XJB-5-131 protects chondrocytes from ferroptosis to alleviate osteoarthritis progression via restoring Pebp1 expression.

Background: Osteoarthritis (OA) is the most common age-related musculoskeletal disease. However, there is still a lack of therapy that can modify OA progression due to the complex pathogenic mechanisms. The aim of the study was to explore the role and mechanism of XJB-5-131 inhibiting chondrocytes ferroptosis to alleviate OA progression. Methods: We treated tert-butyl hydroperoxide (TBHP)-induced ferroptosis of mouse primary chondrocytes with XJB-5-131 in vitro. The intracellular ferroptotic hallmarks, cartilage anabolic and catabolic markers, ferroptosis regulatory genes and proteins were detected. Then we established a mouse OA model via destabilization of the medial meniscus (DMM) surgery. The OA mice were treated with intra-articular injection of XJB-5-131 regularly (2 µM, 3 times per week). After 4 and 8 weeks, we performed micro-CT and histological examination to evaluate the protection role of XJB-5-131 in mouse OA subjects. RNA sequencing analysis was performed to unveil the key downstream gene of XJB-5-131 exerting the anti-ferroptotic effect in OA. Results: XJB-5-131 significantly suppressed TBHP-induced increases of ferroptotic hallmarks (ROS, lipid peroxidation, and Fe2+ accumulation), ferroptotic drivers (Ptgs2, Pgd, Tfrc, Atf3, Cdo1), while restored the expression of ferroptotic suppressors (Gpx4, Fth1). XJB-5-131 evidently promoted the expression of cartilage anabolic and decreased the expression of cartilage catabolic markers. Moreover, intra-articular injection of XJB-5-131 significantly inhibited the expression of Cox2 and Mmp13, while promoted the expression of Col2a1, Gpx4 and Fth1 in DMM-induced mouse articular cartilage. Further, we identified Pebp1 as a potential target of XJB-5-131 by RNA sequencing analysis. The anti-ferroptosis and chondroprotective effects of XJB-5-131 were significantly diminished by Locostatin, a specific antagonist of Pebp1. Conclusion: XJB-5-131 significantly protects chondrocytes from ferroptosis in TBHP-induced mouse primary chondrocytes and DMM surgery-induced OA mice model via restoring the expression of Pebp1. XJB-5-131 is a potential therapeutic drug in the management of OA progression.

Near Infrared Responsive Gold Nanorods Attenuate Osteoarthritis Progression by Targeting TRPV1.

Osteoarthritis (OA) is the most common degenerative joint disease worldwide, with the main pathological manifestation of articular cartilage degeneration. It have been investigated that pharmacological activation of transient receptor potential vanilloid 1 (TRPV1) significantly alleviated cartilage degeneration by abolishing chondrocyte ferroptosis. In this work, in view of the thermal activated feature of TRPV1, Citrate-stabilized gold nanorods (Cit-AuNRs) is conjugated to TRPV1 monoclonal antibody (Cit-AuNRs@Anti-TRPV1) as a photothermal switch for TRPV1 activation in chondrocytes under near infrared (NIR) irradiation. The conjugation of TRPV1 monoclonal antibody barely affect the morphology and physicochemical properties of Cit-AuNRs. Under NIR irradiation, Cit-AuNRs@Anti-TRPV1 exhibited good biocompatibility and flexible photothermal responsiveness. Intra-articular injection of Cit-AuNRs@Anti-TRPV1 followed by NIR irradiation significantly activated TRPV1 and attenuated cartilage degradation by suppressing chondrocytes ferroptosis. The osteophyte formation and subchondral bone sclerosis are remarkably alleviated by NIR-inspired Cit-AuNRs@Anti-TRPV1. Furthermore, the activation of TRPV1 by Cit-AuNRs@Anti-TRPV1 evidently improved physical activities and alleviated pain of destabilization of the medial meniscus (DMM)-induced OA mice. The study reveals Cit-AuNRs@Anti-TRPV1 under NIR irradiation protects chondrocytes from ferroptosis and attenuates OA progression, providing a potential therapeutic strategy for the treatment of OA.

