Induced Chondrocyte Apoptosis Research Articles

NS8593 inhibits sodium nitroprusside-induced chondrocyte apoptosis by mediating the STING signaling pathway

Articular cartilage damage and chondrocyte apoptosis are among the distinguishing features of osteoarthritis. (R)-N-(benzimidazol-2-yl)-1,2,3,4-tetrahydro-1-naphtylamine (NS8593) is a transient receptor potential cation channel subfamily M member 7 (TRPM7) channel inhibitor and was initially considered a potent inhibitor of small-conductance Ca2+-activated K+ channels(SK1-3 or KCa2.1–2.3 channels). Since SK is one of the targets for atrial fibrillation therapy, several studies have been conducted using NS8593 and it has been shown to be effective in improving atrial fibrillation in rats, dogs and horses. Recently, inhibition of TRPM7 has been reported to alleviate articular cartilage destruction. However, the role and mechanism of NS8593 on articular chondrocyte damage is unknown. The purpose of this study was to investigate the effect and mechanism of NS8593 on sodium nitroprusside (SNP)-induced chondrocyte apoptosis in vitro. The results showed that SNP decreased cell viability and induced chondrocyte apoptosis. NS8593 dose-dependently inhibited the SNP-induced decrease in cell viability and reduced chondrocyte apoptosis. In addition, SNP stimulation significantly increased the phosphorylation level of the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING), and NS8593 treatment partially reversed the alteration of STING phosphorylation level. Treatment with the STING inhibitor H-151 inhibited SNP-induced chondrocyte apoptosis. These results suggest that NS8593 may inhibit SNP-induced chondrocyte apoptosis by suppressing the STING signaling pathway.

OSTF1 knockdown mitigates IL-1β-induced chondrocyte injury via inhibiting the NF-κB signaling pathway

Osteoarthritis (OA) is an age-related joint disease characterized by progressive heterogeneous changes in articular cartilage and subchondral bone. Osteoclast stimulating factor 1 (OSTF1) is a small intracellular protein involved in bone formation and bone resorption. However, to our best knowledge, its role in OA is still unclear. In this study, an OA rat model was established by anterior cruciate ligament transection (ALCT). OSTF1 was increased in the cartilage tissues of OA patients and OA rats. Next, the role of OSTF1 in interleukin-1β (IL-1β)-induced chondrocyte apoptosis, inflammation and extracellular matrix degradation was explored through loss of function assays. Strikingly, OSTF1 knockdown relieved IL-1β-induced chondrocyte apoptosis, with decreased cleaved caspase-3 and cleaved PARP levels. Besides, OSTF1 knockdown restrained IL-1β-induced inflammation and degradation of extracellular matrix of chondrocytes. Subsequently, the molecular mechanism of OSTF1 was explored. Transcriptomic analysis revealed the potential gene network map regulated by OSTF1 knockdown. Some differentially expressed genes (DEGs) were involved in regulating the NF-κB signaling pathway. Furthermore, our results demonstrated that OSTF1 knockdown inhibited IL-1β-activated the NF-κB signaling pathway. Ultimately, we analyzed the potential gene network map regulated by OSTF1 and its downstream NF-κB. Bioinformatics analysis showed that 18 DEGs in OSTF1-silenced chondrocytes overlapped with the NF-κB downstream targets. Collectively, our findings indicate that OSTF1 knockdown mitigates IL-1β-induced chondrocyte injury via inhibiting the NF-κB signaling pathway.

Minocycline declines interleukin-1ß-induced apoptosis and matrix metalloproteinase expression in C28/I2 chondrocyte cells: an in vitro study on osteoarthritis.

