Tizoxanide as a novel theraputic candidate for osteoarthritis.

Osteoarthritis (OA) is a frequently seen degenerative joint disease in the elderly. Its pathogenesis is highly related to the local inflammatory reaction and autophagy. Tizoxanide (Tiz), the main active metabolite of nitazoxanide, has proved its anti-inflammatory properties in several diseases. However, the exact role of Tiz in OA remains to explore. In this study, we investigated the anti-arthritic effects and the underlying molecular mechanisms of Tiz on rat OA. The results showed that Tiz could attenuate the IL-1ß-induced inflammatory disorders, cartilage matrix damage and autophagy reduction in rat chondrocytes. Moreover, employment of autophagy inhibitor 3-methyladenine (3-MA) could antagonize the protective effects of Tiz in IL-1ß-treated rat chondrocytes. Additionally, Tiz also inhibited the IL-1ß-induced PI3K/AKT/mTOR and P38/JNK phosphorylation in chondrocytes. In vivo, intra-articular injection of Tiz could significantly alleviate the progression of cartilage damage in rat OA model. Briefly, our study demonstrated the therapeutic potential of Tiz in OA, suggesting that Tiz administration might serve as a promising strategy in OA therapy.

A comprehensive analysis of single-cell RNA transcriptome reveals unique SPP1+ chondrocytes in human osteoarthritis.

Osteoarthritis (OA) has become the most common degenerative disease in the world, which brings a serious economic burden to society and the country. Although epidemiological studies have shown that the occurrence of osteoarthritis is associated with obesity, sex, and trauma, the biomolecular mechanisms for the development and progression of osteoarthritis remain ambiguous. Several studies have drawn a connection between SPP1 and osteoarthritis. SPP1 was first found to be highly expressed in osteoarthritic cartilage, and later more studies have shown that SPP1 is also highly expressed in subchondral bone and synovial in OA patients. However, the biological function of SPP1 remains unclear. Single-cell RNA sequencing (scRNA-seq) is a novel technique that reflects gene expression at the cellular level, making it better depict the state of different cells than ordinary transcriptome data. However, most of the existing chondrocyte scRNA-seq studies focus on the occurrence and development of OA chondrocytes and lack analysis of normal chondrocyte development. Therefore, to better understand the mechanism of OA, scRNA-seq analysis of a larger cell volume containing normal and osteoarthritic cartilage is of great importance. Our study identifies a unique cluster of chondrocytes characterized by high SPP1 expression. The metabolic and biological characteristics of these clusters were further investigated. Besides, in animal models, we found that the expression of SPP1 is spatially heterogeneous in cartilage. Overall, our work provides novel insight into the potential role of SPP1 in OA, which sheds light on understanding the role of SPP1 in OA, promoting the progress of the treatment and prevention in the field of OA.

MCC950, the NLRP3 Inhibitor, Protects against Cartilage Degradation in a Mouse Model of Osteoarthritis.

Osteoarthritis (OA), characterized by chronic systemic low-level inflammation and cartilage degeneration, is a type of arthritis closely associated with aging. Inflammation and aging play a pivotal role in the occurrence and progression of OA. NLRP3 inflammasome is involved in many inflammatory and aging diseases, and NLRP3 inhibitor MCC950 has anti-inflammatory and antisenescence effects on some diseases such as Alzheimer's disease. In the present study, we found that NLRP3 protein was upregulated in human and mouse OA cartilage. Moreover, NLRP3 and Caspase1 expression induced by IL-1ß in chondrocytes was blocked by MCC950. In addition, MCC950 inhibited the expression of inflammatory mediators, matrix-degrading enzymes, senescence marker protein P16 (INK4A), and ß-galactosidase, as well as excessive production of ROS. Meanwhile, MCC950 promoted autophagy-related protein expression and autophagy flux under the inflammatory condition. However, autophagy inhibitor 3-MA reversed anti-inflammatory and anticatabolic effects of MCC950. In in vivo experiments, intra-articular administration of MCC950 further showed its protective effect on cartilage degeneration. Bioinformatic analysis and in vitro experimental results revealed that MCC950 might play a protective role in cartilage by regulating Nrf2/HO-1/NQO1, PI3k/Akt/mTOR, P38/MAPK, and JNK/MAPK pathways. In conclusion, our work demonstrated that NLRP3 inhibitor MCC950 might serve as a promising strategy for OA treatment.

