Rat Chondrocytes Research Articles

Ozone induces autophagy by activating PPARγ/mTOR in rat chondrocytes treated with IL-1β

BackgroundOsteoarthritis (OA) is the main cause of older pain and disability. Intra-articular injections of ozone (O3) commonly have been found to have antioxidative and anti-inflammatory effects to reduce pain and improve function in knee osteoarthritis. It has been reported that reduced autophagy in chondrocytes plays an important role in the development of OA. This study aimed to probe the role of O3 on the autophagy in chondrocytes treated with IL-1β.MethodsPrimary chondrocytes were isolated from Wistar rats cartilage within 3 days. The OA chondrocytes model was induced via treatment with IL-1β for 24 h. Then the cells were treated with O3 and GW9662, the inhibitor of PPARγ. Cell viability was assessed by CCK-8. Further, the cells subjected to Western blot analysis, qRT-PCR and immunofluorescence assay. The numbers of autophagosomes were observed via transmission electron microscopy.Results30 μg/ml O3 improved the viability of chondrocytes treated with IL-1β. The decreased level of autophagy proteins and the numbers of autophagosomes improved in IL-1β-treated chondrocytes with O3 via activating PPARγ/mTOR. In addition, the qRT-PCR results showed that O3 decreased the levels of IL-6, TNF-α and MMP-3, MMP-13 in chondrocytes treated with IL-1β.Conclusions30 μg/ml O3 improved autophagy via activating PPARγ/mTOR signaling and suppressing inflammation in chondrocytes treated with IL-1β.

An Inducible Nitric Oxide Synthase Dimerization Inhibitor Prevents the Progression of Osteoarthritis.

Objective: Osteoarthritis (OA) is a degenerative joint disease. Excessive nitric oxide (NO) mediates the chondrocyte inflammatory response, apoptosis, and extracellular matrix (ECM) degradation during the occurrence and development of OA. NO in chondrocytes is mainly produced by inducible nitric oxide synthase (iNOS). The aim of this study was to design and synthesize an iNOS dimerization inhibitor and evaluate its effects on chondrocyte inflammation and articular cartilage injury in OA via in vitro and in vivo experiments. Design: The title compound 22o was designed, synthesized, and screened based on a previous study. The effects of different concentrations (5, 10, and 20 μM) of compound 22o on chondrocyte inflammatory response and ECM anabolism or catabolism were evaluated by Western blot and real-time quantitative reverse transcription-polymerase chain reaction using the rat chondrocyte model of IL-1β-induced OA. Furthermore, different doses (40 and 80 mg/kg) of compound 22o were administered by gavage to a rat OA model induced by anterior cruciate ligament transection (ACLT), and their protective effects on the articular cartilage were evaluated by histopathology and immunohistochemistry. Results: Compound 22o showed effective iNOS inhibitory activity by inhibiting the dimerization of iNOS. It inhibited the IL-1β-induced expression of cyclooxygenase-2 (COX-2) and matrix metalloproteinase 3 (MMP3) in the chondrocytes, decreased NO production, and significantly increased the expression levels of the ECM anabolic markers, aggrecan (ACAN), and collagen type II (COL2A1). Gavage with compound 22o was found to be effective in the rat OA model induced by ACLT, wherein it regulated the anabolism and catabolism and exerted a protective effect on the articular cartilage. Conclusions: Compound 22o inhibited the inflammatory response and catabolism of the chondrocytes and reduced articular cartilage injury in the rat OA model, indicating its potential as a disease-modifying OA drug.

Echinacoside alleviates osteoarthritis in rats by activating the Nrf2-HO-1 signaling pathway

