Integrated Network Pharmacology and Experimental Validation Approach to Investigate the Mechanisms of Radix Rehmanniae Praeparata - Angelica Sinensis - Radix Achyranthis Bidentatae in Treating Knee Osteoarthritis.

Background: Knee osteoarthritis (KOA) is a persistent degenerative condition characterized by the deterioration of cartilage. The Chinese herbal formula Radix Rehmanniae Praeparata- Angelica Sinensis-Radix Achyranthis Bidentatae (RAR) has often been used in effective prescriptions for KOA as the main functional drug, but its underlying mechanism remains unclear. Therefore, network pharmacology and verification experiments were employed to investigate the impact and mode of action of RAR in the treatment of KOA. Methods: The destabilization of the medial meniscus model (DMM) was utilized to assess the anti-KOA effect of RAR by using gait analysis, micro-computed tomography (Micro-CT), and histology. Primary chondrocytes were extracted from the rib cartilage of a newborn mouse. The protective effects of RAR on OA cells were evaluated using a CCK-8 assay. The antioxidative effect of RAR was determined by measuring reactive oxygen species (ROS), superoxide dismutase (SOD), and glutathione (GSH) production. Furthermore, network pharmacology and molecular docking were utilized to propose possible RAR targets for KOA, which were further verified through experiments. Results: In vivo, RAR significantly ameliorated DMM-induced KOA characteristics, such as subchondral bone sclerosis, cartilage deterioration, gait abnormalities, and the degree of knee swelling. In vitro, RAR stimulated chondrocyte proliferation and the expression of Col2a1, Comp, and Acan. Moreover, RAR treatment significantly reduced ROS accumulation in an OA cell model induced by IL-1ß and increased the activity of antioxidant enzymes (SOD and GSH). Network pharmacology analysis combined with molecular docking showed that Mapk1 might be a key therapeutic target. Subsequent research showed that RAR could downregulate Mapk1 mRNA levels in IL-1ß-induced chondrocytes and DMM-induced rats. Conclusion: RAR inhibited extracellular matrix (ECM) degradation and oxidative stress response via the MAPK signaling pathway in KOA, and Mapk1 may be a core target.

Sericin promotes chondrogenic proliferation and differentiation via glycolysis and Smad2/3 TGF-ß signaling inductions and alleviates inflammation in three-dimensional models.

Knee osteoarthritis is a chronic joint disease mainly characterized by cartilage degeneration. The treatment is challenging due to the lack of blood vessels and nerve supplies in cartilaginous tissue, causing a prominent limitation of regenerative capacity. Hence, we investigated the cellular promotional and anti-inflammatory effects of sericin, Bombyx mori-derived protein, on three-dimensional chondrogenic ATDC5 cell models. The results revealed that a high concentration of sericin promoted chondrogenic proliferation and differentiation and enhanced matrix production through the increment of glycosaminoglycans, COL2A1, COL X, and ALP expressions. SOX-9 and COL2A1 gene expressions were notably elevated in sericin treatment. The proteomic analysis demonstrated the upregulation of phosphoglycerate mutase 1 and triosephosphate isomerase, a glycolytic enzyme member, reflecting the proliferative enhancement of sericin. The differentiation capacity of sericin was indicated by the increased expressions of procollagen12a1, collagen10a1, rab1A, periostin, galectin-1, and collagen6a3 proteins. Sericin influenced the differentiation capacity via the TGF-ß signaling pathway by upregulating Smad2 and Smad3 while downregulating Smad1, BMP2, and BMP4. Importantly, sericin exhibited an anti-inflammatory effect by reducing IL-1ß, TNF-α, and MMP-1 expressions and accelerating COL2A1 production in the early inflammatory stage. In conclusion, sericin demonstrates potential in promoting chondrogenic proliferation and differentiation, enhancing cartilaginous matrix synthesis through glycolysis and TGF-ß signaling pathways, and exhibiting anti-inflammatory properties.

Mume Fructus reduces interleukin-1 beta-induced cartilage degradation via MAPK downregulation in rat articular chondrocytes.

