Apoptosis In Human Chondrocytes Research Articles

Mathematical modeling and application of IL-1β/TNF signaling pathway in regulating chondrocyte apoptosis.

Introduction: Mathematical model can be used to model complex biological processes, and have shown potential in describing apoptosis in chondrocytes. Method: In order to investigate the regulatory mechanisms of TNF signaling pathway in regulating chondrocyte apoptosis, a fractional-order differential equation model is proposed to describe the dynamic behavior and mutual interaction of apoptosis-related genes under the activation of TNF signaling pathway. Compared with the traditional molecular biology techniques, the proposed mathematical modeling has advantages to providing a more comprehensive understanding of the regulatory mechanisms of TNF signaling pathway in chondrocyte apoptosis. Result: In this paper, differentially expressed genes induced by IL-1β in human chondrocyte apoptosis are screened using high-throughput sequencing. It is found that they were significantly enriched in the TNF signaling pathway. Therefore, a mathematical model of the TNF signaling pathway is built. Using real-time PCR experiments, mRNA data is measured and used to identify the model parameters, as well as the correlation coefficient. Finally, the sensitivity of the model parameters is discussed by using numerical simulation methods, which can be used to predict the effects of different interventions and explore the optimal intervention strategies for regulating chondrocyte apoptosis. Discussion: Therefore, fractional-order differential equation modeling plays an important role in understanding the regulatory mechanisms of TNF signaling pathway in chondrocyte apoptosis and its potential clinical applications.

Daurisoline attenuates H2O2-induced chondrocyte autophagy by activating the PI3K/Akt/mTOR signaling pathway

BackgroundOsteoarthritis (OA) is a chronic degenerative joint disease characterized by cartilage degeneration and intra-articular inflammation. Daurisoline (DAS) is an isoquinoline alkaloid isolated from Rhizoma Menispermi, whose antitumor and anti-inflammatory pharmacological effects have been demonstrated, but the effects of DAS on OA have rarely been researched. In this study, we aimed to explore the potential role of DAS in OA and its partial mechanism.Materials and methodsThe cytotoxicity of H2O2 and DAS toward chondrocytes was detected by the Cell Counting Kit-8 assay. Safranin O staining was used to detect chondrocyte phenotype changes. Cell apoptosis was measured by both flow cytometry and quantitative analysis of the protein levels of the apoptosis-related factors Bax, Bcl-2 and cleaved caspase-3 by western blot. Western blotting and immunofluorescence were used to assess the expression of the autophagy-related proteins LC3, Beclin-1 and p62. In addition, key signal pathway targets and matrix-degrading indicators were measured by western blot.ResultsOur results indicated that H2O2 induced human chondrocyte apoptosis and activated autophagy in a dose-dependent manner. DAS treatment dose-dependently reversed the expression of apoptosis-related proteins (Bax, Bcl-2 and cleaved caspase3) and the apoptosis rate induced by H2O2. Western blot and immunofluorescence analyses showed that DAS decreased the H2O2-induced upregulation of the autophagy marker Beclin-1 and the LC3 II/LC3 I ratio and upregulated the p62 protein level. Mechanistically, DAS inhibited autophagy through the activation of the classical PI3K/AKT/mTOR signaling pathway and protected chondrocytes from apoptosis. In addition, DAS alleviated the H2O2-induced degradation of type II collagen and the high expression of matrix metalloproteinase 3 (MMP3) and MMP13.ConclusionOur research demonstrated that DAS alleviated chondrocyte autophagy caused by H2O2 through activation of the PI3K/AKT/mTOR signaling pathway and protected chondrocytes from apoptosis and matrix degradation. In conclusion, these findings suggest that DAS may serve as a promising therapeutic strategy for OA.

Depletion of circ_0128846 ameliorates interleukin-1β-induced human chondrocyte apoptosis and inflammation through the miR-940/PTPN12 pathway