Osteoarthritis (OA) is a degenerative joint disease that occurs with aging. In its late phases, it is determined by the loss of chondrocytes and the breakdown of the extracellular matrix, resulting in pain and functional impairment. Interleukin-1 beta (IL-1β) is increased in the injured joints and contributes to the OA pathobiology by inducing chondrocyte apoptosis and up-regulation of matrix metalloproteinases (MMPs). Here, we aimed to understand whether minocycline could protect chondrocytes against the IL-1β-induced effects. The human C28/I2 chondrocyte cell line was treated with IL-1β or IL-1β plus minocycline. Cell viability/toxicity, cell cycle progression, and apoptosis were assessed with MMT assay and flow cytometry. Expression of apoptotic genes and MMPs were evaluated with qRT-PCR and western blotting. IL-1β showed a significant cytotoxic effect on the C28/I2 chondrocyte cells. The minocycline effective concentration (EC50) significantly protected the C28/I2 cells against the IL-1β-induced cytotoxic effect. Besides, minocycline effectively lowered IL-1β-induced sub-G1 cell population increase, indicating the minocycline anti-apoptotic effect. When assessed by real-time PCR and western blotting, the minocycline treatment group showed an elevated level of Bcl-2 and a significant decrease in the mRNA and protein expression of the apoptotic markers Bax and Caspase-3 and Matrix metalloproteinases (MMPs) such as MMP-3 and MMP-13. In conclusion, IL-1β promotes OA by inducing chondrocyte death and MMPs overexpression. Treatment with minocycline reduces these effects and decreases the production of apoptotic factors as well as the MMP-3 and MMP-13. Minocycline might be considered as an anti-IL-1β therapeutic supplement in the treatment of osteoarthritis. See also the graphical abstract(Fig. 1).

Development of Anti-OSCAR Antibodies for the Treatment of Osteoarthritis.

Osteoarthritis (OA) is the most common joint disease that causes local inflammation and pain, significantly reducing the quality of life and normal social activities of patients. Currently, there are no disease-modifying OA drugs (DMOADs) available, and treatment relies on pain relief agents or arthroplasty. To address this significant unmet medical need, we aimed to develop monoclonal antibodies that can block the osteoclast-associated receptor (OSCAR). Our recent study has revealed the importance of OSCAR in OA pathogenesis as a novel catabolic regulator that induces chondrocyte apoptosis and accelerates articular cartilage destruction. It was also shown that blocking OSCAR with a soluble OSCAR decoy receptor ameliorated OA in animal models. In this study, OSCAR-neutralizing monoclonal antibodies were isolated and optimized by phage display. These antibodies bind to and directly neutralize OSCAR, unlike the decoy receptor, which binds to the ubiquitously expressed collagen and may result in reduced efficacy or deleterious off-target effects. The DMOAD potential of the anti-OSCAR antibodies was assessed with in vitro cell-based assays and an in vivo OA model. The results demonstrated that the anti-OSCAR antibodies significantly reduced cartilage destruction and other OA signs, such as subchondral bone plate sclerosis and loss of hyaline cartilage. Hence, blocking OSCAR with a monoclonal antibody could be a promising treatment strategy for OA.

Multifunctional platinum-tellurium nanorods modified with hyaluronic acid remove reactive oxygen species to improve the photothermal treatment of rheumatoid arthritis

Rheumatoid arthritis (RA) is an autoimmune disease, in which inflammatory macrophages play a crucial role by secreting a large number of proinflammatory factors to aggravate inflammatory response. In addition, reactive oxygen species (ROS) are abundant in hypoxic joints, inducing chondrocyte apoptosis and causing irreversible bone destruction. In this work, we synthesized hyaluronic acid-modified platinum-tellurium nanorods (HPT) as a photothermal agent to target RA joints, ablating inflammatory macrophages and scavenging ROS in the microenvironment. Hyaluronic acid can specifically bind to the CD44 receptors, which are over-expressed on the surface of inflammatory macrophages, achieving active targeting and improving therapy effectiveness. Under the irradiation of an 808 nm laser, thermal energy produced by HPT, which has high photothermal conversion efficiency is sufficient to induce apoptosis of inflammatory macrophages. Simultaneously, platinum particles on the surface of tellurium nanorods can effectively remove a variety of ROS, reducing oxidative stress and alleviating inflammatory response in RA joints. Collagen-induced arthritis (CIA) mice had been used to study the therapeutic potential of HPT. The results revealed that the clinical scores in CIA mice injected with HPT and irradiated with a near-infrared laser were significantly reduced. The inflammatory response was also obviously relieved, when compared with CIA mice injected with PBS. Micro-CT imaging revealed that bone erosion was visibly reduced in the HPT + Laser group. These results demonstrate that multifunctional HPT is promising to serve as a therapeutic platform for the treatment of RA.

