SKP2-mediated ubiquitination and degradation of KLF11 promotes osteoarthritis via modulation of JMJD3/NOTCH1 pathway.

Osteoarthritis (OA) is the main cause of cartilage damage and disability. This study explored the biological function of S-phase kinase-associated protein 2 (SKP2) and Kruppel-like factor 11 (KLF11) in OA progression and its underlying mechanisms. C28/I2 chondrocytes were stimulated with IL-1ß to mimic OA in vitro. We found that SKP2, Jumonji domain-containing protein D3 (JMJD3), and Notch receptor 1 (NOTCH1) were upregulated, while KLF11 was downregulated in IL-1ß-stimulated chondrocytes. SKP2/JMJD3 silencing or KLF11 overexpression repressed apoptosis and extracellular matrix (ECM) degradation in chondrocytes. Mechanistically, SKP2 triggered the ubiquitination and degradation of KLF11 to transcriptionally activate JMJD3, which resulted in activation of NOTCH1 through inhibiting H3K27me3. What's more, the in vivo study found that KLF11 overexpression delayed OA development in rats via restraining apoptosis and maintaining the balance of ECM metabolism. Taken together, ubiquitination and degradation of KLF11 regulated by SKP2 contributed to OA progression by activation of JMJD3/NOTCH1 pathway. Our findings provide promising therapeutic targets for OA.

Histological and immunohistochemical analyses of articular cartilage during onset and progression of pre- and early-stage osteoarthritis in a rodent model.

Early diagnosis and treatment of pre- and early-stage osteoarthritis (OA) is important. However, the cellular and cartilaginous changes occurring during these stages remain unclear. We investigated the histological and immunohistochemical changes over time between pre- and early-stage OA in a rat model of traumatic injury. Thirty-six male rats were divided into two groups, control and OA groups, based on destabilization of the medial meniscus. Histological and immunohistochemical analyses of articular cartilage were performed on days 1, 3, 7, 10, and 14 postoperatively. Cell density of proteins associated with cartilage degradation increased from postoperative day one. On postoperative day three, histological changes, including chondrocyte death, reduced matrix staining, and superficial fibrillation, were observed. Simultaneously, a compensatory increase in matrix staining was observed. The Osteoarthritis Research Society International score increased from postoperative day seven, indicating thinner cartilage. On postoperative day 10, the positive cell density decreased, whereas histological changes progressed with fissuring and matrix loss. The proteoglycan 4-positive cell density increased on postoperative day seven. These findings will help establish an experimental model and clarify the mechanism of the onset and progression of pre- and early-stage traumatic OA.

PPARγ regulates osteoarthritis chondrocytes apoptosis through caspase-3 dependent mitochondrial pathway.

Osteoarthritis (OA) is the most prevalent form of arthritis, characterized by a complex pathogenesis. One of the key factors contributing to its development is the apoptosis of chondrocytes triggered by oxidative stress. Involvement of peroxisome proliferator-activated receptor gamma (PPARγ) has been reported in the regulation of oxidative stress. However, there remains unclear mechanisms that through which PPARγ influences the pathogenesis of OA. The present study aims to delve into the role of PPARγ in chondrocytes apoptosis induced by oxidative stress in the context of OA. Primary human chondrocytes, both relatively normal and OA, were isolated and cultured for the following study. Various assessments were performed, including measurements of cell proliferation, viability and cytotoxicity. Additionally, we examined cell apoptosis, levels of reactive oxygen species (ROS), nitric oxide (NO), mitochondrial membrane potential (MMP) and cytochrome C release. We also evaluated the expression of related genes and proteins, such as collagen type II (Col2a1), aggrecan, inducible nitric oxide synthase (iNOS), caspase-9, caspase-3 and PPARγ. Compared with relatively normal cartilage, the expression of PPARγ in OA cartilage was down-regulated. The proliferation of OA chondrocytes decreased, accompanied by an increase in the apoptosis rate. Down-regulation of PPARγ expression in OA chondrocytes coincided with an up-regulation of iNOS expression, leading to increased secretion of NO, endogenous ROS production, and decrease of MMP levels. Furthermore, we observed the release of cytochrome C, elevated caspase-9 and caspase-3 activities, and reduction of the components of extracellular matrix (ECM) Col2a1 and aggrecan. Accordingly, utilization of GW1929 (PPARγ Agonists) or Z-DEVD-FMK (caspase-3 inhibitor) can protect chondrocytes from mitochondrial-related apoptosis and alleviate the progression of OA. During the progression of OA, excessive oxidative stress in chondrocytes leads to apoptosis and ECM degradation. Activation of PPARγ can postpone OA by down-regulating caspase-3-dependent mitochondrial apoptosis pathway.

