Response In Human Chondrocytes Research Articles

MiR146a up-regulation is involved in small HA oligosaccharides-induced pro-inflammatory response in human chondrocytes

BackgroundSmall 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. MethodsStarting 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. ResultsTreatment of chondrocytes with 6-mer HA showed up-regulation in inflammation parameters such as TLR-4, and CD44 receptors activation, IL-6, IL-1β 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. ConclusionsWe 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 significanceThese results could be useful in develop new therapeutic strategies with the aim to reduce OA and RA incidence.

Overexpression of SOX9 alleviates the progression of human osteoarthritis in vitro and in vivo.

PurposeRecent findings have identified that SOX9 served as a key role during the pathogenesis of osteoarthritis (OA). This study aimed to investigate the mechanisms by which SOX9 regulated the formation of OA in vitro and in vivo.Materials and methodsThe relative expressions of SOX9 in patients with OA and normal fracture of thighbone were analyzed by real-time-PCR. In vitro, IL-1β induced inflammatory response in human chondrocytes was used to evaluate the function of SOX9. The recombinant SOX9 lentivirus vector (Lenti-SOX9) was used to upregulate the expression of SOX9 in cells. ELISA was used to measure the concentration of tumor necrosis factor-α (TNF-α). The protein expressions of SOX9, matrix metalloproteinase-13 (MMP13), Collagen II, Aggrecan and Smad3 were analyzed by Western blot. Cell proliferation and cell apoptosis were detected by CCK-8 assay and flow cytometry, respectively. In vivo, the effect of SOX9 on surgically induced OA mice was evaluated.ResultsThe gene level of SOX9 was remarkably downregulated in patients with OA compared with normal people, while the concentration of TNF-α was upregulated. In addition, IL-1β reduced the expressions of SOX9, Collagen II and Aggrecan and increased the level of MMP13 in chondrocytes. Moreover, Lenti-SOX9 notably inhibited IL-1β-induced growth inhibition and apoptosis in chondrocytes via increasing the expression of Smad3. Finally, Lenti-SOX9 markedly alleviated the symptoms of OA mice in vivo.ConclusionUpregulation of SOX9 inhibited IL-1β-induced inflammatory response via increasing the level Smad3 in human chondrocytes and exhibited therapeutic effect on surgically induced OA mice in vivo. Therefore, SOX9 may serve as a potential target in the treatment of OA in the future.

Knockdown of KIAA1199 suppresses IL-1β-induced cartilage degradation and inflammatory responses in human chondrocytes through the Wnt/β-catenin signalling pathway

The overproduction of proteolytic enzymes and dysregulation of extracellular matrix (ECM) metabolism have been shown to accelerate the degradation process of articular cartilage. The purpose of this study was to investigate the role of KIAA1199 and its association with the pathophysiology of osteoarthritis (OA). We found that the expression of KIAA1199 was significantly upregulated in OA cartilage compared with normal tissues. Serum levels of KIAA1199 were higher in OA patients than in non-OA patients. Furthermore, knockdown of KIAA1199 inhibited interleukin-1 beta (IL-1β)-induced ECM metabolic imbalance by regulating the expression of A disintegrin-like and metalloprotease (reprolysin type) with thrombospondin type 1 motif, 5; matrix metallopeptidase-13; aggrecan; and COL2A1. In addition, silencing of KIAA1199 significantly decreased the expression of inflammatory mediators such as prostaglandin E2, IL-6, and TNF-α. Mechanistic analyses further revealed that IL-1β-induced activation of the Wnt/β-catenin pathway was suppressed during KIAA1199 knockdown. Moreover, KIAA1199 expression was also upregulated in an in vivo rat OA model. Together, these results increase our understanding of the emerging role of KIAA1199 in the process of OA degeneration, and may lead to a novel molecular target to prevent cartilage degradation.

Sauchinone prevents IL-1β-induced inflammatory response in human chondrocytes.