NIR-triggered photodynamic therapy of traumatic heterotopic ossification with a type II collagen-targeted photosensitizer

Traumatic heterotopic ossification (HO) represents an intractable sequela following trauma with no currently effective prophylaxis or treatment. Photodynamic therapy (PDT) is a non-invasive treatment for various proliferative diseases. However, the specific effects of PDT on HO development remain unclear. In this study, the therapeutic potential of a near-infrared (NIR) probe-WL-808, composed of type II collagen-binding peptide (WYRGRL) and a PDT photosensitizer (IR-808), was evaluated for the innovative HO-targeted PDT approach. In vitro studies indicated that WL-808 could induce chondrocyte apoptosis and inhibit cell viability through ROS generation under NIR excitation. In vivo, the efficacy of WL-808-mediated PDT was tested on the tenotomy HO model mice. WL-808 specifically targeted the type II collagen cartilaginous template of HO, promoting cell apoptosis and enhancing extracellular matrix (ECM) degradation under 808 nm NIR excitation, which inhibited the final ectopic bone formation. Moreover, no obvious toxicity or side effects were detected after treatment with WL-808. Taken together, WL-808-mediated PDT significantly diminished ectopic cartilage and subsequent bone formation, providing a new perspective for HO prophylaxis and treatment.

Dapagliflozin suppress endoplasmic reticulum stress mediated apoptosis of chondrocytes by activating Sirt1

ObjectiveOsteoarthritis (OA) is a common joint disease characterized by inflammation and cartilage degeneration. Accumulating evidences support that endoplasmic reticulum (ER) stress induced OA chondrocytes apoptosis. The hypoglycemic and anti-inflammatory properties render Dapagliflozin (DAPA) effective in reducing ER stress on cells. However, its impact and potential mechanisms on the OA pathology are still obscure. The present study aimed to investigate whether DAPA attenuates ER stress in chondrocytes by activating sirt1 and delays the progression of OA. MethodsIn vitro, we first investigated the effect of DAPA on chondrocytes viability with IL-1β or not for 24 or 48 h. Then, chondrocytes were treated with 10 ng/ml IL-1β and 10 μM dapagliflozin with10 μM thapsigargin, 5 μM SRT1460 or not. Chondrocytes apoptosis in each group were detected by Tunel staining and flow cytometric. Immunofluorescence staining was applied to quantify the expression levels of cleaved caspase-3, Sirt1 and CHOP in chondrocytes. Inhibition of ER stress in chondrocytes associated with sirt1 activation were verified by PCR and western blotting. In addition, the effects of DAPA on cartilage were validated by a series of experiments in OA rat model, such as micro-CT, histological and immunohistochemical assay. ResultsThe data demonstrated that DAPA alleviates IL-1β induced ER stress related chondrocytes apoptosis, and PCR and western blotting data confirmed that DAPA inhibits the PERK-eIF2α-CHOP pathway by activating Sirt1. Besides, immunohistochemical results showed that DAPA enhanced the expression of Sirt1 and Collagen II in OA rats, and inhibited the expression of CHOP and cleaved caspase-3. Meanwhile, histological staining and micro-CT photography also confirmed that DAPA alleviated inflammation and cartilage degeneration in OA rat. ConclusionsThe study demonstrated the relationship of ER stress and inflammation in the progression of OA, and verified that DAPA could inhibit PERK-eIF2α-CHOP axis of the ER stress response by activating Sirt1 in IL-1β treated rat chondrocytes and potentially prevent the OA development.

