Objective This study aimed to investigate the potential mechanisms by which lysyl oxidase like 3(LOXL3)affects the autophagy in chondrocytes in osteoarthritis(OA),specifically through the activation of mammalian targe...Objective This study aimed to investigate the potential mechanisms by which lysyl oxidase like 3(LOXL3)affects the autophagy in chondrocytes in osteoarthritis(OA),specifically through the activation of mammalian target of rapamycin complex 1(mTORC1).Methods To establish an OA model,rats underwent anterior cruciate ligament transection(ACLT).Chondrocytes were isolated from cartilage tissues and cultured.Western blotting was performed to assess the expression of LOXL3,Rheb,phosphorylation of p70S6K(p-p70S6K,a downstream marker of mTORC1),and autophagy markers.The autophagy of chondrocytes was observed using an immunofluorescence assay.Results The expression levels of both LOXL3 and Rheb proteins were upregulated in chondrocytes isolated from the OA model cartilage,in comparison to those from the normal cartilage.The silencing of LOXL3 resulted in a decrease in the protein levels of Rheb and p-p70S6K,as well as an increase in the expression of autophagy-related proteins.Additionally,the effect of LOXL3 could be reversed through the silencing of Rheb.The results of the immunofluorescence assay confirmed the impact of LOXL3 and Rheb on chondrocyte autophagy.Conclusion LOXL3 inhibits chondrocyte autophagy by activating the Rheb and mTORC1 signaling pathways.展开更多
Objective To investigate the expression of miRNA-140 in chondrocytes and synovial fluid of osteoarthritis(OA) patients, and explore the relationship between the miRNA-140 expression and OA severity. Methods This study...Objective To investigate the expression of miRNA-140 in chondrocytes and synovial fluid of osteoarthritis(OA) patients, and explore the relationship between the miRNA-140 expression and OA severity. Methods This study enrolled 30 OA patients who underwent total knee arthroplasty for chondrocytes sampling and 30 OA patients who underwent intra-articular injection for synovial fluid sampling. All OA patients were grouped into mild [Kellgren and Lawrence(KL) grade 1-2], moderate(KL grade 3) and severe(KL grade 4), with 10 in each subgroups for each sampling purposes. 7 non-OA patients and 10 patients with knee injury were collected for cartilage and synovial fluid sampling respectively as control groups. Chondrocytes were isolated from the cartilage tissue and cultured in vitro. Quantitative real time PCR for miRNA-140 in chondrocytes and synovial fluid were performed, and the U6 sn RNA was used as internal control. The expression difference of miRNA-140 among groups and correlation between the expression and the KL grade of OA were analysed using one-way ANOVA and Spearman test respectively. Results The expression of miRNA-140 in chondrocytes of knees in OA patients was reduced than that in normal knees, and the between-group difference was statistically significant(F=305.464, P<0.001). miRNA-140 could be detected in synovial fluid of both normal knees and OA knees, its relative expression level was reduced in synovial fluid of OA group compared with normal group, and the between-group difference was statistically significant as well(F=314.245, P<0.001). The relative expression level of miRNA-140 in both chondrocytes and synovial fluid were negatively correlated with the KL grade of OA(r=-0.969, P<0.001; r=-0.970, P<0.001). Conclusion miRNA-140 could be detected in chondrocytes and synovial fluid of OA patients, and its expression was negatively correlated with the severity of OA.展开更多
To identify the osteogenesis genes whose expression is altered in hypertrophic chondrocytes treated with H2O2.Methods Murine chondrogenitor cells(ATDC5)were differentiated into hypertrophic chondrocytes by Insulin-Tra...To identify the osteogenesis genes whose expression is altered in hypertrophic chondrocytes treated with H2O2.Methods Murine chondrogenitor cells(ATDC5)were differentiated into hypertrophic chondrocytes by Insulin-Transferrin-Selenium(ITS)treatment,and then treated with H2O2.Suitable conditions(concentration,time)were determined by using the MTT assay.After total RNA isolation and cDNA synthesis,the levels of 84 genes were determined using the PCR array,whereas quantitative RT-PCR was carried out to validate the PCR array data.Results We identified 9 up-regulated genes and 12 down-regulated genes,encoding proteins with various functions,such as collagen proteins,transcription factors,proteins involved in skeletal development and bone mineral metabolism,as well as cell adhesion molecules.Quantitative RT-PCR confirmed the altered expression of 5 down-regulated genes(Smad2,Smad4,transforming growth factorβreceptor 1,transforming growth factorβreceptor 3,and matrix metalloproteinase 10).Conclusions H2O2 significantly changed the expression of several genes involved in a variety of biological functions.Because of the link between oxidative damage and Kashin-Beck disease,these genes may also be involved in the deep-zone necrosis of the cartilage observed in Kashin-Beck disease.展开更多
Free fatty acids(FFAs), which are elevated with metabolic syndrome, are considered the principal offender exerting lipotoxicity. Few previous studies have reported a causal relationship between FFAs and osteoarthritis...Free fatty acids(FFAs), which are elevated with metabolic syndrome, are considered the principal offender exerting lipotoxicity. Few previous studies have reported a causal relationship between FFAs and osteoarthritis pathogenesis. However, the molecular mechanism by which FFAs exert lipotoxicity and induce osteoarthritis remains largely unknown. We here observed that oleate at the usual clinical range does not exert lipotoxicity while oleate at high pathological ranges exerted lipotoxicity through apoptosis in articular chondrocytes. By investigating the differential effect of oleate at toxic and nontoxic concentrations, we revealed that lipid droplet(LD) accumulation confers articular chondrocytes, the resistance to lipotoxicity. Using high fat diet-induced osteoarthritis models and articular chondrocytes treated with oleate alone or oleate plus palmitate, we demonstrated that articular chondrocytes gain resistance to lipotoxicity through protein kinase casein kinase 2(PKCK2)—six-transmembrane protein of prostate 2(STAMP2)—and fat-specific protein 27(FSP27)-mediated LD accumulation. We further observed that the exertion of FFAs-induced lipotoxicity was correlated with the increased concentration of cellular FFAs freed from LDs, whether FFAs are saturated or not. In conclusion, PKCK2/STAMP2/FSP27-mediated sequestration of FFAs in LD rescues osteoarthritic chondrocytes. PKCK2/STAMP2/FSP27 should be considered for interventions against metabolic OA.展开更多