Selective STAT3 Inhibitor Alantolactone Ameliorates Osteoarthritis via Regulating Chondrocyte Autophagy and Cartilage Homeostasis.

Osteoarthritis (OA), which is identified by chronic pain, impacts the quality of life. Cartilage degradation and inflammation are the most relevant aspects involved in its development. Signal transducer and activator of transcription 3(STAT3), a member of the STATs protein family, is associated with inflammation. Alantolactone (ALT), a sesquiterpene lactone compound, can selectively suppress the phosphorylation of STAT3. However, the pharmacological effect of ALT on OA is still imprecise. In this study, IL-1ß (10 ng/ml) was applied to cartilage chondrocytes, which were treated with different concentrations of Alantolactone for 24 h. The expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2(COX2), matrix metalloproteinases (MMPs) and thrombospondin motifs-5 (ADAMTS5) were detected by western blot. Protein expression of Collagen Ⅱ was observed by western blot, safranin O staining and immunofluorescence. Manifestation of autophagy related proteins such as autophagy-related gene-5 (ATG5), P62, LC3Ⅱ/Ⅰ and PI3K/AKT/mTOR-related signaling molecules were measured by western blot and autophagic flux monitored by confocal microscopy. Expression of STAT3 and NF-κB-related signaling molecules were evaluated by western blot and immunofluorescence. In vivo, 2 mg/kg ALT or equal bulk of vehicle was engaged in the destabilization of medial meniscus (DMM) mouse models by intra-articular injection, the degree of cartilage destruction was classified by Safranin O/Fast green staining. Our findings reported that the enhance of inflammatory factors containing iNOS, COX2, MMPs and ADAMTS5 induced by IL-1ß could be ameliorated by ALT. Additionally, the diminish of Collagen Ⅱ and autophagy which was stimulated by IL-1ß could be alleviated by ALT. Mechanistically, STAT3, NF-κB and PI3K/AKT/mTOR signal pathways might be involved in the effect of ALT on IL-1ß-induced mouse chondrocytes. In vivo, ALT protected cartilage in the DMM mouse model. Overall, this study illustrated that ALT attenuated IL-1ß-induced inflammatory responses, relieved cartilage degeneration and promoted impaired autophagy via restraining of STAT3 and NF-κB signal pathways, implying its auspicious therapeutical effect for OA.

Selonsertib Alleviates the Progression of Rat Osteoarthritis: An in vitro and in vivo Study.

Osteoarthritis (OA) is a prevalent degenerative joint disease. Its development is highly associated with inflammatory response and apoptosis in chondrocytes. Selonsertib (Ser), the inhibitor of Apoptosis Signal-regulated kinase-1 (ASK1), has exhibited multiple therapeutic effects in several diseases. However, the exact role of Ser in OA remains unclear. Herein, we investigated the anti-arthritic effects as well as the potential mechanism of Ser on rat OA. Our results showed that Ser could markedly prevent the IL-1ß-induced inflammatory reaction, cartilage degradation and cell apoptosis in rat chondrocytes. Meanwhile, the ASK1/P38/JNK and NFκB pathways were involved in the protective roles of Ser. Furthermore, intra-articular injection of Ser could significantly alleviate the surgery induced cartilage damage in rat OA model. In conclusion, our work provided insights into the therapeutic potential of Ser in OA, indicating that Ser might serve as a new avenue in OA treatment.

Rhoifolin Ameliorates Osteoarthritis via Regulating Autophagy.

Osteoarthritis (OA) is a common age-related joint disease. Its development has been generally thought to be associated with inflammation and autophagy. Rhoifolin (ROF), a flavanone extracted from Rhus succedanea, has exhibited prominent anti-oxidative and anti-inflammatory properties in several diseases. However the exact role of ROF in OA remains unclear. Here, we investigated the therapeutic effects as well as the underlying mechanism of ROF on rat OA. Our results indicated that ROF could significantly alleviate the IL-1ß-induced inflammatory responses, cartilage degradation, and autophagy downregulation in rat chondrocytes. Moreover, administration of autophagy inhibitor 3-methyladenine (3-MA) could reverse the anti-inflammatory and anti-cartilage degradation effects of ROF. Furthermore, P38/JNK and PI3K/AKT/mTOR signal pathways were involved in the protective effects of ROF. In vivo, intra-articular injection of ROF could notably ameliorate the cartilage damage in rat OA model. In conclusion, our work elucidated that ROF ameliorated rat OA via regulating autophagy, indicating the potential role of ROF in OA therapy.