Tranexamic Acid Causes Chondral Injury Through Chondrocytes Apoptosis Induced by Activating Endoplasmic Reticulum Stress

To investigate whether tranexamic acid (TXA) is cytotoxic in chondrocyte and cartilage tissues, as well as explore the mechanisms behind the possible toxicity in detail. We detected the cell viability of chondrocytes invitro and the change of morphology and specific invivo contents of cartilage after TXA treatment. Furthermore, we detected apoptosis in cartilage. We used apoptosis-specific staining, reactive oxygen species detection, mitochondrial membrane potential detection, flow cytometry, and western blot for apoptosis detection. Finally, we detected the activation of endoplasmic reticulum stress (ERS) in TXA-treated chondrocytes to clarify the mechanism behind chondrocyte apoptosis. TXA presented an increasing toxic effect with increasing concentrations, especially in the 100 mg/mL group. In addition, we found that 50 mg/mL and 100 mg/mL TXA significantly increased apoptosis in cartilage and subchondral bone. TXA could induce chondrocyte apoptosis in cell and protein levels with reactive oxygen species generation and mitochondrial membrane depolarization. An apoptosis inhibitor could inhibit the induced apoptosis. Next, TXA induced calcium overload in chondrocytes and increased ERS-specific protein expression, whereas ERS inhibitor blocked ERS activation and further inhibited chondrocyte apoptosis. We concluded that TXA had a toxic effect on chondrocytes by inducing apoptosis through ERS activation, especially in 50 mg/mL and 100 mg/mL groups. We recommend TXA concentrations of less than 50 mg/mL in joint surgeries. It is still unclear whether TXA has a toxic effect on cartilage when topically used in joint surgeries. The concentration also varies. This study provides additional evidence that TXA at high concentrations will cause cartilage damage, which will help to provide a new understanding of the clinical administration of TXA.

Circ-NFKB1 sponges miR-203a-5p to regulate ERBB4 expression and promotes IL-1β induced chondrocytes apoptosis

BackgroundOsteoarthritis (OA) is a chronic disease of the bones and joints that commonly affects middle-aged and elderly individuals, characterized by the degeneration of articular cartilage and inflammation of the joints. The molecular mechanisms of OA urgently need to be further examined. Our study intended to uncover circ-NFKB1/miR-203a-5p/ERBB4 axis in regulating interleukin-1β (IL-1β) induced chondrocytes apoptosis.MethodsGSE178724, GSE79258 and GSE169077 were downloaded from Gene Expression Omibus (GEO) database and differentially expressed circRNAs, miRNAs and mRNAs were obtained by R software. Annexin V assay was used to determine cell apoptosis rate. ELISA was further performed to identify the inflammation response. Dual-luciferase reporter gene assay was conducted to examine the combination among circ-NFKB1, miR-203a-5p and ERBB4.ResultsOur research demonstrated that circ-NFKB1 and ERBB4 were significantly upregulated through bioinformatic analysis. MiR-203a-5p was significantly downregulated through bioinformatic analysis. Silencing of circ-NFKB1 notably inhibited the IL-1β induced chondrocytes apoptosis and upregulated ERBB4 expression. Through prediction on bioinformatics analysis, miR-203a-5p was the target binding circ-NFKB1, and ERBB4 was the potential target of miR-203a-5p. Subsequently, these changes induced by the silencing of circ-NFKB1 were reversed upon addition of pcDNA/ERBB4.ConclusionsSilencing circ-NFKB1 could sponge miR-203a-5p to regulate ERBB4 expression and alleviate OA progression.

CircTBX5 knockdown modulates the miR-558/MyD88 axis to alleviate IL-1β-induced inflammation, apoptosis and extracellular matrix degradation in chondrocytes via inactivating the NF-κB signaling

BackgroundIt has been widely shared that the dysregulation of circular RNA (circRNA) may contribute to the progression of osteoarthritis (OA). OA is characterized by persistent chondrocyte injury. We aimed to clarify the role of circTBX5 in IL-1β-induced chondrocyte injury.MethodsThe expression of circTBX5, miR-558 and MyD88 mRNA was measured using quantitative real-time PCR (qPCR). Cell viability, proliferation and apoptosis were assessed by CCK-8, EdU or flow cytometry assay. The protein levels of extracellular matrix (ECM)-associated markers, MyD88, IkBα, p65 and phosphorylated IkBα were measured by western blot. The release of inflammatory factors was assessed by ELISA. The targets of circTBX5 were screened by RIP and pull-down assay. The putative binding between miR-558 and circTBX5 or MyD88 was validated by dual-luciferase reporter assay.ResultsCircTBX5 and MyD88 were enhanced, while miR-558 was downregulated in OA cartilage tissues and IL-1β-treated C28/I2 cells. IL-1β induced C28/I2 cell injury by impairing cell viability and proliferation and promoting cell apoptosis, ECM degradation and inflammatory response, while circTBX5 knockdown alleviated IL-1β induced injury. CircTBX5 bound to miR-558 to regulate IL-1β induced cell injury. In addition, MyD88 was a target of miR-558, and circTBX5 targeted miR-558 to positively regulate MyD88 expression. MiR-558 enrichment attenuated IL-1β induced injury by sequestering MyD88 expression. Moreover, circTBX5 knockdown weakened the activity of NF-κB signaling, while miR-558 inhibition or MyD88 overexpression recovered the activity of NF-κB signaling.ConclusionCircTBX5 knockdown modulated the miR-558/MyD88 axis to alleviate IL-1β induced chondrocyte apoptosis, ECM degradation and inflammation via inactivating the NF-кB signaling pathway.