Osteoarthritis is the most prevalent type of degenerative arthritis. It is characterized by persistent pain, joint dysfunction, and physical disability. Pain relief and inflammation control are prioritised during osteoarthritis treatment Mume Fructus (Omae), a fumigated product of the Prunus mume fruit, is used as a traditional medicine in several Asian countries. However, its therapeutic mechanism of action and effects on osteoarthritis and articular chondrocytes remain unknown. In this study, we analyzed the anti-osteoarthritis and articular regenerative effects of Mume Fructus extract on rat chondrocytes. Mume Fructus treatment reduced the interleukin-1ß-induced expression of matrix metalloproteinase 3, matrix metalloproteinase 13, and a disintegrin and metalloproteinase with thrombospondin type 1 motifs 5. Additionally, it enhanced collagen type II alpha 1 chain and aggrecan accumulation in rat chondrocytes. Furthermore, Mume Fructus treatment regulated the inflammatory cytokine levels, mitogen-activated protein kinase phosphorylation, and nuclear factor-kappa B activation. Overall, our results demonstrated that Mume Fructus inhibits osteoarthritis progression by inhibiting the nuclear factor-kappa B and mitogen-activated protein kinase pathways to reduce the levels of inflammatory cytokines and prevent cartilage degeneration. Therefore, Mume Fructus may be a potential therapeutic option for osteoarthritis.

TGF-ß2 Induces Ribosome Activity, Alters Ribosome Composition and Inhibits IRES-Mediated Translation in Chondrocytes.

Alterations in cell fate are often attributed to (epigenetic) regulation of gene expression. An emerging paradigm focuses on specialized ribosomes within a cell. However, little evidence exists for the dynamic regulation of ribosome composition and function. Here, we stimulated a chondrocytic cell line with transforming growth factor beta (TGF-ß2) and mapped changes in ribosome function, composition and ribosomal RNA (rRNA) epitranscriptomics. 35S Met/Cys incorporation was used to evaluate ribosome activity. Dual luciferase reporter assays were used to assess ribosomal modus. Ribosomal RNA expression and processing were determined by RT-qPCR, while RiboMethSeq and HydraPsiSeq were used to determine rRNA modification profiles. Label-free protein quantification of total cell lysates, isolated ribosomes and secreted proteins was done by LC-MS/MS. A three-day TGF-ß2 stimulation induced total protein synthesis in SW1353 chondrocytic cells and human articular chondrocytes. Specifically, TGF-ß2 induced cap-mediated protein synthesis, while IRES-mediated translation was not (P53 IRES) or little affected (CrPv IGR and HCV IRES). Three rRNA post-transcriptional modifications (PTMs) were affected by TGF-ß2 stimulation (18S-Gm1447 downregulated, 18S-ψ1177 and 28S-ψ4598 upregulated). Proteomic analysis of isolated ribosomes revealed increased interaction with eIF2 and tRNA ligases and decreased association of eIF4A3 and heterogeneous nuclear ribonucleoprotein (HNRNP)s. In addition, thirteen core ribosomal proteins were more present in ribosomes from TGF-ß2 stimulated cells, albeit with a modest fold change. A prolonged stimulation of chondrocytic cells with TGF-ß2 induced ribosome activity and changed the mode of translation. These functional changes could be coupled to alterations in accessory proteins in the ribosomal proteome.

TPX2 upregulates MMP13 to promote the progression of lipopolysaccharide-induced osteoarthritis.

Purpose: This study seeks to identify potential clinical biomarkers for osteoarthritis (OA) using bioinformatics and investigate OA mechanisms through cellular assays. Methods: Differentially Expressed Genes (DEGs) from GSE52042 (four OA samples, four control samples) were screened and analyzed with protein-protein interaction (PPI) analysis. Overlapping genes in GSE52042 and GSE206848 (seven OA samples, and seven control samples) were identified and evaluated using Gene Set Enrichment Analysis (GSEA) and clinical diagnostic value analysis to determine the hub gene. Finally, whether and how the hub gene impacts LPS-induced OA progression was explored by in vitro experiments, including Western blotting (WB), co-immunoprecipitation (Co-IP), flow cytometry, etc. Result: Bioinformatics analysis of DEGs (142 up-regulated and 171 down-regulated) in GSE52042 identified two overlapping genes (U2AF2, TPX2) that exhibit significant clinical diagnostic value. These genes are up-regulated in OA samples from both GSE52042 and GSE206848 datasets. Notably, TPX2, which AUC = 0.873 was identified as the hub gene. In vitro experiments have demonstrated that silencing TPX2 can alleviate damage to chondrocytes induced by lipopolysaccharide (LPS). Furthermore, there is a protein interaction between TPX2 and MMP13 in OA. Excessive MMP13 can attenuate the effects of TPX2 knockdown on LPS-induced changes in OA protein expression, cell growth, and apoptosis. Conclusion: In conclusion, our findings shed light on the molecular mechanisms of OA and suggested TPX2 as a potential therapeutic target. TPX2 could promote the progression of LPS-induced OA by up-regulating the expression of MMP13, which provides some implications for clinical research.

Botulinum toxin A attenuates osteoarthritis development via inhibiting chondrocyte ferroptosis through SLC7Al1/GPX4 axis.