Objective Osteoarthritis (OA) is a progressive disease characterized by degeneration of cartilage and echinacoside (Ech) has anti-inflammatory and antioxidant effects in various human diseases. This study aimed to reveal the effect and potential mechanism of Ech on OA. Materials and methods The in vitro OA model was established by rat chondrocytes treated with IL-1β, and the in vivo OA model was established by anterior cruciate ligament transaction. The effect of Ech on the viability, inflammatory response, extracellular matrix (ECM) degradation, and oxidative stress of IL-1β-treated rat chondrocytes were evaluated by Cell Counting Kit-8 assay, enzyme-linked immunosorbent assay, quantitative real-time PCR, Western blot, and immunofluorescence assay. Meanwhile, the mechanism of Ech was assessed using Western blot, Cell Counting Kit-8 assay, enzyme-linked immunosorbent assay, and immunofluorescence analysis. Moreover, the function of Ech in vivo was analyzed in rat models of OA. Results Functionally, Ech enhanced the viability of rat chondrocytes, repressed the inflammatory response and ECM degradation of rat chondrocytes induced by IL-1β with restrained oxidative stress. Mechanically, Ech repressed IL-1β-induced chondrocyte injury by activating the Nrf2/HO-1 signaling pathway. Meanwhile, Ech alleviated the degree of articular cartilage injury in rats and exerted protective effects on the rat model of OA in vivo. Discussion and conclusions Ech alleviated OA in rats by activating the Nrf2-HO-1 signaling pathway.

GDF11 inhibits abnormal adipogenesis of condylar chondrocytes in temporomandibular joint osteoarthritis.

AimsAbnormal lipid metabolism is involved in the development of osteoarthritis (OA). Growth differentiation factor 11 (GDF11) is crucial in inhibiting the differentiation of bone marrow mesenchymal stem cells into adipocytes. However, whether GDF11 participates in the abnormal adipogenesis of chondrocytes in OA cartilage is still unclear.MethodsSix-week-old female mice were subjected to unilateral anterior crossbite (UAC) to induce OA in the temporomandibular joint (TMJ). Histochemical staining, immunohistochemical staining (IHC), and quantitative real-time polymerase chain reaction (qRT-PCR) were performed. Primary condylar chondrocytes of rats were stimulated with fluid flow shear stress (FFSS) and collected for oil red staining, immunofluorescence staining, qRT-PCR, and immunoprecipitation analysis.ResultsAbnormal adipogenesis, characterized by increased expression of CCAAT/enhancer-binding protein α (CEBPα), fatty acid binding protein 4 (FABP4), Perilipin1, Adiponectin (AdipoQ), and peroxisome proliferator-activated receptor γ (PPARγ), was enhanced in the degenerative cartilage of TMJ OA in UAC mice, accompanied by decreased expression of GDF11. After FFSS stimulation, there were fat droplets in the cytoplasm of cultured cells with increased expression of PPARγ, CEBPα, FABP4, Perilipin1, and AdipoQ and decreased expression of GDF11. Exogenous GDF11 inhibited increased lipid droplets and expression of AdipoQ, CEBPα, and FABP4 induced by FFSS stimulation. GDF11 did not affect the change in PPARγ expression under FFSS, but promoted its post-translational modification by small ubiquitin-related modifier (SUMOylation). Local injection of GDF11 alleviated TMJ OA-related cartilage degeneration and abnormal adipogenesis in UAC mice.ConclusionAbnormal adipogenesis of chondrocytes and decreased GDF11 expression were observed in degenerative cartilage of TMJ OA. GDF11 supplementation effectively inhibits the adipogenesis of chondrocytes and thus alleviates TMJ condylar cartilage degeneration. GDF11 may inhibit the abnormal adipogenesis of chondrocytes by affecting the SUMOylation of PPARγ.Cite this article: Bone Joint Res 2022;11(7):453–464.

Wnt3a knockdown promotes collagen type II expression in rat chondrocytes.

Osteoarthritis (OA) is a chronic condition caused by cartilage degradation, and there are currently no effective methods for preventing the progression of this disease; gene therapy is a relatively novel method for treating arthritis. Decreased collagen type II (Col2) expression within the cartilage matrix is an important factor for the development of OA, and Wnt3a serves a significant role in cartilage homeostasis. The present study assessed whether Wnt3a knockdown promoted Col2 expression in chondrocytes. Lentivirus-introduced small interfering RNA was used to knock down the expression of Wnt3a in primary rat chondrocytes, and then IL-1β treatment was used to establish an OA chondrocyte model. The expression of target genes (Wnt3a, Col2, MMP-13 and β-catenin) was analyzed using reverse transcription-quantitative PCR, western blotting and immunocytochemistry. There was significantly less MMP-13 and β-catenin expression in the Wnt3a knockdown cells compared with the other controls. Col2 expression was significantly higher in the Wnt3a-knockdown cells compared with the control cells, indicating that knockdown of Wnt3a may promote Col2 expression. Consequently, Wnt3a was indicated to be an important factor in cartilage homeostasis, and Wnt3a knockdown may serve as a novel method for OA therapy.

