Chondrocyte Dysfunction Research Articles

Comet assay for quantification of the increased DNA damage burden in primary human chondrocytes with aging and osteoarthritis.

It is known that chondrocytes from joints with osteoarthritis (OA) exhibit high levels of DNA damage, but the degree to which chondrocytes accumulate DNA damage during “normal aging” has not been established. The goal of this study was to quantify the DNA damage present in chondrocytes obtained from cadaveric donors of a wide age range, and to compare the extent of this damage to OA chondrocytes. The alkaline comet assay was used to measure the DNA damage in normal cartilage from the ankle (talus) and the knee (femur) of cadaveric donors, as well as in OA chondrocytes obtained at the time of total knee replacement. Chondrocytes from younger donors (<45 years) had less DNA damage than older donors (>70 years) as assessed by the percentage of DNA in the comet “tail”. In donors between 50 and 60 years old, there was increased DNA damage in chondrocytes from OA cartilage as compared to cadaveric. Talar chondrocytes from 23 donors between the ages of 34 and 78 revealed a linear increase in DNA damage with age (R 2 = 0.865, p < 0.0001). A “two‐tailed” comet assay was used to demonstrate that most of the accumulated damage is in the form of strand breaks as opposed to alkali‐labile base damage. Chondrocytes from young donors required 10 Gy irradiation to recapitulate the DNA damage present in chondrocytes from older donors. Given the potential for DNA damage to contribute to chondrocyte dysfunction and senescence, this study supports the investigation of mechanisms by which hypo‐replicative cell types accumulate high levels of damage.

Osteoarthritis-Induced Metabolic Alterations of Human Hip Chondrocytes.

Osteoarthritis (OA) alters chondrocyte metabolism and mitochondrial biology. We explored whether OA and non-OA chondrocytes show persistent differences in metabolism and mitochondrial function and different responsiveness to cytokines and cAMP modulators. Hip chondrocytes from patients with OA or femoral neck fracture (non-OA) were stimulated with IL-1β, TNF, forskolin and opioid peptides. Mediators released from chondrocytes were measured, and mitochondrial functions and glycolysis were determined (Seahorse Analyzer). Unstimulated OA chondrocytes exhibited significantly higher release of IL-6, PGE2 and MMP1 and lower production of glycosaminoglycan than non-OA chondrocytes. Oxygen consumption rates (OCR) and mitochondrial ATP production were comparable in unstimulated non-OA and OA chondrocytes, although the non-mitochondrial OCR was higher in OA chondrocytes. Compared to OA chondrocytes, non-OA chondrocytes showed stronger responses to IL-1β/TNF stimulation, consisting of a larger decrease in mitochondrial ATP production and larger increases in non-mitochondrial OCR and NO production. Enhancement of cAMP by forskolin prevented IL-1β-induced mitochondrial dysfunction in OA chondrocytes but not in non-OA chondrocytes. Endogenous opioids, present in OA joints, influenced neither cytokine-induced mitochondrial dysfunction nor NO upregulation. Glycolysis was not different in non-OA and OA chondrocytes, independent of stimulation. OA induces persistent metabolic alterations, but the results suggest upregulation of cellular mechanisms protecting mitochondrial function in OA.

WITHDRAWN: OPA1 protected against IL-1β-induced chondrocyte dysfunction by alleviating mitochondrial dysfunction and oxidative stress through activation of Parkin-mediated mitophagy

