Chondrocyte Apoptosis In Osteoarthritis Research Articles

LINC02288 promotes chondrocyte apoptosis and inflammation through miR-374a-3p targeting RTN3.

Dysregulation of long non-coding RNAs (lncRNAs) is related to the occurrence of osteoarthritis (OA). In the present study, we explored the role of LINC02288 and its regulatory mechanism in OA development. GSE113825 was obtained from Gene Expression Omnibus (GEO) database and analyzed to identify the differentially expressed lncRNAs in OA. Gene enrichment analyses and Kyoto Encyclopedia of Genes and Genomes biological process analysis were performed through Metascape (http://metascape.org/gp). The interactions among LINC02288, miR-374a-3p and RTN3 were determined using RNA immunoprecipitation (RIP) assays and dual luciferase reporter assays. Chondrocyte apoptosis was examined using flow cytometry. Western blot assays were conducted to assess the pro-apoptotic and anti-apoptotic markers. We identified a total of 4,491 differentially expressed lncRNAs. We focused on LINC02288 as the top-ranked up-regulated lncRNA in OA as indicated by a significant p-value. LINC02288 was significantly up-regulated, which was further verified by a real-time polymerase chain reaction. Down-regulation of LINC02288 significantly reduced the apoptosis of OA chondrocytes induced by interleukin-1β and the production of pro-inflammatory cytokines. These effects were further verified in an OA rat model. An RIP assay and dual luciferase assay further confirmed that LINC02288 served as a sponge of miR-374a-3p. Moreover, the overexpression of RTN3 could partially reverse the effects of LINC02288 knockdown, mediating inhibitory effects on chondrocyte apoptosis and the inflammatory response. Down-regulation of LINC02288 alleviated OA development in an in vivo OA animal model. Our findings indicate that LINC02288 contributes to OA progression by targeting the miR-374a-3p/RTN3 axis, which might provide a promising molecular therapy strategy for OA.

Hydrogen sulfide inhibits endoplasmic reticulum stress through the GRP78/mTOR pathway in rat chondrocytes subjected to oxidative stress.

The activation of oxidative stress is a primary cause of chondrocyte apoptosis in osteoarthritis (OA). The 78-kDa glucose-regulated protein (GRP78)/mammalian target of rapamycin (mTOR) signaling pathway has been demonstrated to be linked with the endoplasmic reticulum (ER) and autophagy. Hydrogen sulfide (H2S) has been reported to exert antioxidant effects. The present study investigated oxidative stress levels via 2′,7′-dichlorofluorescin diacetate and MitoSOX staining, apoptosis rates via flow cytometry and the expression levels of ER stress-related proteins in GYY4137 (donor of H2S)-treated chondrocytes (CHs). CHs were isolated from the bilateral hip joints of male rats to examine mitochondrial permeability transition pore opening- and mTOR signaling pathway-related proteins. The results demonstrated that tert-Butyl hydroperoxide (TBHP) increased CH apoptosis, and treatment with GYY4137 ameliorated TBHP-mediated the generation of ROS and CH apoptosis. Moreover, TBHP-treated CHs displayed elevated ER stress sensor expression levels and apoptotic rates; however, the TBHP-induced protein expression levels were decreased following GYY4137 treatment. In the present study, treatment with either GYY4137 or transfection with GRP78 siRNA both suppressed the activation of p-P70S6k and p-mTOR. H2S played an important role in regulating ER stress in TBHP-stimulated CHs. GYY4137 promoted autophagy, which was accompanied by the inhibition of ER stress. On the whole, the present study demonstrates that TBHP-induced oxidative stress stimulates ER interactions and CH apoptosis, which are suppressed by exogenous H2S via modulating the GRP78/mTOR signaling pathway.

Polysaccharide from Angelica sinensis attenuates SNP-induced apoptosis in osteoarthritis chondrocytes by inducing autophagy via the ERK1/2 pathway