Mouth breathing induces condylar remodelling and chondrocyte apoptosis via both the extrinsic and mitochondrial pathways in male adolescent rats

The prevalence of mouth breathing is high in children and adolescents. It causes various changes to the respiratory tract and, consequently, craniofacial growth deformities. However, the underlying mechanisms contributing to these effects are obscure. Herein, we aimed to study the effects of mouth breathing on chondrocyte proliferation and death in the condylar cartilage and morphological changes in the mandible and condyle. Additionally, we aimed to elucidate the mechanisms underlying chondrocyte apoptosis and investigate any variations in the related pathways. Subchondral bone resorption and decreased condylar cartilage thickness were observed in mouth-breathing rats; further, mRNA expression levels of Collagen II, Aggrecan, and Sox 9 were lower in the mouth breathing group, while those of matrix metalloproteinase 9 increased. TdT-mediated dUTP nick end labelling staining and immunohistochemistry analyses showed that apoptosis occurred in the proliferative and hypertrophic layers of cartilage in the mouth breathing group. TNF, BAX, cytochrome c, and cleaved-caspase-3 were highly expressed in the condylar cartilage of the mouth-breathing rats. These results suggest that mouth breathing leads to subchondral bone resorption, cartilage layer thinning, and cartilage matrix destruction, inducing chondrocyte apoptosis via both the extrinsic and mitochondrial apoptosis pathways.

Trimethylamine-N-oxide sensitizes chondrocytes to mechanical loading through the upregulation of Piezo1.

Mechanical strain plays a crucial role in chondrocyte apoptosis and osteoarthritis (OA) disease progression through Piezo1. Trimethylamine-N-oxide (TMAO) is a diet-derived metabolite that correlates positively with multiple chronic diseases. Herein, we explored the potential role of TMAO in sensitizing chondrocytes to Piezo1-mediated mechanotransduction. The cytotoxicity of TMAO on chondrocytes was assayed. Piezo1 expression was measured after TMAO intervention. Pathological mechanical loading or Yoda1 (a specific Piezo1 channel activator) was administered in chondrocytes. The calcium levels and cytoskeleton in chondrocytes were observed by fluorescence microscopy. Flow cytometry, western blotting, and mitochondrial membrane potential assays were utilized to evaluate apoptosis. A rat OA model was constructed by anterior cruciate ligament transection. Hematoxylin-eosin staining, Safranin-O/Fast Green staining, immunochemistry, and TUNEL were applied to estimate OA severity. TMAO intervention alone did not affect chondrocyte viability up to 600μM. TMAO significantly increased Piezo1 expression and up-regulated intracellular calcium levels, further leading to cytoskeletal damage. Mechanical strain or Yoda1 treatment significantly induced chondrocyte apoptosis. Notably, TMAO intervention further aggravated chondrocyte apoptosis and cartilage destruction under pathological mechanical loading. TMAO significantly up-regulated Piezo1 expression and sensitized chondrocytes to mechanical loading, which may be closely related to the pathogenesis of OA.

PTEN inhibitor VO-OHpic protects endplate chondrocytes against apoptosis and calcification via activating Nrf-2 signaling pathway.

