The Effect of Nivalenol on Metabolism of Cultured Chondrocytes and the Protection of Selenium

Objective: To investigate the possible effect of nivalenol on metabolism ofthe cultured chondrocytes and the protection of selenium. Methods: The quantitative analyses ofmetabolism in single- layer cultured chondrocytes were performed by biocliemical means and theimpairment of DNA was observed by both of the single cell microgel electrophoresis assay and theagarose gel electrophoresis assay. Results: In the media containing different concentrations ofnivalenol (0. 000 5-0. 020 0 mg/L), the amounts of DNA and proteoglycan in matrix of thechondrocytes were decreased. The syn-thesis of protein was reduced and the impairment of DNAdeteriorated with the increase of the concentrations of nivalenol in tlte given dose. When seleniumwas added into the media, the impairment by nivalenol was decreased. In the media containingdifferent concentrations of nivalenol, however, the lipid peroxidation of the chondrocytes was notaffected by nivalenol, yet the amount of lipid peroxides significantly declined. Conclusion:Nivalenol may evidently cause impairment of the chondrocytes when its concentrations are in thepresent experimental range. Selenium can protect cultured cliondrocytes, but cannot prevent theirDNA from being impaired.

LOXL3 Inhibits Autophagy of Chondrocytes by Activating Rheb in Osteoarthritis

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.

Matrix stiffening promotes chondrocyte senescence and the osteoarthritis development through downregulating HDAC3

Extracellular matrix(ECM)stiffening is a typical characteristic of cartilage aging,which is a quintessential feature of knee osteoarthritis(KOA).However,little is known about how ECM stiffening affects chondrocytes and other molecules downstream.This study mimicked the physiological and pathological stiffness of human cartilage using polydimethylsiloxane(PDMS)substrates.It demonstrated that epigenetic Parkin regulation by histone deacetylase 3(HDAC3)represents a new mechanosensitive mechanism by which the stiffness matrix affected chondrocyte physiology.We found that ECM stiffening accelerated cultured chondrocyte senescence in vitro,while the stiffness ECM downregulated HDAC3,prompting Parkin acetylation to activate excessive mitophagy and accelerating chondrocyte senescence and osteoarthritis(OA)in mice.Contrarily,intra-articular injection with an HDAC3-expressing adeno-associated virus restored the young phenotype of the aged chondrocytes stimulated by ECM stiffening and alleviated OA in mice.The findings indicated that changes in the mechanical ECM properties initiated pathogenic mechanotransduction signals,promoted the Parkin acetylation and hyperactivated mitophagy,and damaged chondrocyte health.These results may provide new insights into chondrocyte regulation by the mechanical properties of ECM,suggesting that the modification of the physical ECM properties may be a potential OA treatment strategy.

Mechanism of acupoint penetration acupuncture therapy regulating chondrocyte autophagy via the PI3K/Akt-mTOR pathway in KOA rats

Objective To investigate whether acupoint penetration acupuncture(APA)could regulate chondrocyte autophagy and apoptosis via the PI3K/Akt-mTOR signaling pathway to reduce cartilage degeneration in knee osteoarthritis(KOA)rats.Methods KOA was induced in rats via intra-articular injection of sodium iodoacetate resolution.Forty male Sprague-Dawley rats were randomly assigned to blank control,model,APA,electro-acupuncture(EA),and sham model groups(n=8)and those in the APA and EA groups received their respective therapies.Following completion of the treatment course,histological examinations of cartilage and muscle were conducted.Levels of apoptosis-and autophagy-related factors,including Bax,Bcl-2,mTOR,ULK-1,and Beclin-1 protein,and mRNAs were assessed.Additionally,β-endorphin(β-EP)concentrations in the brain and serum were measured.Results Histological analysis revealed that APA alleviated cartilage and muscle damage compared with the model group.APA inhibited cartilage degeneration by modulating the expression of apoptosis-and autophagy-related proteins and mRNA,thus preventing chondrocyte apoptosis.In the APA group,Bax and mTOR protein levels were significantly lower than those in the model group(both P=.024).Conversely,the Bcl-2 expression level was significantly higher than that in the EA group(P=.035).Additionally,ULK-1 expression was significantly lower than that in the EA group(P=.045).The mRNA level of Bax was significantly higher than that in the blank control group(P<.001).However,Beclin-1 levels were significantly higher than those in both the model and EA groups(both P<.001).ELISA results showed a significant decrease in the concentration ofβ-EP in the brains of the rats in the APA group compared with those in the model group(P=.032).Conclusions APA reduced osteoarthritis-related pain and alleviated cartilage damage by upregulating chondrocyte autophagy and down-regulating apoptosis via signaling pathways involving PI3K/Akt-mTOR in KOA rats.