ObjectiveChondrocyte apoptosis plays a vital role in osteoarthritis (OA) progression. Angelica sinensis polysaccharide (ASP), a traditional Chinese medicine, possesses anti-inflammatory and anti-apoptotic properties in chondrocytes. This study aimed to determine the protective role of ASP on sodium nitroprusside (SNP)-induced chondrocyte apoptosis, and explore the underlying mechanism.MethodHuman primary chondrocytes isolated from the articular cartilage of OA patients were treated with SNP alone or in combination with different doses of ASP. Cell viability and apoptosis were assessed, and apoptosis-related proteins including Bcl-2 and Bax were detected. Autophagy levels were evaluated by light chain 3 (LC3) II immunofluorescence staining, mRFP-GFP-LC3 fluorescence localization, and western blot (LC3II, p62, Beclin-1, Atg5). Meanwhile, activation of the ERK 1/2 pathway was determined by western blot. The autophagy inhibitors, 3-methyladenine (3-MA), chloroquine (CQ), and a specific inhibitor of ERK1/2, SCH772984, were used to confirm the autophagic effect of ASP.ResultsThe results showed that SNP-induced chondrocyte apoptosis was significantly rescued by ASP, whereas ASP alone promoted chondrocyte proliferation. The anti-apoptotic effect of ASP was related to the enhanced autophagy and depended on the activation of the ERK1/2 pathway.ConclusionASP markedly rescued SNP-induced apoptosis by activating ERK1/2-dependent autophagy in chondrocytes, and it made ASP as a potential therapeutic supplementation for OA treatment.

LncRNA HOTTIP leads to osteoarthritis progression via regulating miR-663a/ Fyn-related kinase axis

BackgroundLong non-coding RNA (lncRNA) has been implicated in the progression of osteoarthritis (OA). This study was aimed to explore the role and molecular mechanism of lncRNA HOXA terminal transcriptional RNA (HOTTIP) in the development of OA.MethodsThe expression of HOTTIP, miR-663a and Fyn-related kinase (FRK) in the OA articular cartilage and OA chondrocyte model induced by IL-1β was determined by qRT-PCR. CCK-8, colony formation and flow cytometry were used to determine the cell proliferation and apoptosis of OA chondrocytes. The specific molecular mechanism of HOTTIP in OA chondrocytes was determined by dual luciferase reporter assay, qRT-PCR, western blotting and RNA pull-down.ResultsThe expression of HOTTIP and FRK were up-regulated, while miR-663a was down-regulated in OA cartilage tissues. Knockdown of HOTTIP decreased the proliferation and induced the apoptosis of OA cartilage model cells, while overexpression of HOTTIP increased the proliferation and reduced the apoptosis of OA cartilage model cells. Moreover, HOTTIP could bind to miR-663a as competitive endogenous RNA. Inhibition of miR-663a expression could alleviate the effect of HOTTIP knockdown on the proliferation and apoptosis of OA cartilage model cells. Furthermore, FRK was found to be a direct target of miR-663a, which could markedly down-regulate the expression of FRK in OA chondrocytes, while HOTTIP could remarkably up-regulate the expression of FRK. In addition, miR-663a inhibition increased the proliferation and reduced the apoptosis of OA cells, while FRK knockdown reversed the effect of miR-663a inhibition on the proliferation and apoptosis of OA cells. Meanwhile, overexpression of miR-663a decreased the proliferation and induced the apoptosis of OA cells, while overexpression of FRK reversed the effect of miR-663a overexpression on the proliferation and apoptosis of OA cells.ConclusionHOTTIP was involved in the proliferation and apoptosis of OA chondrocytes via miR-663a/ FRK axis, and HOTTIP/miR-663a/FRK might be a potential target for the treatment of OA.

MicroRNA-130b downregulation potentiates chondrogenic differentiation of bone marrow mesenchymal stem cells by targeting SOX9.

Osteoarthritis (OA) is a chronic health condition. MicroRNAs (miRs) are critical in chondrocyte apoptosis in OA. We aimed to investigate the mechanism of miR-130b in OA progression. Bone marrow mesenchymal stem cells (BMSCs) and chondrocytes were first extracted. Chondrogenic differentiation of BMSCs was carried out and verified. Chondrocytes were stimulated with interleukin (IL)-1β to imitate OA condition in vitro. The effect of miR-130b on the viability, inflammation, apoptosis, and extracellular matrix of OA chondrocytes was studied. The target gene of miR-130b was predicted and verified. Rescue experiments were performed to further study the underlying downstream mechanism of miR-130b in OA. miR-130b first increased and drastically reduced during chondrogenic differentiation of BMSCs and in OA chondrocytes, respectively, while IL-1β stimulation resulted in increased miR-130b expression in chondrocytes. miR-130b inhibitor promoted chondrogenic differentiation of BMSCs and chondrocyte growth and inhibited the levels of inflammatory factors. miR-130b targeted SOX9. Overexpression of SOX9 facilitated BMSC chondrogenic differentiation and chondrocyte growth, while siRNA-SOX9 contributed to the opposite trends. Silencing of SOX9 significantly attenuated the pro-chondrogenic effects of miR-130b inhibitor on BMSCs. Overall, miR-130b inhibitor induced chondrogenic differentiation of BMSCs and chondrocyte growth by targeting SOX9.