Chondroprotective and antiarthritic effects of galangin in osteoarthritis: An in vitro and in vivo study.

Osteoarthritis (OA) is a common degenerative joint disease blamed for pain and disability in the elderly. Galangin (GAL) is a natural flavonoid that exhibits anti-inflammatory properties in various inflammation diseases. However, the role of GAL in OA remains unclear. In this study, we investigate the role of GAL in the progress and development of OA in vitro and vivo. The results showed that IL-1ß exposure resulted in increased expression of iNOS, COX-2, MMP1, MMP3, MMP13 and ADAMTS5 in rat chondrocytes. However, co-treatment with GAL significantly decreased theses inflammatory cytokines and catabolic factors expression. In addition, GAL reduced IL-1ß-induced degradation of collagen II and aggrecan in chondrocytes. Furthermore, GAL significantly suppressed IL-1ß-induced Akt phosphorylation and NF-κB activation in rat chondrocytes. In vivo, intra-articular injection of GAL could also reduce the cartilage degradation in the ACLT rat model. This study reveals galangin may act as a promising novel agent in the treatment of OA.

Tomatidine suppresses inflammation in primary articular chondrocytes and attenuates cartilage degradation in osteoarthritic rats.

In this study, we investigated whether the anti-inflammatory effects of tomatidine alleviate osteoarthritis (OA)-related pathology in primary articular chondrocytes and a rat OA model. STITCH database analysis identified 22 tomatidine-target genes that were enriched in 78 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Moreover,39 of the 105 OA-related KEGG pathways were related to tomatidine-target genes. The top two OA-related KEGG pathways with tomatidine-target genes were the MAPK and neutrophin signaling pathways. Pretreating primary chondrocytes with tomatidine suppressed interleukin-1ß (IL-1ß)-induced expression of iNOS, COX-2, MMP1, MMP3, MMP13, and ADAMTS-5. Tomatidine also suppressed IL-1ß-induced degradation of collagen-II and aggrecan proteins by inhibiting NF-κB and MAPK signaling. In a rat OA model, histological and immunohistochemical analyses showed significantly less cartilage degeneration in thetibiofemoral joints of rats treated for 12 weeks with tomatidine after OA induction (experimental group) than in untreated OA group rats. However, micro-computed tomography (µ-CT) showed that tomatidine did not affect remodeling of the subchondral bone at the tibial plateau. These data shows that tomatidine suppresses IL-1ß-induced inflammation in primary chondrocytes by inhibiting the NF-κB and MAPK signaling pathways, and protects against cartilage destruction in a rat OA model.

Neferine Inhibits Expression of Inflammatory Mediators and Matrix Degrading Enzymes in IL-1ß-Treated Rat Chondrocytes via Suppressing MAPK and NF-κB Signaling Pathways.

Osteoarthritis (OA), in which inflammation plays a crucial role, is the most common joint disease characterized by cartilage degradation. Neferine (Nef), a dibenzyl isoquinoline alkaloid, has shown its anti-inflammatory effects on other inflammatory diseases. Therefore, we hypothesized that Nef might also have an anti-inflammatory effect on OA and explored its effect on IL-1ß-treated rat chondrocytes. Sprague Dawley (SD) rat chondrocytes were stimulated with IL-1ß (10 ng/ml) and Nef (1, 5, and 10 µM) or IL-1ß (10 ng/ml) alone for 24 h. Expression of inducible nitric oxide synthase (iNOS), cyclooxygenase 2 (COX-2), matrix metalloproteinases (MMPs), and thrombospondin motifs-5 (ADAMTS5) was determined by quantitative real-time PCR and Western blotting. Expression of collagen II and aggrecan was examined by Western blotting, immunofluorescence, and safranin O staining. In addition, activation of MAPK and NF-κB signaling pathway was examined by Western blotting, and p65 nuclear translocation was evaluated by immunofluorescence. Nef reduced expression of inflammatory regulators (iNOS and COX-2) in IL-1ß-treated chondrocytes. Expression of IL-1ß-induced major catabolic enzymes (MMP3, MMP13, and ADAMTS5) was inhibited by Nef. Meanwhile, downregulation of collagen II and aggrecan expression was also ameliorated. Furthermore, Nef dampened abnormal activation of MAPK and NF-κB signaling pathway triggered by IL-1ß. Overall, the results above showed that Nef inhibited IL-1ß-induced excess production of inflammatory and catabolic factors in rat chondrocytes via inhibiting the MAPK and NF-κB pathways, suggesting a promising pharmacotherapy for OA.