BackgroundPrevious evidence has suggested that circular RNA (circRNA) is abnormally expressed in osteoarthritis (OA). However, the underlying mechanism of circRNA in OA progression remains unclear. The study aims to reveal the mechanism of circ_0128846 regulating OA. MethodsHuman chondrocytes (C28/I2 cells) were treated with interleukin-1β (IL-1β) to mimic an OA cell model. The expression levels of circ_0128846, miR-940 and protein tyrosine phosphatase 12 (PTPN12) were detected by qRT-PCR. Protein expression was checked by Western blotting. Cell viability, proliferation, and apoptosis were analyzed by a cell counting kit-8 assay, 5-Ethynyl-2′-deoxyuridine (EdU) assay and flow cytometry analysis, respectively. The production of tumor necrosis factor-α (TNF-α) and IL-6 was determined by an enzyme-linked immunosorbent assay (ELISA). The binding relationship between miR-940 and circ_0128846 or PTPN12 was identified by dual-luciferase reporter assay and RNA immunoprecipitation assay. ResultsCirc_0128846 and PTPN12 expression were significantly upregulated, whereas miR-940 was downregulated in the cartilage tissues of OA patients and IL-1β-treated C28/I2 cells compared with controls. IL-1β treatment inhibited C28/I2 cell proliferation and induced cell apoptosis and the production of inflammatory factors, TNF-α and IL-6; however, these effects were partly reversed after circ_0128846 depletion. In terms of mechanism, circ_0128846 acted as a miR-940 sponge, and miR-940 combined with PTPN12. Also, circ_0128846 depletion partly ameliorated IL-1β-induced C28/I2 cell disorders through miR-940. PTPN12 overexpression also partly relieved miR-940-mediated effects in IL-1β-treated C28/I2 cells. Further, circ_0128846 induced PTPN12 expression by interacting with miR-940. ConclusionCirc_0128846 regulated human chondrocyte proliferation, apoptosis and inflammation through the miR-940/PTPN12 pathway in OA.

N6 -methyladenosine-modified circRNA RERE modulates osteoarthritis by regulating β-catenin ubiquitination and degradation.

N6 -methyladenosine (m6A) is one of the most abundant internal RNA modifications. We investigated the role of m6A-modified circRERE in osteoarthritis (OA) and its mechanism. CircRERE and IRF2BPL were screened by microarrays. The role of m6A-modification in circRERE was examined by methylated RNA precipitation and morpholino oligo (MOs) treatment. The axis of circRERE/miR-195-5p/IRF2BPL/β-catenin was determined using flow cytometry, western blotting and immunofluorescence in human chondrocytes (HCs) and corroborated using a mouse model of destabilization of medial meniscus (DMM) with intra-articular (IA) injection of adeno-associated viruses (AAV). CircRERE was decreased in OA cartilage and chondrocytes compared with control. CircRERE downregulation was likely attributed to its increased m6A modification prone to endoribonucleolytic cleavage by YTHDF2-HRSP12-RNase P/MRP in OA chondrocytes. MOs transfection targeting HRSP12 binding motifs in circRERE partially reversed decreased circRERE expression and increased apoptosis in HCs treated with IL-1β for 6h. CircRERE exerted chondroprotective effects by targeting miR-195-5p/IRF2BPL, thus regulating the ubiquitination and degradation of β-catenin. CircRere (mouse homologue) overexpression by IA-injection of AAV-circRere into mice attenuated the severity of DMM-induced OA, whereas AAV-miR-195a-5p or AAV-sh-Irf2bpl reduced the protective effects. The detrimental effects of AAV-sh-Irf2bpl on DMM-induced OA were substantially counteracted by ICG-001, an inhibitor of β-catenin. Our study is a proof-of-concept demonstration for targeting m6A-modified circRERE and its target miR-195-5p/IRF2BPL/β-catenin as potential therapeutic strategies for OA treatment.

Upregulation of osteoprotegerin inhibits tert-butyl hydroperoxide-induced apoptosis of human chondrocytes.

Necrosis of the femoral head (NFH) is an orthopedic disease characterized by a severe lack of blood supply to the femoral head and a marked increase in intraosseous pressure. NFH is associated with numerous factors, such as alcohol consumption and hormone levels. The present study focused on the expression levels of osteoprotegerin (OPG) in NFH and the effect of OPG overexpression on chondrocyte apoptosis. The results demonstrated that OPG expression was markedly decreased in the femoral head of patients with NFH compared with normal femoral heads. Lentivirus-mediated overexpression of OPG in human chondrocytes reversed the decrease in cell viability and the increase in reactive oxygen species production induced by an oxidative stress-inducing factor, tert-butyl hydroperoxide. Flow cytometry and TUNEL assays revealed that OPG overexpression inhibited the apoptosis of chondrocytes. In addition, it was revealed that OPG exerted its anti-apoptotic effect mainly by promoting Bcl-2 expression and Akt phosphorylation and inhibiting caspase-3 cleavage and Bax expression. The present study revealed that OPG may be an important regulator of NFH.