Spinosin ameliorates osteoarthritis through enhancing the Nrf2/HO-1 signaling pathway.

Osteoarthritis (OA) is a common degenerative joint disease in the elderly, while oxidative stress-induced chondrocyte degeneration plays a key role in the pathologic progression of OA. One possible reason is that the expression of nuclear factor erythroid 2-related factor 2 (Nrf2), which acts as the intracellular defense factor against oxidative stress, is significantly inhibited in chondrocytes. Spinosin (SPI) is a potent Nrf2 agonist, but its effect on OA is still unknown. In this study, we found that SPI can alleviate tert-Butyl hydroperoxide (TBHP)-induced extracellular matrix degradation of chondrocytes. Additionally, SPI can effectively activate Nrf2, heme oxygenase-1 (HO-1), and NADPH quinone oxidoreductase 1 (NQO1) in chondrocytes under the TBHP environment. When Nrf2 was silenced by siRNA, the cartilage protective effect of SPI was also weakened. Finally, SPI showed good alleviative effects on OA in mice. Thus, SPI can ameliorate oxidative stress-induced chondrocyte dysfunction and exhibit a chondroprotective effect through activating the Nrf2/HO-1 pathway, which may provide a novel and promising option for the treatment of OA.

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.

DDX5 inhibits hyaline cartilage fibrosis and degradation in osteoarthritis via alternative splicing and G-quadruplex unwinding.

Hyaline cartilage fibrosis is typically considered an end-stage pathology of osteoarthritis (OA), which results in changes to the extracellular matrix. However, the mechanism behind this is largely unclear. Here, we found that the RNA helicase DDX5 was dramatically downregulated during the progression of OA. DDX5 deficiency increased fibrosis phenotype by upregulating COL1 expression and downregulating COL2 expression. In addition, loss of DDX5 aggravated cartilage degradation by inducing the production of cartilage-degrading enzymes. Chondrocyte-specific deletion of Ddx5 led to more severe cartilage lesions in the mouse OA model. Mechanistically, weakened DDX5 resulted in abundance of the Fn1-AS-WT and Plod2-AS-WT transcripts, which promoted expression of fibrosis-related genes (Col1, Acta2) and extracellular matrix degradation genes (Mmp13, Nos2 and so on), respectively. Additionally, loss of DDX5 prevented the unfolding Col2 promoter G-quadruplex, thereby reducing COL2 production. Together, our data suggest that strategies aimed at the upregulation of DDX5 hold significant potential for the treatment of cartilage fibrosis and degradation in OA.

MiR-653-5p drives osteoarthritis pathogenesis by modulating chondrocyte senescence.

BACKGROUND: Due to the unclear pathogenesis of osteoarthritis (OA), effective treatment for this ailment is presently unavailable. Accumulating evidence points to chondrocyte senescence as a key driver in OA development. This study aims to identify OA-specific microRNAs (miRNAs) targeting chondrocyte senescence to alleviate OA progression. METHODS: We screened and identified miRNAs differentially expressed in OA and normal cartilage, then confirmed the impact of miR-653-5p on chondrocyte functions and senescence phenotypes through in vitro experiments with overexpression/silencing. We identified interleukin 6 (IL-6) as the target gene of miR-653-5p and confirmed the regulatory influence of miR-653-5p on the IL-6/JAK/STAT3 signaling pathway through gain/loss-of-function studies. Finally, we assessed the therapeutic efficacy of miR-653-5p on OA using a mouse model with destabilization of the medial meniscus. RESULTS: MiR-653-5p was significantly downregulated in cartilage tissues and chondrocytes from OA patients. Overexpression of miR-653-5p promoted chondrocyte matrix synthesis and proliferation while inhibiting chondrocyte senescence. Furthermore, bioinformatics target prediction and the luciferase reporter assays identified IL-6 as a target of miR-653-5p. Western blot assays demonstrated that miR-653-5p overexpression inhibited the protein expression of IL-6, the phosphorylation of JAK1 and STAT3, and the expression of chondrocyte senescence phenotypes by regulating the IL-6/JAK/STAT3 signaling pathway. More importantly, the cartilage destruction was significantly alleviated and chondrocyte senescence phenotypes were remarkably decreased in the OA mouse model treated by agomiR-653-5p compared to the control mice. CONCLUSIONS: MiR-653-5p showed a significant decrease in cartilage tissues of individuals with OA, leading to an upregulation of chondrocyte senescence phenotypes in the articular cartilage. AgomiR-653-5p emerges as a potential treatment approach for OA. These findings provide further insight into the role of miR-653-5p in chondrocyte senescence and the pathogenesis of OA.