Circular RNA circANKRD36 regulates Casz1 by targeting miR-599 to prevent osteoarthritis chondrocyte apoptosis and inflammation.

Osteoarthritis (OA) is an ageing‐related disease characterized by articular cartilage degradation and joint inflammation. circRNA has been known to involve in the regulation of multiple inflammatory diseases including OA. However, the mechanism underlying how circRNA regulates OA remains to be elucidated. Here, we report circANKRD36 prevents OA chondrocyte apoptosis and inflammation by targeting miR‐599, which specifically degrades Casz1. We performed circRNA sequencing in normal and OA tissues and found the expression of circANKRD36 is decreased in OA tissues. circANKRD36 is also reduced in IL‐1β–treated human chondrocytes. FACS analysis and Western blot showed that the knockdown of circANKRD36 promotes the apoptosis and inflammation of chondrocytes in IL‐1β stress. We then found miR‐599 to be the target of circANKRD36 and correlate well with circANKRD36 both in vitro and in vivo. By database analysis and luciferase assay, Casz1 was found to be the direct target of miR‐599. Casz1 helps to prevent apoptosis and inflammation of chondrocytes in response to IL‐1β. In conclusion, our results proved circANKRD36 sponge miR‐599 to up‐regulate the expression of Casz1 and thus prevent apoptosis and inflammation in OA.

Corrigendum] Panaxnotoginseng saponins prevent senescence and inhibit apoptosis by regulating the PI3K‑AKT‑mTOR pathway in osteoarthritic chondrocytes.

Following the publication of the above article, the authors have realized that the first author, who analyzed the data, inadvertently made a mistake by over‑estimating the background of the blots in calculating the intensity of the histograms for the western blots shown in Fig.6A andE. This error led to subsequent differences in the actual quantification and statistics from the originally submitted results. Accordingly, to further verify the conclusions reported in the study, the authors repeated these experiments independently, and the results were found to be consistent with the same changes in trends as originally observed. The revised version of Fig.6, containing the new western blot data for Fig.6A and E and new histograms showing the quantification of these data, is shown on the next page. The authors are grateful to the Editor of International Journal of Molecular Medicine for allowing them the opportunity to publish this Corrigendum, and apologize to the readership for any inconvenience caused. [the original article was published in International Journal of Molecular Medicine 45: 1225-1236, 2020; DOI: 10.3892/ijmm.2020.4491].

Oxidative Stress Induces Chondrocyte Apoptosis through Caspase-Dependent and Caspase-Independent Mitochondrial Pathways and the Antioxidant Mechanism of Angelica Sinensis Polysaccharide

Introduction Chondrocyte apoptosis is considered one of the pathogenic factors of osteoarthritis (OA), but its importance in the pathogenesis of OA remains unclear. Recent research adds progress to the knowledge that the mitochondrial signaling pathway mediates chondrocyte apoptosis in OA. Method Rat chondrocyte exposed to H2O2 was used as the experimental oxidative stress model. Chondrocyte viability was tested by cell counting kit-8 (CCK-8) assay. Cell apoptosis and ROS were tested by flow cytometry. Contents of malondialdehyde (MDA), catalase (CAT), caspase-3, caspase-9, cytochrome C, superoxide dismutase (SOD)-2, and adenosine triphosphate (ATP) were evaluated by biochemical detection. The expressions of related genes and proteins were assessed by quantitative polymerase chain reaction (qPCR) and western blot. Results H2O2 provokes oxidative stress and decreases the viability of chondrocyte, which leads to the release of cytochrome C and inhibition of SOD-2 activity. The damage of mitochondrion disturbs the energy metabolism of chondrocyte and eventually induces chondrocyte apoptosis through the mitochondrial pathway. Furthermore, pretreated with anglicasinensis polysaccharide (ASP) or caspase inhibitors increase the expression of Bcl-2 and Bcl-xL but do not work for the expression of Bax and Bad. Conclusion Oxidative stress induces chondrocyte apoptosis through caspase-dependent and caspase-independent mitochondrial pathways. ASP protects chondrocyte from H2O2-induced oxidative stress and subsequent cell injury through its antioxidant effect by inhibiting the caspase pathway.