Osteoarthritis (OA) is a prevalent joint degenerative disease, resulting in a significant societal burden. However, there is currently a lack of effective treatment option available. Previous studies have suggested that Botulinum toxin A (BONT/A), a macromolecular protein extracted from Clostridium Botulinum, may improve the pain and joint function in OA patients, but the mechanism remains elusive. This study was to investigate the impact and potential mechanism of BONT/A on OA in vivo and in vitro experiment. LPS increased the levels of ROS, Fe2+and Fe3+, as well as decreased GSH levels, the ratio of GSH / GSSH and mitochondrial membrane potential. It also enhanced the degeneration of extracellular matrix (ECM) and altered the ferroptosis-related protein expression in chondrocytes. BONT/A rescued LPS-induced decrease in collagen type II (Collagen II) expression and increase in matrix metalloproteinase 13 (MMP13), mitigated LPS-induced cytotoxicity in chondrocytes, abolished the accumulation of ROS and iron, upregulated GSH and the ratio of GSH/ GSSH, improved mitochondrial function, and promoted SLC7A11/GPX4 anti-ferroptosis system activation. Additionally, intra-articular injection of BONT/A inhibited the degradation of cartilage in OA model rats. This chondroprotective effect of BONT/A was reversed by erastin (a classical ferroptosis agonist) and enhanced by liproxstatin-1 (a classic ferroptosis inhibitor). Our research confirms that BONT/A alleviates the OA development by inhibiting the ferroptosis of chondrocytes, which revealed to be a potential therapeutic mechanism for BONT/A treating the OA.

Glucosamine and Silibinin Alter Cartilage Homeostasis through Glycosylation and Cellular Stresses in Human Chondrocyte Cells.

Osteoarthritis is more prevalent than any other form of arthritis and is characterized by the progressive mechanical deterioration of joints. Glucosamine, an amino monosaccharide, has been used for over fifty years as a dietary supplement to alleviate osteoarthritis-related discomfort. Silibinin, extracted from milk thistle, modifies the degree of glycosylation of target proteins, making it an essential component in the treatment of various diseases. In this study, we aimed to investigate the functional roles of glucosamine and silibinin in cartilage homeostasis using the TC28a2 cell line. Western blots showed that glucosamine suppressed the N-glycosylation of the gp130, EGFR, and N-cadherin proteins. Furthermore, both glucosamine and silibinin differentially decreased and increased target proteins such as gp130, Snail, and KLF4 in TC28a2 cells. We observed that both compounds dose-dependently induced the proliferation of TC28a2 cells. Our MitoSOX and DCFH-DA dye data showed that 1 µM glucosamine suppressed mitochondrial reactive oxygen species (ROS) generation and induced cytosol ROS generation, whereas silibinin induced both mitochondrial and cytosol ROS generation in TC28a2 cells. Our JC-1 data showed that glucosamine increased red aggregates, resulting in an increase in the red/green fluorescence intensity ratio, while all the tested silibinin concentrations increased the green monomers, resulting in decreases in the red/green ratio. We observed increasing subG1 and S populations and decreasing G1 and G2/M populations with increasing amounts of glucosamine, while increasing amounts of silibinin led to increases in subG1, S, and G2/M populations and decreases in G1 populations in TC28a2 cells. MTT data showed that both glucosamine and silibinin induced cytotoxicity in TC28a2 cells in a dose-dependent manner. Regarding endoplasmic reticulum stress, both compounds induced the expression of CHOP and increased the level of p-eIF2α/eIF2α. With respect to O-GlcNAcylation status, glucosamine and silibinin both reduced the levels of O-GlcNAc transferase and hypoxia-inducible factor 1 alpha. Furthermore, we examined proteins and mRNAs related to these processes. In summary, our findings demonstrated that these compounds differentially modulated cellular proliferation, mitochondrial and cytosol ROS generation, the mitochondrial membrane potential, the cell cycle profile, and autophagy. Therefore, we conclude that glucosamine and silibinin not only mediate glycosylation modifications but also regulate cellular processes in human chondrocytes.

Thyroid hormone induces ossification and terminal maturation in a preserved OA cartilage biomimetic model.