Proteolysis of the pericellular matrix: Pinpointing the role and involvement of matrix metalloproteinases in early osteoarthritic remodeling.

The pericellular matrix (PCM) serves a critical role in signal transduction and mechanoprotection in chondrocytes. Osteoarthritis (OA) leads to a gradual deterioration of the cartilage, marked by a shift in the spatial arrangement of chondrocytes from initially isolated strands to large cell clusters in end-stage degeneration. These changes coincide with progressive enzymatic breakdown of the PCM. This study aims to assess the role and involvement of specific matrix metalloproteinases (MMPs) in PCM degradation during OA. We selected cartilage samples from 148 OA patients based on the predominant spatial chondrocyte patterns. The presence of various MMPs (-1,-2,-3,-7,-8,-9,-10,-12,-13) was identified by multiplexed immunoassays. For each pattern and identified MMP, the levels and activation states (pro-form vs. active form) were measured by zymograms and western blots. The localization of these MMPs was determined using immunohistochemical labeling. To verify these results, healthy cartilage was exposed to purified MMPs, and the consecutive structural integrity of the PCM was analyzed through immunolabeling and proximity ligation assay. Screening showed elevated levels of MMP-1,-2,-3,-7, and -13, with their expression profile showing a clear dependency of the degeneration stage. MMP-2 and -7 were localized in the PCM, whereas MMP-1,-7, and -13 were predominantly intracellular. We found that MMP-2 and -3 directly disrupt collagen type VI, and MMP-3 and -7 destroy perlecan. MMP-2, -3, and -7 emerge as central players in early PCM degradation in OA. With the disease's initial stages already displaying elevated peaks in MMP expression, this insight may guide early targeted therapies to halt abnormal PCM remodeling. STATEMENT OF SIGNIFICANCE: Osteoarthritis (OA) causes a gradual deterioration of the articular cartilage, accompanied by a progressive breakdown of the pericellular matrix (PCM). The PCM's crucial function in protecting and transmitting signals within chondrocytes is impaired in OA. By studying 148 OA-patient cartilage samples, the involvement of matrix metalloproteinases (MMPs) in PCM breakdown was explored. Findings highlighted elevated levels of certain MMPs linked to different stages of degeneration. Notably, MMP-2, -3, and -7 were identified as potent contributors to early PCM degradation, disrupting key components like collagen type VI and perlecan. Understanding these MMPs' roles in initiating OA progression, especially in its early stages, provides insights into potential targets for interventions to preserve PCM integrity and potentially impeding OA advancement.

Lumbar instability remodels cartilage endplate to induce intervertebral disc degeneration by recruiting osteoclasts via Hippo-CCL3 signaling.

Degenerated endplate appears with cheese-like morphology and sensory innervation, contributing to low back pain and subsequently inducing intervertebral disc degeneration in the aged population.1 However, the origin and development mechanism of the cheese-like morphology remain unclear. Here in this study, we report lumbar instability induced cartilage endplate remodeling is responsible for this pathological change. Transcriptome sequencing of the endplate chondrocytes under abnormal stress revealed that the Hippo signaling was key for this process. Activation of Hippo signaling or knockout of the key gene Yap1 in the cartilage endplate severed the cheese-like morphological change and disc degeneration after lumbar spine instability (LSI) surgery, while blocking the Hippo signaling reversed this process. Meanwhile, transcriptome sequencing data also showed osteoclast differentiation related gene set expression was up regulated in the endplate chondrocytes under abnormal mechanical stress, which was activated after the Hippo signaling. Among the discovered osteoclast differentiation gene set, CCL3 was found to be largely released from the chondrocytes under abnormal stress, which functioned to recruit and promote osteoclasts formation for cartilage endplate remodeling. Over-expression of Yap1 inhibited CCL3 transcription by blocking its promoter, which then reversed the endplate from remodeling to the cheese-like morphology. Finally, LSI-induced cartilage endplate remodeling was successfully rescued by local injection of an AAV5 wrapped Yap1 over-expression plasmid at the site. These findings suggest that the Hippo signaling induced osteoclast gene set activation in the cartilage endplate is a potential new target for the management of instability induced low back pain and lumbar degeneration.