Mitophagy affects mitochondrial function and intracellular oxidative stress, and plays essential roles in apoptosis and extracellular matrix (ECM) degradation in osteoarthritis (OA) development. Optic atrophy 1 (OPA1) has been verified to protect cell against damage by inducing mitophagy. However, the function and underlying mechanism of OPA1 in OA remains largely unknown. Here, interleukin (IL)-1β-treated chondrocytes were used to mimic pathological environment of OA progress in vitro. Our results showed that the expression of OPA1 was decreased in cartilage tissue of OA patients and IL-1β-treated chondrocytes. Overexpression of OPA1 inhibited the IL-1β-induced chondrocyte apoptosis and ECM degradation, while OPA1 knockdown significantly exacerbated the effects of IL-1β on chondrocyte apoptosis and ECM degradation. Moreover, OPA1 overexpression enhanced mitochondrial membrane potential, ATP production, the expression of mitochondrial dynamics-related genes and antioxidant enzyme activity, indicating that OPA1 attenuated the IL-1β-induced mitochondrial dysfunction and oxidative stress. Interestingly, the expression of mitophagy-related proteins in IL-1β-induced cells was up-regulated by OPA1 overexpression. Furthermore, Silencing Parkin reversed the activated effect of OPA1 on mitophagy-related proteins, suggesting that Parkin signaling was involved in the mitophagy induced by OPA1. the protective roles of OPA1 in IL-1β-induced mitochondrial dysfunction, oxidative stress, apoptosis and ECM underproduction were suppressed by silencing Parkin. Taken together, OPA1 protected the IL-1β-induced chondrocytes against apoptosis and ECM degradation by alleviating mitochondrial dysfunction and oxidative stress through induction of Parkin-mediated mitophagy. These findings suggest the protective effects of OPA1 in OA.

Circular RNA from phosphodiesterase 4D can attenuate chondrocyte apoptosis and matrix degradation under OA milieu induced by IL-1β via circPDE4D/miR-4306/SOX9 Cascade

Background Phosphodiesterase 4D (PDE4D) is a novel molecular therapeutic agent for human diseases, including Alzheimer’s disease, ischemic stroke, asthma, and cancers. Circular RNA from PDE4D (circPDE4D; ID hsa_circ_0072568) was one of the most downregulated circRNAs in OA patients. However, its precise role in OA-related chondrocytes was largely unknown. Methods Expressions of circPDE4D, microRNA (miR)-4306, and sex-determining region Y-box 9 (SOX9) were measured by quantitative real-time PCR; protein levels of SOX9 and proteins related to apoptosis and extracellular matrix (ECM) were detected by Western blotting. Cell apoptosis was assessed by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, 5-ethynyl-2′-deoxyuridine and Annexin V-fluorescein isothiocyanate apoptosis assays. MiR-4306 response elements were predicted by bioinformatics algorithm and identified using dual-luciferase reporter, RNA immunoprecipitation, and biotin-coupled miRNA capture assays. Results CircPDE4D was markedly downregulated in OA cartilages and interleukin (IL)-1β-stressed human normal chondrocytes (HNC). Ectopic expression of circPDE4D rescued cell viability, proliferation, and expressions of B-cell lymphoma/leukemia-2 (Bcl-2) and Collagen type II α1 in IL-1β-insulted HNC, and meanwhile declined apoptosis rate and levels of Bcl-2-associated X protein, cleaved caspase-3, cleaved poly (ADP-ribose) polymerase-1, matrix metalloproteinase-13, ADAM metallopeptidase with thrombospondin type 1 motif 5, IL-6, and IL-8. CircPDE4D and SOX9 were competing endogenous RNAs (ceRNAs) for miR-4306, and circPDE4D could positively regulate SOX9 expression via miR-4306. Conclusion CircPDE4D and miR-4306 were important regulators in regulating IL-1β-induced HNC apoptosis and matrix degradation via regulating the key transcription factor SOX9, suggesting a novel circPDE4D/miR-4306/SOX9 ceRNA pathway in OA-related chondrocyte dysfunction.

RPL38 knockdown inhibits the inflammation and apoptosis in chondrocytes through regulating METTL3-mediated SOCS2 m6A modification in osteoarthritis.