Exosomes derived from miR-155-5p-overexpressing synovial mesenchymal stem cells prevent osteoarthritis via enhancing proliferation and migration, attenuating apoptosis, and modulating extracellular matrix secretion in chondrocytes.

Synovial mesenchymal stem cells (SMSCs) have the potential to attenuate osteoarthritis (OA)-induced injury. The role and mechanism of SMSC-derived exosomes (SMSC-Exos), pivotal paracrine factors of stem cells, in OA-associated injury remain unclear. We aimed to confirm the effect of SMSC-Exos with specific modifications on OA-induced damage and to investigate the potential molecular mechanisms. Exosomes derived from miR-155-5p-overexpressing SMSCs (SMSC-155-5p-Exos) and SMSCs (SMSC-Exos) were isolated and characterized. CCK-8, Transwell, and Western blot analyses were used to detect proliferation, migration, extracellular matrix (ECM) secretion, and apoptosis of osteoarthritic chondrocytes. The therapeutic effect of exosomes in a mouse model of OA was examined using immunohistochemical staining and OARSI scores. SPSS 17.0 and GraphPad software were used for all statistical analyses in this study. The SMSC-Exos enhanced the proliferation and migration and inhibited the apoptosis of osteoarthritic chondrocytes but had no effect on ECM secretion. The miR-155-5p-overexpressing exosomes showed common characteristics of exosomes in vitro and further promoted ECM secretion by targeting Runx2. Thus, the SMSC-155-5p-Exos promoted proliferation and migration, suppressed apoptosis and enhanced ECM secretion of osteoarthritic chondrocytes, and effectively prevented OA in a mouse model. In addition, overexpression of Runx2 partially reversed the effect of the SMSC-155-5p-Exos on osteoarthritic chondrocytes. Given the insufficient effect of the SMSC-Exos on the ECM secretion of osteoarthritic chondrocytes, we modified the SMSM-Exos and demonstrated that the SMSC-155-5p-Exos could prevent OA. Exosomes derived from modified SMSCs may be a new treatment strategy to prevent OA. Graphical abstract.

OPN Deficiency Increases the Severity of Osteoarthritis Associated with Aberrant Chondrocyte Senescence and Apoptosis and Upregulates the Expression of Osteoarthritis-Associated Genes.

Objectives A recent work has reported that the elevated osteopontin (OPN) levels in the articular cartilage and synovial fluid are correlated with the progressive osteoarthritis (OA) joint damage, and OPN has a protective effect against OA by suppressing the expressions of OA-associated genes. The present study examined whether the OPN deficiency was susceptible to OA through the regulation of chondrocyte senescence and apoptosis and the expressions of OA-associated genes. Methods The mRNA levels of COL2A1 and OPN were compared between human OA chondrocytes and normal chondrocytes. The effects of OPN siRNA on the SA-β-Gal expressions and the percentage of apoptotic chondrocytes were examined by using SA-β-Gal staining and apoptosis assay, and the effects on the expressions of COL2A1 and OA-associated genes (COL10A1, IL-1β, TNF-ɑ, MMP-13, and ADAMTS5) were examined by western blot analysis and quantitative real-time RT-PCR. Furthermore, an in vivo OA model was established to examine the effects of OPN siRNA on the senescence and apoptosis of OA chondrocytes and the expressions of OA-associated genes. Results The mRNA levels of COL2A1 and OPN were decreased in knee OA chondrocytes in comparison with those in normal chondrocytes. The OPN deficiency enhanced the senescence and apoptosis of OA chondrocytes and increased the expressions of COL10A1, IL-1β, TNF-ɑ, MMP-13, and ADAMTS5 but decreased the expression of COL2A1. Meanwhile, OPN deficiency could result in severe, accelerated OA in vivo, which was also associated with enhanced senescence and apoptosis of chondrocytes and elevated expressions of OA-associated genes. Conclusions The findings of this study suggest that the OPN deficiency can result in accelerated OA, which is associated with enhanced senescence and apoptosis of OA chondrocytes and the upregulated expressions of OA-associated genes.

LncRNA RMRP knockdown promotes proliferation and inhibits apoptosis in osteoarthritis chondrocytes by miR-206/CDK9 axis.