Sauchinone is one of the active lignan isolated from Saururus chinensis, which has been considered to possess various pharmacological activities, such as antitumor, hepatoprotective, antioxidant, and anti-inflammatory effects. However, the functional roles of sauchinone in interleukin-1 beta (IL-1β)-stimulated human osteoarthritis (OA) chondrocytes are still unknown. Thus, in this study, we investigated the anti-inflammatory effects of sauchinone in IL-1β-stimulated chondrocytes. Our results demonstrated that sauchinone significantly attenuated NO and PGE2 production, as well as inhibited iNOS and COX-2 expression in IL-1β-stimulated OA chondrocytes. In addition, sauchinone efficiently inhibited IL-1β-induced MMP-3 and MMP-13 release in human OA chondrocytes. Furthermore, sauchinone significantly attenuated the activation of NF-κB in human OA chondrocytes. In conclusion, we showed for the first time that sauchinone inhibited inflammatory response in IL-1β-stimulated human chondrocytes probably through inhibiting the activation of NF-κB signaling pathway. These data suggest that sauchinone may be a potential agent in the treatment of OA.

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-1β, 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.

Leptin induces MMP1/13 and ADAMTS 4 expressions through bone morphogenetic protein-2 autocrine effect in human chondrocytes.

The induction of bone morphogenetic protein (BMP)2 in injured and arthritis articular cartilage has been proposed, but the precise mechanism has not been clearly clarified. Our previous study has found that leptin could stimulate the BMP2 autocrine effect to increase the anabolic collagen II expression when it initiates the catabolic response in human chondrocytes. It has been suggested that this BMP2 autocrine effect contributes to a reparative role in leptin-stimulated human chondrocytes. In this study, we further determined whether this BMP2 autocrine effect also affect the expressions of catabolic matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinase with thrombospondin motif (ADAMTS). Human primary and SW1353 chondrocytes were used in this study. It was shown that leptin could induce the expressions of MMP1, 3, and 13 and ADAMTS4 and 5 in both human primary and SW1353 chondrocytes. Leptin-increased MMP1/13 (not MMP3) and ADAMTS4 (not ADAMTS5) expressions were affected by the leptin-upregulated BMP2 and its specific downstream Smad1/5 signaling. Moreover, both HDAC3 and 4 are involved in regulating leptin-induced BMP2 upregulation and then affect MMP1 and 13 and ADAMTS4 expression. Both HDAC3 and 4 also affect leptin-increased MMP3 mRNA expression but not through BMP2 autocrine effect of leptin induction. Our results further elucidated the role of BMP2 autocrine effect in matrix-degrading enzymes expressions under leptin stimulation. The findings in this study provide new insights into the possible mechanism of BMP2 induction in leptin-stimulated chondrocytes and in leptin-induced OA development.

201 - Butein: A Plant Derived Small Molecule Activates Autophagy and Suppresses IL-1β Induced Oxidative Stress, Mitochondrial Dysfunction and Inflammatory Response in Human Chondrocytes

Osteoarthritis (OA) is the most common joint disease worldwide and is characterized by pain and stiffness due to the progressive loss of articular cartilage. There is increasing evidence that support a role of high levels of oxidative stress, increased mitochondrial dysfunction, inflammation and decline in autophagy in the pathogenesis of OA. Currently there is no disease modifying drug available for the treatment of OA. Autophagy is a cellular defense mechanism against stress-induced accumulation of protein aggregates and dysfunctional subcellular organelles. Here, we studied the effect of a plant derived small molecule, Butein, on human chondrocytes and cartilage explants (isolated from donors with and without known history of OA) against IL-1β induced oxidative stress, mitochondrial dysfunction and expression of inflammatory mediators (Cyclooxygenase 2 (COX-2), inducible Nitric Oxide Synthase (iNOS) and interleukin-6 (IL-6) in vitro . Butein blocked the IL-1β induced disruption of mitochondrial membrane potential and mitochondrial dysfunction and suppressed the generation of total and mitochondrial ROS and RNS levels in normal and OA chondrocytes. Additionally, IL-1β induced expression of iNOS, COX-2 and IL-6 was also inhibited by Butein in both normal and OA chondrocytes and cartilage explants. Butein activated AMPKα and inhibited mTOR activity to activate autophagy in human chondrocytes as evidenced by the increased levels of LC3-II, decreased levels of p62/SQSTM1 and increased numbers of autophagosomes. Activation of autophagy was critical to the observed chondroprotective effects as inhibition of autophagy abrogated the protective effects of Butein. These results suggest that Butein may be developed as a therapeutic agent for the prevention and or treatment of OA.