OBJECTIVE: To characterize aspects of triiodothyronine (T3) induced chondrocyte terminal maturation within the molecular osteoarthritis pathophysiology using the previously established T3 human ex vivo osteochondral explant model. DESIGNS: RNA-sequencing was performed on explant cartilage obtained from OA patients (n = 8), that was cultured ex vivo with or without T3 (10 ng/ml), and main findings were validated using RT-qPCR in an independent sample set (n = 22). Enrichment analysis was used for functional clustering and comparisons with available OA patient RNA-sequencing and GWAS datasets were used to establish relevance for OA pathophysiology by linking to OA patient genomic profiles. RESULTS: Besides the upregulation of known hypertrophic genes EPAS1 and ANKH, T3 treatment resulted in differential expression of 247 genes with main pathways linked to extracellular matrix and ossification. CCDC80, CDON, ANKH and ATOH8 were among the genes found to consistently mark early, ongoing and terminal maturational OA processes in patients. Furthermore, among the 37 OA risk genes that were significantly affected in cartilage by T3 were COL12A1, TNC, SPARC and PAPPA. CONCLUSIONS: RNA-sequencing results show that metabolic activation and recuperation of growth plate morphology are induced by T3 in OA chondrocytes, indicating terminal maturation is accelerated. The molecular mechanisms involved in hypertrophy were linked to all stages of OA pathophysiology and will be used to validate disease models for drug testing.

Analgesic, Anti-Inflammatory, and Chondroprotective Activities of Siraitia grosvenorii Residual Extract.

Inflammation is crucial to osteoarthritis (OA) pathogenesis. The aim of this study was to evaluate Siraitia grosvenorii residue extract (NHGRE) obtained by extracting S. grosvenorii fruits with water as a potential food supplement for treating arthritis based on its analgesic, anti-inflammatory, and chondroprotective effects and the remaining residue with 70% ethanol. We observed the analgesic activity of NHGRE based on the acetic acid-induced writhing response in mice, examined its anti-inflammatory efficacy against carrageenan-induced paw oedema in mice, and investigated its effect on inflammatory cytokine expression in interleukin (IL)-1ß-induced SW1353 cells. Furthermore, we determined its effects on cartilage protection in interleukin-1ß (IL-1ß)-treated SW1353 cells. NHGRE at 200 mg/kg significantly reduced the acetic acid-induced writhing response and prevented oedema formation in the carrageenan-induced paw oedema model. In IL-1ß-induced SW1353 cells, NHGRE at 400 µg/mL reduced the expression of inflammation mediators such as tumour necrosis factor (TNF)-α (55.3%), IL-6 (35.4%), and prostaglandin E2 (PGE2) (36.9%) and down-regulated the expression of matrix metalloproteinase (MMP)-1 (38.6%), MMP-3 (29.3%), and MMP-13 (44.8%). Additionally, it restored degraded collagen II levels in chondrocytes. NHGRE plays a protective role in chondrocytes by regulating Nuclear factor kappa B (NF-κB) activation. Overall, NHGRE may be a useful therapeutic agent for OA by controlling pain, oedema formation, and inflammation-related mechanisms.

Response of Articular Cartilage to Hyperosmolar Stress: Report of an Ex Vivo Injury Model.

BACKGROUND: Physiological 0.9% saline is commonly used as an irrigation fluid in modern arthroscopy. There is a growing body of evidence that a hyperosmolar saline solution has chondroprotective effects, especially if iatrogenic injury occurs. PURPOSE: To (1) corroborate the superiority of a hyperosmolar saline solution regarding chondrocyte survival after mechanical injury and (2) observe the modulatory response of articular cartilage to osmotic stress and injury. STUDY DESIGN: Controlled laboratory study. METHODS: Osteochondral explants were isolated from bovine stifle joints and exposed to either 0.9% saline (308 mOsm) or hyperosmolar saline (600 mOsm) and then damaged with a sharp dermatome blade to attain a confined full-thickness cartilage injury site, incubated in the same fluids for another 3 hours, and transferred to chondropermissive medium for further culture for 1 week. Chondrocyte survival was assessed by confocal imaging, while the cellular response was evaluated over 1 week by relative gene expression for apoptotic and inflammatory markers and mediator release into the medium. RESULTS: The full-thickness cartilage cut resulted in a confined zone of cell death that mainly affected superficial zone chondrocytes. Injured samples that were exposed to hyperosmolar saline showed less expansion of cell death in both the axial (P < .007) and the coronal (P < .004) plane. There was no progression of cell death during the following week of culture. Histological assessment revealed an intact cartilage matrix and normal chondrocyte morphology. Inflammatory and proapoptotic genes were upregulated on the first days postexposure with a notable downregulation toward day 7. Mediator release into the medium was concentrated on day 3. CONCLUSION: This in vitro cartilage injury model provides further evidence for the chondroprotective effect of a hyperosmolar saline irrigation fluid, as well as novel data on the capability of articular cartilage to quickly regain joint homeostasis after osmotic stress and injury. CLINICAL RELEVANCE: Raising the osmolarity of an irrigating solution may be a simple and safe strategy to protect articular cartilage during arthroscopic surgery.