Objective To investigate the effect on the structure of reestablished cartilage in vitro and CD44 expression on chondrocytes and compare the inducing effect on the reestablished cartilage in vitro between cortical bon...Objective To investigate the effect on the structure of reestablished cartilage in vitro and CD44 expression on chondrocytes and compare the inducing effect on the reestablished cartilage in vitro between cortical bone matrix gelatin and cancellous bone matrix gelatin. Methods To plant human fetal chondrocytes on the BMG, the damage of the cultured chondrocytes was observed by the optical microscope (HE staining). The immunohistochemistry of CD44 was quantitative analysis by the image collection and analysis system. Results With the increasing concentration of T 2 toxin, the damage of chondroytes was more and more evident and CD44 expression was lowered. After adding selenium, the damage was relieved and CD44 expression increased. The density of chondrocytes on the cortical bone matrix gelatin was much higher than that on the cancellous bone matrix gelatin. Conclusion T 2 toxin can lower the CD44 expression on the chondrocytes and adding selenium can relieve the damage caused by T 2toxin and increased CD44 expression. The inducing effect on reestablished cartilage in vitro of cortical bone matrix gelatin was much higher than that of cancellous bone matrix gelatin.展开更多
Objective: To study the adverse effects of advanced glycation end products(AGEs) on chondrocytes and the role of autophagy in this process. Methods: Chondrocytes were harvested from the human articular cartilage tissu...Objective: To study the adverse effects of advanced glycation end products(AGEs) on chondrocytes and the role of autophagy in this process. Methods: Chondrocytes were harvested from the human articular cartilage tissues in surgery. AGEs were administered during chondrocytes culture. The rapamycin was used to induce autophagy. The cell viability was determined by 3-[4,5-dimethylthiazol2-yl]-2,5-diphenyl tetrazolium bromide(MTT) assay.The expression of tumor necrosis factor-α(TNF-α) and nuclear factor-κ B(NF-κ B) was detected by quantitative real-time polymerase chain reaction. The reactive oxygen species(ROS) production and apoptosis of the chondrocytes were determined by fluorescent probe and flow cytometer, respectively. Results: The chondrocytes viability was significantly reduced after 12 h incubation with AGEs(P<0.01)). In contrast, rapamycin pretreatment increased the chondrocytes viability through autophagy. AGEs increased TNF-α and NF-κ B mRNA expression of chondrocytes and autophagy receded or proceeded the change. AGEs increased intracellular ROS accumulation and autophagy reversed the change. AGEs accelerated chondrocytes apoptosis and autophagy suspended apoptosis. Conclusions: Accumulation of AGEs may have an adverse role for chondrocytes by increasing TNF-α and NF-κB expression, ROS accumulation and apoptosis; meanwhile, autophagy ameliorates the AGEsinduced adverse effects.展开更多
Objective: To study a comprehensive proteomic analysis of celecoxib in oseteoarthritis (OA) chondrocytes. Methods: OA chondrocytes were stimulated with celecoxib, IL-1β and IL-1β together with celecoxib. Proteins we...Objective: To study a comprehensive proteomic analysis of celecoxib in oseteoarthritis (OA) chondrocytes. Methods: OA chondrocytes were stimulated with celecoxib, IL-1β and IL-1β together with celecoxib. Proteins were extracted from the cells and subjected to 2-dimensional differential image gel electrophoresis (2D-DIGE). Proteins of interest were identified by mass spectrometry. Results: Eighty-six protein spots showed significantly different intensities with each reagent or reagent combination. AAA+ protein, HSP47/Serpin, cAMP-dependent protein kinase type II-beta regulatory subunit, alpha-actin-4 and tubulin decreased with the addition of celecoxib, while apolipoprotein A-V, glutamate carboxipeptide 2, mitochondrial stress-70 protein, sorting nexin-9 and GRP78 increased with the addition of celecoxib. GRP78 is a stress protein and may be chondroprotective. Celecoxib modulated IL-1β stimulated chondrocytes, and CD200R and moesin were identified as such resulting proteins. Conclusion: Protein profiles of OA chondrocytes changed after administration of celecoxib. Further investigation is needed to elucidate the function of each protein in OA chondrocytes.展开更多
In order to investigate the possibility of expression of exogenous gene in transduced articular chondrocytes, plasmid pcDNA3 rhBMP7 was delivered to cultured chondrocytes. Through immunohistochemical staining and RT P...In order to investigate the possibility of expression of exogenous gene in transduced articular chondrocytes, plasmid pcDNA3 rhBMP7 was delivered to cultured chondrocytes. Through immunohistochemical staining and RT PCR assay, the expression of rhBMP7 gene was detected. And the bioactivity of transgene expression product was detected through MTT assay as well. It was confirmed that exogenous gene could be expressed efficiently in transduced chondrocytes and the transgene expression product had obvious bioactivity. The present study provided a theoretical basis for gene therapy on the problems of articular cartilge.展开更多
Objective To observe the effect of growth differentiation factor-5 (GDF-5) on the growth and anabolic metabolism of articular chondrocytes. Methods The articular chondrocytes isolated from rats were treated with vario...Objective To observe the effect of growth differentiation factor-5 (GDF-5) on the growth and anabolic metabolism of articular chondrocytes. Methods The articular chondrocytes isolated from rats were treated with various concentrations of rmGDF-5, and the growth of chondrocytes measured by MTT assay, the cellular cartilage matrices formation detected sulfated glycosaminoglycan by Alcian blue staining and type Ⅱcollagen by RT-PCR. Results After 7 days culture, MTT assay showed that GDF-5 enhanced the growth of chondrocytes in a dose-dependent manner, RT-PCR showed that GDF-5 clearly induced the synthesis of type Ⅱ collagen because of the col2a1 mRNA band more and more strong in a dose-dependent. Chondrocytes were cultured with GDF-5 for 14 days, the intensity of Alcian blue staining was greatly enhanced, especially, at a high concentration of 1000ng/mL, and GDF-5 enhanced the accumulation of the Alcian blue-stainable material in a concentration-dependent manner and in a does-dependent manner. Conclusion GDF-5 enhanced the growth of mature articular chondrocytes, and stimulated the cellular cartilage matrices formation in mono-layer culture.展开更多