Regulation of circADAMTS6-miR-324-5p-PIK3R3 ceRNA pathway may be a novel mechanism of IL-1β-induced osteoarthritic chondrocytes.

A disintegrin and metallopeptidase with thrombospondin type 1 motif 6 (ADAMTS6)-derived circular RNA (circADAMTS6; hsa_circ_0008667) is a novel regulator in interleukin (IL)-1β-induced apoptosis of human chondrocytes (HCs). Here, we planned to probe into its role and mechanism underlying HCs injury in osteoarthritis. Real time-quantitative PCR and immunoblotting estimated the abundance of RNA and protein, respectively. Cell proliferation and apoptosis were measured by WST-8, EdU, fluorescein isothiocyanate, and caspase3/7 activity assays. Levels of inflammatory cytokines (IL-6 and tumor necrosis factor-α), apoptosis-related proteins (Bcl-2 and Bcl-2-associated X protein), extracellular matrix (ECM)-related proteins (matrix metalloproteinase-13 and collagen type II alpha-1), and PI3K/AKT/mTOR signaling pathway-related proteins (AKT, mTOR, phosphorylated-AKT, and phosphorylated-mTOR) were evaluated by enzyme-linked immunosorbent assays and immunoblotting. Target relationship was confirmed by dual-luciferase reporter, Argonaute-2 immunoprecipitation and RNA pull-down assays. Abundances of circADAMTS6 and phosphoinositide-3-kinase regulatory subunit 3 (PIK3R3) were underexpressed, and microRNA (miR)-324-5p was elevated in human osteoarthritic tissues and IL-1β-induced HCs. Overexpressing circADAMTS6 and inhibiting miR-324-5p enhanced proliferation and ECM synthesis, but suppressed apoptosis and inflammatory response in IL-1β-challenged HCs. Besides, silencing circADAMTS6 caused similar effects of IL-1β stress on HCs. Mechanically, there was a direct interaction between miR-324-5p and circADAMTS6 or PIK3R3, and IL-1β-induced activation of PI3K/AKT/mTOR signaling pathway was suppressed by circADAMTS6 overexpression and miR-324-5p silencing. Furthermore, counteractive effects of miR-324-5p upregulation on circADAMTS6 overexpression and PIK3R3 knockdown on miR-324-5p silencing were observed. CircADAMTS6-miR-324-5p-PIK3R3 axismight participate in IL-1β-induced HCs dysfunction via competing endogenous RNA mechanism and thePI3K/AKT/mTOR signaling pathway.

The lncRNA MIAT/miR-181a-5p axis regulates osteopontin (OPN)-mediated proliferation and apoptosis of human chondrocytes in osteoarthritis.

Osteoarthritis (OA) is a slow-progressing degenerative joint disease mainly characterized by progressive cartilage loss and subchondral bone remodeling. Osteopontin (OPN) is a matrix extracellular glyco-phosphoprotein capable of regulating the expression levels of multiple factors linked with OA pathogenesis. This study explores the upstream regulatory molecular mechanism of OPN on proliferation and apoptosis of human chondrocytes in OA. Chondrocytes were isolated from OA cartilage and identified by toluidine blue staining and immunofluorescent staining of type II collagen. An MTT assay was used for cell viability, and a BrdU assay was applied for DNA synthesis. Cell apoptosis was detected by a flow cytometry assay. A lncRNA MIAT/miR-181a-5p/OPN axis regulating OA chondrocyte proliferation and apoptosis were identified. miR-181a-5p directly targeted OPN and inhibited OPN expression in OA chondrocytes. miR-181a-5p overexpression inhibited OA chondrocyte viability, suppressed DNA synthesis, and promoted apoptosis. OPN overexpression exerted opposite effects on OA chondrocytes and significantly attenuated the roles of miR-181a-5p overexpression in OA chondrocytes. A total of six long non-coding RNAs (lncRNAs) were predicted to target miR-181a-5p, and MIAT was the most up-regulated in OA cartilage tissues among the six lncRNAs. Through direct targeting, MIAT inhibited miR-181a-5p expression. MIAT silencing inhibited cell viability, suppressed DNA synthesis, and promoted cell apoptosis. Moreover, miR-181a-5p inhibition partially reversed the effects of MIAT silencing on OA chondrocytes. The lncRNA MIAT/miR-181a-5p/OPN axis could modulate OA chondrocyte proliferation and apoptosis. The comprehensive function of this axis on OA requires further in vivo and clinical investigations.