Protective effects of Erdosteine on interleukin-1ß-stimulated inflammation via inhibiting the activation of MAPK, NF-κB, and Wnt/ß-catenin signaling pathways in rat osteoarthritis.

Osteoarthritis (OA), a degenerative arthropathy, is featured with progressive degradation of cartilage and a chondrocyte inflammatory response. Erdosteine (ER) showed the anti-oxidant properties and various anti-inflammatory effects in various diseases. However, whether it protects against OA remains unknown. In this study, we explore the potential therapeutic properties of ER on IL-1ß-stimulated rat chondrocytes and its underlying mechanism in vitro and vivo. Cell viability, pro-inflammatory cytokines and the degradation of ECM biomarkers were tested to determine the effects of ER at 10, 20, and 40 µM doses on IL-1ß-induced rat chondrocytes for 24 h in virto. In vivo, intra-articular injections of 50 µl of 100 mg/ml ER twice a week for 8 weeks. The results showed ER significantly suppressed the expressions of IL-1ß-induced the production of inflammatory factors in a dose-dependent pattern (4.30-fold decrease in COX-2, p < 0.05; 4.77-fold decrease in iNOS, p < 0.05 at 40 µM in protein levels). Moreover, ER could attenuate the degradation of ECM in IL-1ß-induced rat chondrocytes by repressing the expression of OA-related factors (2.40-fold decrease in ADAMTS-5, p < 0.05; 3.12-fold decrease in MMP1, p < 0.05; 3.97-fold decrease in MMP3, p < 0.05; and 2.62-fold decrease in MMP-13, p < 0.05 at 40 µM in protein levels). Furthermore, our study revealed that ER could inhibit the activations of IL-1ß-induced MAPK and Wnt/ß-catenin. Besides, ER could suppress the process of IL-1ß-induced P65 from the cytoplasm into the nucleus. In vivo, ER delaied the osteoarthritis progression in rat OA models. Collectively, ER might become a new therapeutic agent for OA.

p-Coumaric Acid Attenuates IL-1ß-Induced Inflammatory Responses and Cellular Senescence in Rat Chondrocytes.

Osteoarthritis (OA) is a common chronic inflammatory joint disease characterized by cartilage degradation. p-Coumaric acid (PCA), a dietary phenolic compound, has exerted anti-inflammatory and anti-oxidative activities in various diseases. However, the effects of PCA on OA have not been reported. In the present study, we aimed to investigate the effects of PCA on interleukin-1ß(IL-1ß)-induced inflammatory responses and cellular senescence in rat chondrocytes. Our results revealed that PCA remarkably downregulated IL-1ß-induced inflammatory factors such as COX2 and iNOS and cartilage-degrading enzymes like matrix metalloproteinases (MMP1, MMP3, and MMP13) and aggrecanases (ADAMTS4 and ADAMTS5) in chondrocytes. The IL-1ß-induced degradation of cartilage matrix (collagen II and aggrecan) could also be suppressed by PCA. Besides, PCA treatment effectively inhibited the IL-1ß-induced p16INK4a protein expression and SAß-gal activities in vitro. Mechanism analysis showed that PCA suppressed IL-1ß-induced activation of mitogen-activated protein kinase (MAPK) and nuclear factor-kappaB (NF-κB) pathways. In vivo, we also found that PCA could alleviate the development of OA in a rat model. Altogether, our findings implicate that p-coumaric acid attenuates IL-1ß-induced inflammatory responses and cellular senescence via inhibition of the MAPK and NF-κB signaling pathway in chondrocytes, and p-coumaric acid may be a promising candidate for the treatment of osteoarthritis.

Protease-activated receptor 2 (PAR-2) antagonist AZ3451 as a novel therapeutic agent for osteoarthritis.