Glucose metabolism is fundamental for the functions of all tissues,including cartilage.Despite the emerging evidence related to glucose metabolism in the regulation of prenatal cartilage development,little is known ab...Glucose metabolism is fundamental for the functions of all tissues,including cartilage.Despite the emerging evidence related to glucose metabolism in the regulation of prenatal cartilage development,little is known about the role of glucose metabolism and its biochemical basis in postnatal cartilage growth and homeostasis.We show here that genetic deletion of the glucose transporter Glutl in postnatal cartilage impairs cell proliferation and matrix production in growth plate(GPs)but paradoxically increases cartilage remnants in the metaphysis,resulting in shortening of long bones.On the other hand,articular cartilage(AC)with Glutl deficiency presents diminished cellularity and loss of proteoglycans,which ultimately progress to cartilage fibrosis.Moreover,predisposition to Glutl deficiency severely exacerbates injury-induced osteoarthritis.Regardless of the disparities in glucose metabolism between GP and AC chondrocytes under normal conditions,both types of chondrocytes demonstrate metabolic plasticity to enhance glutamine utilization and oxidation in the absence of glucose availability.However,uncontrolled glutamine flux causes collagen overmodification,thus affecting extracellular matrix remodeling in both cartilage compartments.These results uncover the pivotal and distinct roles of Glutl-mediated glucose metabolism in two of the postnatal cartilage compartments and link some cartilage abnormalities to altered glucose/glutamine metabolism.展开更多
Objective To investigate the vascular endothelial growth factor(VEGF)expression level by chondrocytes isolated from patients with osteoarthritis (OA) in hip or femoral neck fracture (FNF) and explore the effect of syn...Objective To investigate the vascular endothelial growth factor(VEGF)expression level by chondrocytes isolated from patients with osteoarthritis (OA) in hip or femoral neck fracture (FNF) and explore the effect of synovial fluid from OA展开更多
Tissue engineering techniques for cartilage re-pair to heal defects in joint surfaces is a clinical practice. Harvested autologous chondrocytes are expanded in culture and delivered in a suitable carrier medium back i...Tissue engineering techniques for cartilage re-pair to heal defects in joint surfaces is a clinical practice. Harvested autologous chondrocytes are expanded in culture and delivered in a suitable carrier medium back into the patient>s joint de-fect. The defect is then subsequently filled by new cartilage. Whether the cells in the repair tissue originate from the engineered tissue of the host or are derived from the surrounding original cartilage remains a relevant question for the ap-plied therapy. To answer this several methods exist to track cells, such as transfection of cells with LacZ carrying viruses, radio labeling with 111 IN or 51 Cr or fluorescent labeling with FDA. However, these techniques have drawbacks such as they may influence cellular properties, are radioactive and or quickly lose their tracking ability. New fluorescent probes are easier to handle and do not to interfere with cells. PKH 26劌 is a relatively new cell-labeling agent, but few data exist on the application of this dye in chondrocytes in vitro and in vivo. 5-chloromethylfluorescein diacetate - CMFDA (&amp;amp;amp;amp;amp;amp;amp;amp;amp;#168;cell tracker green〔) is an established fluores-cent probe for imaging the dynamic processes of cell proliferation in vitro and in vivo. Likewise, several studies exist on different cell types. However, little data are available for chondro-cytes. The first aim of the study was to evaluate qualitative differences in fluorescence pattern after labeling of articular, auricular and costal chondrocytes. Secondly, we evaluated the influ-ence of labeling with CMFDA on cellular adhe-sion properties. The third aim was to compare the duration of cell labeling of chondrocytes of different origin with established CMFDA as stan-dard and PKH 26潴 for 3 cell generations in vitro and 12 weeks in vivo. We show that chondro-cytes from different origin can be labeled effec-tively with both PKH 26潴 and CMFDA. The PKH 26潴 labeled articular chondrocytes maintained fluorescence longer than CMFDA in vitro and in vivo. A higher percentage of articular chondro-cytes remained stained at 63 days than auricular or costal chondrocytes.展开更多
The progressive destruction of condylar cartilage is a hallmark of the temporomandibular joint(TMJ) osteoarthritis(OA);however, its mechanism is incompletely understood. Here, we show that Kindlin-2, a key focal adhes...The progressive destruction of condylar cartilage is a hallmark of the temporomandibular joint(TMJ) osteoarthritis(OA);however, its mechanism is incompletely understood. Here, we show that Kindlin-2, a key focal adhesion protein, is strongly detected in cells of mandibular condylar cartilage in mice. We find that genetic ablation of Kindlin-2 in aggrecan-expressing condylar chondrocytes induces multiple spontaneous osteoarthritic lesions, including progressive cartilage loss and deformation, surface fissures, and ectopic cartilage and bone formation in TMJ. Kindlin-2 loss significantly downregulates the expression of aggrecan, Col2a1 and Proteoglycan 4(Prg4), all anabolic extracellular matrix proteins, and promotes catabolic metabolism in TMJ cartilage by inducing expression of Runx2and Mmp13 in condylar chondrocytes. Kindlin-2 loss decreases TMJ chondrocyte proliferation in condylar cartilages. Furthermore,Kindlin-2 loss promotes the release of cytochrome c as well as caspase 3 activation, and accelerates chondrocyte apoptosis in vitro and TMJ. Collectively, these findings reveal a crucial role of Kindlin-2 in condylar chondrocytes to maintain TMJ homeostasis.展开更多