Bifunctional TRPV1 Targeted Magnetothermal Switch to Attenuate Osteoarthritis Progression.

Transient receptor potential vanilloid family member 1 (TRPV1) has been revealed as a therapeutic target of osteoarthritis (OA), the most common deteriorating whole joint disease, by impeding macrophagic inflammation and chondrocytes ferroptosis. However, the clinical application for capsaicin as the TRPV1 agonist is largely limited by its chronic toxicity. To address this issue, we developed a bifunctional controllable magnetothermal switch targeting TRPV1 for the alleviation of OA progression by coupling of magnetic nanoparticles (MNPs) to TRPV1 monoclonal antibodies (MNPs-TRPV1). Under the alternating magnetic field (AMF) stimulation, MNPs-TRPV1 locally dissipated heat, which was sufficient to trigger the opening and activation of TRPV1, and effectively impeded macrophagic inflammation and chondrocyte ferroptosis. This magnetothermal modulation of TRPV1 simultaneously attenuated synovitis and cartilage degeneration in mice incurred by destabilization of medial meniscus surgery, indicating the delayed OA progression. Furthermore, MNPs-TRPV1 with AMF exposure remarkably reduced knee pain sensitivity, alleviated the crippled gait, and improved spontaneous ambulatory activity performance in the mice OA model. Overall, this work provides a potential pathogenesis-based precise OA therapy with temporally and spatially magnetothermal modulation of TRPV1 in a controllable manner.

Microtubule stabilization targeting regenerative chondrocyte cluster for cartilage regeneration.

Purpose: Chondrocytes (CHs) in cartilage undergo several detrimental events during the development of osteoarthritis (OA). However, the mechanism underlying CHs regeneration involved in pathogenesis is largely unknown. The aim of this study was to explore the underlying mechanism of regeneration of CHs involved in the pathological condition and the potential therapeutic strategies of cartilage repair. Methods and Materials: CHs were isolated from human cartilage in different OA stages and the high-resolution cellular architecture of human osteoarthritis was examined by applying single-cell RNA sequencing. The analysis of gene differential expression and gene set enrichment was utilized to reveal the relationship of cartilage regeneration and microtubule stabilization. Microtubule destabilizer (nocodazole) and microtubule stabilizer (docetaxel) treated-human primary CHs and rats cartilage defect model were used to investing the effects and downstream signaling pathway of microtubule stabilization on cartilage regeneration. Results: CHs subpopulations were identified on the basis of their gene markers and the data indicated an imbalance caused by an increase in the degeneration and disruption of CHs regeneration in OA samples. Interestingly, the CHs subpopulation namely CHI3L1+ CHs, was characterized by the cell regenerative capacity, stem cell potency and the activated microtubule (MT) process. Furthermore, the data indicated that MT stabilization was effective in promoting cartilage regeneration in rats with cartilage injury model by inhibiting YAP activity. Conclusion: These findings lead to a new understanding of CHs regeneration in the OA pathophysiology context and suggest that MT stabilization is a promising therapeutic target for OA and cartilage injury.

Molecular crosstalk between articular cartilage, meniscus, synovium, and subchondral bone in osteoarthritis.