Cartilage endplate (CEP) degeneration and calcification is an important contributor to the onset and pathogenesis of intervertebral disc degeneration (IDD). However, the underlying mechanisms of CEP degeneration remain elusive, let alone according treatment strategies to prevent CEP degeneration. Phosphatase and tensin homolog (PTEN) is a tumor suppressor gene that promotes cell apoptosis, and recent studies indicated that PTEN is overexpressed in degenerated intervertebral disc. However, whether direct inhibition of PTEN attenuates CEP degeneration and IDD development remains largely unknown. In the present study, our in vivo experiments demonstrated that VO-OHpic could attenuate IDD progression and CEP calcification. We also found that VO-OHpic inhibited oxidative stress induced chondrocytes apoptosis and degeneration by activating Nrf-2/HO-1 pathway, thus promoted parkin mediated mitophagy process and inhibited chondrocytes ferroptosis, alleviated redox imbalance and eventually improved cell survival. Nrf-2 siRNA transfection significantly reversed the protective effect of VO-OHpic on endplate chondrocytes. In conclusion, our study demonstrated that inhibition of PTEN with VO-OHpic attenuates CEP calcification and IDD progression. Moreover, VO-OHpic protects endplate chondrocytes against apoptosis and degeneration via activating Nrf-2/HO-1 mediated mitophagy process and ferroptosis inhibition. Our results suggest that VO-OHpic may be a potential effective medicine for IDD prevention and treatment.

Effect of pulsed electromagnetic fields on mesenchymal stem cell-derived exosomes in inhibiting chondrocyte apoptosis

The study aims to explore the effect of mesenchymal stem cells-derived exosomes (MSCs-Exo) on staurosporine (STS)-induced chondrocyte apoptosis before and after exposure to pulsed electromagnetic field (PEMF) at different frequencies. The AMSCs were extracted from the epididymal fat of healthy rats before and after exposure to the PEMF at 1 mT amplitude and a frequency of 15, 45, and 75 Hz, respectively, in an incubator. MSCs-Exo was extracted and identified. Exosomes were labeled with DiO fluorescent dye, and then co-cultured with STS-induced chondrocytes for 24 h. Cellular uptake of MSC-Exo, apoptosis, and the protein and mRNA expression of aggrecan, caspase-3 and collagenⅡA in chondrocytes were observed. The study demonstrated that the exposure of 75 Hz PEMF was superior to 15 and 45 Hz PEMF in enhancing the effect of exosomes in alleviating chondrocyte apoptosis and promoting cell matrix synthesis. This study lays a foundation for the regulatory mechanism of PEMF stimulation on MSCs-Exo in inhibiting chondrocyte apoptosis, and opens up a new direction for the prevention and treatment of osteoarthritis.

IL-6, IL-1β and TNF-α regulation of the chondrocyte phenotype: a possible mechanism of haemophilic cartilage destruction

ABSTRACTObjective:Proinflammatory cytokines are considered to be one of the key causes of haemophilic cartilage destruction by inducing chondrocyte apoptosis and extracellular matrix degradation. However, few studies have focused on how proinflammatory cytokines regulate the phenotypic changes of chondrocytes, which may be an important factor in haemophilic cartilage degradation pathogenesis. More understanding is needed about the effect of proinflammatory cytokines on phenotypic changes of the chondrocyte. The objective of this study was to examine how IL-6, TNF-α and IL-1β regulate the chondrocyte phenotype, which may be an important factor in haemophilic cartilage degradation pathogenesis.Methods:HUM-iCell-s018 chondrocytes were treated with increasing concentrations of TNF-α, IL-6 or IL-1β (0, 1, 5, 10 ng/ml) for 24 h, then FGF23 and SOX9 expression was determined by qRT-PCR and WB, respectively.Results:We found that TNF-α, IL-6 and IL-1β induced FGF23 and suppressed SOX9 expression in chondrocytes in a dose-dependent manner. IL-1β had a stronger regulatory effect on FGF23, while TNF-α and IL-6 had stronger regulatory effects on SOX9.Conclusions:These findings suggest that IL-6, IL-1β and TNF-α may be involved in haemophilic cartilage destruction pathogenesis by altering the chondrocyte phenotype through modulation of FGF23 and SOX9 gene expression.

Pentosan polysulfate sodium promotes redifferentiation to the original phenotype in micromass-cultured canine articular chondrocytes and exerts molecular weight-dependent effects.