Ribosomal protein L38 (RPL38) was found upregulated in osteoarthritic peripheral blood mononuclear cells, however, its role in progression of osteoarthritis has not been characterized. The protein levels of RPL38 and SOCS2 in cartilage tissues from OA patients and controls were detected with Western blotting. IL-1β was used to stimulate primary chondrocytes to establish an OA cell model, and RPL38 siRNA (si-RPL38) was transfected into chondrocytes to investigate the effect of RPL38 knockdown on cell viability, apoptosis, inflammatory factor secretion and extracellular matrix degradation. Then, the mechanism that RPL38 regulate the SOCS2 expression and SOCS2-induced chondrocyte dysfunction was explored. The methyltransferase-like 3 (METTL3)-mediated m6A modification of SOCS2 mRNA was confirmed, and the interaction of RPL38 and METTL3 was verified. Moreover, the effects of SOCS2 overexpression on IL-1β-induced chondrocyte dysfunction and SOCS2 knockdown on the restoration of chondrocyte function by siRPL38 were investigated. Finally, RPL38 was knocked down in vivo and its role in OA progression was validated. RPL38 was upregulated and SOCS2 was downregulated in OA cartilages. RPL38 knockdown or SOCS2 overexpression either attenuated IL-1β-induced chondrocyte apoptosis, inflammatory cytokine secretion, and ECM degradation. RPL38 directly interacted with METTL3 and it inhibited SOCS2 expression through METTL3-mediated m6A modification. SOCS2 knockdown activated the JAK2/STAT3 proinflammatory pathway and reversed the effects of RPL38 knockdown on IL-1β-induced chondrocyte apoptosis, inflammation and ECM degradation. RPL38 knockdown alleviated cartilage tissue damage and ECM degradation in OA mice. RPL38 knockdown inhibited osteoarthritic chondrocyte dysfunction and alleviated OA progression through promoting METTL3-m6A-mediated SOCS2 expression.

Epigallocatechin-3-O-gallate ameliorates oxidative stress-induced chondrocyte dysfunction and exerts chondroprotective effects via the Keap1/Nrf2/ARE signaling pathway.

Oxidative stress-induced degeneration and dysfunction of chondrocytes play a key role in the pathological progression of osteoarthritis (OA), a common degenerative joint disease in the elderly. Epigallocatechin-3-O-gallate (EGCG) increases Nrf2-mediated antioxidase expression levels. We aimed to determine the effects of EGCG on C28/I2human chondrocytes subjected to interleukin-1β (IL-1β)-induced oxidative stress. EGCG suppressed IL-1β-induced oxidative stress, as indicated by decreased malondialdehyde (MDA) and reactive oxygen species (ROS) generation. Additionally, EGCG attenuated the IL-1β-induced reduction in cartilage matrix generated by chondrocytes by upregulating collagen II, aggrecan, sulfated proteoglycans, and SRY-box transcription factor 9 (SOX9). EGCG reversed the IL-1β-induced increased cyclooxygenase 2 (COX2), inducible nitric oxide synthase (iNOS), collagen X, and matrix metalloproteinases (MMPs). Furthermore, EGCG inhibited apoptosis and senescence of IL-1β-treated chondrocytes, as indicated by the decrease in mitochondrial membrane potential and senescence-associated β-galactosidase-positive cells, respectively. Mechanically, EGCG upregulated nuclear factor erythroid 2-related factor 2 (Nrf2), oxygenase-1 (HO-1), and NADPH quinone oxidoreductase1 (NQO1). The antioxidant and chondroprotective effects of EGCG were blocked by ML385, a Keap1/Nrf2/ARE signaling pathway inhibitor. Thus, EGCG ameliorated oxidative stress-induced chondrocyte dysfunction and exerted chondroprotective effects via Keap1/Nrf2/ARE signaling. This provides a novel perspective on the molecular mechanisms underlying the therapeutic effects of EGCG on OA.