Cyaonoside A-loaded composite hydrogel microspheres to treat osteoarthritis by relieving chondrocyte inflammation.

Cyaonoside A (CyA), derived from the natural Chinese medicine, Cyathula officinalis Kuan, which was for a long time used to treat knee injuries and relieve joint pain in traditional Chinese medicine, showed an unclear mechanism for protecting cartilage. In addition, CyA was poorly hydrosoluble and incapable of being injected directly into the joint cavity, which limited its clinical application. This study reveals that CyA resisted IL-1ß-mediated chondrogenic inflammation and apoptosis. Next, transcriptome sequencing is used to explore the potential mechanisms underlying CyA regulation of MSC chondrogenic differentiation. Based on these findings, CyA-loaded composite hydrogel microspheres (HLC) were developed and they possessed satisfactory loading efficiency, a suitable degradation rate and good biocompatibility. HLC increased chondrogenic anabolic gene (Acan, COL2A, and SOX9) expression, while downregulating the expression of the catabolic marker MMP13 in vitro. In the osteoarthritis mouse model, HLC demonstrated promising therapeutic capabilities by protecting the integrity of articular cartilage. In conclusion, this study provides insights into the regulatory mechanisms of CyA for chondrocytes and proposes a composite hydrogel microsphere-based advanced therapeutic strategy for osteoarthritis.

Esculin suppresses the PERK-eIF2α-CHOP pathway by enhancing SIRT1 expression in oxidative stress-induced rat chondrocytes, mitigating osteoarthritis progression in a rat model.

OBJECTIVE: Osteoarthritis (OA) is a degenerative disease characterized by the gradual degeneration of chondrocytes, involving endoplasmic reticulum (ER) stress. Esculin is a natural compound with antioxidant, anti-inflammatory and anti-tumor properties. However, its impact on ER stress in OA therapy has not been thoroughly investigated. We aim to determine the efficiency of Esculin in OA treatment and its underlying mechanism. METHODS: We utilized the tert-butyl hydroperoxide (TBHP) to establish OA model in chondrocytes. The expression of SIRT1, PERK/eIF2α pathway-related proteins, apoptosis-associated proteins and ER stress-related proteins were detected by Western blot and Real-time PCR. The apoptosis was evaluated by flow cytometry and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining. X-ray imaging, Hematoxylin & Eosin staining, Safranin O staining and immunohistochemistry were used to assess the pharmacological effects of Esculin in the anterior cruciate ligament transection (ACLT) rat OA model. RESULTS: Esculin downregulated the expression of PERK/eIF2α pathway-related proteins, apoptosis-associated proteins and ER stress-related proteins, while upregulated the expression of SIRT1 and Bcl2 in the TBHP-induced OA model in vitro. It was coincident with the results of TUNEL staining and flow cytometry. We further confirmed the protective effect of Esculin in the rat ACLT-related model. CONCLUSION: Our results suggest the potential therapeutic value of Esculin on osteoarthritis. It probably inhibits the PERK-eIF2α-ATF4-CHOP pathway by upregulating SIRT1, thereby mitigating endoplasmic reticulum stress and protecting chondrocytes from apoptosis.

Molecular mechanism of thiram-induced abnormal chondrocyte proliferation via lncRNA MSTRG.74.1-BNIP3 axis.

Thiram, a widely used organic pesticide in agriculture, exhibits both bactericidal and insecticidal effects. However, prolonged exposure to thiram has been linked to bone deformities and cartilage damage, contributing to the development of tibial dyschondroplasia (TD) in broilers and posing a significant threat to global agricultural production. TD, a prevalent nutritional metabolic disease, manifests as clinical symptoms like unstable standing, claudication, and sluggish movement in affected broilers. In recent years, there has been growing recognition of the regulatory role of long non-coding RNA (lncRNA) in tibial cartilage formation among broilers through diverse signaling pathways. This study employs in vitro experimental models, growth performance analysis, and clinical observation to assess broilers' susceptibility to thiram pollution. Transcriptome sequencing analysis revealed a significant elevation in the expression of lncRNA MSTRG.74.1 in both the con group and the thiram-induced in vitro group. The results showed that lncRNA MSTRG.74.1 plays a pivotal role in influencing the proliferation and abnormal differentiation of chondrocytes. This regulation occurs through the negative modulation of apoptotic genes, including Bax, Cytc, Bcl2, Apaf1, and Caspase3, along with genes Atg5, Beclin1, LC3b, and protein p62. Moreover, the overexpression of lncRNA MSTRG.74.1 was found to regulate broiler chondrocyte development by upregulating BNIP3. In summary, this research sheds light on thiram-induced abnormal chondrocyte proliferation in TD broilers, emphasizing the significant regulatory role of the lncRNA MSTRG.74.1-BNIP3 axis, which will contribute to our understanding of the molecular mechanisms underlying TD development in broilers exposed to thiram.