Pentosan polysulfate sodium (PPS) is a heparin-like polysaccharide that is applied as a therapeutic treatment for osteoarthritis (OA) in animals. This study investigated the efficacy of different molecular weights PPS (1,500-7,000 Da) on the phenotype regulatory and chondrogenic properties of canine articular chondrocytes. The cytotoxicity of PPS on chondrocytes was assessed using flow cytometry and 3-(4,5-dimehylthiazolyl-2)-2,5-diphenyltetrazolium bromide assay. After 72 hr of exposure, PPS did not induce chondrocyte apoptosis, regardless of molecular weight. In addition, chondrogenic properties were determined according to the mRNA and protein levels in micromass-cultured chondrocytes. Quantitative polymerase chain reaction analysis confirmed that PPS promotes a chondrogenic phenotype in chondrocytes in a molecular weight-dependent manner, with significant upregulation of collagen type II alpha 1 chain, aggrecan, and SRY-box transcription factor 9 (SOX9) mRNA levels relative to those in the control. However, the collagen type I alpha 2 chain mRNA level simultaneously increased after 7,000 Da PPS treatment. PPS exposure also increased collagen type II and SOX9 protein production in a molecular weight-dependent manner and inhibited Akt phosphorylation in chondrocytes. Alcian blue staining indicated that PPS treatment enhanced proteoglycan deposition in micromass cultures, with stronger effects observed in 5,000 and 7,000 Da groups. Overall, these results indicate that PPS exerts protective effects on the chondrocyte phenotype and may represent a potential therapeutic target for OA treatment. Increasing the molecular weight of PPS could enhance these anabolic effects.

Effect of Flax lignans on apoptosis of growth plate chondrocytes in rats

To compare the effects of different concentrations of linolenin on inhibiting apoptosis of chondrocytes in the growth plate, and to screen the optimal concentration of linolenin, so as to provide theoretical support for delaying epiphyseal closure and promoting long bone growth in rats. Two 4-week-old male SD rats (SPF grade) with a body mass of 80 g were selected. The growth plate cartilage of rat tibia and femur was dissected and isolated in vitro to obtain growth plate chondrocytes for culture. The chondrocytes were observed and identified by inverted phase contrast microscope and typeⅡ collagen immunofluorescence test, and then 20 ng/ml IL-1β was used to induce apoptosis of growth plate chondrocytes as model group, and added with 1, 10, 20, 40 μM linolenin as the experimental group, and 5 μM letrozole as the positive control group. The cells were cultured for 24 and 48 hours respectively. The drug promoted cell proliferation was observed by MTT method, and the drug inhibited cell apoptosis was detected by flow cytometry. Contents 1, 10, 20, 40 μM could promote cell proliferation in varying degrees, and the principle was that the drug inhibits IL-1β induced chondrocyte apoptosis in the growth plate, and the optimal concentration of drugs to inhibit apoptosis was 20 μM. The appropriate concentration of linseed lignans can significantly inhibit the apoptosis of chondrocytes in the growth plate of rats, and the optimal drug concentration is 20 μM. It provides possibility for delayed bone closure and longer growth time to promote bone growth during development.

3,3'-diindolylmethane inhibits LPS-induced human chondrocytes apoptosis and extracellular matrix degradation by activating PI3K-Akt-mTOR-mediated autophagy.

Osteoarthritis (OA) is a chronic degenerative joint disease characterized by articular cartilage destruction. The pathological mechanisms are complex; in particular, inflammation, autophagy, and apoptosis are often involved. 3,3-Diindolylmethane (DIM), a phytoconstituent extracted from cruciferous vegetables, has various effects such as anti-inflammatory, antioxidant and anti-apoptotic. However, the effects of DIM on osteoarthritic chondrocytes remain undetermined. In this study, we simulated a lipopolysaccharide (LPS)-induced osteoarthritis model in human primary chondrocytes. We found that LPS stimulation significantly inhibited autophagy, induced chondrocyte apoptosis and extracellular matrix (ECM) degradation, which could be ameliorated by DIM. DIM inhibited the expression of a disintegrin and metalloproteinase with thrombospondin motif 5 (ADAMTS-5), matrix metalloproteinase 13 (MMP13), cleaved caspase-3, Bax, and p62, and increased the expression level of collagen II, aggrecan, Bcl-2, light chain 3 Ⅱ (LC3 Ⅱ), and beclin-1. Mechanistic studies showed that DIM increased chondrocyte autophagy levels by inhibiting the activation of PI3K/AKT/mTOR pathway. In mice destabilization of the medial meniscus (DMM) model, immunohistochemical analysis showed that DIM inhibited the expression of p-PI3K and cleaved caspase-3, increased the expression of LC3 Ⅱ. Furthermore, DIM relieved joint cartilage degeneration. In conclusion, our findings demonstrate for the first time that DIM inhibits LPS-induced chondrocyte apoptosis and ECM degradation by regulating the PI3K/AKT/mTOR-autophagy axis and delays OA progression in vivo.