WITHDRAWN: Brucine restores sodium nitroprusside-induced chondrocyte dysfunction by suppressing the GSK-3β/β-catenin pathway

The dysfunction of chondrocytes is thought to play a role in the initiation and progression of osteoarthritis (OA). Brucine possesses wide pharmacological activities. But the protective mechanism of the brucine on chondrocytes remains unclear. This study is aimed to determine the therapeutic effects of brucine on the mouse chondrocyte OA model by sodium nitroprusside (SNP). The primary chondrocytes were obtained from the knee articular cartilage of a healthy suckling mouse donor. The cultured chondrocytes were divided into the control group, SNP group, brucine group, brucine-SNP group, brucine-SNP-GSK-3β antagonist group (brucine-SNP- group), and brucine-SNP-GSK-3β agonist group (brucine-SNP-GSK-3β+ group). After 24 h, the chondrocytes from different treated groups were collected to detect chondrocyte proliferation and ultrastructure, regulation factors, apoptosis, oxidative stress, and GSK-3β/β-catenin pathway. Compared to the SNP group, chondrocyte proliferation, and regulation factors were promoted, and chondrocyte apoptosis, oxidative stress, and the GSK-3β/β-catenin pathway were inhibited by brucine. It indicates that the adverse effect of SNP is reversed by the brucine on the chondrocyte. Compared to the brucine-SNP group, the effect of brucine on the chondrocyte proliferation, regulation factothe apoptosis, and oxidative stress were promoted by the GSK-3β antagonist. It indicates that the chondrocyte is protected agairucine through buying the GSK-3β/β-catenin pathway.

Ellagic acid attenuates interleukin-1β-induced oxidative stress and exerts protective effects on chondrocytes through the Kelch-like ECH-associated protein 1 (Keap1)/ Nuclear factor erythroid 2-related factor 2 (Nrf2) pathway

ABSTRACT Osteoarthritis (OA) is the most prevalent type of degenerative joint disease, and its pathological progression is highly associated with oxidative stress. Natural antioxidants can attenuate oxidative stress and chondrocyte injury, suggesting that antioxidants have potential applications in the management of OA. Ellagic acid (EA), a natural polyphenol derived from fruits or nuts, exerts antioxidant and anti-inflammatory effects in diseases related to oxidative stress. Herein, we investigated the effects of EA on interleukin-1β (IL-1β)-induced oxidative stress and degeneration in C28/I2 human chondrocytes. EA efficiently suppressed IL-1β-induced oxidative stress and ameliorated oxidative stress-induced dysfunction of chondrocytes, as indicated by the promotion of cartilage matrix secretion. Moreover, EA remarkably suppressed cell apoptosis and senescence, and reduced the expression of proinflammatory factors and metalloproteinases, suggesting that EA could alleviate chondrocyte injury under oxidative stress. Mechanistically, EA upregulated the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) as well as its downstream targets NADPH quinone oxidoreductase 1 and heme oxygenase-1. ML385, a specific Keap1/Nrf2 pathway inhibitor, blocked the antioxidant and chondroprotective effects of EA. Our findings demonstrated that EA could attenuate oxidative stress and exert protective effects on chondrocytes by upregulating the Keap1/Nrf2 signaling pathway.

Reduced Levels of H2S in Diabetes-Associated Osteoarthritis Are Linked to Hyperglycaemia, Nrf-2/HO-1 Signalling Downregulation and Chondrocyte Dysfunction.

Different findings indicate that type 2 diabetes is an independent risk factor for osteoarthritis (OA). However, the mechanisms underlying the connection between both diseases remain unclear. Changes in the balance of hydrogen sulphide (H2S) are thought to play an important role in the pathogenesis of diabetes and its complications, although its role is still controversial. In this study, we examined the modulation of H2S levels in serum and chondrocytes from OA diabetic (DB) and non-diabetic (non-DB) patients and in cells under glucose stress, in order to elucidate whether impairment in H2S-mediated signalling could participate in the onset of DB-related OA. Here, we identified a reduction in H2S synthesis in the cartilage from OA-DB patients and in cells under glucose stress, which is associated with hyperglycaemia-mediated dysregulation of chondrocyte metabolism. In addition, our results indicate that H2S is an inductor of the Nrf-2/HO-1 signalling pathway in cartilage, but is also a downstream target of Nrf-2 transcriptional activity. Thereby, impairment of the H2S/Nrf-2 axis under glucose stress or DB triggers chondrocyte catabolic responses, favouring the disruption of cartilage homeostasis that characterizes OA pathology. Finally, our findings highlight the benefits of the use of exogeneous sources of H2S in the treatment of DB-OA patients, and warrant future clinical studies.