Protopine protects chondrocytes from undergoing ferroptosis by activating Nrf2 pathway.

Osteoarthritis is a highly prevalent joint disease; however, effective treatments are lacking. Protopine (PTP) is an isoquinoline alkaloid with potent anti-inflammatory and antioxidant properties; however, it has not been studied in osteoarthritis. This study aimed to investigate whether PTP can effectively protect chondrocytes from ferroptosis. Primary mouse chondrocytes were treated with tert-butyl hydroperoxide (TBHP) to simulate oxidative stress in an in vitro model of osteoarthritis. Two concentrations of PTP (10 and 20 µg/mL) were validated for in vitro experiments. Cellular inflammation and metabolism were detected using RT-qPCR and western blotting (WB). Ferroptosis was assessed via WB, qPCR, reactive oxygen species (ROS) levels, lipid ROS, and immunofluorescence staining. In vitro, PTP significantly ameliorated chondrocyte inflammation and cytolytic metabolism and significantly suppressed chondrocyte ferroptosis through the activation of the Nrf2 pathway. The anterior cruciate ligament transection (ACLT) mouse model was used to validate the in vivo effects of PTP. The joint cartilage was assessed using the Osteoarthritis Research Society International (OARSI) score, Safranin O staining, and immunohistochemistry. The intra-articular administration of PTP alleviated cartilage inflammation and ferroptosis, as evidenced by the expression of MMP3, MMP13, COL2A1, GPX4, and Nrf2. Overall, we find that PTP exerted anti-ferroptosis and anti-inflammatory effects on chondrocytes to protect the articular cartilage.

Engineered Exosomes with ATF5-Modified mRNA Loaded in Injectable Thermogels Alleviate Osteoarthritis by Targeting the Mitochondrial Unfolded Protein Response.

Osteoarthritis (OA) progression is highly associated with chondrocyte mitochondrial dysfunction and disorders of catabolism and anabolism of the extracellular matrix (ECM) in the articular cartilage. The mitochondrial unfolded protein response (UPRmt), which is an integral component of the mitochondrial quality control (MQC) system, is essential for maintaining chondrocyte homeostasis. We successfully validated the pivotal role of activating transcription factor 5 (ATF5) in upregulating the UPRmt, mitigating IL-1ß-induced inflammation and mitochondrial dysfunction, and promoting balanced metabolism in articular cartilage ECM, proving its potential as a promising therapeutic target for OA. Modified mRNAs (modRNAs) have emerged as novel and efficient gene delivery vectors for nucleic acid therapeutic approaches. In this study, we combined Atf5-modRNA (modAtf5) with engineered exosomes derived from bone mesenchymal stem cells (ExmodAtf5) to exert cytoprotective effects on chondrocytes in articular cartilage via Atf5. However, the rapid localized metabolization of ExmodAtf5 limits its application. PLGA-PEG-PLGA (Gel), an injectable thermosensitive hydrogel, was used as a carrier of ExmodAtf5 (Gel@ExmodAtf5) to achieve a sustained release of ExmodAtf5. In vitro and in vivo, the use of Gel@ExmodAtf5 was shown to be a highly effective strategy for OA treatment. The in vivo therapeutic effect of Gel@ExmodAtf5 was evidenced by the preservation of the intact cartilage surface, low OARSI scores, fewer osteophytes, and mild subchondral bone sclerosis and cystic degeneration. Consequently, the combination of ExmodAtf5 and PLGA-PEG-PLGA could significantly enhance the therapeutic efficacy and prolong the exosome release. In addition, the mitochondrial protease ClpP enhanced chondrocyte autophagy by modulating the mTOR/Ulk1 pathway. As a result of our research, Gel@ExmodAtf5 can be considered to be effective at alleviating the progression of OA.

Gubi Zhitong formula alleviates osteoarthritis in vitro and in vivo via regulating BNIP3L-mediated mitophagy.