YAP plays a protective role in T-2 toxin-induced inhibition of chondrocyte proliferation and matrix degradation

Previous research has shown that T-2 toxin can damage cartilage, resulting in a disease phenotype similar to osteoarthritis. The precise molecular mechanism by which T-2 toxin causes chondrocyte injury, however, is unknown. The purpose of this study was to look into the role of YAP in T-2 toxin-induced rat chondrocyte injury. Based on research results, T-2 toxin decreased the levels of collagen II and PCNA while increasing the expression of matrix metalloproteinase MMP13. These findings supported the T-2 toxin's detrimental effect on chondrocytes. YAP's role in T-2 toxin-induced chondrocyte injury was also investigated. Total YAP and related nuclear proteins were found to decrease as the concentration of T-2 toxin increased. While PYAP expression was not significantly altered in response to T-2 toxin, the PYAP/YAP ratio decreased as the T-2 toxin concentration increased, implying that the HIPPO signaling pathway was activated. Furthermore, the YAP-specific inhibitor Verteporfin was used to investigate the role of YAP in T-2 toxin-induced chondrocyte injury. YAP inhibition increased MMP13 expression while decreasing COL2 and PCNA levels. In summary, the current study found that T-2 toxin decreased the levels of COL2 and PCNA while increasing the expression of MMP13 in chondrocytes after inhibiting YAP, providing a new insight into the mechanism of T-2 toxin-induced cartilage damage.

Upregulation of P2Y12 inhibits chondrocyte apoptosis in lumbar osteoarthritis through the PI3K/AKT signaling pathway.

Lumbar facet osteoarthritis (FJOA) is a major cause of severe lower back pain and disability worldwide. However, the mechanism underlying cartilage degeneration in FJOA remains unclear. The purpose of this study was to investigate the regulation and mechanism of P2Y12 on chondrocyte apoptosis in FJOA. The experimental rats were randomly divided into non-operation (n = 20) and operation groups (n = 20). In the operation group, Sodium iodoacetate (MIA, Sigma, 200mg/mL) was injected into the right L4/5 facet process using a blunt nanoneedle 26 (WPI, Sarasota, FL, USA) under the control of an injection pump. The final injection volume was 5µL and the injection rate was 2µL/min. The facet joint was removed four weeks after surgery. After the operation, samples were stored at -80°C until further use, whereby the right facet joints in each group were tested. Hematoxylin and eosin (HE) and iron-red solid green staining were used to observe the degeneration of articular chondrocytes in rats. Immunohistochemistry and western blotting were used to observe the expressions of P2Y12, Matrix metalloproteinase 13 (MMP13), Collagen II (COL2), and other cartilage degeneration and apoptosis-related genes. Co-localization of P2Y12-cleaved caspase-3 in the apoptosis model was detected by dual-standard immunofluorescence staining. Apoptosis was also detected by flow cytometry and TUNEL assay.P2Y12 is highly expressed in OA cartilage tissue, and inhibits IL-1β -induced chondrocyte apoptosis through PI3K/AKT signaling pathway, thus playing a certain protective role on cartilage.

PTH (1-34) enhances the therapeutic effect of bone marrow mesenchymal stem cell-derived exosomes by inhibiting proinflammatory cytokines expression on OA chondrocyte repair in vitro