GROWTH DIFFERENTIATION FACTOR-5 STIMULATES THE GROWTH AND ANABOLIC METABOLISM OF ARTICULAR CHONDROCYTES

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.

Exploring the effect of Bushen Bitong recipe-containing serum on IL-1β-induced chondrocyte apoptosis based on SOX9/NF-κB/MMP-13 signaling pathway

Objective:To observe the effect and possible mechanism of action of Bushen Bitong recipe(BSBT)containing serum on IL-1β-induced chondrocyte apoptosis.Methods:Generation 3 rat chondrocytes were randomized into Control,IL-1β,IL-1β+BSBT(L),IL-1β+BSBT(M),and IL-1β+BSBT(H)groups(5%,10%and 15%BSBT-containing serum),and then 24h after intervention respectively,the cell proliferation and Apoptosis rate;Western blot detected the expression levels of Bcl-2,BAX,Caspase-3,SOX9,NF-κB p65,MMP-13 proteins in chondrocytes.ELISA detected the levels of TNF-α,IL-6,and bFGF in the supernatants of chondrocyte culture.Results:Compared with Control group,cell proliferation activity decreased,apoptosis rate increased,NF-κB p65,MMP-13 protein level and TNF-α,IL-6 level increased,and SOX9 protein level and bFGF level decreased in IL-1βgroup;compared with IL-1βgroup,different concentrations of BSBT-containing serum group,cell proliferation activity increased,and apoptosis rate decreased.NF-κB p65,MMP-13 protein level and TNF-α,IL-6 level decreased,SOX9 protein level and bFGF level increased;compared with IL-1β+BSBT(L)group,cell proliferation activity increased,apoptosis rate decreased in IL-1β+BSBT(M)and IL-1β+BSBT(H)groups,and NF-κB p65,MMP-13 protein level and TNF-αlevel decreased.13 protein levels and TNF-αand IL-6 levels decreased,and SOX9 protein levels and bFGF levels increased.Conclusion:BSBT-containing serum may promote IL-1β-induced proliferation of chondrocytes,reduce apoptosis,improve the microenvironment of chondrocytes,and promote cartilage repair through the SOX9/NF-κB/MMP-13 signaling pathway.

AAV-mediated expression of p65shRNA and bone morphogenetic protein 4 synergistically enhances chondrocyte regeneration

BACKGROUND:Adeno-associated virus(AAV)gene therapy has been proven to be reliable and safe for the treatment of osteoarthritis in recent years.However,given the complexity of osteoarthritis pathogenesis,single gene manipulation for the treatment of osteoarthritis may not produce satisfactory results.Previous studies have shown that nuclear factorκB could promote the inflammatory pathway in osteoarthritic chondrocytes,and bone morphogenetic protein 4(BMP4)could promote cartilage regeneration.OBJECTIVE:To test whether combined application of AAV-p65shRNA and AAV-BMP4 will yield the synergistic effect on chondrocytes regeneration and osteoarthritis treatment.METHODS:Viral particles containing AAV-p65-shRNA and AAV-BMP4 were prepared.Their efficacy in inhibiting inflammation in chondrocytes and promoting chondrogenesis was assessed in vitro and in vivo by transfecting AAV-p65-shRNA or AAV-BMP4 into cells.The experiments were divided into five groups:PBS group;osteoarthritis group;AAV-BMP4 group;AAV-p65shRNA group;and BMP4-p65shRNA 1:1 group.Samples were collected at 4,12,and 24 weeks postoperatively.Tissue staining,including safranin O and Alcian blue,was applied after collecting articular tissue.Then,the optimal ratio between the two types of transfected viral particles was further investigated to improve the chondrogenic potential of mixed cells in vivo.RESULTS AND CONCLUSION:The combined application of AAV-p65shRNA and AAV-BMP4 together showed a synergistic effect on cartilage regeneration and osteoarthritis treatment.Mixed cells transfected with AAV-p65shRNA and AAV-BMP4 at a 1:1 ratio produced the most extracellular matrix synthesis(P<0.05).In vivo results also revealed that the combination of the two viruses had the highest regenerative potential for osteoarthritic cartilage(P<0.05).In the present study,we also discovered that the combined therapy had the maximum effect when the two viruses were administered in equal proportions.Decreasing either p65shRNA or BMP4 transfected cells resulted in less collagen II synthesis.This implies that inhibiting inflammation by p65shRNA and promoting regeneration by BMP4 are equally important for osteoarthritis treatment.These findings provide a new strategy for the treatment of early osteoarthritis by simultaneously inhibiting cartilage inflammation and promoting cartilage repair.