Saikosaponin A inhibits IL-1β-induced inflammatory mediators in human osteoarthritis chondrocytes by activating LXRα

Saikosaponin a (SSa), one of the main active components of Bupleurum falcatum, has been reported to have anti-inflammatory effect. In the present study, we investigated the anti-inflammatory effect of SSa on IL-1β-stimulated human osteoarthritis chondrocytes. The cells were pretreated with SSa 12 h before IL-1β treatment. The production of PGE2 and NO were detected by ELISA and Griess method. The levels of MMP1, MMP3, and MMP13 were measured by ELISA and qRT-PCR. The expression of NF-κB and LXRα were tested by western blot analysis. The results showed that SSa inhibited IL-1β-induced PGE2 and NO production in a concentration-dependent manner. SSa also suppressed IL-1β-induced MMP1, MMP3, and MMP13 production. Furthermore, SSa significantly attenuated IL-1β-induced phosphorylation levels of NF-κB p65 and IκBα. SSa also up-regulated the expression of LXRα. The inhibition of SSa on PGE2, NO, MMP1, MMP3, and MMP13 production were reversed by LXRα siRNA or GGPP, the inhibitor of LXRα. In conclusion, our results demonstrated that SSa inhibited inflammatory responses in human chondrocytes in vitro. SSa might be a potential therapeutic drug for osteoarthritis.

Knockdown of PRMT1 suppresses IL-1β-induced cartilage degradation and inflammatory responses in human chondrocytes through Gli1-mediated Hedgehog signaling pathway.

Osteoarthritis (OA) is characterized by articular cartilage degradation and joint inflammation. The purpose of the present study is to elucidate the role of the specific function of PRMT1 in chondrocytes and its association with the pathophysiology of OA. We observed that the expression of PRMT1 was apparently upregulated in OA cartilage, as well as in chondrocytes stimulated with IL-1β. Additionally, knockdown of PRMT1 suppressed interleukin 1 beta (IL-1β)-induced extracellular matrix (ECM) metabolic imbalance by regulating the expression of MMP-13, ADAMTS-5, COL2A1, and ACAN. Furthermore, silencing of PRMT1 dramatically declined the production of prostaglandin E2 (PGE2) and nitric oxide as well as the level of pro-inflammatory cytokine IL-6 and TNF-α. Mechanistic analyses further revealed that IL-1β-induced activation of the Hedgehog/Gli-1 signaling is suppressed upon PRMT1 knockdown. However, the effects of inhibition of PRMT1-mediated IL-1β-induced cartilage matrix degradation and inflammatory response in OA chondrocytes were obviously abolished by Hedgehog agonist Purmorphamine (Pur). Our data collectively suggest that silencing of PRMT1 exerts anti-catabolic and anti-inflammatory effects on IL-1β-induced chondrocytes via suppressing the Gli-1 mediated Hedgehog signaling pathway, indicating that PRMT1 plays a critical role in OA development and serves as a promising therapeutic target for OA.

Hyperglycemia and Hyperinsulinemia-Like Conditions Independently Induce Inflammatory Responses in Human Chondrocytes