BackgroundThe effects of bone marrow mesenchymal stem cells (BMSCs) during the treatment of cartilage damage have been proven to be attributed to paracrine mechanisms, particularly the effect of exosomes. Exosomes from different batches are inhomogeneous, and different treatment effects are observed between samples. The purpose of this research was to find more effective and homogeneous exosomes for the repair of chondrocytes in osteoarthritis (OA). We observed the potential effects and possible mechanisms of exosomes derived from parathyroid hormone (PTH) (1-34)-preconditioned BMSCs (ExoPTH) in the alleviation of OA.Materials and methodsExosomes derived from BMSCs (ExoBMSC) and ExoPTH were isolated by differential centrifugation. Primary rat chondrocytes were used to establish the OA model by interleukin 1 beta (IL-1β) in vitro. The effects of these two types of exosomes on OA chondrocyte proliferation, migration, apoptosis, and extracellular matrix formation were measured and compared. We observed changes in IL-2, TNF-α, and IL-6 levels via Western blotting (WB), and quantitative real-time PCR (qRT–PCR).ResultsWe successfully extracted ExoBMSC and ExoPTH and established an IL-1β-induced OA model in primary chondrocytes from rats. Our study showed that IL-2, TNF-α, and IL-6 levels increased significantly in OA chondrocytes; however, both ExoBMSC and ExoPTH reduced the levels of IL-2, TNF-α, and IL-6. In addition, ExoPTH exhibited stronger anti-inflammatory effects. ExoPTH had a more marked effect on proliferation, migration, and production of the extracellular matrix (Col-II) in OA chondrocytes than ExoBMSC at 24 h.ConclusionExoPTH increased the migration, proliferation, and chondral matrix formation of OA chondrocytes in vitro. In OA chondrocyte therapy, the potential mechanism of ExoPTH might involve the inhibition of production of proinflammatory cytokines. Although the two types of exosomes had some similar effects, most effects of ExoPTH were better than those of ExoBMSC, so ExoPTH may have a better ability to alleviate OA.

Effects of Diosmin on IL-1β- Induced Inflammatory Response in Primary Rat Chondrocytes

Effects of Diosmin on IL-1β- Induced Inflammatory Response in Primary Rat Chondrocytes

Chaenomeles Fructus (CF), the Fruit of Chaenomeles sinensis Alleviates IL-1β Induced Cartilage Degradation in Rat Articular Chondrocytes

Osteoarthritis (OA) causes persistent pain, joint dysfunction, and physical disability. It is the most prevalent type of degenerative arthritis, affecting millions of people worldwide. OA is currently treated with a focus on pain relief, inflammation control, and artificial joint surgery. Hence, a therapeutic agent capable of preventing or delaying the progression of OA is needed. OA is strongly associated with the degeneration of the articular cartilage and changes in the ECM, which are primarily associated with a decrease in proteoglycan and collagen. In the progress of articular cartilage degradation, catabolic enzymes, such as matrix metalloproteinases (MMPs), are activated by IL-1β stimulation. Given the tight relationship between IL-1β and ECM (extra-cellular matrix) degradation, this study examined the effects of Chaenomeles Fructus (CF) on IL-1β-induced OA in rat chondrocytes. The CF treatment reduced IL-1β-induced MMP3/13 and ADAMTS-5 production at the mRNA and protein levels. Similarly, CF enhanced col2a and aggrecan accumulation and chondrocyte proliferation. CF inhibited NF-κB (nuclear factor kappa B) activation, nuclear translocation induced by IL-1β, reactive oxygen species (ROS) production, and ERK phosphorylation. CF demonstrated anti-OA and articular regeneration effects on rat chondrocytes, thus, suggesting that CF is a viable and fundamental therapeutic option for OA.

The protective effect of mangiferin on osteoarthritis: An in vitro and in vivo study.