BACKGROUND: Osteoarthritis (OA) is characterized by degeneration of articular cartilage, leading to joint pain and dysfunction. Gubi Zhitong formula (GBZTF), a traditional Chinese medicine formula, has been used in the clinical treatment of OA for decades, demonstrating definite efficacy. However, its mechanism of action remains unclear, hindering its further application. METHODS: The ingredients of GBZTF were analyzed and performed with liquid chromatography-mass spectrometry (LC-MS). 6 weeks old SD rats were underwent running exercise (25 m/min, 80 min, 0°) to construct OA model with cartilage wear and tear. It was estimated by Micro-CT, Gait Analysis, Histological Stain. RNA-seq technology was performed with OA Rats' cartilage, and primary chondrocytes induced by IL-1ß (mimics OA chondrocytes) were utilized to evaluated and investigated the mechanism of how GBZTF protected OA cartilage from being damaged with some functional experiments. RESULTS: A total of 1006 compounds were identified under positive and negative ion modes by LC-MS. Then, we assessed the function of GBZTF through in vitro and vivo. It was found GBZTF could significantly up-regulate OA rats' limb coordination and weight-bearing capacity, and reduce the surface and sub-chondral bone erosions of OA joints, and protect cartilage from being destroyed by inflammatory factors (iNOS, IL-6, IL-1ß, TNF- α, MMP13, ADAMTS5), and promote OA chondrocytes proliferation and increase the S phage of cell cycle. In terms of mechanism, RNA-seq analysis of cartilage tissues revealed 1,778 and 3,824 differentially expressed genes (DEGs) in model vs control group and GBZTF vs model group, respectively. The mitophagy pathway was most significantly enriched in these DEGs. Further results of subunits of OA chondrocytes confirmed that GBZTF could alleviate OA-associated inflammation and cartilage damage through modulation BCL2 interacting protein 3-like (BNIP3L)-mediated mitophagy. CONCLUSION: The therapeutic effectiveness of GBZTF on OA were first time verified in vivo and vitro through functional experiments and RNA-seq, which provides convincing evidence to support the molecular mechanisms of GBZTF as a promising therapeutic decoction for OA.

The Role of Copper-Induced M2 Macrophage Polarization in Protecting Cartilage Matrix in Osteoarthritis.

BACKGROUND The pathological mechanism of osteoarthritis is still unclear. The regulation of the immune microenvironment has been of growing interest in the progression and treatment of osteoarthritis. Macrophages with different phenotypes, producing different cytokines, have been linked to the mechanism of cartilage injury in osteoarthritis. Copper ions play a role in the immune response and are involved in the pathological mechanisms of osteoarthritis by affecting the metabolism of the cartilage matrix. Bioactive glass (BG) is an osteogenic material with superior biocompatibility. Here, we report on the regulatory behavior of macrophages using a copper-based composite BG material. MATERIAL AND METHODS Cu-BGC powder was prepared by sol-gel method, and scaffolds were fabricated and characterized using 3D printing. Macrophage cultures grown with Cu-BGC were examined for cell culture and proliferation. The effect of Cu-BGC on the degradation metabolism of chondrocytes, cultured in the environment of inflammatory cytokine IL-1ß, was determined. In addition, the morphology of macrophages, secretion of inflammatory cytokines, and expression of surface markers were examined. RESULTS The results show that Cu-BGC promotes macrophage proliferation at a range of concentrations and increases the secretion of anti-inflammatory cytokines while inhibiting proinflammatory cytokines. At the same time, M2-type cell surface markers are definitely expressed and the morphology of macrophages is altered. In addition, Cu-BGC inhibited the degradation metabolism of chondrocytes in the inflammatory environment induced by IL-1ß. CONCLUSIONS These results suggest that Cu-BGC induced macrophage polarization into an M2 type anti-inflammatory phenotype, and inhibition of immune injury response may play a role in delaying cartilage matrix damage in osteoarthritis.

Icariin inhibits chondrocyte ferroptosis and alleviates osteoarthritis by enhancing the SLC7A11/GPX4 signaling.