MiR9 inhibits 6-mer HA-induced cytokine production and apoptosis in human chondrocytes by reducing NF-kB activation

The present study aimed to investigate the expression of miR9 and its correlation with cytokines, proteolytic enzymes and apoptosis in an experimental model of 6-mer HA induced inflammation in human chondrocytes.Human articular chondrocytes, transfected with a miR-9 mimic and miR-9 inhibitor, were stimulated with 6-mer HA in presence/absence of a specific NF-kB inhibitor.6-mer HA induced a significant increase of TLR-4, CD44, IL-8, IL-18, MMP-9, ADAMTS-5, BAX and BCL-2 mRNAs expression and the related proteins, as well as NF-kB activation, associated with a significant up regulation of miR-9. In chondrocytes transfected with the miR-9 mimic before 6-mer HA treatment we found a decrease of such inflammatory cytokines, metalloproteases and pro-apoptotic molecules, while we found them increased in chondrocytes transfected with the miR9 inhibitor before 6-mer HA stimulation. The activities of TLR-4 and CD44, up regulated by 6-mer HA, were not modified by miR9 mimic/inhibitor, while the NF-kB activation was significantly affected.We suggested that the up regulation of miR9, induced by 6-mer HA, could be a cellular attempt to limit cell damage during inflammation.

MiR-520c-3p regulates IL-1\u03b2-stimulated human chondrocyte apoptosis and cartilage degradation by targeting GAS2

BackgroundMicroRNAs (miRNAs) have been shown to be associated with osteoarthritis (OA) progression. This study aimed to explore the role of miR-520c-3p in OA progression.MethodsExpression levels of miR-520c-3p and Growth arrest-specific 2 (GAS2) were detected using quantitative real-time PCR. The proliferation and apoptosis of cells were measured using cell counting kit 8 (CCK8) assay and flow cytometry. Furthermore, the protein levels of apoptosis-related markers, extracellular degradation markers, inflammatory response markers, and GAS2 were tested using quantitative real-time polymerase chain reaction (RT-PCR) and western blot (WB) analysis. In addition, the interaction between miR-520c-3p and GAS2 was examined using dual luciferase reporter assay.ResultsGAS2 was highly expressed, and miR-520c-3p was lowly expressed in OA cartilage tissues. miR-520c-3p could promote the proliferation and inhibit the apoptosis and inflammation of OA chondrocytes. miR-520c-3p could be sponged by GAS2, and its inhibitor could reverse the regulation of GAS2 on the biological functions of OA chondrocytes. GAS2 was a target of miR-520c-3p, which was identified by bioinformatic analysis and dual-luciferase reporter assay. Overexpression of GAS2 could inhibit the proliferation and promoted the apoptosis and inflammation of OA chondrocytes.ConclusionOur data showed that miR-520c-3p might regulate the GAS2 to inhibit the progression of OA.

Estradiol activates ERK phosphorylation by binding to ERβ to inhibit proliferation and promote apoptosis of human chondrocytes

To explore the effect of estradiol (E2) binding to its receptor ERβ on the proliferation and apoptosis of C28I2 cells. We cloned the sequence of ESR2 into a recombinant adenovirus plasmid (pAd-ESR2) and packaged the plasmid in HEK293 cells. Normal human chondrocyte C28I2 cells were transfected with Ad-ESR2 or small interfering RNA targeting ESR2-siRNA (ESR2-siRNA), and the effects of treatment with DMSO or E2 on the expression of the proteins associated with endoplasmic reticulum (ER) stress and cell apoptosis were determined using Western blotting. qRT-PCR was used to detect the expressions of proliferation-related marker genes, and an EdU kit and flow cytometry were used to assess cell proliferation and apoptosis. We also tested the effects of U0126 (an ERK pathway inhibitor) and E2, alone or in combination, on ER stress, apoptosis and the ERK signaling pathway in C28I2 cells infected with Ad-ESR2 using Western blotting. Overexpression of Ad-ESR2 in C28I2 cells significantly promoted the expressions of IRE1α, PERK, XBP1s, and cleaved caspase-12, inhibited proliferation related marker genes PCNA, cyclin B1, cyclin D1, and decreased the level of ERK phosphorylation following E2 treatment (all P < 0.05). Interference of ESR2 caused significant reduction in the expressions of ER stress-related proteins and apoptosis-related proteins, up-regulated the genes related to cell proliferation, and increased intracellular pERK/ERK ratio in C28I2 cells. The effect of E2 binding to ERβ, which promoted the expressions of ER stress associated proteins and apoptosis related proteins, was obviously antagonized by treatment of the cells with U0126. The binding of E2 to ERβ promotes ER stress and apoptosis in human chondrocytes by activating ERK pathway phosphorylation inhibit cell proliferation.