To elucidate the mechanisms by which type 2 Diabetes Mellitus (DM2) constitutes a risk factor for the development and progression of osteoarthritis (OA), this work determined whether high glucose and/or high insulin, the hallmarks of DM2, are capable of activating the transcription factor, Nuclear Factor-κB (NF-κB), which plays a critical role in OA by inducing the expression of pro-inflammatory and catabolic genes. For this, we analyzed NF-κB activation by measuring the nuclear levels of p65 by western blot. As readouts of NF-κB activity, Interleukin-1β, Tumor Necrosis Factor-α, and inducible nitric oxide synthase (iNOS) expression were analyzed by real time RT-PCR and western blot. Culture of the human chondrocytic cell line, C28-I2, in high glucose (30 mM) increased nuclear NF-κB p65 levels in a time-dependent manner, relative to cells cultured in medium containing 10 mM glucose (regular culture medium). High glucose-induced NF-κB activation was inhibited by co-treatment with its specific inhibitor, Bay 11-7082, 5 µM. Culture of primary human chondrocytes under high glucose for 24 h increased IL-1β and TNF-α mRNA levels by 97% (p = 0.0066) and 85% (p = 0.0045), respectively, while iNOS mRNA and protein levels and NO production increased by 61% (p = 0.0017), 148% (p = 0.0089), and 70% (p = 0.049), respectively, relative to chondrocytes maintained in 10 mM glucose. Treatment of chondrocytic cells with 100 nM insulin was also sufficient to increase nuclear NF-κB p65 levels, independently of the glucose concentration in the culture medium. This study shows that hyperglycemia and hyperinsulinemia are independently sufficient to induce inflammatory responses in human chondrocytes, namely by activating NF-κB. This can be a relevant mechanism by which DM type 2 and other conditions associated with impaired glucose and insulin homeostasis, like obesity and the metabolic syndrome, contribute to the development and progression of OA.

ChondroprotectiveEffectsofGinsenosideRg1in HumanOsteoarthritisChondrocytesandaRatModel ofAnteriorCruciateLigamentTransection.

This study aimed to assess whether Ginsenoside Rg1 (Rg1) inhibits inflammatory responses in human chondrocytes and reduces articular cartilage damage in a rat model of osteoarthritis (OA). Gene expression and protein levels of type II collagen, aggrecan, matrix metalloproteinase (MMP)-13 and cyclooxygenase-2 (COX-2) were determined in vitro by quantitative real-time-polymerase chain reaction and Western blotting. Prostaglandin E2 (PGE2) amounts in the culture medium were determined by enzyme-linked immunosorbent assay (ELISA). For in vivo assessment, a rat model of OA was generated by anterior cruciate ligament transection (ACLT). Four weeks after ACLT, Rg1 (30 or 60 mg/kg) or saline was administered by gavage once a day for eight consecutive weeks. Joint damage was analyzed by histology and immunohistochemistry. Ginsenoside Rg1 inhibited Interleukin (IL)-1β-induced chondrocyte gene and protein expressions of MMP-13, COX-2 and PGE2, and prevented type II collagen and aggrecan degradation, in a dose-dependent manner. Administration of Ginsenoside Rg1 to OA rats attenuated cartilage degeneration, and reduced type II collagen loss and MMP-13 levels. These findings demonstrated that Ginsenoside Rg1 can inhibit inflammatory responses in human chondrocytes in vitro and reduce articular cartilage damage in vivo, confirming the potential therapeutic value of Ginsenoside Rg1 in OA.

TWIST1 induces MMP3 expression through up-regulating DNA hydroxymethylation and promotes catabolic responses in human chondrocytes

The objective was to investigate the levels of TWIST1 in normal and OA cartilage and examine its role in regulating gene expression in chondrocytes. Human cartilage tissues and chondrocytes were obtained at autopsy from normal knee joints and from OA-affected joints at the time of total knee arthroplasty. TWIST1 expression was increased in human OA knee cartilage compared to normal knee cartilage. TWIST1 induced matrix metalloproteinase 3 (MMP3) expression without direct binding to MMP3 promoter and increased the 5-hydroxymethylcytosine (5hmC) level at the MMP3 promoter. The effect of TWIST1 on expression of TET family (TET1, 2 and 3) was measured in stable TWIST1 transfected TC28 cells, and TET1 expression was up-regulated. TWIST1 dependent upregulation of Mmp3 expression was suppressed in Tet triple KO fibroblast derived from mouse ES cells. Increased TWIST1 expression is a feature of OA-affected cartilage. We identified a novel mechanism of catabolic reaction where TWIST1 up-regulates MMP3 expression by enriching 5hmC levels at the MMP3 promoter via TET1 induction. These findings implicate TWIST1 as an important factor regulating OA related gene expression. Clarifying epigenetic mechanisms of 5hmC induced by TWIST1 is a critical molecule to understanding OA pathogenesis.