Advanced Glycation End Products Induced Mitochondrial Dysfunction of Chondrocytes through Repression of AMPKα-SIRT1-PGC-1α Pathway

Introduction: Our previous studies have demonstrated advanced glycation end products (AGEs) was an important mediator in osteoarthritis (OA) which may induce mitochondrial dysfunction. AMP-activated protein kinase (AMPK), sirtuin 1 (SIRT1), and its downstream target peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) are the critical sensors that regulate mitochondrial biogenesis and have been recognized as therapeutic targets in OA. This study was designed to test whether AGEs caused mitochondrial dysfunction through modulation of AMPKα/SIRT1/PGC-1α. Methods: We knocked down or overexpressed AMPKα, SIRT1, and PGC-1α by small interfering RNA or plasmid DNA transfection, respectively. Mitochondrial membrane potential (△Ψ) was detected by tetraethylbenzimidazolyl carbocyanine iodide (JC-1) fluorescence probe. Results: The results showed that AGEs impaired △Ψ, intracellular ATP level, and mitochondrial DNA content, linked to decreased AMPKα, SIRT1, and PGC-1α expression in chondrocyte. AMPKα pharmacologic activation or overexpression of AMPKα, SIRT1, and PGC-1α reversed impairments of mitochondrial biogenesis, oxidative stress, and inflammation in AGEs-induced chondrocytes. However, AMPKα activation using AICAR had decreased capacity to increase each of those same effect readouts in AGEs-treated SIRT1-siRNA or PGC-1α-siRNA chondrocyte. Conclusion: Taken together, AGEs reduced the AMPKα/SIRT1/PGC-1α signaling in chondrocytes, leading to mitochondrial dysfunction as a result of increased oxidative stress, inflammation, and apoptosis. These results indicated that target AMPK may be as a novel therapeutic strategy for AGEs-related OA prevention.

De novo serine synthesis regulates chondrocyte proliferation during bone development and repair

The majority of the mammalian skeleton is formed through endochondral ossification starting from a cartilaginous template. Cartilage cells, or chondrocytes, survive, proliferate and synthesize extracellular matrix in an avascular environment, but the metabolic requirements for these anabolic processes are not fully understood. Here, using metabolomics analysis and genetic in vivo models, we show that maintaining intracellular serine homeostasis is essential for chondrocyte function. De novo serine synthesis through phosphoglycerate dehydrogenase (PHGDH)-mediated glucose metabolism generates nucleotides that are necessary for chondrocyte proliferation and long bone growth. On the other hand, dietary serine is less crucial during endochondral bone formation, as serine-starved chondrocytes compensate by inducing PHGDH-mediated serine synthesis. Mechanistically, this metabolic flexibility requires ATF4, a transcriptional regulator of amino acid metabolism and stress responses. We demonstrate that both serine deprivation and PHGDH inactivation enhance ATF4 signaling to stimulate de novo serine synthesis and serine uptake, respectively, and thereby prevent intracellular serine depletion and chondrocyte dysfunction. A similar metabolic adaptability between serine uptake and de novo synthesis is observed in the cartilage callus during fracture repair. Together, the results of this study reveal a critical role for PHGDH-dependent serine synthesis in maintaining intracellular serine levels under physiological and serine-limited conditions, as adequate serine levels are necessary to support chondrocyte proliferation during endochondral ossification.

C10orf10/DEPP activates mitochondrial autophagy and maintains chondrocyte viability in the pathogenesis of osteoarthritis.