Excavating bioactivities of nanozyme to remodel microenvironment for protecting chondrocytes and delaying osteoarthritis

Osteoarthritis (OA) is the main cause of disability in the elderly. Effective intervention in the early and middle stage of osteoarthritis can greatly prevent or slow down the development of the disease, and reduce the probability of joint replacement. However, there is to date no effective intervention for early and middle-stage OA. OA microenvironment mainly destroys the balance of oxidative stress, extracellular matrix synthesis and degradation of chondrocytes under the joint action of biological and mechanical factors. Herein, hollow Prussian blue nanozymes (HPBzymes) were designed via a modified hydrothermal template-free method. The aim of this study was to investigate the effects of HPBzymes on chondrocytes and the progression of OA. The intrinsic bioactivities of HPBzymes were excavated in vitro and in vivo, remodeling microenvironment for significantly protecting chondrocytes and delaying the progression of traumatic OA by inhibiting reactive oxygen species (ROS) and Rac1/nuclear factor kappa-B (NF-κB) signaling in a rat model. HPBzyme significantly diminished interleukin (IL)-1β-stimulated inflammation, extracellular matrix degradation, and apoptosis of human chondrocytes. HPBzyme attenuated the expression of Rac1 and the ROS levels and prevented the release and nuclear translocation of NF-κB. Deeply digging the intrinsic bioactivities of nanozyme with single component to remodel microenvironment is an effective strategy for ROS-associated chronic diseases. This study reveals that excavating the bioactivities of nanomedicine deserves attention for diagnosis and treatment of severe diseases.

CircADAMTS6/miR-431-5p axis regulate interleukin-1β induced chondrocyte apoptosis.

Growing evidence suggests that circular RNAs (circRNAs) are involved in the development of osteoarthritis (OA). The present study aimed to explore the CircADAMTS6/miR-431-5p axis with respect to regulating interleukin-1β (IL-1β) induced chondrocyte apoptosis. We first evaluated the differentially expressed circRNAs between normal chondrocytes and interleukin (IL)-1β-stimulated chondrocytes. Then, bioinformatic analysis was performed to identify the role and function of circADAMTS6. Small interfering RNA-expressing or overexpressing circADAMTS6 lentiviral vectors were used for transduction of chondrocytes. Annexin-V-fluorescein isothiocyanate (FITC) double staining was performed to measure the apoptotic rate of the chondrocytes in each group. Finally, a dual luciferase reporter assay was performed to identify the target relationship between circADAMTS6 and miR-431-5p. After treatment with IL-1β, circADAMTS6 was down-regulated compared to the normal chondrocyte group. The overexpression of circADAMTS6 inhibited apoptosis in human chondrocytes, as indicated by annexin-V-FITC double staining. However, overexpression of miR-431-5p had the opposite effect. A dual luciferase reporter assay indicated that circADAMTS6 could directly binding with miR-431-5p. Our findings demonstrate that the circADAMTS6/miR-431-5p axis comprises a new target for OA. Bioinformatic analysis suggested that circADAMTS6 acted as a sponge of miR-431-5p.

MicroRNA-675-3p regulates IL-1β-stimulated human chondrocyte apoptosis and cartilage degradation by targeting GNG5

Growing evidence has indicated that microRNAs (miRNAs) are modulators of osteoarthritis (OA) development and progression. In this study, we first evaluated the anti-apoptosis and chondroprotective effects of microRNA-675-3p (miR-675-3p) on interleukin-1β (IL-1β)-stimulated human chondrocytes. The overexpression of miR-675-3p inhibited apoptosis and cartilage matrix degradation and promoted cell proliferation in human chondrocytes. Target gene prediction and luciferase reporter assays suggested that G-protein subunit γ 5 (GNG5) may be the target gene of miR-675-3p. The overexpression of miR-675-3p inhibited IL-1β-stimulated chondrocyte apoptosis, and this effect was reversed by the overexpression of GNG5. Finally, we used bioinformatic tools and biological methods to show that the long noncoding RNA X-inactive specific transcript (lncRNA XIST) could bind to miR-675-3p, which affects the expression of GNG5 mRNA. Our findings may substantiate miR-675-3p as a new treatment for OA.