Aberrant methylation and expression of TNXB promote chondrocyte apoptosis and extracullar matrix degradation in hemophilic arthropathy via AKT signaling.

Recurrent joint bleeding in hemophilia patients frequently causes hemophilic arthropathy (HA). Drastic degradation of cartilage is a major characteristic of HA, but its pathological mechanisms has not yet been clarified. In HA cartilages, we found server matrix degradation and increased expression of DNA methyltransferase proteins. We thus performed genome-wide DNA methylation analysis on human HA (N=5) and osteoarthritis (OA) (N=5) articular cartilages, and identified 1228 differentially methylated regions (DMRs) associated with HA. Functional enrichment analyses revealed the association between DMR genes (DMGs) and extracellular matrix (ECM) organization. Among these DMGs, Tenascin XB (TNXB) expression was down-regulated in human and mouse HA cartilages. The loss of Tnxb in F8-/- mouse cartilage provided a disease-promoting role in HA by augmenting cartilage degeneration and subchondral bone loss. Tnxb knockdown also promoted chondrocyte apoptosis and inhibited phosphorylation of AKT. Importantly, AKT agonist showed chondroprotective effects following Tnxb knockdown. Together, our findings indicate that exposure of cartilage to blood leads to alterations in DNA methylation, which is functionally related to ECM homeostasis, and further demonstrate a critical role of TNXB in HA cartilage degeneration by activating AKT signaling. These mechanistic insights allow development of potentially new strategies for HA cartilage protection.

Development and functional evaluation of a hyaluronic acid coated nano-formulation with kaempferol as a novel intra-articular agent for Knee Osteoarthritis treatment.

Knee osteoarthritis (OA) involves articular cartilage degradation driven mainly by inflammation. Kaempferol (KM), known for its anti-inflammatory property, holds potential for OA treatment. This study investigated the potential of hyaluronic acid (HA)-coated gelatin nanoparticles loaded with KM (HA-KM GNP) for treating knee OA. KM was encapsulated into gelatin nanoparticles (KM GNP) and then coated with HA to form HA-KM GNPs. Physical properties were characterized, and biocompatibility and cellular uptake were assessed in rat chondrocytes. Anti-inflammatory and chondrogenic properties were evaluated using IL-1ß-stimulated rat chondrocytes, compared with HA-coated nanoparticles without KM (HA GNP) and KM alone. Preclinical efficacy was tested in an anterior cruciate ligament transection (ACLT)-induced knee OA rat model treated with intra-articular injection of HA-KM GNP. Results show spherical HA-KM GNPs (88.62 ± 3.90 nm) with positive surface charge. Encapsulation efficiency was 98.34 % with a sustained release rate of 18 % over 48 h. Non-toxic KM concentration was 2.5 µg/mL. In IL-1ß-stimulated OA rat chondrocytes, HA-KM GNP significantly down-regulated RNA expression of IL-1ß, TNF-α, COX-2, MMP-9, and MMP-13, while up-regulating SOX9 compared to HA GNP, and KM. In vivo imaging demonstrated significantly higher fluorescence intensity within rat knee joints for 3 hours post HA-KM GNP injection compared with KM GNP (185.2% ± 34.1% vs. 45.0% ± 16.7%). HA-KM GNP demonstrated significant effectiveness in reducing subchondral sclerosis, attenuating inflammation, inhibiting matrix degradation, restoring cartilage thickness, and reducing the severity of OA in the ACLT rat model. In conclusion, HA-KM GNP holds promise for knee OA therapy.

Vitamin K2 ameliorates osteoarthritis by suppressing ferroptosis and extracellular matrix degradation through activation GPX4's dual functions.