Osteoarthritis (OA), the most prevalent joint disease, is characterized by the progressive loss of articular cartilage. Autophagy, a lysosomal degradation pathway, maintains cellular homeostasis, and autophagic dysfunction in chondrocytes is a hallmark of OA pathogenesis. However, the cause of autophagic dysfunction in OA chondrocytes remains incompletely understood. Recent studies have reported that decidual protein induced by progesterone (C10orf10/DEPP) positively regulates autophagic functions. In this study, we found that DEPP was involved in mitochondrial autophagic functions of chondrocytes, as well as in OA pathogenesis. DEPP expression decreased in human OA chondrocytes in the absence or presence of pro-inflammatory cytokines, and was induced by starvation, hydrogen peroxide (H2 O2 ), and hypoxia (cobalt chloride). For functional studies, DEPP knockdown decreased autophagic flux induced by H2 O2 , whereas DEPP overexpression increased autophagic flux and maintained cell viability following H2 O2 treatment. DEPP was downregulated by knockdown of forkhead box class O (FOXO) transcription factors and modulated the autophagic function regulated by FOXO3. In an OA mouse model by destabilization of the medial meniscus, DEPP-knockout mice exacerbated the progression of cartilage degradation with TUNEL-positive cells, and chondrocytes isolated from knockout mice were decreased autophagic flux and increased cell death following H2 O2 treatment. Subcellular fractionation analysis revealed that mitochondria-located DEPP activated mitochondrial autophagy via BCL2 interacting protein 3. Taken together, our data demonstrate that DEPP is a major stress-inducible gene involved in the activation of mitochondrial autophagy in chondrocytes, and maintains chondrocyte viability during OA pathogenesis. DEPP represents a potential therapeutic target for enhancing autophagy in patients with OA.

Galectin-3 facilitates inflammation and apoptosis in chondrocytes through upregulation of the TLR-4-mediated oxidative stress pathway in TC28a2 human chondrocyte cells.

Osteoarthritis (OA) is a common degenerative joint disease. The pathological changes of chondrocytes involve oxidative stress, the pro-inflammatory response, and pro-apoptotic events. Galectin-3 (Gal-3) is a 35 kDa protein with a special chimeric structure. Gal-3 participates in the progression of many diseases, such as cancer metastasis and heart failure. A previous study demonstrated that Gal-3 expression in human cartilage with OA is increased. However, the role of Gal-3 in chondrocyte dysfunction in joints is still unclear. In this study, we applied Gal-3 (5-20 μg/ml) to TC28a2 human chondrocyte cells for 24 h to induce chondrocyte dysfunction. We found that Gal-3 upregulated TLR-4 and MyD88 expression and NADPH oxidase, thereby increasing intracellular ROS in the chondrocytes. Gal-3 increased phosphorylated MEK1/2 and ERK levels, and promoted NF-κB activity. This activation of NF-κB was reduced by silencing TLR-4 and NOX-2. In addition, Gal-3 caused apoptosis of chondrocytes through the mitochondrial-dependent pathway via the TLR-4/NADPH oxidase/MAPK axis. Our study proves the pathogenic role of Gal-3 in Gal-3-induced chondrocyte dysfunction and injuries.

FoxO3a cooperates with RUNX1 to promote chondrogenesis and terminal hypertrophic of the chondrogenic progenitor cells

FoxO transcription factors (FoxOs) have recently been shown to protect against chondrocyte dysfunction and modulate cartilage homeostasis in osteoarthritis. The mechanism underlying of FoxOs regulate chondrocyte differentiation remains unknown. Runt related transcription factor 1 (RUNX1) mediated both chondrocyte and osteoblast differentiation. Our data showed that FoxO3a and RUNX1 are co-expressed in ATDC5 cells and undifferentiated mesenchyme cells and have similar high levels in chondrocytes undergoing transition from proliferation to hypertrophy. Overexpression of FoxO3a in ATDC5 cells or mouse mesenchymal cells resulted in a potent induction of the chondrocyte differentiation markers. Knockdown FoxO3a or RUNX1 potently inhibits the expressions of chondrocyte differentiation markers, including Sox9, Aggrecan, Col2, and hypertrophic chondrocyte markers including RUNX2, ColX, MMP13 and ADAMTs-5 in ATDC5 cells. Co-immunoprecipitation showed that FoxO3a binds the transcriptional regulator RUNX1. Immunohistochemistry showed that FoxO3a and RUNX1 are highly co-expressed in the proliferative chondrocytes of the growth plates in the hind limbs of newborn mice. Collectively, we revealed that FoxO3a cooperated with RUNX1 promoted chondrocyte differentiation through enhancing both early chondrogenesis and terminal hypertrophic of the chondrogenic progenitor cells, indicating FoxO3a interacting with RUNX1 may be a therapeutic target for the treatment of osteoarthritis and other bone diseases.