Exosome modification to better alleviates endoplasmic reticulum stress induced chondrocyte apoptosis and osteoarthritis

Osteoarthritis (OA) is characterized by cartilage matrix degeneration and chondrocyte apoptosis. Prolonged endoplasmic reticulum (ER) stress participates in chondrocyte apoptosis and cartilage degeneration in OA progression. miR-486-5p could suppress the apoptosis of nucleus pulposus cells and cardiomyocyte, yet whether miR-486-5p modified exosomes could modulate ER stress and apoptosis of chondrocytes remain unknown. We validated the increased inflammation and ER stress in OA synovium and cartilage, and the inhibition of ER stress could attenuate the IL-1β induced chondrocyte apoptosis. Administration of exogenous miR-486-5p could inhibit the ER stress, alleviate chondrocytes apoptosis and promote matrix regeneration. In comparison with direct administration of miR-486-5p and miR-486-5p overexpressing ADSCs, miR-486-5p modified exosomes indicated a better effect in modulating chondrocyte homeostasis. MiR-486-5p containing exosomes could also regulate macrophage polarization. Our IVIS imaging data validated that intraarticular injection of miR-486-5p containing exosomes could sustain for at least 7 days. MiR-486-5p containing exosomes showed a better effect on alleviating rats OA compared with direct administration of miR-486-5p and miR-486-5p overexpressing ADSCs. Our data demonstrated that miR-486-5p modified exosomes have a better effect on alleviating chondrocyte apoptosis and osteoarthritis. This study provides evidence of this efficient strategy of exosomal miRNA delivery and the miRNA-based therapy for OA.

Melatonin promotes sirtuin 1 expression and inhibits IRE1α–XBP1S–CHOP to reduce endoplasmic reticulum stress–mediated apoptosis in chondrocytes

Osteoarthritis (OA) is the most common chronic disease characterized by a loss of chondrocytes and the degeneration of cartilage. Inflammation plays an important role in the pathogenesis and progression of OA via the activation of the endoplasmic reticulum (ER) stress signaling pathway. In this study, we stimulated human primary chondrocytes with lipopolysaccharide (LPS) to reduce cell viability and induce chondrocyte apoptosis. LPS–stimulated human primary chondrocytes induced ER stress and significantly upregulated the ER chaperone glucose–regulated protein 78 (GRP78) and increased the expression level of C/EBP–homologous protein (CHOP), a key mediator of ER stress––induced apoptosis. Interestingly, melatonin treatment attenuated ER stress–mediated chondrocyte apoptosis. Melatonin inhibited the expression of cleaved caspase-3, cleaved caspase-10, Bax, CHOP, GRP78, cleaved caspase-4, phospho–inositol–requiring enzyme 1α (P-IRE1α), and spliced X-box-binding protein 1 (XBP1S). In an anterior cruciate ligament transection mouse model of OA, melatonin (50 and 150 mg/kg) dose–dependently relieved joint cartilage degeneration and inhibitied of chondrocyte apoptosis. Immunohistochemical analysis indicated that melatonin could promote SIRT1 the expression and inhibit CHOP and cleaved caspase-3 expression in OA mice. In conclusion, our findings demonstrate for the first time that melatonin inhibits the IRE1α-XBP1S-CHOP signaling pathway by promoting the expression of SIRT1 in LPS-treated human chondrocytes and delaying OA progression in vivo.