The etiology of osteoarthritis (OA) has been discussed widely, but the molecular mechanisms beneath OA aggravation have not yet been investigated in detail. This study focused on the role of lncRNA RMRP (RMRP) on OA progression. We found that the expression of RMRP was significantly increased in cartilage tissues of patients with OA. CCK-8 and colony formation assays showed that RMRP knockdown promoted proliferation of chondrocytes treated with IL-1β. Flow cytometry and caspase-3 activity analysis indicated that RMRP silence inhibited apoptosis of chondrocytes treated with IL-1β. Moreover, luciferase reporter, RNA pull-down and RIP assays showed that RMRP competing with miR-206. Additionally, CDK9 acted as a direct target of miR-206. Moreover, rescue assays indicated that miR-206 inhibitor or pcDNA-CDK9 reversed the effects of RMRP suppression on the proliferation and apoptosis of chondrocytes. Taken together, our results indicated that RMRP knockdown could promote proliferation and inhibit apoptosis in OA chondrocytes via the miR-206/CDK9 axis.

Long non-coding RNA PCAT-1 regulates apoptosis of chondrocytes in osteoarthritis by sponging miR-27b-3p.

Osteoarthritis (OA) is a non-inflammatory degenerative disease, with progressive damages on the articular cartilages. In recent years, researchers have paid many efforts in the diagnostics and treatments of OA. However, no effective therapeutic method has been revealed to help inhibit the development of OA. Herein, we studied the roles and associations of PCAT-1 and miR-27-3p in the pathogenesis OA. OA articular cartilages and healthy articular cartilages were isolated for investigation. The chondrocytes were isolated from articular cartilage samples. QRT-PCR and western blotting were used for the detection of expression of genes and proteins. cell Titer 96® AQueous one proliferation kit was applied for detect cell viability of Chondrocytes transfected with negative control vector, pcDNA3.1 PCAT-1 plasmid or siRNA against PCAT-1. RNA pull-down assays and Luciferase reporter assay were used to confirm the connection. SPSS 17.0 was employed for statistical analysis. We found that the expressions of PCAT-1 were up-regulated in OA chondrocytes compared with normal chondrocytes. si-PCAT-1 suppressed apoptotic OA chondrocytes. Over-expression of PCAT-1 enhanced the apoptosis of normal chondrocytes. In addition, the online database and luciferase assay confirmed that PCAT-1 could directly target miR-27b-3p. PCAT-1 could promote the apoptosis of OA and normal chondrocytes through binding with miR-27b-3p. Based on the comparisons and analysis, we could conclude that lncRNA PCAT-1 regulated the apoptosis of chondrocytes through sponging miR-27b-3p in OA. PCAT-1 has potential values to act as a new therapeutic target for OA patients.

MiR-145 targeting BNIP3 reduces apoptosis of chondrocytes in osteoarthritis through Notch signaling pathway.

The purpose of this study was to explore the effect of micro ribonucleic acid (miR)-145 on the apoptosis of chondrocytes in osteoarthritis (OA), and to research the association between its targeting on B-cell lymphoma-2 (Bcl-2)/adenovirus E1B 19 kDa interacting protein 3 (BNIP3) and Notch signaling pathway and chondrocyte apoptosis. The mouse model of OA was established via surgery, and chondrocytes were isolated and cultured in vitro. Then, the chondrocytes were transfected with miR-145 inhibitor, miR-145 mimics, miR-negative control (NC), BNIP3-siRNA and BNIP3-vector, respectively, with those normally cultured as the control. After that, the expression levels of miR-145 and BNIP3 in cells were detected via quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR), the apoptosis rate was detected via flow cytometry, and the apoptosis level was detected using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. Moreover, the target gene sequences were predicted and compared using the software, and the BNIP3 Luciferase reporter vectors containing predicted target sites for miR-145 were constructed. Finally, the protein expressions of BNIP3, Notch1, and P21 were determined through Western blotting. The results of qRT-PCR showed that in OA chondrocytes, the expression of miR-145 was lower than that in normal chondrocytes (p<0.05), while the mRNA and protein expressions of BNIP3 were higher than those in normal chondrocytes (p<0.05). According to flow cytometry, the apoptosis rate was (4.4±0.6)% in normal cartilage tissues and (29.2±2.1)% in OA cartilage tissues. Overexpression of miR-145 significantly reduced chondrocyte apoptosis (p<0.05), while overexpression of BNIP3 markedly increased chondrocyte apoptosis (p<0.05). In addition, the Luciferase reporter system showed that miR-145 mimics evidently inhibited BNIP3 (p<0.05) and suppressed the Notch signaling pathway (p<0.05), while BNIP3 enhanced the expression of Notch signaling pathway (p<0.05). MiR-145 can reduce OA-induced chondrocyte apoptosis through targeted inhibition on BNIP3 and regulation on Notch signaling pathway.