Mangiferin is a kind of polyphenol chemical compound separated from these herbal medicines of Mangifera indica L., Anemarrhena asphodeloides Bge. and Belamcanda chinensis L., which has anti-inflammatory, anti-virus, and other physiological activities without toxic effects. Osteoarthritis (OA) is a chronic disease that is also a kind of arthritis disease in which articular cartilage or bones under the joint is damaged. In addition, artificial replacements are required in severe cases. At present, there are not too much researches on the potential biological activities of mangiferin that plays a protective role in the treatment of OA. In this study, we evaluated the protective effect of mangiferin on osteoarthritis (OA) in vitro and in vivo. First, the effect of different concentrations of mangiferin on rat chondrocytes was determined by MTT assay. Second, the effects of mangiferin on the expression levels of matrix metalloproteinase (MMP)-13, TNF alpha, Collagen II, Caspase-3, and cystatin-C in interleukin-1beta (IL-1beta)-induced rat chondrocytes were examined by the real-time polymerase chain reaction in vitro, meanwhile the effects of mangiferin on the nuclear factor kappa-B (NF-kappaB) signaling pathway were also investigated by Western Blot. Finally, the anti-osteoarthritic protective effect of mangiferin was evaluated in the rat model by anterior cruciate ligament transection (ACLT) combined with bilateral ovariectomy-induced OA in vivo. The results showed that the mangiferin was found to inhibit the expression of MMP-13, TNF-alpha, and Caspase-3 which also increased the expression of Collagen II and cystatin-C in IL 1beta induced rat chondrocytes. In addition, IL-1beta-induced activation of nuclear factor kappa-B (NF-kappaB) and the degradation of inhibitor of kappaB (IkappaB)-alpha were suppressed by mangiferin. For the in vivo study in a rat model of OA, 100 microl of mangiferin was administered by intra-articular injections for rats, the results showed that the cartilage degradation was suppressed by mangiferin through Micro CT and Histological Examination. According to both in vitro and in vivo results, mangiferin has a protective effect in the treatment of OA which may be a promising therapeutic agent for OA.

Fuzi decoction ameliorates pain and cartilage degeneration of osteoarthritic rats through PI3K-Akt signaling pathway and its clinical retrospective evidence

BackgroundOsteoarthritis (OA) is a difficult disease but the clinic lacks effective therapy. As a classic formula of traditional Chinese medicine (TCM), Fuzi decoction (FZD) has been clinically applied for treating OA-related syndromes, but its anti-OA efficacy and mechanism remain unclear. PurposeTo experimentally and clinically determine the anti-OA efficacy of FZD and clarify the underlying mechanism. MethodsUPLC/MS/MS was applied to identify the main components of FZD. A monoiodoacetate (MIA)-induced OA rat model was employed to evaluate the in vivo efficacy of FZD against OA, by using pain behavior assessment, histopathological observation, and immunohistochemical analysis. Primary rat chondrocytes were isolated to determine the in vitro effects of FZD by using cell viability assay, wound healing assay, and real-time PCR (qPCR) analysis on anabolic/catabolic mRNA expressions. RNA sequencing (RNA-seq) and network pharmacology analysis were conducted and the overlapping data were used to predict the mechanism of FZD, followed by verification with qPCR and Western blot assays. Finally, a retrospective analysis was performed to confirm FZDʹs efficacy and safety in OA patients. ResultsThe UPLC/MS/MS result showed that FZD contained atractylenolide I, benzoylhypaconitine, benzoylmesaconitine, benzoylaconitine, hypaconitine, mesaconitine, aconitine, lobetyolin, paeoniflorin, and pachymic acid. The in vivo data showed that FZD restored the cartilage degeneration in MIA-induced OA rats by ameliorating pain behavior parameters, recovering histopathological alterations, benefitting cartilage anabolism (up-regulating Col2 expression), and suppressing catabolism (down-regulating MMP13 and Col10 expressions). The in vitro data showed that FZD increased cell viability and wound healing capacity of chondrocytes, and restored the altered expressions of anabolic and catabolic genes of chondrocytes. The overlapping results of RNA-seq and network pharmacology analysis suggested that PI3K/Akt signaling mediated the anti-OA mechanism of FZD, which was verified by qPCR and Western blot experiments. Clinically, the anti-OA efficacy and safety of FZD were confirmed by the retrospective analysis on OA patients. ConclusionThe scientific innovation of this study was the determination of anti-OA efficacy of FZD by experimental and clinical evidence and the discovery of its mechanism by integrated RNA-seq, network pharmacology, and molecular experiments, which suggests FZD as a promising TCM agency for OA treatment.

Efficient TGF-β1 Delivery to Articular Chondrocytes In Vitro Using Agro-Based Liposomes.