Hsp22 Inhibits Oxidative Stress-Induced Endplate Chondrocyte Apoptosis by Regulating Mitochondrial Pathway

Background:Facet joint degeneration (FJD), which is also called facet joint syndrome (FJS), has become one of the most commonly seen etiological factors for lumbago. Cartilage lesion triggered by lumbar facet joint (LFJ) degeneration might be related to mitochondrial impairment, but the its underlying mechanism remains unclear.Materials and methods:The endplate chondrocytes were induced by hydrogen peroxide (H2O2) to mimic the pathological conditions of oxidative stress. Enzyme linked immunosorbent assay (ELISA) were used for the evaluation of reactive oxygen species (ROS). Adenosine-triphosphate (ATP) level was assessed using ATP detection, along with the detection the expression of cytochrome C in mitochondria (mito-cyt c) and in cell cytoplasm (cyto-cyt c) and cleaved caspase 3 by Western blot analysis. TUNEL assay was conducted for the measurement of cell apoptosis in endplate chondrocytes. Reverse transcription-polymerase chain reaction (RT-qPCR) was used to verify the expression of heat shock protein 22 (HSP22) and the transfection efficiency of HSP22 interference plasmid.Results:It was found that H2O2promoted the mitochondrial dysfunction, ROS generation and cell apoptosis in endplate chondrocytes. Moreover, HSP22 was down-regulated in H2O2-induced endplate chondrocytes, and interference of HSP22 decreased the ROS production, increased the ATP level and promoted the cell apoptosis, resulting in the enhanced impairment of endplate chondrocytes. Additionally, mitochondrial ROS inhibitor (Mito-TEMPO) ameliorated the injury effects of HSP22 silencing in the H2O2-induced endplate chondrocytes.Conclusion:In conclusion, HSP22 inhibits oxidative stress-induced endplate chondrocyte apoptosis by regulating mitochondrial pathway, possibly providing novel guidance direction for the treatment of LFJ degeneration.

Metabolic reprogramming in chondrocytes to promote mitochondrial respiration reduces downstream features of osteoarthritis

Metabolic dysfunction in chondrocytes drives the pro-catabolic phenotype associated with osteoarthritic cartilage. In this study, substitution of galactose for glucose in culture media was used to promote a renewed dependence on mitochondrial respiration and oxidative phosphorylation. Galactose replacement alone blocked enhanced usage of the glycolysis pathway by IL1β-activated chondrocytes as detected by real-time changes in the rates of proton acidification of the medium and changes in oxygen consumption. The change in mitochondrial activity due to galactose was visualized as a rescue of mitochondrial membrane potential but not an alteration in the number of mitochondria. Galactose-replacement reversed other markers of dysfunctional mitochondrial metabolism, including blocking the production of reactive oxygen species, nitric oxide, and the synthesis of inducible nitric oxide synthase. Of more clinical relevance, galactose-substitution blocked downstream functional features associated with osteoarthritis, including enhanced levels of MMP13 mRNA, MMP13 protein, and the degradative loss of proteoglycan from intact cartilage explants. Blocking baseline and IL1β-enhanced MMP13 by galactose-replacement in human osteoarthritic chondrocyte cultures inversely paralleled increases in markers associated with mitochondrial recovery, phospho-AMPK, and PGC1α. Comparisons were made between galactose replacement and the glycolysis inhibitor 2-deoxyglucose. Targeting intermediary metabolism may provide a novel approach to osteoarthritis care.