Biomimetic sulphated alginate hydrogels suppress IL-1β-induced inflammatory responses in human chondrocytes.

Loss of articular cartilage from ageing, injury or degenerative disease is commonly associated with inflammation, causing pain and accelerating degradation of the cartilage matrix. Sulphated glycosaminoglycans (GAGs) are involved in the regulation of immune responses in vivo, and analogous polysaccharides are currently being evaluated for tissue engineering matrices to form a biomimetic environment promoting tissue growth while suppressing inflammatory and catabolic activities. Here, we characterise physical properties of sulphated alginate (S-Alg) gels for use in cartilage engineering scaffolds, and study their anti-inflammatory effects on encapsulated chondrocytes stimulated with IL-1β. Sulphation resulted in decreased storage modulus and increased swelling of alginate gels, whereas mixing highly sulphated alginate with unmodified alginate resulted in improved mechanical properties compared to gels from pure S-Alg. S-Alg gels showed extensive anti-inflammatory and anti-catabolic effects on encapsulated chondrocytes induced by IL-1β. Cytokine-stimulated gene expression of pro-inflammatory markers IL-6, IL-8, COX-2 and aggrecanase ADAMTS-5 were significantly lower in the sulphated gels compared to unmodified alginate gels. Moreover, sulphation of the microenvironment suppressed the protein expression of COX-2 and NF-κB as well as the activation of NF-κB and p38-MAPK. The sulphated alginate matrices were found to interact with IL-1β, and proposed to inhibit inflammatory induction by sequestering cytokines from their receptors. This study shows promising potential for sulphated alginates in biomimetic tissue engineering scaffolds, by reducing cytokine-mediated inflammation and providing a protective microenvironment for encapsulated cells.

Sucrose, But Not Glucose, Blocks IL1-β-Induced Inflammatory Response in Human Chondrocytes by Inducing Autophagy via AKT/mTOR Pathway.

Pathogenesis of osteoarthritis (OA) is multifactorial but interleukin-1β (IL-1β) is known to be an important mediator of cartilage degradation. Autophagy is an essential cellular homeostasis mechanism and has been proposed to protect against cartilage degradation and chondrocyte death under pathological conditions. We investigated the role of autophagy activated by sucrose, a natural disaccharide, in suppressing inflammatory mediator's expression and cell death under pathological conditions in human chondrocytes. Autophagy activation was investigated by Western blotting for LC3 and Beclin-1, immunofluorescence staining for LC3 puncta, and measuring autophagic flux. Activation of mTOR, AKT, and P70S6K was evaluated by Western blotting. Chondrocyte apoptosis was evaluated by propidium iodide (PI) staining using flowcytometry, expression of Bax by Western blotting, gene expression by TaqMan assays and caspase 3/7 activity was measured using a luminescence-based assay. We found that sucrose-induced active autophagy in OA chondrocytes in vitro was dependent on the activation of AKT/mTOR/P70S6K signaling pathways but was independent of reactive oxygen species (ROS) production. Sucrose activated autophagy blocked IL-1β-induced apoptosis and mRNA expression of MMP-13, COX-2, and IL-6 in human OA chondrocytes. Glucose or fructose, the two metabolites of sucrose, failed to induce autophagy indicating that autophagy was specifically mediated by sucrose. In conclusion, sucrose attenuated IL-1β induced apoptosis and the expression of catabolic mediators by inducing autophagy, and the autophagy in part was mediated through the activation of AKT/mTOR/P70S6K signaling pathway in human OA chondrocytes. J. Cell. Biochem. 118: 629-639, 2017. © 2016 Wiley Periodicals, Inc.

The Role of PPARγ in Advanced Glycation End Products-Induced Inflammatory Response in Human Chondrocytes.