Vitamin K2 (VK2) is an effective compound for anti-ferroptosis and anti-osteoporosis, and Semen sojae praeparatum (Dandouchi in Chinese) is the main source of VK2. Chondrocyte ferroptosis and extracellular matrix (ECM) degradation playing a role in the pathogenesis of osteoarthritis (OA). Glutathione peroxidase 4 (GPX4) is the intersection of two mechanisms in regulating OA progression. But no studies have elucidated the therapeutic effects and mechanisms of VK2 on OA. This study utilized an in vivo rat OA model created via anterior cruciate ligament transection (ACLT) and an in vitro chondrocyte oxidative damage model induced by TBHP to investigate the protective effects and mechanisms of action of VK2 in OA. Knee joint pain in mice was evaluated using the Von Frey test. Micro-CT and Safranin O-Fast Green staining were employed to observe the extent of damage to the tibial cartilage and subchondral bone, while immunohistochemistry and PCR were used to examine GPX4 levels in joint cartilage. The effects of VK2 on rat chondrocyte viability were assessed using CCK-8 and flow cytometry assays, and chondrocyte morphology was observed with toluidine blue and alcian blue staining. The impact of VK2 on intracellular ferroptosis-related markers was observed using fluorescent staining and flow cytometry. Protein expression changes were detected by immunofluorescence and Western blot analysis. Furthermore, specific protein inhibitors were applied to confirm the dual-regulatory effects of VK2 on GPX4. VK2 can increase bone mass and cartilage thickness in the subchondral bone of the tibia, and reduce pain and the OARSI score induced by OA. Immunohistochemistry results indicate that VK2 exerts its anti-OA effects by regulating GPX4 to delay ECM degradation. VK2 can inhibit the activation of the MAPK/NFκB signaling pathway caused by reduced expression of intracellular GPX4, thereby decreasing ECM degradation. Additionally, VK2 can reverse the inhibitory effect of RSL3 on GPX4, increase intracellular GSH content and the GSH/GSSG ratio, reduce MDA content, and rescue chondrocyte ferroptosis. The protective mechanism of VK2 may involve its dual-target regulation of GPX4, reducing chondrocyte ferroptosis and inhibiting the MAPK/NFκB signaling pathway to decelerate the degradation of the chondrocyte extracellular matrix.

Osteophyte Cartilage as a Potential Source for Minced Cartilage Implantation: A Novel Approach for Articular Cartilage Repair in Osteoarthritis.

Osteoarthritis (OA) is a common joint disorder characterized by cartilage degeneration, often leading to pain and functional impairment. Minced cartilage implantation (MCI) has emerged as a promising one-step alternative for large cartilage defects. However, the source of chondrocytes for MCI remains a challenge, particularly in advanced OA, as normal cartilage is scarce. We performed in vitro studies to evaluate the feasibility of MCI using osteophyte cartilage, which is present in patients with advanced OA. Osteophyte and articular cartilage samples were obtained from 22 patients who underwent total knee arthroplasty. Chondrocyte migration and proliferation were assessed using cartilage fragment/atelocollagen composites to compare the characteristics and regenerative potential of osteophytes and articular cartilage. Histological analysis revealed differences in cartilage composition between osteophytes and articular cartilage, with higher expression of type X collagen and increased chondrocyte proliferation in the osteophyte cartilage. Gene expression analysis identified distinct gene expression profiles between osteophytes and articular cartilage; the expression levels of COL2A1, ACAN, and SOX9 were not significantly different. Chondrocytes derived from osteophyte cartilage exhibit enhanced proliferation, and glycosaminoglycan production is increased in both osteophytes and articular cartilage. Osteophyte cartilage may serve as a viable alternative source of MCI for treating large cartilage defects in OA.

Matrix stiffening promotes chondrocyte senescence and the osteoarthritis development through downregulating HDAC3.

Extracellular matrix (ECM) stiffening is a typical characteristic of cartilage aging, which is a quintessential feature of knee osteoarthritis (KOA). However, little is known about how ECM stiffening affects chondrocytes and other molecules downstream. This study mimicked the physiological and pathological stiffness of human cartilage using polydimethylsiloxane (PDMS) substrates. It demonstrated that epigenetic Parkin regulation by histone deacetylase 3 (HDAC3) represents a new mechanosensitive mechanism by which the stiffness matrix affected chondrocyte physiology. We found that ECM stiffening accelerated cultured chondrocyte senescence in vitro, while the stiffness ECM downregulated HDAC3, prompting Parkin acetylation to activate excessive mitophagy and accelerating chondrocyte senescence and osteoarthritis (OA) in mice. Contrarily, intra-articular injection with an HDAC3-expressing adeno-associated virus restored the young phenotype of the aged chondrocytes stimulated by ECM stiffening and alleviated OA in mice. The findings indicated that changes in the mechanical ECM properties initiated pathogenic mechanotransduction signals, promoted the Parkin acetylation and hyperactivated mitophagy, and damaged chondrocyte health. These results may provide new insights into chondrocyte regulation by the mechanical properties of ECM, suggesting that the modification of the physical ECM properties may be a potential OA treatment strategy.

Tributyltin chloride induces chondrocyte damage through the activation of NLRP3­mediated inflammation and pyroptosis.