Introduction: Intracellular calcium concentration ([Ca2+]i) is a critical parameter in cellular homeostasis, including articular chondrocytes. Perturbed [Ca2+]i of chondrocytes may be associated with joint disease. Th...Introduction: Intracellular calcium concentration ([Ca2+]i) is a critical parameter in cellular homeostasis, including articular chondrocytes. Perturbed [Ca2+]i of chondrocytes may be associated with joint disease. The objective of the study was to compare large animal models for investigating Ca2+ homeostasis in chondrocytes. Materials and Methods: The gross anatomy of the metacarpophalangeal joint (MCP) of cattle and sheep was compared, along with the effect of various manoeuvres used to study the mechanisms of Ca2+ homeostasis in chondrocytes from load-bearing areas. The gross anatomy was observed before and after dissection, and internal architecture was examined after sectioning. Cartilage thickness was measured with a digital micrometer. Chondrocyte yield was determined after isolation. Chondrocytes were incubated with Fura-2 and Ca2+i followed in different extracellular conditions. A hypotonic shock (HTS) was used to mimic removal of a load. Results: The results showed that ovids and bovids were skeletally immature and aspects of Ca2+ homeostasis were similar. Ovine chondrocytes had higher resting fluorescence, consistent with elevated resting Ca2+ levels. Results from ion substitution experiments were consistent with a role for Na+/Ca2+ exchange, and swelling-induced Ca2+ enters into the cytoplasm via the plasma membrane and intracellular stores. Conclusions: Ca2+ homeostasis in chondrocytes from both species behaved in a similar manner to HTS and ion substitutions. Differences in resting [Ca2+]i could be associated with species, stage of maturation, or Fura-2 itself and require further investigation. These findings contribute to our understanding of the physiology of articular cartilage in different species, and their potential use as models for studying joint disease in humans.展开更多
Objective To investigate the effects of nivalenol(NIV) and selenium(Se) on the metabolism of aggrecan in the cultured chondrocytes,and to explore the mechanism involved in cartilage aggrecan catabolism in the process ...Objective To investigate the effects of nivalenol(NIV) and selenium(Se) on the metabolism of aggrecan in the cultured chondrocytes,and to explore the mechanism involved in cartilage aggrecan catabolism in the process of Kashin-Beck Disease(KBD).Method Aggrecan mRNA expression was studied by RT-PCR.The concentration of GlcUA in culture medium was determined by diphenylenimine-sulfuric acid method.Result NIV significantly decrease aggrecan mRNA expression.That Se can partially antagonize the effect of NIV on aggrecan mRNA expression.The content of GlcUA in medium with NIV was high er than that in other groups.Conclusion NIV could inhibit chondrocyte synthesis aggrecan,promote the loss of aggrecan from cartilage.All the effects result in the metabolic disorder of the cartilage aggrecan,which eventually leads to irreversible mechani cal destruction of the cartilage.It suggested that Se can partially alleviate the effects of NIV on chondrocytes cultured in vitro.展开更多
From this article,we can find that nivalenol could damage the DNA,disturb aggrecan mRNA expression in chondro cytes and further induce metabolic disturbance of cartilage extracellular matrix,which may play an importan...From this article,we can find that nivalenol could damage the DNA,disturb aggrecan mRNA expression in chondro cytes and further induce metabolic disturbance of cartilage extracellular matrix,which may play an important role during patho genesis of KBD.Se supplementation can resist the injury of nivalenol to chondrocytes,but its action is limited.展开更多
Osteoarthritis(OA) is an age-related disorder that is strongly associated with chondrocyte senescence. The causal link between disruptive PTEN/Akt signaling and chondrocyte senescence and the underlying mechanism are ...Osteoarthritis(OA) is an age-related disorder that is strongly associated with chondrocyte senescence. The causal link between disruptive PTEN/Akt signaling and chondrocyte senescence and the underlying mechanism are unclear. In this study, we found activated Akt signaling in human OA cartilage as well as in a mouse OA model with surgical destabilization of the medial meniscus.Genetic mouse models mimicking sustained Akt signaling in articular chondrocytes via PTEN deficiency driven by either Col2a1-Cre or Col2a1-Cre^(ERT2) developed OA, whereas restriction of Akt signaling reversed the OA phenotypes in PTEN-deficient mice.Mechanistically, prolonged activation of Akt signaling caused an accumulation of reactive oxygen species and triggered chondrocyte senescence as well as a senescence-associated secretory phenotype, whereas chronic administration of the antioxidant N-acetylcysteine suppressed chondrocyte senescence and mitigated OA progression in PTEN-deficient mice. Therefore,inhibition of Akt signaling by PTEN is required for the maintenance of articular cartilage. Disrupted Akt signaling in articular chondrocytes triggers oxidative stress-induced chondrocyte senescence and causes OA.展开更多
Osteoarthritis(OA)is a degenerative disease characterized by matrix degradation and cell death leading to a gradual loss of articular cartilage integrity.As a bacterial synthesis of quinine,pyrroloquinoline quinone(PQ...Osteoarthritis(OA)is a degenerative disease characterized by matrix degradation and cell death leading to a gradual loss of articular cartilage integrity.As a bacterial synthesis of quinine,pyrroloquinoline quinone(PQQ)is a strong redox cofactor with a variety of biological benefits,including antioxidant,anti-inflammation-induced mitochondrial metabolism regulation.This study was designed to investigate the effect of PQQ on TNF-α-induced mitochondrial damage in chondrocytes.Chondrocytes isolated from C57BL/6 mice were exposed to TNF-α50 ng/mL,TNF-α50 ng/mL+PQQ 10µmol/L for 24 h.Then,morphological study,functional study and mechanism study were taken.The results revealed TNF-α-induced chondrocyte mitochondrion damage could be reduced by application of PQQ,evidenced by elevated number of mitochondria,well-kept mtDNA integrity,preserved ATP level,reestablished mitochondrial membrane potential,and prevented mitochondrial function.The present work strongly suggests that the mitochondrion is an important target for OA chondrocyte damage induced by TNF-αand the PQQ protection from this damage ameliorates mitochondrial dysfunction induced by TNF-α.PQQ might be a potential chemical for OA intervention.展开更多
基金the National Natural Science Foundation of China(No.81702187)Natural Science Foundation of Jiangxi Province(No.20202BAB206019)+4 种基金Science Fund for Distinguished Young Scholars of Jiangxi Province(No.20224ACB216018)Scientific Talents Grants of Jiangxi Province(No.S2018LQCQ0800)Scientific Grants of Health Commission of Jiangxi Province(No.20194048)Scientific Innovation Talents Grants of Ganzhou(No.2019-60-08)Leading Talents Grants and Ph.D.Programs Foundation of Ganzhou People’s Hospital(No.Bsqd2019003)and Academic leaders Program of Ganzhou Institutes of Health.