AIMS: Osteoarthritis (OA) is a common degenerative joint disease worldwide, which is characterized by articular cartilage lesions. With more understanding of the disease, OA is considered to be a disorder of the whole joint. However, molecular communication within and between tissues during the disease process is still unclear. In this study, we used transcriptome data to reveal crosstalk between different tissues in OA. METHODS: We used four groups of transcription profiles acquired from the Gene Expression Omnibus database, including articular cartilage, meniscus, synovium, and subchondral bone, to screen differentially expressed genes during OA. Potential crosstalk between tissues was depicted by ligand-receptor pairs. RESULTS: During OA, there were 626, 97, 1,060, and 2,330 differentially expressed genes in articular cartilage, meniscus, synovium, and subchondral bone, respectively. Gene Ontology enrichment revealed that these genes were enriched in extracellular matrix and structure organization, ossification, neutrophil degranulation, and activation at different degrees. Through ligand-receptor pairing and proteome of OA synovial fluid, we predicted ligand-receptor interactions and constructed a crosstalk atlas of the whole joint. Several interactions were reproduced by transwell experiment in chondrocytes and synovial cells, including TNC-NT5E, TNC-SDC4, FN1-ITGA5, and FN1-NT5E. After lipopolysaccharide (LPS) or interleukin (IL)-1ß stimulation, the ligand expression of chondrocytes and synovial cells was upregulated, and corresponding receptors of co-culture cells were also upregulated. CONCLUSION: Each tissue displayed a different expression pattern in transcriptome, demonstrating their specific roles in OA. We highlighted tissue molecular crosstalk through ligand-receptor pairs in OA pathophysiology, and generated a crosstalk atlas. Strategies to interfere with these candidate ligands and receptors may help to discover molecular targets for future OA therapy.Cite this article: Bone Joint Res 2022;11(12):862-872.

Blocking TRPV4 Ameliorates Osteoarthritis by Inhibiting M1 Macrophage Polarization via the ROS/NLRP3 Signaling Pathway.

Osteoarthritis (OA) is a low-level inflammatory disease in which synovial macrophage M1 polarization exacerbates the progression of synovitis and OA. Notedly, the ROS (reactive oxygen species) level in macrophages is intimately implicated in macrophage M1 polarization. TRPV4 (transient receptor potential channel subfamily V member 4), as an ion channel, plays a pivotal role in oxidative stress and inflammation. In this study, we investigated the role of TRPV4 in OA progression and M1 macrophage polarization. Male adult Sprague-Dawley (SD) rats underwent a medial meniscus radial transection operation to create an OA model in vivo and RAW 264.7 cells were intervened with 100 ng/mL LPS (lipopolysaccharide) to induce M1-polarized macrophages in vitro. We demonstrated that the infiltration of M1 synovial macrophages and the expression of TRPV4 were increased significantly in OA synovium. In addition, intra-articular injection of HC067074 (a specific inhibitor of TRPV4) alleviated the progression of rat OA and significantly decreased synovial macrophage M1 polarization. Further mechanisms suggested that ROS production by M1 macrophages was decreased after TRPV4 inhibition. In addition, NLRP3 (pyrin domain containing protein 3) as a downstream effector of ROS in M1-polarized macrophage, was significantly suppressed following TRPV4 inhibition. In conclusion, this study discovered that inhibition of TRPV4 delays OA progression by inhibiting M1 synovial macrophage polarization through the ROS/NLRP3 pathway.

Articular fibrocartilage-targeted therapy by microtubule stabilization.

The fibrocartilage presented on the joint surface was caused by cartilage injury or degeneration. There is still a lack of effective strategies for fibrocartilage. Here, we hypothesized that the fibrocartilage could be viewed as a raw material for the renewal of hyaline cartilage and proposed a previously unidentified strategy of cartilage regeneration, namely, "fibrocartilage hyalinization." Cytoskeleton remodeling plays a vital role in modifying the cellular phenotype. We identified that microtubule stabilization by docetaxel repressed cartilage fibrosis and increased the hyaline cartilage extracellular matrix. We further designed a fibrocartilage-targeted negatively charged thermosensitive hydrogel for the sustained delivery of docetaxel, which promoted fibrocartilage hyalinization in the cartilage defect model. Moreover, the mechanism of fibrocartilage hyalinization by microtubule stabilization was verified as the inhibition of Sparc (secreted protein acidic and rich in cysteine). Together, our study suggested that articular fibrocartilage-targeted therapy in situ was a promising strategy for hyaline cartilage repair.