PKR Promotes Oxidative Stress and Apoptosis of Human Articular Chondrocytes by Causing Mitochondrial Dysfunction through p38 MAPK Activation-PKR Activation Causes Apoptosis in Human Chondrocytes.

Osteoarthritis (OA) is one of the most common types of arthritis in the elderly people. It has been known that chondrocyte apoptosis occurs in OA cartilage; however, the detailed molecular mechanism remains unclear. In the current study, we aimed to elucidate the role of double-stranded RNA-dependent protein kinase R (PKR) in the TNF-α-caused apoptosis in chondrocytes. Human articular chondrocytes were digested from cartilages of OA subjects who accepted arthroplastic knee surgery. Our results showed that phosphorylation of p38 MAPK was increased after TNF-α stimulation or PKR activation using poly (I:C), and TNF-α-induced p38 MAPK upregulation was inhibited by PKR inhibition, suggesting phosphor-p38 MAPK was regulated by PKR. Moreover, we found that PKR participated in the p53-dependent destruction of AKT following activation of p38 MAPK. The inhibition of AKT led to the reduced expression of PGC-1α, which resulted in mitochondrial dysfunction and increased oxidative stress. We showed that the reduction of oxidative stress using antioxidant Mito TEMPO lowered the TNF-α-induced caspase-3 activation and TUNEL-positive apoptotic cells. The diminished apoptotic response was also observed after repression of PKR/p38 MAPK/p53/AKT/PGC-1α signaling. Taken together, we demonstrated that the aberrant mitochondrial biogenesis and increased oxidative stress in chondrocytes after TNF-α stimulation were mediated by PKR, which may contribute to the chondrocyte apoptosis and cartilage degeneration in OA.

Inhibition of lysosomal function enhances inflammatory gene expression, induce oxidative stress and apoptosis in human chondrocytes

Purpose: Lysosomes are the major catabolic organelle of the cells and are essential for the clearance of autophagosomes. Loss of lysosomal function with age has been associated with mitochondrial dysfunction, oxidative stress and apoptosis of the cells. However, the role of lysosomes in chondrocytes homeostasis under pathological conditions has not been explored. Here, we used Chloroquine (CQ) and Bafilomycin A1 (Baf) to inhibit lysosomal function and investigated its consequences on chondrocyte homeostasis and viability in vitro. Methods: Human articular chondrocytes and cartilage explants were prepared from OA knee cartilage obtained from donors undergoing knee arthroplasty at the SUMMA Health Barberton Hospital. Human normal cartilage was obtained from NDRI. The expression of lysosomal associated membrane protein 1 (LAMP1) in normal and OA cartilage was determined by immunohistochemistry. Primary human OA chondrocytes or cartilage explants were maintained in complete DMEM/F12 and treated with CQ or Baf for 24 h or indicated time in DMEM/F12 medium without serum. Induction of apoptosis in chondrocytes treated with CQ or Baf was determined by measuring the activity of LDH released in the culture supernatant, caspase 3/7-luciferase reporter assay and TUNEL staining. The level of IL-1β and IL-6 was determined by TaqMan assays. The level of reactive oxygen species (ROS) was determined by DHR123 staining followed by flow cytometry and MitoSOX Red staining followed by confocal microscopy. The loss of mitochondrial membrane potential was determined by JC-1 dye staining and ATP levels were determined using a kit. The accumulation of dysfunctional mitochondria was determined by staining with MitoTracker Deep Red followed by flow cytometry. The signaling pathway activation was investigated by immunoblot analysis of NFκB and MAPK pathways and its involvement was confirmed by using small molecule inhibitors of the induced pathway. The role of ROS in CQ and Baf induced expression of IL-1β and IL-6 was determined by using anti-oxidants, MitoTempo and Trolox. Results: The expression of LAMP1 was highly downregulated in OA cartilage compared to normal cartilage. CQ and Baf significantly induced apoptosis of primary human OA chondrocytes as determined by LDH activity assay, caspase3/7 luciferase reporter assay and TUNEL staining. Similar results were obtained with mouse chondrocytes. Human OA cartilage explants as well as mouse femoral head cartilage explant culture treated with CQ or Baf showed significant increase in LDH release and TUNEL staining. The CQ and Baf induced inhibition of lysosomal function resulted in the loss of mitochondrial membrane potential, excessive levels of ROS, lower levels of ATP and the accumulation of dysfunctional mitochondria in the treated chondrocytes. Treatment of primary human OA chondrocytes with CQ or Baf significantly increased the gene and protein expression of IL-1β and IL-6. Investigation of the signaling pathways showed no role of NFκB signaling in the CQ and Baf induced upregulation of IL-1β and IL-6 expression. However, the JNK-MAPK, but not ERK- or p38-MAPK pathway, was activated in response to Baf treatment. Inhibition of JNK activation by using a specific inhibitor abrogated the upregulation of IL-1β and IL-6 expression in Baf treated human chondrocytes. The use of anti-oxidants, MitoTempo and Trolox also suppressed the CQ and Baf induced expression of IL-1β and IL-6. JNK inhibition and treatment with MitoTempo or Trolox failed to suppress apoptosis suggesting that this was independent of ROS levels and JNK pathway in chondrocytes with inhibited lysosomal function. Conclusions: We demonstrate here that inhibition of lysosomal function promotes inflammation, oxidative stress and chondrocyte apoptosis through JNK activation. Our data suggest that protecting lysosomal function may be a therapeutic target for OA management.