文摘Objective This study aimed to investigate the potential mechanisms by which lysyl oxidase like 3(LOXL3)affects the autophagy in chondrocytes in osteoarthritis(OA),specifically through the activation of mammalian target of rapamycin complex 1(mTORC1).Methods To establish an OA model,rats underwent anterior cruciate ligament transection(ACLT).Chondrocytes were isolated from cartilage tissues and cultured.Western blotting was performed to assess the expression of LOXL3,Rheb,phosphorylation of p70S6K(p-p70S6K,a downstream marker of mTORC1),and autophagy markers.The autophagy of chondrocytes was observed using an immunofluorescence assay.Results The expression levels of both LOXL3 and Rheb proteins were upregulated in chondrocytes isolated from the OA model cartilage,in comparison to those from the normal cartilage.The silencing of LOXL3 resulted in a decrease in the protein levels of Rheb and p-p70S6K,as well as an increase in the expression of autophagy-related proteins.Additionally,the effect of LOXL3 could be reversed through the silencing of Rheb.The results of the immunofluorescence assay confirmed the impact of LOXL3 and Rheb on chondrocyte autophagy.Conclusion LOXL3 inhibits chondrocyte autophagy by activating the Rheb and mTORC1 signaling pathways.
基金Supported by the National Natural Science Foundation of China(No.81672219No.81601936)the Science and Technology Support Program of Sichuan province(No.2014SZ0023-2)
文摘Objective To investigate the expression of miRNA-140 in chondrocytes and synovial fluid of osteoarthritis(OA) patients, and explore the relationship between the miRNA-140 expression and OA severity. Methods This study enrolled 30 OA patients who underwent total knee arthroplasty for chondrocytes sampling and 30 OA patients who underwent intra-articular injection for synovial fluid sampling. All OA patients were grouped into mild [Kellgren and Lawrence(KL) grade 1-2], moderate(KL grade 3) and severe(KL grade 4), with 10 in each subgroups for each sampling purposes. 7 non-OA patients and 10 patients with knee injury were collected for cartilage and synovial fluid sampling respectively as control groups. Chondrocytes were isolated from the cartilage tissue and cultured in vitro. Quantitative real time PCR for miRNA-140 in chondrocytes and synovial fluid were performed, and the U6 sn RNA was used as internal control. The expression difference of miRNA-140 among groups and correlation between the expression and the KL grade of OA were analysed using one-way ANOVA and Spearman test respectively. Results The expression of miRNA-140 in chondrocytes of knees in OA patients was reduced than that in normal knees, and the between-group difference was statistically significant(F=305.464, P<0.001). miRNA-140 could be detected in synovial fluid of both normal knees and OA knees, its relative expression level was reduced in synovial fluid of OA group compared with normal group, and the between-group difference was statistically significant as well(F=314.245, P<0.001). The relative expression level of miRNA-140 in both chondrocytes and synovial fluid were negatively correlated with the KL grade of OA(r=-0.969, P<0.001; r=-0.970, P<0.001). Conclusion miRNA-140 could be detected in chondrocytes and synovial fluid of OA patients, and its expression was negatively correlated with the severity of OA.
基金Supported by the National Natural Science Foundation of China(81573102 and 81273006)the Natural Science Fund Projects of Shaanxi Province(2017JM812)
文摘To identify the osteogenesis genes whose expression is altered in hypertrophic chondrocytes treated with H2O2.Methods Murine chondrogenitor cells(ATDC5)were differentiated into hypertrophic chondrocytes by Insulin-Transferrin-Selenium(ITS)treatment,and then treated with H2O2.Suitable conditions(concentration,time)were determined by using the MTT assay.After total RNA isolation and cDNA synthesis,the levels of 84 genes were determined using the PCR array,whereas quantitative RT-PCR was carried out to validate the PCR array data.Results We identified 9 up-regulated genes and 12 down-regulated genes,encoding proteins with various functions,such as collagen proteins,transcription factors,proteins involved in skeletal development and bone mineral metabolism,as well as cell adhesion molecules.Quantitative RT-PCR confirmed the altered expression of 5 down-regulated genes(Smad2,Smad4,transforming growth factorβreceptor 1,transforming growth factorβreceptor 3,and matrix metalloproteinase 10).Conclusions H2O2 significantly changed the expression of several genes involved in a variety of biological functions.Because of the link between oxidative damage and Kashin-Beck disease,these genes may also be involved in the deep-zone necrosis of the cartilage observed in Kashin-Beck disease.