Effects of Nivalenol and Selenium on the Metabolism of Aggrecan in the Cultured Human Fetal Chondrocytes

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.

Summary of Effects on Aggrecan Metabolism of Chondrocytes by Nivalenol and Selenium

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.

Altered Gene Expression in Articular Chondrocytes of Smad3^(ex8/ex8) Mice, Revealed by Gene Profiling Using Microarrays

It has been previously reported that small mother against decapentaplegic 3 （Smad3） gene knockout （Smad3^ex8/ex8） mice displays phenotypes similar to human osteoarthritis, as characterized by abnormal hypertrophic differentiation of articular chondrocytes. To further clarify the crucial target genes that mediate transformation growth factor-β （TGF-β）/Smad3 signals on articular chondrocytes differentiation and investigate the underlying molecular mechanism of osteoarthritis, microarrays were used to perform comparative transcriptional profiling in the articular cartilage between Smad3^ex8/ex8and wild-type mice on day five after birth. The gene profding results showed that the activity of bone morphogenetic protein （BMP） and TGF-β/cell division cycle 42 （Cdc42） signaling pathways were enhanced in Smad3^ex8/ex8 chondrocytes. Moreover, there was altered gene expression in growth hormone/insulin-like growth factor 1 （Igfl） axis and fibroblast growth factor （Fgf） signaling pathway. Notably, protein synthesis related genes and electron transport chain related genes were upregulated in Smad3^ex8/ex8 chondrocytes, implying that accelerated protein synthesis and enhanced cellular respiration might contribute to hypertrophic differentiation of articular chondrocytes and the pathogenesis of osteoarthritis.

Increase of TNFα-stimulated Osteoarthritic Chondrocytes Apoptosis and Decrease of Matrix Metalloproteinases 9 by NF-κB Inhibition

Objective To investigate the in vitro effect of caffeic acid phenethyl ester （CAPE）, a NF-KB inhibitor, on the apoptosis of osteoarthritic （OA） chondrocytes and on the regulation of the gelatinases matrix metalloproteinase 2 （MMP-2） and matrix metalloproteinase 9 （MMP-9）. Methods Annexin V-FITC/propidium iodide （PI） labeling and western blotting were used to observe and determine the apoptosis in TNFa-stimulated primary cultured osteoarthritic chondrocytes. Also, gelatin zymography was applied to examine MMP-2 and MMP-9 activities in supernatants. Results it was confirmed by both flow cytometry and western blotting that chondrocytes from OA patients have an apoptotic background. Use of CAPE in combination with 10 ng/mL of TNFa for 24 h facilitated the apoptosis. MMP-9 in the supernatant could be autoactivated （from proMMP-9 to active MMP-9）, and the physiologic calcium concentration （2.5 mmol/L） could delay the autoactivation of MMP-9. The activities of MMP-2 and MMP-9 in the fresh supernatant increased significantly in response to stimulation by 10 ng/mL of TNFa for 24 h. The stimulatory effect of TNFa just on proMMP-9 was counteracted significantly by CAPE. Conclusion NF-KB could prevent chondrocytes apoptosis though its activation was attributed to the increase of proMMP-9 activity induced by TNFa （a pro-apoptotic factor）. Therefore, therapeutic NF-KB inhibitor was a ＇double-edged swords＇ to the apoptosis of chondrocytes and the secretion of MMP-9.

Expression of miRNA-140 in Chondrocytes and Synovial Fluid of Knee Joints in Patients with Osteoarthritis

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.

Sustained Akt signaling in articular chondrocytes causes osteoarthritis via oxidative stress-induced senescence in mice

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.