Nupr1 regulates palmitate-induced apoptosis in human articular chondrocytes.

Obesity, a major risk factor for the development of osteoarthritis (OA), is associated with increased circulating levels of free fatty acids (FFA). However, the role of these FFAs in OA pathophysiology is not clearly understood. In the present study, we found that palmitate treatment of human primary articular chondrocytes increased the expression of ER stress markers [activating transcription factor 4 (ATF4), C/EBP homologous protein (CHOP)] and apoptosis markers [cytochrome c and cleaved caspase-3 (CC3)]. Palmitate treatment also increased the expression of Nuclear protein 1 (Nupr1) and tribbles related protein 3 (TRB3), which are known negative regulators of cell survival pathways. Knockdown of Nupr1 or CHOP expression inhibited palmitate mediated increased expression of TRB3 and CC3, indicating that Nupr1 and CHOP cooperate to regulate cell survival and apoptotic pathways in human chondrocytes. Nupr1 knockdown had no effect on CHOP expression whereas CHOP knockdown abolished the palmitate-mediated Nupr1 expression, indicating that CHOP is functional upstream to Nupr1 in this pathway. Moreover, overexpression of Nupr1 markedly increased the basal expression of pro-apoptotic molecules, including cytochrome c and CC3. Taken together, our study demonstrates that Nupr1 plays a crucial role in palmitate-induced apoptosis in human chondrocytes and Nupr1 as a potential novel drug target for the treatment of OA.

Tormentic acid inhibits IL-1β-induced chondrocyte apoptosis by activating the PI3K/Akt signaling pathway.

Interleukin-1β (IL-1β) accelerates degradation of the cartilage matrix and induces apoptosis of chondrocytes. Tormentic acid (TA) is a triterpene isolated from the stem bark of the Vochysiadivergens plant, which has been demonstrated to exert invitro inhibitory activity against hepatocyte apoptosis. However, the effects of TA on IL‑1β‑induced apoptosis of human chondrocytes remain unclear. Therefore, the present study investigated the in vitro effects of TA on human osteoarthritic chondrocyte apoptosis cultivated in the presence of IL‑1β. Human chondrocytes were pretreated with or without various concentrations of TA and then co‑incubated in the absence or presence of IL‑1β for 24h. Cell viability was determined using the MTT assay, and cell apoptosis was detected using a Nucleosome ELISA kit. Caspase‑3 activity was detected using a caspase‑3 colorimetric assay kit. The levels of B‑cell lymphoma 2 (Bcl‑2)‑associated X protein (Bax), Bcl‑2, phosphorylated (p)‑phosphoinositide 3‑kinase (PI3K), PI3K, p‑protein kinase B (Akt) and Akt were measured by western blotting. The results revealed that pretreatment with TA inhibited IL‑1β‑induced cytotoxicity and apoptosis in chondrocytes. In addition, TA pretreatment increased B‑cell lymphoma (Bcl)‑2 expression, and decreased caspase‑3 activity and Bax expressionin human chondrocytes. In addition, pretreatment with TA markedly increased the expression of p‑PI3K and p‑Akt in IL‑1β‑induced chondrocytes. Collectively, these results indicate that TA inhibits IL‑1β‑induced chondrocyte apoptosis by activating the PI3K/Akt signaling pathway. Therefore, TA may be considered a potential therapeutic target for the treatment of osteoarthritis.

MiR-26a and miR-26b mediate osteoarthritis progression by targeting FUT4 via NF-κB signaling pathway.