BACKGROUND: Chondrocyte ferroptosis plays a critical role in the pathogenesis of osteoarthritis (OA), regulated by the SLC7A11/GPX4 signaling pathway. Icariin (ICA), a flavonoid glycoside, exhibits strong anti-inflammatory and antioxidant activities. This study investigated whether ICA could modulate the SLC7A11/GPX4 signaling to inhibit chondrocyte ferroptosis and alleviate OA. PURPOSE: The objective was to explore the impact of ICA on chondrocyte ferroptosis in OA and its modulation of the SLC7A11/GPX4 signaling pathway. METHODS: The anti-ferroptosis effects of ICA were evaluated in an interleukin-1ß (IL-1ß)-treated SW1353 cell model, using Ferrostatin-1 (Fer-1) and Erastin (Era) as ferroptosis inhibitor and inducer, respectively, along with GPX4 knockdown via lentivirus-based shRNA. Additionally, the therapeutic efficacy of ICA on OA-related articular cartilage damage was assessed in rats through histopathology and immunohistochemistry (IHC). RESULTS: IL-1ß treatment upregulated the expression of OA-associated matrix metalloproteinases (MMP3 and MMP1), a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS-5), and increased intracellular ROS, lipid ROS, and MDA levels while downregulating collagen II and SOX9 expression in SW1353 cells. ICA treatment countered the IL-1ß-induced upregulation of MMPs and ADAMTS-5, restored collagen II and SOX9 expression, and reduced intracellular ROS, lipid ROS, and MDA levels. Furthermore, IL-1ß upregulated P53 but downregulated SLC7A11 and GPX4 expression in SW1353 cells, effects that were mitigated by ICA or Fer-1 treatment. Significantly, ICA also alleviated Era-induced ferroptosis, whereas it had no effect on GPX4-silenced SW1353 cells. In vivo, ICA treatment reduced articular cartilage damage in OA rats by partially restoring collagen II and GPX4 expression, inhibiting cartilage extracellular matrix (ECM) degradation and chondrocyte ferroptosis. CONCLUSION: ICA treatment mitigated chondrocyte ferroptosis and articular cartilage damage by enhancing the SLC7A11/GPX4 signaling, suggesting its potential as a therapeutic agent for OA interventions.

Chondroitin Sulfate/Hyaluronic Acid-Blended Hydrogels Suppress Chondrocyte Inflammation under Pro-Inflammatory Conditions.

Osteoarthritis is characterized by enzymatic breakdown of the articular cartilage via the disruption of chondrocyte homeostasis, ultimately resulting in the destruction of the articular surface. Decades of research have highlighted the importance of inflammation in osteoarthritis progression, with inflammatory cytokines shifting resident chondrocytes into a pro-catabolic state. Inflammation can result in poor outcomes for cells implanted for cartilage regeneration. Therefore, a method to promote the growth of new cartilage and protect the implanted cells from the pro-inflammatory cytokines found in the joint space is required. In this study, we fabricate two gel types: polymer network hydrogels composed of chondroitin sulfate and hyaluronic acid, glycosaminoglycans (GAGs) known for their anti-inflammatory and prochondrogenic activity, and interpenetrating networks of GAGs and collagen I. Compared to a collagen-only hydrogel, which does not provide an anti-inflammatory stimulus, chondrocytes in GAG hydrogels result in reduced production of pro-inflammatory cytokines and enzymes as well as preservation of collagen II and aggrecan expression. Overall, GAG-based hydrogels have the potential to promote cartilage regeneration under pro-inflammatory conditions. Further, the data have implications for the use of GAGs to generally support tissue engineering in pro-inflammatory environments.

Tanshinone IIA alleviates IL-1ß-induced chondrocyte apoptosis and inflammation by regulating FBXO11 expression.

OBJECTIVE: This study explored the pharmacological mechanism of Tanshinone IIA (TAN IIA) in the treatment of Osteoarthritis (OA), which provided a certain reference for further research and clinical application of Tan IIA in OA. METHODS: CHON-001 cells were stimulated with 10 µg/mL IL-1ß for 48 h and treated with 10 µM TAN IIA for 48 h. Cellular viability and apoptosis were evaluated by CCK-8 assay and flow cytometry, and Cleaved caspase-3 was measured by Immunoblot assay and RT-qPCR. TNF-α, IL-6, and iNOS in CHON-001 cells were determined by RT-qPCR and ELISA. To further verify the effect of TAN IIA on OA, a rat model of OA in vivo was established by right anterior cruciate ligament transection. TAN IIA was administered at 50 mg/kg or 150 mg/kg for 7 weeks. The degree of cartilage destruction in OA rats was observed by TUNEL and HE staining. Cleaved caspase-3 and FBXO11 were measured by immunohistochemical staining, RT-qPCR, and Immunoblot. TNF-α, IL-6, and iNOS in chondrocytes of OA rats were detected by ELISA. RESULTS: IL-1ß stimulated CHON-001 cell apoptosis and inflammation, and TAN IIA had anti-apoptosis and anti-inflammatory effects on IL-1ß-regulated CHON-001 cells. TAN IIA down-regulated FBXO11 and inhibited PI3K/AKT and NF-κB pathways, thereby alleviating apoptotic and inflammatory reactions in CHON-001 cells under IL-1ß treatment. Moreover, TAN IIA treatment improved chondrocyte apoptosis and inflammations in OA rats. CONCLUSION: TAN IIA inhibits PI3K/Akt and NF-κB pathways by down-regulating FBXO11 expression, alleviates chondrocyte apoptosis and inflammation, and delays the progression of OA.