Low expression of CircRNA HIPK3 promotes osteoarthritis chondrocyte apoptosis by serving as a sponge of miR-124 to regulate SOX8.

CircRNA, a type of circular RNA, has recently been shown to be a potential target for osteoarthritis (OA). Circular RNA HIPK3 (CircHIPK3) is reported to be abnormally expressed in various disease tissues and affects the occurrence and development of the disease. However, the role and underlying mechanism of CircRNA HIPK3 in osteoarthritis are still unclear. The purpose of this study is to explore the effect of CircRNA HIPK3 on osteoarthritis and analyze its regulatory mechanism. We took human OA tissues, normal knee cartilage, human OA chondrocytes and normal chondrocytes as the research objects. Real Time-quantitative Polymerase Chain Reaction (RT-qPCR) was used to detect CircHIPK3 expression level, its target gene miRNA 124 (miR-124) and downstream target molecule SOX8. Flow cytometry analysis was applied to discover the apoptosis of CircHIPK3 and miR-124 on OA cartilage in different transfection situations. Moreover, Western blot and RT-qPCR were used to detect the expression of caspase-3 in OA chondrocytes. The binding site of CircRNA HIPK3 and miR-124, miR-124, and SOX8 were verified by using Dual-Luciferase assay. High expressed CircHIPK3 and low expressed miR-124 were found in OA tissues and OA chondrocytes. In addition, the Dual-Luciferase report showed CircHIPK3 acted as a sponge of miR-124 in OA chondrocytes. CircHIPK3 and miR-124 expression in OA tissue were confirmed to be negatively correlated. To our surprise, knocking down CircHIPK3 and transfected miR-124 mimics both inhibited the apoptosis of OA chondrocytes. Further experiments verified that the downstream target molecule of miR-124 was SOX8 in OA chondrocytes. Besides, miR-124 inhibitors reversed the knockdown of CircHIPK3 while si-SOX8 reversed the miR-124 inhibitors effect of apoptosis on OA chondrocytes. Our results demonstrated that CircHIPK3 was significantly upregulated in OA cartilage tissue and cells. Low expression of CircHIPK3 promoted the apoptosis of OA chondrocytes by promoting miR-124 to suppress SOX8 expression. The molecular mechanism of CircHIPK3 in present study is expected to provide new ideas for the treatment of osteoarthritis.

Ubiquitin conjugating enzyme E2 M promotes apoptosis in osteoarthritis chondrocytes via Wnt/β-catenin signaling

In this study, the role of ubiquitin conjugating enzyme E2 M (UBE2M) and molecular mechanisms associated with osteoarthritis (OA) were explored. Cartilage tissues and corresponding healthy tissues from OA patients were isolated. Our data suggested that the expression level of UBE2M in OA patients was significantly higher compared to that in healthy individuals (P < 0.01). The apoptosis of human OA chondrocytes was inhibited when silencing UBE2M and increased when overexpressing UBE2M. XAV939, as a tankyrase 1 inhibitor, could block the signaling pathway of Wnt/β-catenin, which significantly reversed the change introduced by UBE2M. The expression level of cytoplasmic β-catenin in siUBE2M cells dramatically increased, and the expression levels of nuclear β-catenin, cleaved caspase-3 (C-caspase-3), and MMP13 remarkably downregulated. Moreover, the ubiquitination of Axin was enhanced by the overexpression of UBE2M. The expression level of Axin significantly decreased in OA chondrocytes with UBE2M overexpression and increased after MG132 treatment. Moreover, UBE2M enhanced the apoptosis of OA chondrocytes by activating the Axin-dependent Wnt/β-catenin pathway. In this process, UBE2M downregulated Axin in an ubiquitination-dependent degradation pathway and subsequently activated Wnt/β-catenin signaling.