Tributyltin chloride (TBTC) is known to have effects and mechanisms in various diseases; however, whether TBTC is detrimental to joints and causes osteoarthritis (OA), as well as its underlying mechanism, has not yet been fully elucidated. The present study explored the effects of TBTC on rat chondrocytes, as well as on mouse OA. The toxicity of TBTC toward rat chondrocytes was detected using a lactate dehydrogenase (LDH) leakage assay and cell viability was evaluated using the Cell Counting Kit­8 assay. The results showed that TBTC decreased the viability of rat chondrocytes and increased the LDH leakage rate in a concentration­dependent manner. Moreover, compared with in the control group, TBTC increased the expression levels of interleukin (IL)­1ß, IL­18, matrix metalloproteinase (MMP)­1, MMP­13, NLR family pyrin domain containing 3 (NLRP3), caspase­1, PYD and CARD domain containing, and gasdermin D in chondrocytes. Furthermore, knockdown of NLRP3 reversed the TBTC­induced increases in LDH leakage and NLRP3 inflammasome­associated protein levels. In vivo, TBTC exacerbated cartilage tissue damage in mice from the OA group, as evidenced by the attenuation of safranin O staining. In conclusion, TBTC may aggravate OA in mice by promoting chondrocyte damage and inducing pyroptosis through the activation of NLRP3 and caspase­1 signaling. The present study demonstrated that TBTC can cause significant damage to the articular cartilage; therefore, TBTC contamination should be strictly monitored.

GPX7 reduces chondrocyte inflammation and extracellular matrix degradation triggered by IL­1ß, via a mechanism mediated by ferroptosis.

During osteoarthritis (OA), chondrocytes become highly active, with increased matrix synthesis and inflammatory cytokine­induced catabolic pathways. Early intervention strategies targeting pathological changes may attenuate or halt disease progression. The present study aimed to reveal the role of glutathione peroxidase (GPX)7 in OA. For this purpose, a research model was established by inducing C28/I2 human chondrocytes with interleukin (IL)­1ß, and the expression level of GPX7 was determined. To explore its roles, C28/I2 cells were transfected to gain GPX7 overexpression. The effects of GPX7 overexpression on intracellular inflammation, extracellular matrix (ECM) degradation, apoptosis and ferroptosis were then evaluated. In addition, the cells were treated with the ferroptosis inducer, erastin, and its effects on the aforementioned phenotypes were assessed. The level of GPX7 was decreased in response to IL­1ß treatment, and GPX7 overexpression suppressed cellular inflammation, ECM degradation and apoptosis. Moreover, the reduction of lipid peroxidation, ferrous ions and transferrin indicated that GPX7 overexpression inhibited ferroptosis. Subsequently, inflammation, ECM degradation and apoptosis were found to be promoted in the cells upon treatment with erastin. These findings suggested that the regulatory role of GPX7 may be mediated by a pathway involving ferroptosis. On the whole, the present study revealed that GPX7 reduces IL­1ß­induced chondrocyte inflammation, apoptosis and ECM degradation partially through a mechanism involving ferroptosis. The results of the present study lay a theoretical foundation for subsequent OA­related research and may enable the development of translational strategies for the treatment of OA.

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.

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.

Multi-omics analysis of a case of congenital microtia reveals aldob and oxidative stress associated with microtia etiology.

BACKGROUND: Microtia is reported to be one of the most common congenital craniofacial malformations. Due to the complex etiology and the ethical barrier of embryonic study, the precise mechanisms of microtia remain unclear. Here we report a rare case of microtia with costal chondrodysplasia based on bioinformatics analysis and further verifications on other sporadic microtia patients. RESULTS: One hundred fourteen deleterious insert and deletion (InDel) and 646 deleterious SNPs were screened out by WES, candidate genes were ranked in descending order according to their relative impact with microtia. Label-free proteomic analysis showed that proteins significantly different between the groups were related with oxidative stress and energy metabolism. By real-time PCR and immunohistochemistry, we further verified the candidate genes between other sporadic microtia and normal ear chondrocytes, which showed threonine aspartase, cadherin-13, aldolase B and adiponectin were significantly upregulated in mRNA levels but were significantly lower in protein levels. ROS detection and mitochondrial membrane potential (∆ Ψ m) detection proved that oxidative stress exists in microtia chondrocytes. CONCLUSIONS: Our results not only spot new candidate genes by WES and label-free proteomics, but also speculate for the first time that metabolism and oxidative stress may disturb cartilage development and this might become therapeutic targets and potential biomarkers with clinical usefulness in the future.

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.