Osteoarthritis (OA) is a highly prevalent joint disorder blamed for pain and disability in older individuals. It's commonly accepted that inflammation, apoptosis, autophagy and cellular senescence participate in the progress of OA. Protease activated receptor 2 (PAR2), a member of the G-protein coupled receptors, is involved in the regulation of various inflammation diseases. Previous studies have identified PAR2 as a potential therapeutic target for the treatment of OA. Here, we investigated the role of PAR2 antagonist AZ3451 in inflammation response, apoptosis, autophagy and cellular senescence during OA. We confirmed that PAR2 expression was significantly up-regulated in OA articular cartilage tissues as well as in interleukin 1ß (IL-1ß) stimulated chondrocytes. We demonstrated AZ3451 could prevent the IL-1ß-induced inflammation response, cartilage degradation and premature senescence in chondrocytes. Further study showed that AZ3451 attenuated chondrocytes apoptosis by activating autophagy in vitro. The P38/MAPK, NF-κB and PI3K/AKT/mTOR pathways were involved in the protective effect of AZ3451. In vivo, we found that intra-articular injection of AZ3451 could ameliorate the surgery induced cartilage degradation in rat OA model. Our work provided a better understanding of the mechanism of PAR2 in OA, and indicated that PAR2 antagonist AZ3451 might serve as a promising strategy for OA treatment.

NOV/CCN3 induces cartilage protection by inhibiting PI3K/AKT/mTOR pathway.

Osteoarthritis (OA), an age-related degenerative joint disease, is pathologically characterized by articular cartilage degeneration and synovial inflammation. Nephroblastoma overexpressed (NOV or CCN3), a matricellular protein, is a primary member of the CCN family (Cyr61, Ctgf, NOV) of proteins and is involved in various inflammatory disorders. Previous studies reported that CCN3 might play a therapeutic role in OA. However, the underlying mechanism remains unclear. In this study, we confirmed the expression of CCN3 was decreased in human and rat OA articular cartilage. Recombinant CCN3 ameliorated the IL-1ß-induced matrix catabolism, as demonstrated by MMP1, MMP3, MMP13, ADAMTS5 and iNOS expression, in vitro. In addition, the degradation of cartilage matrix such as collagen 2 and aggrecan could be reversed by CCN3. Furthermore, we found CCN3 promoted autophagy as Atg5, Beclin1 and LC3-II expression were increased. High-mobility group box 1 was negatively correlated with CCN3 in IL-1ß-induced osteoarthritis responses, and HMGB1 is involved in the protective effect of CCN3 in OA. Moreover, CCN3 overexpression decreased the expression of HMGB1 and reversed the IL-1ß induced MMPs production. Additionally, recombinant CCN3 or CCN3 overexpression attenuated the activation of PI3K/AKT/mTOR pathway induced by IL-1ß. Our study presents new mechanisms of CCN3 in osteoarthritis and indicates that CCN3 can serve as a novel potential therapeutic target for osteoarthritis.

Vanillic acid attenuates cartilage degeneration by regulating the MAPK and PI3K/AKT/NF-κB pathways.

Osteoarthritis (OA) is a frequently seen arthropathy that features cartilage loss and destruction. Vanillic acid (VA), is a well-known flavonoid, which possesses various pharmacological activities. However, the effects of Vanillic acid on articular cartilage destruction and the pathogenesis of OA remain unknown. The present study observed that VA attenuated OA progression in vivo and vitro. VA inhibited the expression of inflammation responses, including cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), matrix metalloproteinases (MMPs) and aggrecanase -2(ADAMTS5). Moreover, the major markers of hypertrophic chondrocytes such as Collagen X, Runt related transcription factor 2 (Runx2) and Vascular endothelial growth factor (VEGFA) were also reduced by VA. In addition, the interleukin-1ß (IL-1ß) -stimulated collagen 2 and aggrecan destruction were suppressed by VA. Moreover, VA concentration -dependently inhibited IL-1ß induced production of High-mobility group box 1 (HMGB1), a classic damage-associated molecular pattern (DAMP) molecule with strong pro-inflammatory effects in OA. Meanwhile, we revealed that VA suppressed the IL-1ß induced activation of MAPK and PI3K/AKT/NF-κB pathways. In vivo, VA alleviated osteoarthritis progression in a rat OA model. Collectively, our results demonstrate that VA could potentially be a new candidate for OA therapy.