Progranulin Protects Chondrocytes Induced by Lipopolysaccharide via Regulating the Focal Adhesion Kinase (FAK)/Mitogen-Activated Protein Kinase (MAPK) Pathway

Osteoarthritis (OA) is frequently associated with loss of articular cartilage and is more common in elderly patients. Progranulin (PGRN) is a chondrogenic factor. However, the role of PGRN in inflammatory articular chondrocyte arthritis and related mechanisms has not been elucidated. In vitro cultured chondrocytes were divided into control group, LPS group that was treated with 1 μg/ml lipopolysaccharide (LPS), and PGRN group, in which LPS-stimulated chondrocytes were treated with PGRN (5 μM and 10 μM). The survival rate of chondrocytes was detected by tetrazolium salt colorimetry (MTT method). MMP-3, tissue metalloproteinase inhibitor 1 (TIMP-1), and MMP-3/TIMP-1 ratio were assessed by Western blot. The expressions of FAK and MAPK were detected by Western blot. TNF-α and IL-β secretion was evaluated by ELISA. Chondrocyte survival rate was decreased, Caspase 3 activity increased, MMP-3 expression upregulated, TIMP-1 expression reduced, MMP-3/TIMP-1 ratio elevated, FAK and MAPK expressions downregulated, and TNF-α and IL-1β secretions enhanced in LPS group (P &lt; 0.05). PGRN significantly promoted the survival of LPS-treated chondrocytes, attenuated Caspase 3 activity, decreased MMP-3 level, enhanced TIMP-1 level, decreased MMP-3/TIMP-1 ratio, upregulated FAK and MAPK, and inhibited TNF-α and IL-1β secretions dose dependently (P &lt; 0.05). PGRN can reduce the apoptosis of osteoarthritic chondrocytes, promote cell proliferation, reduce secretion of inflammatory factors, and delay the progression of osteoarthritis possibly through regulating FAK/MAPK pathway.

LINC00623/miR-101/HRAS axis modulates IL-1β-mediated ECM degradation, apoptosis and senescence of osteoarthritis chondrocytes.

Chondrocyte apoptosis and extracellular matrix (ECM) degeneration have been implicated in the pathogenesis of osteoarthritis (OA). Based on previously reported microarray analysis, HRAS (Harvey rat sarcoma viral oncogene homolog), a member of the RAS protein family, was chosen as a potential regulator of OA chondrocyte apoptosis and ECM degradation. HRAS expression was downregulated in OA tissues, particularly in mild-OA tissues. HRAS overexpression partially attenuated IL-1β-induced OA chondrocyte apoptosis and ECM degradation. Similar to HRAS, the long non-coding RNA LINC00623 was downregulated in OA tissues. LINC00623 knockdown enhanced IL-1β-induced OA chondrocyte apoptosis and ECM degradation, which could be partially reversed by HRAS overexpression. It has been reported that lncRNAs act as ceRNAs of miRNAs to exert their function. Herein, miR-101 was predicted to bind to both LINC00623 and HRAS, which was further confirmed by luciferase reporter and RIP assays. LINC00623 competed with HRAS for miR-101 binding, therefore reducing the inhibitory effect of miR-101 on HRAS expression. More importantly, the effect of LINC00623 was partially eliminated by miR-101 inhibition. Overall, the LINC00623/miR-101/HRAS axis modulates OA chondrocyte apoptosis, senescence and ECM degradation through MAPK signaling, which might play a critical role in OA development.

Panax notoginseng saponins prevent senescence and inhibit apoptosis by regulating the PI3K‑AKT‑mTOR pathway in osteoarthritic chondrocytes.