The effects of interleukin-1β in modulating osteoclast-conditioned medium's influence on gelatinases in chondrocytes through mitogen-activated protein kinases

Osteoarthritis is recognised to be an interactive pathological process involving the cartilage, subchondral bone and synovium. The signals from the synovium play an important role in cartilage metabolism, but little is known regarding the influence of the signalling from bone. Additionally, the collagenases and stromelysin-1 are involved in cartilage catabolism through mitogen-activated protein kinase （MAPK） signalling, but the role of the gelatinases has not been elucidated. Here, we studied the influence of osteoclastic signals on chondrocytes by characterising the expression of interleukin-1β （IL-1β）-induced gelatinases through MAPK signalling. We found that osteoclast-conditioned media attenuated the gelatinase activity in chondrocytes. However, IL-1β induced increased levels of gelatinase activity in the conditioned media group relative to the mono-cultured chondrocyte group. More specifically, IL-1β restored high levels of gelatinase activity in c-Jun N-terminal kinase inhibitor-pretreated chondrocytes in the conditioned media group and led to lower levels of gelatinase activity in extracellular signal-regulated kinase or p38 inhibitor-pretreated chondrocytes. Gene expression generally correlated with protein expression. Taken together, these results show for the first time that signals from osteoclasts can influence gelatinase activity in chondrocytes. Furthermore, these data show that IL-11~ restores gelatinase activity through MAPK inhibitors; this information can help to increase the understanding of the gelatinase modulation in articular cartilage.

Gene expression profile of hypertrophic chondrocytes treated with H2O2:A Preliminary investigation

Objective To identify the osteogenesis genes whose expression is altered in hypertrophic chondrocytes treated with H2 O2.Methods Murine chondrogenitor cells(ATDC5) were differentiated into hypertrophic chondrocytes by InsulinTransferrin-Selenium(ITS) treatment, and then treated with H2 O2. Suitable conditions(concentration, time) were determined by using the MTT assay. After total RNA isolation and cD NA 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 factor β receptor 1, transforming growth factor β receptor 3, and matrix metalloproteinase 10). Conclusions H2 O2 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.

T-2 toxin-induced apoptosis involving Fas,p53,Bcl-xL,Bcl-2,Bax and caspase-3 signaling pathways in human chondrocytes

Objective:To investigate the effects of T-2 toxin on expressions of Fas,p53,Bcl-xL,Bcl-2,Bax and caspase-3 on human chondrocytes.Methods:Human chondrocytes were treated with T-2 toxin(1～20 ng/ml)for 5 d.Fas,p53 and other apoptosis-related proteins such as Bax,Bcl-2,Bcl-xL,caspase-3 were determined by Western blot analysis and their mRNA expressions were determined by reverse transcriptase-polymerase chain reaction(RT-PCR).Results:Increases in Fas,p53 and the pro-apoptotic factor Bax protein and mRNA expressions and a decrease of the anti-apoptotic factor Bcl-xL were observed in a dose-dependent manner after exposures to 1～20 ng/ml T-2 toxin,while the expression of the anti-apoptotic factor Bcl-2 was unchanged.Meanwhile,T-2 toxin could also up-regulate the expressions of both pro-caspase-3 and caspase-3 in a dose-dependent manner.Conclusion:These data suggest a possible underlying molecular mechanism for T-2 toxin to induce the apoptosis sig- naling pathway in human chondrocytes by regulation of apoptosis-related proteins.

In vitro effects of sodium hyaluronate on the proliferation and the apoptosis in chondrocytes from patients with Kashin-Beck disease and osteoarthritis

Objective：To identify the in vitro effects of sodium hyaluronate（HA） on the proliferation and the apoptosis of chondrocytes from patients with Kashin-Beck disease（KBD） and osteoarthritis（OA）. Methods：Samples of articular cartilages from KBD and OA patients, as well as healthy volunteers（6 subjects in each of the 3 groups） were dissected, digested with collagenase and the cells cultured in monolayers. Chondrocytes from each sample were assigned to an untreated group and two HA-treated groups： H0（no HA）, H100（HA, 0.1 g/L） and H500（HA, 0.5 g/L）. The first passage chondrocytes were used to observe proliferation using the MTT assay, and apoptosis by flow cytometry through Annexin V/PI staining. Results：HA promoted proliferation of chondrocytes in all the three groups, and.in KBD and OA groups, for cells cultured for 4 and 6 days, H500 significantly promoted the cell proliferation. The apoptotic rates of both KBD and OA group chondrocytes were in the order H500 〈 HA100 〈 H0. Conclusion：Sodium hyaluronate administration has a dosedependent in vitro effect to promote proliferation and inhibit apoptosis of chondrocytes from patients with KBD and OA.