Osteoarthritis (OA) is the most common joint disease, characterized by articular cartilage degradation and changes in all other joint tissues. MicroRNAs (miRNAs) play an important role in mediating the main risk factors for OA. This study aimed to investigate the effect of miR-26a/26b on the proliferation and apoptosis of human chondrocytes by targeting fucosyltransferase 4 (FUT4) through NF-κB signaling pathway. We revealed the differential expression profiles of FUT4 and miR-26a/26b in the articular cartilage tissues of OA patients and normal people. The ability of miR-26a/26b to specifically interact with the 3'UTR of FUT4 was demonstrated via a luciferase reporter assay in chondrocytes. Further results showed altered levels of miR-26a/26b and FUT4 could regulate the process of IL-1β-induced extracellular matrix degradation in chondrocytes. Forced miR-26a/26b expression was able to affect chondrocytes proliferation and apoptosis, while altered expression of FUT4 in chondrocytes modulated progression upon transfection with miR-26a/26b mimic or inhibitor. In OA mice, the overexpression of miR-26a/26b by intra-articular injection significantly attenuated OA progression. In addition, regulating FUT4 expression markedly modulated the activity of NF-κB signaling pathway, and this effect could be reversed by miR-26a/26b. In short, miR-26a/-26b/FUT4/NF-κB axis may serve as a predictive biomarker and a potential therapeutic target in OA treatment.

Expression of miR-206 in Human Knee Articular Chondrocytes and Effects of miR-206 on Proliferation and Apoptosis of Articular Chondrocytes

BackgroundIncreasing evidence has demonstrated that microRNAs regulate the development of cartilage and osteogenesis. Whether miR-206 participates in the development of human articular cartilage remains largely unknown. This study aimed to investigate the role of miR-206 in human chondrocytes. MethodsExpression of miR-206 was initially assessed in human osteoarthritis (OA) tissues and articular chondrocytes through quantitative real-time polymerase chain reaction. The effects of miR-206 on proliferation and apoptosis of human chondrocytes were assessed by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) double staining assay. Then, the effects of miR-206 on type II collagen alpha 1 (Col2a1), aggrecan, runt-related transcription factor 2 (RUNX2) and matrix metalloproteinase13 (MMP13) were examined with quantitative real-time polymerase chain reaction and Western blot analysis. ResultsMiR-206 was significantly increased in human OA tissues and chondrocytes. MiR-206 significantly inhibited the proliferation of chondrocytes, but promoted apoptosis. Expression of Col2a1 and aggrecan were dramatically decreased, and the expression of RUNX2 and MMP13 were significantly increased when miR-206 was overexpressed. ConclusionsMiR-206 may participate in cartilage degradation in OA. Manipulation of the expression of miR-206 in human chondrocytes may be a novel therapeutic strategy for the treatment of OA.

Piezo1 protein induces the apoptosis of human osteoarthritis-derived chondrocytes by activating caspase-12, the signaling marker of ER stress.

The present study was carried out to determine whether the mechanically activated cation channel Piezo1 protein plays a role as a signaling pathway which causes the apoptosis of human chondrocytes. The chondrocytes were isolated, cultured, and then subjected to mechanical stretch force for 0, 2, 12, 24 and 48 h, respectively. The expression levels of Piezo1 and the apoptosis-related protein caspase-12 were assessed by reverse transcription-quantitative polymerase chain reaction, as well as the apoptosis-related genes, B cell lymphoma/leukemia-2 (Bcl-2), Bcl-associated X protein (Bax) and Bcl-2-associated death promoter (BAD). Lactate dehydrogenase (LDH) activity was used to discern dead cells. Piezo1 expression was determined by immunofluorescence. In addition, Piezo1 inhibitor, GsMTx4, was used to block the mechanically activated (MA) cation channel Piezo1, and served as a positive control. The results showed that the osteoarthritis (OA)-derived chondrocytes showed a tendency to undergo late-stage apoptosis under compressive loading. Piezo1 and caspase-12 were significantly upregulated under static compressive stimuli and the expression was related to the rate of apoptosis of the OA-derived chondrocytes during compressive loading. The expression of caspase-12 and late-stage apoptosis of the human OA-derived chondrocytes were repressed by GsMTx4, the specific inhibitor of Piezo1, while the expression of Piezo1 and the induction of the apoptosis of the OA-derived chondrocytes during compressive loading was not totally blocked. Thus, we conclude that Piezo1 plays an important role in the apoptosis of human OA-derived chondrocytes through a caspase-12-dependent pathway. The expression of Piezo1 protein was not totally inhibited by GsMTx4.