ObjectiveAdvances made in the past ten years highlight the notion that peroxisome proliferator-activated receptors gamma (PPARγ) has protective properties in the pathophysiology of osteoarthritis (OA). The aim of this study was to define the roles of PPARγ in AGEs-induced inflammatory response in human chondrocytes.MethodsPrimary human chondrocytes were stimulated with AGEs in the presence or absence of neutralizing antibody against RAGE (anti-RAGE), MAPK specific inhibitors and PPARγ agonist pioglitazone. The expression of IL-1, MMP-13, TNF-α, PPARγ, nuclear NF-κB p65 and cytosol IκBα was determined by western blotting and real-time PCR.ResultsAGEs could enhance the expression of IL-1, TNF-α, and MMP-13, but the level of PPARγ was decreased in a time- and dose-dependent manner, which was inhibited by anti-RAGE, SB203580 (P38 MAPK specific inhibitor) and SP600125 (a selective inhibitor of JNK). PPARγ agonist pioglitazone could inhibit the effects of AGEs-induced inflammatory response and PPARγ down-regulation. In human chondrocytes, AGEs could induce cytosol IκBα degradation and increase the level of nuclear NF-κB p65, which was inhibited by PPARγ agonist pioglitazone.ConclusionsIn primary human chondrocytes, AGEs could down-regulate PPARγ expression and increase the inflammatory mediators, which could be reversed by PPARγ agonist pioglitazone. Activation of RAGE by AGEs triggers a cascade of downstream signaling, including MAPK JNK/ p38, PPARγ and NF-κB. Taken together, PPARγ could be a potential target for pharmacologic intervention in the treatment of OA.

PPARγ is involved in the hyperglycemia-induced inflammatory responses and collagen degradation in human chondrocytes and diabetic mouse cartilages.

Diabetic hyperglycemia has been suggested to play a role in osteoarthritis. Peroxisome proliferator-activated receptor-γ (PPARγ) was implicated in several pathological conditions including diabetes and inflammation. The detailed effects and mechanisms of hyperglycemia on cartilage damage still need to be clarified. Here, we investigated the role of PPARγ in hyperglycemia-triggered chondrocyte/cartilage damages using a human chondrocyte culture model and a diabetic mouse model. Human chondrocytes were cultured and treated with high concentration of glucose (30 mM) to mimic hyperglycemia in the presence or absence of pioglitazone, a PPARγ agonist. Streptozotocin (STZ) was used to induce mouse diabetes. Our data showed that high glucose induced the protein expressions of cyclooxygenase-2 (COX-2) and production of prostaglandin-E2 (PGE2 ), interleukin-6 (IL-6), and metalloproteinase-13 (MMP-13), but decreased the protein expression of collagen II and PPARγ in human chondrocytes. These alterations in high glucose-treated human chondrocytes could be reversed by pioglitazone in a dose-dependent manner. Moreover, pioglitazone administration could also significantly reverse the hyperglycemia, formation of AGEs, productions of IL-6 and MMP-13, and cartilage damage in STZ-induced diabetic mice. Taken together, these findings suggest that hyperglycemia down-regulates PPARγ expression and induces inflammatory and catabolic responses in human chondrocytes and diabetic mouse cartilages.

IL-36α: a novel cytokine involved in the inflammatory response in human chondrocytes

IL-36α: a novel cytokine involved in the inflammatory response in human chondrocytes

EGb761 inhibits inflammatory responses in human chondrocytes and shows chondroprotection in osteoarthritic rat knee

Osteoarthritis (OA) is a degenerative joint disease involving a combination of cartilage degradation and inflammation. EGb761, a standardized extract of Ginkgo biloba leaves, holds an anti-inflammatory potency. Here, we determined whether EGb761 could inhibit lipopolysaccharide (LPS)- and IL-1β-induced inflammatory responses in human articular chondrocytes and apply the chondroprotection in OA rats. We found that LPS markedly induced the productions of PGE2 and NO and the protein expressions of COX-2 and iNOS in human chondrocytes. LPS was also seen to up-regulate the expressions of toll-like receptor-4 (TLR4), its downstream signal TNF receptor-associated factor 6 (TRAF6), and nuclear factor (NF)-κB signaling. These LPS-induced inflammatory responses were efficaciously reversed by EGb761 and its active components quercetin and kampferol. The similar results could be observed by using IL-1β as an in vitro model to mimic an inflammatory response. In an OA rat model, PGE2 and NO levels in blood, the histological alterations, and COX-2 and nitrotyrosine expressions in cartilages were markedly increased, which were effectively reversed by EGb761. Our results suggested that EGb761 exerts the anti-inflammatory effects on human articular chondrocytes and OA rats.