Effect of Lentivirus-mediated uPA Silencing on the Proliferation and Apoptosis of Chondrocytes and the Expression of MMPs

The lentivirus-mediated u PA interference in the proliferation, apoptosis, and secretion of osteoarthritic chondrocytes was examined in this study. Cells were obtained from the cartilage tissues of New Zealand white rabbits. They were cultured with interleukin（IL）-1β（10 ng/m L） for 24 h and then divided into three groups： u PA-si RNA group（cells transfected with u PA-si RNA lentiviruses）, blank control group（untreated cells）, and negative control group（cells transfected with empty vectors）. Western blotting and real-time quantitative reverse transcription-PCR（RT-QPCR） were performed to detect the protein and m RNA expression levels of u PA, MMP-1, MMP-3, MMP-9, MMP-10, MMP-13 and MMP-14 in osteoarthritic chondrocytes. Cell Counting Kit-8, flow cytometry, and colony formation assay were used to examine the proliferation and apoptosis of chondrocytes. The results showed that after u PA-si RNA transfection, the protein and mR NA expression levels of uP A, MMP-1, MMP-3, MMP-9, MMP-10, MMP-13, and MMP-14 were significantly decreased（P〈0.05 for MMP-1, MMP-9, MMP-10 and MMP-14, P〈0.01 for u PA, MMP-3 and MMP-13）. Cell proliferation and colony formation rate were significantly higher and the cell apoptosis rate was significantly lower in u PA-si RNA group than in control groups（P〈0.01）. The proportion of cells in G0/G1 phase was markedly increased and that in the S phase decreased, and the cell cycle was arrested at the G1/S phase in the control group. In the u PAsi RNA group, the proportion of cells in the S phase was significantly increased, resulting in a different proportion of cells in cell cycle phase（P〈0.01）. It was suggested that the down-regulation of uP A gene could inhibit the expression of MMPs protein and cell apoptosis, increase the proliferation and colony formation of osteoarthritic chondrocytes.

Inhibitory Effects of SRT1720 on the Apoptosis of Rabbit Chondrocytes by Activating SIRT1 via p53/bax and NF-κB/PGC-1αPathways

SRT1720, a new discovered drug, was reported to activate silent information regulator 1（SIRT1） and inhibit the chondrocyte apoptosis. However, the underlying mechanism remains elusive. In the present study, the chondrocytes were extracted from the cartilage tissues of New Zealand white rabbits, cultured in the presence of sodium nitroprusside（SNP）（2.5 mmol/L） and divided into five groups： 1, 5, 10, and 20 μmol/L SRT1720 groups and blank control group（0 μmol/L SRT1720）. MTT assay was used to detect the chondrocyte viability and proliferation, and DAPI staining and flow cytometry to measure the chondrocyte apoptosis. The expression levels of SIRT1, p53, NF-κB/p65, Bax, and peroxisome proliferator-activated receptor gamma coactivator 1-α（PGC-1α） were detected by Western blotting and the expression levels of SIRT1, type Ⅱ collagen, and aggrecan m RNA by RT-PCR. The results showed that in the SRT1720-treated groups, the nuclei of chondrocytes were morphologically intact and had uniform chromatin. In the blank control group, nuclear rupture into debris was observed in chondrocytes. With the SRT1720 concentration increasing, the chondrocyte viability increased, the apoptosis rate decreased, the protein expression levels of SIRT1 and PGC-1α and the m RNA expression levels of type Ⅱ collagen and aggrecan increased（P〈0.05）, and the expression levels of p53, NF-κB and bax decreased（P〈0.05）. It was suggested that SRT1720 inhibits chondrocyte apoptosis by activating the expression of SIRT1 via p53/bax and NF-κB/PGC-1α pathways.

Dietary fat-associated osteoarthritic chondrocytes gain resistance to lipotoxicity through PKCK2/STAMP2/FSP27

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.