMPK) affects NO production and mitochondrial function. Isolated human OA chondrocytes were stimulated with IL-1β in combination with/without forskolin, L-NIL, AMPK activator or inhibitor. The release of NO, IL-6, PGE₂, MMP3, and the expression of iNOS were measured by ELISA or Western blot. Parameters of mitochondrial respiration were measured using a seahorse analyzer. IL-1β significantly induced NO release and mitochondrial dysfunction. Inhibition of iNOS by L-NIL prevented IL-1β-induced NO release and mitochondrial dysfunction but not IL-1β-induced release of IL-6, PGE₂, and MMP3. Enhancement of cAMP by forskolin reduced IL-1β-induced NO release and prevented IL-1β-induced mitochondrial impairment. Activation of AMPK increased IL-1β-induced NO production and the negative impact of IL-1β on mitochondrial respiration, whereas inhibition of AMPK had the opposite effects. NO is critically involved in the IL-1β-induced impairment of mitochondrial respiration in human OA chondrocytes. Increased intracellular cAMP or inhibition of AMPK prevented both IL-1β-induced NO release and mitochondrial dysfunction.     

The Interaction between microRNAs and the Wnt/β-Catenin Signaling Pathway in Osteoarthritis

Osteoarthritis (OA) is a chronic disease affecting the whole joint, which still lacks a disease-modifying treatment. This suggests an incomplete understanding of underlying molecular mechanisms. The Wnt/β-catenin pathway is involved in different pathophysiological processes of OA. Interestingly, both excessive stimulation and suppression of this pathway can contribute to the pathogenesis of OA. microRNAs have been shown to regulate different cellular processes in different diseases, including the metabolic activity of chondrocytes and osteocytes. To bridge these findings, here we attempt to give a conclusive overview of microRNA regulation of the Wnt/β-catenin pathway in bone and cartilage, which may provide insights to advance the development of miRNA-based therapeutics for OA treatment.     

Ameliorative Effects of PACAP against Cartilage Degeneration. Morphological,Immunohistochemical and Biochemical Evidence from in Vivo and in Vitro Models of Rat Osteoarthritis

Osteoarthritis (OA); the most common form of degenerative joint disease, is associated with variations in pro-inflammatory growth factor levels, inflammation and hypocellularity resulting from chondrocyte apoptosis. Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide endowed with a range of trophic effects in several cell types; including chondrocytes. However; its role in OA has not been studied. To address this issue, we investigated whether PACAP expression is affected in OA cartilage obtained from experimentally-induced OA rat models, and then studied the effects of PACAP in isolated chondrocytes exposed to IL-1β in vitro to mimic the inflammatory milieu of OA cartilage. OA induction was established by histomorphometric and histochemical analyses. Changes in PACAP distribution in cartilage, or its concentration in synovial fluid (SF), were assessed by immunohistochemistry and ELISA. Results showed that PACAP abundance in cartilage tissue and SF was high in healthy controls. OA induction decreased PACAP levels both in affected cartilage and SF. In vitro, PACAP prevented IL-1β-induced chondrocyte apoptosis, as determined by MTT assay; Hoechst staining and western blots of apoptotic-related proteins. These changes were also accompanied by decreased i-NOS and COX-2 levels, suggesting an anti-inflammatory effect. Altogether, these findings support a potential role for PACAP as a chondroprotective agent for the treatment of OA.