基金supported by a grant from the National Research Foundation of Korea(NRF)funded by the Korean Government(MISP)(No.2015R1A2A1A10051603)
文摘Free fatty acids(FFAs), which are elevated with metabolic syndrome, are considered the principal offender exerting lipotoxicity. Few previous studies have reported a causal relationship between FFAs and osteoarthritis pathogenesis. However, the molecular mechanism by which FFAs exert lipotoxicity and induce osteoarthritis remains largely unknown. We here observed that oleate at the usual clinical range does not exert lipotoxicity while oleate at high pathological ranges exerted lipotoxicity through apoptosis in articular chondrocytes. By investigating the differential effect of oleate at toxic and nontoxic concentrations, we revealed that lipid droplet(LD) accumulation confers articular chondrocytes, the resistance to lipotoxicity. Using high fat diet-induced osteoarthritis models and articular chondrocytes treated with oleate alone or oleate plus palmitate, we demonstrated that articular chondrocytes gain resistance to lipotoxicity through protein kinase casein kinase 2(PKCK2)—six-transmembrane protein of prostate 2(STAMP2)—and fat-specific protein 27(FSP27)-mediated LD accumulation. We further observed that the exertion of FFAs-induced lipotoxicity was correlated with the increased concentration of cellular FFAs freed from LDs, whether FFAs are saturated or not. In conclusion, PKCK2/STAMP2/FSP27-mediated sequestration of FFAs in LD rescues osteoarthritic chondrocytes. PKCK2/STAMP2/FSP27 should be considered for interventions against metabolic OA.
文摘Objective To investigate the effect on the structure of reestablished cartilage in vitro and CD44 expression on chondrocytes and compare the inducing effect on the reestablished cartilage in vitro between cortical bone matrix gelatin and cancellous bone matrix gelatin. Methods To plant human fetal chondrocytes on the BMG, the damage of the cultured chondrocytes was observed by the optical microscope (HE staining). The immunohistochemistry of CD44 was quantitative analysis by the image collection and analysis system. Results With the increasing concentration of T 2 toxin, the damage of chondroytes was more and more evident and CD44 expression was lowered. After adding selenium, the damage was relieved and CD44 expression increased. The density of chondrocytes on the cortical bone matrix gelatin was much higher than that on the cancellous bone matrix gelatin. Conclusion T 2 toxin can lower the CD44 expression on the chondrocytes and adding selenium can relieve the damage caused by T 2toxin and increased CD44 expression. The inducing effect on reestablished cartilage in vitro of cortical bone matrix gelatin was much higher than that of cancellous bone matrix gelatin.
文摘Objective: To study the adverse effects of advanced glycation end products(AGEs) on chondrocytes and the role of autophagy in this process. Methods: Chondrocytes were harvested from the human articular cartilage tissues in surgery. AGEs were administered during chondrocytes culture. The rapamycin was used to induce autophagy. The cell viability was determined by 3-[4,5-dimethylthiazol2-yl]-2,5-diphenyl tetrazolium bromide(MTT) assay.The expression of tumor necrosis factor-α(TNF-α) and nuclear factor-κ B(NF-κ B) was detected by quantitative real-time polymerase chain reaction. The reactive oxygen species(ROS) production and apoptosis of the chondrocytes were determined by fluorescent probe and flow cytometer, respectively. Results: The chondrocytes viability was significantly reduced after 12 h incubation with AGEs(P<0.01)). In contrast, rapamycin pretreatment increased the chondrocytes viability through autophagy. AGEs increased TNF-α and NF-κ B mRNA expression of chondrocytes and autophagy receded or proceeded the change. AGEs increased intracellular ROS accumulation and autophagy reversed the change. AGEs accelerated chondrocytes apoptosis and autophagy suspended apoptosis. Conclusions: Accumulation of AGEs may have an adverse role for chondrocytes by increasing TNF-α and NF-κB expression, ROS accumulation and apoptosis; meanwhile, autophagy ameliorates the AGEsinduced adverse effects.
文摘Objective: To study a comprehensive proteomic analysis of celecoxib in oseteoarthritis (OA) chondrocytes. Methods: OA chondrocytes were stimulated with celecoxib, IL-1β and IL-1β together with celecoxib. Proteins were extracted from the cells and subjected to 2-dimensional differential image gel electrophoresis (2D-DIGE). Proteins of interest were identified by mass spectrometry. Results: Eighty-six protein spots showed significantly different intensities with each reagent or reagent combination. AAA+ protein, HSP47/Serpin, cAMP-dependent protein kinase type II-beta regulatory subunit, alpha-actin-4 and tubulin decreased with the addition of celecoxib, while apolipoprotein A-V, glutamate carboxipeptide 2, mitochondrial stress-70 protein, sorting nexin-9 and GRP78 increased with the addition of celecoxib. GRP78 is a stress protein and may be chondroprotective. Celecoxib modulated IL-1β stimulated chondrocytes, and CD200R and moesin were identified as such resulting proteins. Conclusion: Protein profiles of OA chondrocytes changed after administration of celecoxib. Further investigation is needed to elucidate the function of each protein in OA chondrocytes.
文摘In order to investigate the possibility of expression of exogenous gene in transduced articular chondrocytes, plasmid pcDNA3 rhBMP7 was delivered to cultured chondrocytes. Through immunohistochemical staining and RT PCR assay, the expression of rhBMP7 gene was detected. And the bioactivity of transgene expression product was detected through MTT assay as well. It was confirmed that exogenous gene could be expressed efficiently in transduced chondrocytes and the transgene expression product had obvious bioactivity. The present study provided a theoretical basis for gene therapy on the problems of articular cartilge.