Oleate Prevents Palmitate-Induced Mitochondrial Dysfunction in Chondrocytes.

The association between obesity and osteoarthritis (OA) in joints not subjected to mechanical overload, together with the relationship between OA and metabolic syndrome, suggests that there are systemic factors related to metabolic disorders that are involved in the metabolic phenotype of OA. The aim of this work is study the effects of palmitate and oleate on cellular metabolism in an “in vitro” model of human chondrocytes. The TC28a2 chondrocyte cell line was used to analyze the effect of palmitate and oleate on mitochondrial and glycolytic function, Adenosine triphosphate (ATP) production and lipid droplets accumulation. Palmitate, but not oleate, produces mitochondrial dysfunction observed with a lower coupling efficiency, maximal respiration and spare respiratory capacity. Glycolytic function showed lower rates both glycolytic capacity and glycolytic reserve when cells were incubated with fatty acids (FAs). The production rate of total and mitochondrial ATP showed lower values in chondrocytes incubated with palmitic acid (PA). The formation of lipid droplets increased in FA conditions, being significantly higher when the cells were incubated with oleic acid (OL). These results may help explain, at least in part, the close relationship of metabolic pathologies with OA, as well as help to elucidate some of the factors that can define a metabolic phenotype in OA.

Punicalin alleviates TNF-α- and IL-1β-induced chondrocyte dysfunction and cartilage metabolism via mediating FOXO3 signaling axis.

Forkhead box O3 (FOXO3) transcription factor is involved in chondral homeostasis of normal, aging and osteoarthritis (OA) cartilage. At present, we aimed to investigate whether FOXO3 is a target of punicalin to prevent IL-1β- and TNF-α-induced chondrocyte dysfunction in vitro and in vivo models. Cell and mouse models of chondrocyte dysfunction were established to determine the pharmacological value of hydrolyzable tannin, punicalin, which was extracted from the pomegranate. FOXO3 protein levels in the nucleus and cytoplasm were analysed using western blot. Safranine O staining was performed to evaluate the expansion of growth plate and chondrocyte differentiation in IL-1β- and TNF-α-treated mice. In IL-1β- and TNF-α-treated chondrocytes and mice, IL-1β and TNF-α evoked phosphorylation and nucleocytoplasmic shuttling of FOXO3, as well as reduced FOXO3 expression levels in the nucleus. However, punicalin treatment repressed FOXO3 phosphorylation and cytoplasmic transfer. Punicalin treatment improved IL-1β and TNF-α-induced growth inhibition and apoptosis of chondrocyte and the abnormal expansion of growth plate and hypertrophic zone. Moreover, punicalin could maintain the normal phenotype of chondrocyte via mediating multiple gene expression. Punicalin showed a beneficial effect on IL-1β- and TNF-α-stimulated chondrocytes and cartilaginous metabolic disorders via preserving the transcriptional activity of FOXO3. PRACTICAL APPLICATIONS: Our study presents a prospective adjuvant therapeutic drug, punicalin, to prevent inflammation-related cartilage injury and chondrocyte dysfunction.

Biological effect of APPA -apocynin and paeonol- in human articular chondrocytes

Purpose: Osteoarthritis (OA) is the most common rheumatological disease and a major cause of pain and disability in older adults. Currently no treatment is available that alters the course of OA. Mitochondrial dysfunction in chondrocytes is associated with the molecular dysregulation underlying OA, and has been proposed as a potential therapeutic target. APPA, a combination of apocynin (AP) and paeonol (PA), has the potential capacity to protect mitochondria from injury. Aim To study the biological effect of APPA in human articular chondrocytes.

Synovial fluid mitochondrial DNA as a biomarker after naturally occurring intra-articular fracture

Synovial fluid mitochondrial DNA as a biomarker after naturally occurring intra-articular fracture