Panaxnotoginseng saponins (PNS) are active extracts obtained from the P.notoginseng plant. PNS exhibit various anti‑inflammatory, anti‑oxidant and anti‑aging pharmacological properties in some cells. However, the effects of PNS on senescence and apoptosis in chondrocytes have not been studied to date. In the present study, whether PNS could limit tumor necrosis factor (TNF)‑α‑induced senescence and apoptosis in chondrocytes and whether they could slow down cartilage degeneration in a surgery‑induced rat osteoarthritis(OA) model by regulating the phosphatidyl inositol3 kinase(PI3K)‑protein kinaseB (AKT)‑mammalian target of rapamycin(mTOR) signaling pathway was examined. A potential mechanism underlying these effects was further elucidated. The present invitro experiments showed that PNS significantly inhibited senescence and apoptosis in OA chondrocytes and prevented a decrease in the mitochondrial membrane potential and excessive mitochondrial permeability. In addition, the expression levels of autophagy‑related proteins and the anti‑apoptotic protein Bcl‑2 were significantly increased in PNS‑treated OA chondrocytes, but the expression levels of Bax and caspase‑3 were decreased; these effects were concentration‑dependent. TNF‑α significantly increased the expression of p‑PI3K/p‑AKT/p‑mTOR in OA chondrocytes, whereas PNS reduced PI3K, AKT and mTOR phosphorylation. The results of the invivo experiments demonstrated that PNS significantly inhibited the PI3K‑AKT‑mTOR signaling pathway and collagenII degradation, as well as reduced matrix metalloproteinase (MMP)‑3 and MMP‑13 expression in chondrocytes in a rat OA model, thus attenuating cartilage destruction in OA. The results obtained in the rat model were consistent with the invitro experimental results. Furthermore, histological analyses and ultrastructural observations confirmed these results. Taken together, the results of the present study demonstrated that PNS may protect osteoarthritic chondrocytes from senescence and apoptosis by inhibiting the PI3K‑AKT pathway, thus delaying the degradation of articular cartilage.

Identification of microRNA‑363‑3p as an essential regulator of chondrocyte apoptosis in osteoarthritis by targeting NRF1 through the p53‑signaling pathway.

Osteoarthritis (OA) is a degenerative joint disease that affects the physical, and mental health of middle-aged and elderly people. The aims of the present study were to determine the biological function and molecular mechanisms of miR-363-3p in chondrocyte apoptosis. Exploration of the molecular mechanisms of OA may be helpful in the understand of the causes, and facilitating the prevention and treatment of OA. In the present study, the expression of nuclear respiratory factor1 (NRF1) was downregulated in the articular cartilage of OA rats in vivo and lipopolysaccharide (LPS)-treated chondrocytes in vitro. MicroRNAs (miRNA) are regulators of gene expression in the progression of OA. TargetScan software was used to predict that NRF1 was a potential target for miRNA (miR)-363, and this was confirmed in subsequent experiments. The expression of miR-363-3p was negatively correlated with the expression of NRF1, and its expression was significantly upregulated in OA model rats and in LPS-induced chondrocytes compared with the expression in the respective controls. In addition, the overexpression of miR-363-3p increased the levels of interleukin (IL)-1β, IL-6 and tumor necrosis factor-α in vivo, and was demonstrated to promote chondrocyte injury and apoptosis by Safranin O staining and TUNEL. Moreover, the inhibition of miR-363-3p expression increased the expression of NRF1 and protected chondrocytes from apoptosis in vitro and in vivo, whereas the overexpression of miR-363-3p downregulated NRF1 expression and promoted LPS-induced chondrocyte apoptosis through the p53 pathway in vitro. The results of this study suggested that miR-363-3p-mediated inhibition of NRF1may be associated with chondrocyte apoptosis in OA.

MiR93-5p inhibits chondrocyte apoptosis in osteoarthritis by targeting lncRNA CASC2

BackgroundIt has been reported that miR-93-5p and long non-coding RNA (lncRNA) Cancer Susceptibility 2 (CASC2) play opposite roles in regulating chondrocyte apoptosis, indicating the possible interaction between them. This study aimed to investigate the interaction between miR-93-5p and lncRNA CASC2 in chondrocyte apoptosis, which plays critical roles in osteoarthritis (OA).MethodsThe interaction between CASC2 and miR-93-5p was analyzed by dual luciferase assay and overexpression experiments. Levels of CASC2 and miR-93-5p in plasma sample from OA patients and healthy controls were measured by RT-qPCR. The roles of CASC2 and miR-93-5p in regulating the apoptosis of chondrocyte induced by LPS were analyzed by cell apoptosis assay.ResultsThrough bioinformatics analysis we observed the potential interaction between CASC2 and miR-93-5p, which was confirmed by dual luciferase assay. In OA patients, miR-93-5p was downregulated, while CASC2 was upregulated, and they were inversely correlated. LPS treatment led to downregulated miR-93-5p and upregulated CASC2. Overexpression of miR-93-5p led to the downregulated CASC2 in chondrocytes. Under LPS treatment, CASC2 overexpression promoted the apoptosis of chondrocyte. MiR-93-5p overexpression played an opposite role and attenuated the effects of CASC2 overexpression.ConclusionMiR-93-5p was downregulated in OA may inhibit LPS-induced chondrocyte apoptosis by targeting lncRNA CASC2.