文摘Objective To observe the effect of growth differentiation factor-5 (GDF-5) on the growth and anabolic metabolism of articular chondrocytes. Methods The articular chondrocytes isolated from rats were treated with various concentrations of rmGDF-5, and the growth of chondrocytes measured by MTT assay, the cellular cartilage matrices formation detected sulfated glycosaminoglycan by Alcian blue staining and type Ⅱcollagen by RT-PCR. Results After 7 days culture, MTT assay showed that GDF-5 enhanced the growth of chondrocytes in a dose-dependent manner, RT-PCR showed that GDF-5 clearly induced the synthesis of type Ⅱ collagen because of the col2a1 mRNA band more and more strong in a dose-dependent. Chondrocytes were cultured with GDF-5 for 14 days, the intensity of Alcian blue staining was greatly enhanced, especially, at a high concentration of 1000ng/mL, and GDF-5 enhanced the accumulation of the Alcian blue-stainable material in a concentration-dependent manner and in a does-dependent manner. Conclusion GDF-5 enhanced the growth of mature articular chondrocytes, and stimulated the cellular cartilage matrices formation in mono-layer culture.
基金This work was supported by the following NIH/NIAMS grants:R01 grants(AR069605 and AR079100 to RJ.O.as well as AR075860 and AR077616 to J.S.),an R21 grant(AR077226 to J.S.),a P30 Core Center grant(AR057235 to Musculoskeletal Research Center)an NCI grant(R35ES028365 to G.P.).
文摘Glucose metabolism is fundamental for the functions of all tissues,including cartilage.Despite the emerging evidence related to glucose metabolism in the regulation of prenatal cartilage development,little is known about the role of glucose metabolism and its biochemical basis in postnatal cartilage growth and homeostasis.We show here that genetic deletion of the glucose transporter Glutl in postnatal cartilage impairs cell proliferation and matrix production in growth plate(GPs)but paradoxically increases cartilage remnants in the metaphysis,resulting in shortening of long bones.On the other hand,articular cartilage(AC)with Glutl deficiency presents diminished cellularity and loss of proteoglycans,which ultimately progress to cartilage fibrosis.Moreover,predisposition to Glutl deficiency severely exacerbates injury-induced osteoarthritis.Regardless of the disparities in glucose metabolism between GP and AC chondrocytes under normal conditions,both types of chondrocytes demonstrate metabolic plasticity to enhance glutamine utilization and oxidation in the absence of glucose availability.However,uncontrolled glutamine flux causes collagen overmodification,thus affecting extracellular matrix remodeling in both cartilage compartments.These results uncover the pivotal and distinct roles of Glutl-mediated glucose metabolism in two of the postnatal cartilage compartments and link some cartilage abnormalities to altered glucose/glutamine metabolism.
文摘Objective To investigate the vascular endothelial growth factor(VEGF)expression level by chondrocytes isolated from patients with osteoarthritis (OA) in hip or femoral neck fracture (FNF) and explore the effect of synovial fluid from OA
文摘Tissue engineering techniques for cartilage re-pair to heal defects in joint surfaces is a clinical practice. Harvested autologous chondrocytes are expanded in culture and delivered in a suitable carrier medium back into the patient>s joint de-fect. The defect is then subsequently filled by new cartilage. Whether the cells in the repair tissue originate from the engineered tissue of the host or are derived from the surrounding original cartilage remains a relevant question for the ap-plied therapy. To answer this several methods exist to track cells, such as transfection of cells with LacZ carrying viruses, radio labeling with 111 IN or 51 Cr or fluorescent labeling with FDA. However, these techniques have drawbacks such as they may influence cellular properties, are radioactive and or quickly lose their tracking ability. New fluorescent probes are easier to handle and do not to interfere with cells. PKH 26劌 is a relatively new cell-labeling agent, but few data exist on the application of this dye in chondrocytes in vitro and in vivo. 5-chloromethylfluorescein diacetate - CMFDA (&amp;amp;amp;amp;amp;amp;amp;amp;amp;#168;cell tracker green〔) is an established fluores-cent probe for imaging the dynamic processes of cell proliferation in vitro and in vivo. Likewise, several studies exist on different cell types. However, little data are available for chondro-cytes. The first aim of the study was to evaluate qualitative differences in fluorescence pattern after labeling of articular, auricular and costal chondrocytes. Secondly, we evaluated the influ-ence of labeling with CMFDA on cellular adhe-sion properties. The third aim was to compare the duration of cell labeling of chondrocytes of different origin with established CMFDA as stan-dard and PKH 26潴 for 3 cell generations in vitro and 12 weeks in vivo. We show that chondro-cytes from different origin can be labeled effec-tively with both PKH 26潴 and CMFDA. The PKH 26潴 labeled articular chondrocytes maintained fluorescence longer than CMFDA in vitro and in vivo. A higher percentage of articular chondro-cytes remained stained at 63 days than auricular or costal chondrocytes.
基金supported, in part, by the National Key Research and Development Program of China Grants (2019YFA0906004)the National Natural Science Foundation of China Grants (81991513, 81870532, 82172375)+1 种基金the Guangdong Provincial Science and Technology Innovation Council Grant (2017B030301018)the Shenzhen Municipal Science and Technology Innovation Council Grant (20200925150409001)。
文摘The progressive destruction of condylar cartilage is a hallmark of the temporomandibular joint(TMJ) osteoarthritis(OA);however, its mechanism is incompletely understood. Here, we show that Kindlin-2, a key focal adhesion protein, is strongly detected in cells of mandibular condylar cartilage in mice. We find that genetic ablation of Kindlin-2 in aggrecan-expressing condylar chondrocytes induces multiple spontaneous osteoarthritic lesions, including progressive cartilage loss and deformation, surface fissures, and ectopic cartilage and bone formation in TMJ. Kindlin-2 loss significantly downregulates the expression of aggrecan, Col2a1 and Proteoglycan 4(Prg4), all anabolic extracellular matrix proteins, and promotes catabolic metabolism in TMJ cartilage by inducing expression of Runx2and Mmp13 in condylar chondrocytes. Kindlin-2 loss decreases TMJ chondrocyte proliferation in condylar cartilages. Furthermore,Kindlin-2 loss promotes the release of cytochrome c as well as caspase 3 activation, and accelerates chondrocyte apoptosis in vitro and TMJ. Collectively, these findings reveal a crucial role of Kindlin-2 in condylar chondrocytes to maintain TMJ homeostasis.