Targeting macrophagic SHP2 for ameliorating osteoarthritis via TLR signaling.

Osteoarthritis (OA), in which M1 macrophage polarization in the synovium exacerbates disease progression, is a major cause of cartilage degeneration and functional disabilities. Therapeutic strategies of OA designed to interfere with the polarization of macrophages have rarely been reported. Here, we report that SHP099, as an allosteric inhibitor of src-homology 2-containing protein tyrosine phosphatase 2 (SHP2), attenuated osteoarthritis progression by inhibiting M1 macrophage polarization. We demonstrated that M1 macrophage polarization was accompanied by the overexpression of SHP2 in the synovial tissues of OA patients and OA model mice. Compared to wild-type (WT) mice, myeloid lineage conditional Shp2 knockout (cKO) mice showed decreased M1 macrophage polarization and attenuated severity of synovitis, an elevated expression of cartilage phenotype protein collagen II (COL2), and a decreased expression of cartilage degradation markers collagen X (COL10) and matrix metalloproteinase 3 (MMP3) in OA cartilage. Further mechanistic analysis showed thatSHP099 inhibited lipopolysaccharide (LPS)-induced Toll-like receptor (TLR) signaling mediated by nuclear factor kappa B (NF-κB) and PI3K-AKT signaling. Moreover, intra-articular injection of SHP099 also significantly attenuated OA progression, including joint synovitis and cartilage damage. These results indicated that allosteric inhibition of SHP2 might be a promising therapeutic strategy for the treatment of OA.

Intraarticular injection of SHP2 inhibitor SHP099 promotes the repair of rabbit full-thickness cartilage defect.

BACKGROUND: Cartilage repair has been a challenge in the field of orthopaedics for decades, highlighting the significance of investigating potential therapeutic drugs. In this study, we explored the effect of the SHP2 inhibitor SHP099, a small-molecule drug, on cartilage repair. METHODS: Human synovial mesenchymal stem cells (SMSCs) were isolated, and their three-way differentiation potential was examined. After treatment with chondrogenic medium, the chondrogenic effect of SHP099 on SMSCs was examined by western blot, qPCR, and immunofluorescence (IF). Micro-mass culture was also used to detect the effect of SHP099. To explore the chondrogenic effects of SHP099 in vivo, full-thickness cartilage defects with microfractures were constructed in the right femoral trochlea of New Zealand White rabbits. Intraarticular injection of SHP099 or normal saline was performed twice a week for 6 weeks. Cartilage repair was evaluated by haematoxylin and eosin (HE) staining and safranin O/fast green staining. Immunohistochemistry (IHC) for collagen II (COL2) was also conducted to verify the abundance of cartilage extracellular matrix after SHP099 treatment. The mechanism involving yes-associated protein (YAP) and WNT signalling was investigated in vitro. RESULTS: SMSCs isolated from human synovium have optimal multi-differentiation potential. SHP099 increased chondrogenic marker (SOX9, COL2) expression and decreased hypertrophic marker (COL10, RUNX2) expression in SMSCs. In micro-mass culture, the SHP099-induced cartilage tissues had a better result of Safranin O and Toluidine blue staining and are enriched in cartilage-specific collagen II. Inhibition of YAP and WNT signalling was also observed. Moreover, compared to the normal saline group at 6 weeks, intraarticular injection of SHP099 resulted in better defect filling, forming increased hyaline cartilage-like tissue with higher levels of glycosaminoglycan (GAG) and COL2. CONCLUSION: SHP099 promotes the repair of rabbit full-thickness cartilage defects, representing a potential therapeutic drug for cartilage repair. THE TRANSLATIONAL POTENTIAL OF THIS ARTICLE: This study provides evidence that SHP2 inhibition promotes chondrogenesis and the repair of cartilage in defect area, which could be a novel therapeutic approach for cartilage repair.