Response of human chondrocytes and mesenchymal stromal cells to a decellularized human dermis

BackgroundAlthough progress has been made in the treatment of articular cartilage lesions, they are still a major challenge because current techniques do not provide satisfactory long-term outcomes. Tissue engineering and the use of functional biomaterials might be an alternative regenerative strategy and fulfill clinical needs. Decellularized extracellular matrices have generated interest as functional biologic scaffolds, but there are few studies on cartilage regeneration. The aim of this study was to evaluate in vitro the biological influence of a newly developed decellularized human dermal extracellular matrix on two human primary cultures.MethodsNormal human articular chondrocytes (NHAC-kn) and human mesenchymal stromal cells (hMSC) from healthy donors were seeded in polystyrene wells as controls (CTR), and on decellularized human dermis batches (HDM_derm) for 7 and 14 days. Cellular proliferation and differentiation, and anabolic and catabolic synthetic activity were quantified at each experimental time. Histology and scanning electron microscopy were used to evaluate morphology and ultrastructure.ResultsBoth cell cultures had a similar proliferation rate that increased significantly (p < 0.0005) at 14 days. In comparison with CTR, at 14 days NHAC-kn enhanced procollagen type II (CPII, p < 0.05) and aggrecan synthesis (p < 0.0005), whereas hMSC significantly enhanced aggrecan synthesis (p < 0.0005) and transforming growth factor-beta1 release (TGF-β1, p < 0.0005) at both experimental times. Neither inflammatory stimulus nor catabolic activity induction was observed. By comparing data of the two primary cells, NHAC-kn synthesized significantly more CPII than did hMSC at both experimental times (p < 0.005), whereas hMSC synthesized more aggrecan at 7 days (p < 0.005) and TGF-β1 at both experimental times than did NHAC-kn (p < 0.005).ConclusionsThe results obtained showed that in in vitro conditions HDM_derm behaves as a suitable scaffold for the growth of both well-differentiated chondrocytes and undifferentiated mesenchymal cells, thus ensuring a biocompatible and bioactive substrate. Further studies are mandatory to test the use of HDM_derm with tissue engineering to assess its therapeutic and functional effectiveness in cartilage regeneration.

Regulation of inflammatory response in human chondrocytes by lentiviral mediated RNA interference against S100A10

The aim of the present study was to evaluate the effects of S100A10 silencing on the inflammatory response in human chondrocytes (HCs).The inflammation induced by lipopolysaccharide (LPS) was investigated in HCs in which the S100A10 was blocked with a lentiviral shRNA vector. A lentiviral shRNA vector targeting S100A10 was constructed and packaged to effectively block S100A10 expression in HCs. HCs were infected with the lentivirus. S100A10 expression levels in HCs were detected by western blot analysis. Enzyme-linked immunosorbent assay (ELISA) was employed to evaluate the change of cytokine secretion levels. The effects of S100A10 silencing on the activation of mitogen-activated protein kinases (MAPKs) and NF-κB signaling pathway were also determined by western blot analysis. In addition, fluo-3-AM was used to demonstrate the change in calcium mobilization. Lentivirus effectively infected the HCs and inhibited the expression of S100A10. HCs with downregulated S100A10 showed significantly decreased production of inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-10. S100A10 silencing markedly suppressed the activation of MAPKs induced by LPS. Furthermore, the calcium concentration increase in HCs stimulated by LPS was also inhibited by S100A10 knockdown. Our investigation demonstrated that S100A10 might be considered as a potential target for anti-inflammatory treatment.