文摘Introduction: Intracellular calcium concentration ([Ca2+]i) is a critical parameter in cellular homeostasis, including articular chondrocytes. Perturbed [Ca2+]i of chondrocytes may be associated with joint disease. The objective of the study was to compare large animal models for investigating Ca2+ homeostasis in chondrocytes. Materials and Methods: The gross anatomy of the metacarpophalangeal joint (MCP) of cattle and sheep was compared, along with the effect of various manoeuvres used to study the mechanisms of Ca2+ homeostasis in chondrocytes from load-bearing areas. The gross anatomy was observed before and after dissection, and internal architecture was examined after sectioning. Cartilage thickness was measured with a digital micrometer. Chondrocyte yield was determined after isolation. Chondrocytes were incubated with Fura-2 and Ca2+i followed in different extracellular conditions. A hypotonic shock (HTS) was used to mimic removal of a load. Results: The results showed that ovids and bovids were skeletally immature and aspects of Ca2+ homeostasis were similar. Ovine chondrocytes had higher resting fluorescence, consistent with elevated resting Ca2+ levels. Results from ion substitution experiments were consistent with a role for Na+/Ca2+ exchange, and swelling-induced Ca2+ enters into the cytoplasm via the plasma membrane and intracellular stores. Conclusions: Ca2+ homeostasis in chondrocytes from both species behaved in a similar manner to HTS and ion substitutions. Differences in resting [Ca2+]i could be associated with species, stage of maturation, or Fura-2 itself and require further investigation. These findings contribute to our understanding of the physiology of articular cartilage in different species, and their potential use as models for studying joint disease in humans.
基金supported by the Scientific and Technological Research Project of the Education Department of Shaanxi Province(12JK0703)the Natural Science Foundation of Shaanxi Provincial Health Department(2012D75)foundation of Xi'an Medical University(11FZ26)
文摘Objective To investigate the effects of nivalenol(NIV) and selenium(Se) on the metabolism of aggrecan in the cultured chondrocytes,and to explore the mechanism involved in cartilage aggrecan catabolism in the process of Kashin-Beck Disease(KBD).Method Aggrecan mRNA expression was studied by RT-PCR.The concentration of GlcUA in culture medium was determined by diphenylenimine-sulfuric acid method.Result NIV significantly decrease aggrecan mRNA expression.That Se can partially antagonize the effect of NIV on aggrecan mRNA expression.The content of GlcUA in medium with NIV was high er than that in other groups.Conclusion NIV could inhibit chondrocyte synthesis aggrecan,promote the loss of aggrecan from cartilage.All the effects result in the metabolic disorder of the cartilage aggrecan,which eventually leads to irreversible mechani cal destruction of the cartilage.It suggested that Se can partially alleviate the effects of NIV on chondrocytes cultured in vitro.
基金supported by the Scientific and Technological Research Project of the Education Department of Shaanxi Province(12JK0703)the Natural Science Foundation of Shaanxi Provincial Health Department(2012D75)the foundation of Xi’an Medical University(11FZ26)
文摘From this article,we can find that nivalenol could damage the DNA,disturb aggrecan mRNA expression in chondro cytes and further induce metabolic disturbance of cartilage extracellular matrix,which may play an important role during patho genesis of KBD.Se supplementation can resist the injury of nivalenol to chondrocytes,but its action is limited.
基金supported by grants from the State Key Program of National Natural Science of China (31630093)the National Natural Science Foundation of China (31571512, 31871476, and 81241062)+1 种基金the Beijing Nova Program (Z161100004916146)the National Basic Research Program of China (2012CB966904)
文摘Osteoarthritis(OA) is an age-related disorder that is strongly associated with chondrocyte senescence. The causal link between disruptive PTEN/Akt signaling and chondrocyte senescence and the underlying mechanism are unclear. In this study, we found activated Akt signaling in human OA cartilage as well as in a mouse OA model with surgical destabilization of the medial meniscus.Genetic mouse models mimicking sustained Akt signaling in articular chondrocytes via PTEN deficiency driven by either Col2a1-Cre or Col2a1-Cre^(ERT2) developed OA, whereas restriction of Akt signaling reversed the OA phenotypes in PTEN-deficient mice.Mechanistically, prolonged activation of Akt signaling caused an accumulation of reactive oxygen species and triggered chondrocyte senescence as well as a senescence-associated secretory phenotype, whereas chronic administration of the antioxidant N-acetylcysteine suppressed chondrocyte senescence and mitigated OA progression in PTEN-deficient mice. Therefore,inhibition of Akt signaling by PTEN is required for the maintenance of articular cartilage. Disrupted Akt signaling in articular chondrocytes triggers oxidative stress-induced chondrocyte senescence and causes OA.
基金the National Natural Science Foundation of China(No.81171760).
文摘Osteoarthritis(OA)is a degenerative disease characterized by matrix degradation and cell death leading to a gradual loss of articular cartilage integrity.As a bacterial synthesis of quinine,pyrroloquinoline quinone(PQQ)is a strong redox cofactor with a variety of biological benefits,including antioxidant,anti-inflammation-induced mitochondrial metabolism regulation.This study was designed to investigate the effect of PQQ on TNF-α-induced mitochondrial damage in chondrocytes.Chondrocytes isolated from C57BL/6 mice were exposed to TNF-α50 ng/mL,TNF-α50 ng/mL+PQQ 10µmol/L for 24 h.Then,morphological study,functional study and mechanism study were taken.The results revealed TNF-α-induced chondrocyte mitochondrion damage could be reduced by application of PQQ,evidenced by elevated number of mitochondria,well-kept mtDNA integrity,preserved ATP level,reestablished mitochondrial membrane potential,and prevented mitochondrial function.The present work strongly suggests that the mitochondrion is an important target for OA chondrocyte damage induced by TNF-αand the PQQ protection from this damage ameliorates mitochondrial dysfunction induced by TNF-α.PQQ might be a potential chemical for OA intervention.