Identification of Transcription Factor Networks during Mouse Hindlimb Development.

Mammalian hindlimb development involves a variety of cells and the regulation of spatiotemporal molecular events, but regulatory networks of transcription factors contributing to hindlimb morphogenesis are not well understood. Here, we identified transcription factor networks during mouse hindlimb morphology establishment through transcriptome analysis. We used four stages of embryonic hindlimb transcription profiles acquired from the Gene Expression Omnibus database (GSE30138), including E10.5, E11.5, E12.5 and E13.5, to construct a gene network using Weighted Gene Co-expression Network Analysis (WGCNA), and defined seven stage-associated modules. After filtering 7625 hub genes, we further prioritized 555 transcription factors with AnimalTFDB3.0. Gene ontology enrichment showed that transcription factors of different modules were enriched in muscle tissue development, connective tissue development, embryonic organ development, skeletal system morphogenesis, pattern specification process and urogenital system development separately. Six regulatory networks were constructed with key transcription factors, which contribute to the development of different tissues. Knockdown of four transcription factors from regulatory networks, including Sox9, Twist1, Snai2 and Klf4, showed that the expression of limb-development-related genes was also inhibited, which indicated the crucial role of transcription factor networks in hindlimb development.

Microtubule Stabilization Enhances the Chondrogenesis of Synovial Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) are well known for their multi-directional differentiation potential and are widely applied in cartilage and bone disease. Synovial mesenchymal stem cells (SMSCs) exhibit a high proliferation rate, low immunogenicity, and greater chondrogenic differentiation potential. Microtubule (MT) plays a key role in various cellular processes. Perturbation of MT stability and their associated proteins is an underlying cause for diseases. Little is known about the role of MT stabilization in the differentiation and homeostasis of SMSCs. In this study, we demonstrated that MT stabilization via docetaxel treatment had a significant effect on enhancing the chondrogenic differentiation of SMSCs. MT stabilization inhibited the expression of Yes-associated proteins (YAP) and the formation of primary cilia in SMSCs to drive chondrogenesis. This finding suggested that MT stabilization might be a promising therapeutic target of cartilage regeneration.

Antiviral activity of PHA767491 against human herpes simplex virus in vitro and in vivo.

BACKGROUND: Herpes simplex virus (HSV) is a common human pathogen that causes a variety of diseases, including oral-labial, genital lesions and life-threatening encephalitis. The antiviral nucleoside analogues such as acyclovir are currently used in anti-HSV therapies; however, clinical overuse of these drugs has led to the emergence of drug-resistant viral strains. Hence, there is an urgent need to develop new anti-HSV agents. METHODS: To identify novel anti-HSV-1 compounds, we screened the LOPAC small scale library of 1280 bioactive compounds to identify inhibitors of HSV-1-induced necroptosis. Further experiments including western blot analysis, Q-PCR analysis and immunohistochemistry were performed to explore the antiviral mechanism of the compounds. RESULTS: Here, we identified PHA767491 as a new inhibitor of HSV. PHA767491 potently blocked the proliferation of HSV in cells, as well as HSV induced cell death. Further, we found that PHA767491 strongly inhibited HSV infection post viral entry. Moreover, PHA767491 reduced the expression of viral genes required for DNA synthesis including UL30/42 DNA polymerase and UL5/8/52 helicase-primase complex. The essential immediate early (IE) genes such as ICP4 and ICP27 are critical for the expression of the early and late genes. Of note, PHA767491 inhibited the expression of all IE genes of both HSV-1 and HSV-2. Importantly, PHA767491 reduced viral titers in the tissues from the mice infected with HSV-1. Consistently, immunohistochemistry analysis showed that PHA767491 dramatically attenuated expression of viral protein gB in the livers. CONCLUSIONS: Taken together, PHA767491 has potent anti-HSV activity by inhibiting viral replication both in vitro and in mouse model. Thus, PHA767491 could be a promising agent for the development of new anti-HSV therapy.