The low efficiency in transfecting rat- and human-derived chondrocytes have been hampering developments in the field of cartilage biology. Transforming growth factor (TGF)-β1 has shown positive effects on chondrocytes, but its applications remain limited due to its short half-life, low stability and poor penetration into cartilage. Naturally derived liposomes have been shown to be promising delivery nanosystems due to their similarities with biological membranes. Here, we used agro-based rapeseed liposomes, which contains a high level of mono- and poly-unsaturated fatty acids, to efficiently deliver encapsulated TGF-β1 to rat chondrocytes. Results showed that TGF-β1 encapsulated in nano-sized rapeseed liposomes were safe for chondrocytes and did not induce any alterations of their phenotype. Furthermore, the controlled release of TGF-β1 from liposomes produced an improved response in chondrocytes, even at low doses. Altogether, these outcomes demonstrate that agro-based nanoliposomes are promising drug carriers.

Chondroprotective Effects of 4,5-Dicaffeoylquinic Acid in Osteoarthritis through NF-κB Signaling Inhibition.

Osteoarthritis (OA) is characterized by cartilage degradation, inflammation, and pain. The dicaffeoylquinic acid (diCQA) isomer, 4,5-diCQA, exhibits antioxidant activity and various other health-promoting benefits, but its chondroprotective effects have yet to be elucidated. In this study, we aimed to investigate the chondroprotective effects of 4,5-diCQA on OA both in vitro and in vivo. Primary rat chondrocytes were pre-treated with 4,5-diCQA for 1 h before stimulation with interleukin (IL)-1β (5 ng/mL). The accumulation of nitrite, PGE2, and aggrecan was observed using the Griess reagent and ELISA. The protein levels of iNOS, COX-2, MMP-3, MMP-13, ADMATS-4, MAPKs, and the NF-κB p65 subunit were measured by Western blotting. In vivo, the effects of 4,5-diCQA were evaluated for 2 weeks in a destabilization of the medial meniscus (DMM)-surgery-induced OA rat model. 4,5-diCQA significantly inhibited IL-1β-induced expression of nitrite, iNOS, PGE2, COX-2, MMP-3, MMP-13, and ADAMTS-4. 4,5-diCQA also decreased the IL-1β-induced degradation of aggrecan. It also suppressed the IL-1β-induced phosphorylation of MAPKs and translocation of the NF-κB p65 subunit to the nucleus. These findings indicate that 4,5-diCQA inhibits DMM-surgery-induced cartilage destruction and proteoglycan loss in vivo. 4,5-diCQA may be a potential therapeutic agent for the alleviation of OA progression. In this study, diclofenac was set to be administered once every two days, but it showed an effect on OA. These results may be used as basic data to suggest a new dosing method for diclofenac.

The Expression of MDM2 Gene Promoted Chondrocyte Proliferation in Rats with Osteoarthritis via the Wnt/β-Catenin Pathway.

This study aimed to investigate the regulatory mechanism of MDM2 gene expression on cartilage cell proliferation in Osteoarthritis (OA) rats. For this purpose, 22 SD rats were randomly divided into normal control (10 cases) and treated (12 cases) groups. Treated group was used for OA modelling with the modified Hulth method. After a week, RT-PCR was used to detect MDM2 in cartilage tissue of rats, Wnt 1, Wnt 3 a, Wnt 10 b and β-catenin genes mRNA expression. Rat chondrocytes were isolated and cultured, and the recombinant eukaryotic expression vector pcDNA3.1 myc-siRNA-MDM2-β-catenin and co-expression plasmid pcDNA3.1 myc-siRNA-MDM2-β-catenin was used to transfect chondrocytes and the proliferation and related gene expression levels of the transfected chondrocytes were detected by MTT method and RT-PCR. The results showed that compared with the control group, MDM2, Wnt 1, Wnt 3 a, Wnt 10b and β-catenin genes in OA rat cartilage constructed by Hulth method were increased (p<0.05). The pcDNA3.1 myc-beta-catenin transfection slowed down the proliferation of OA chondrocytes, different from the non-transfected OA group (p<0.001), and increased Wnt 1, Wnt 3a, Wnt 10b and β-catenin genes expression compared with the Control group (p<0.05), but did not affect the expression of MDM2. The transfection of siRNA-MDM2 was opposite to pcDNA3.1 myc-β-catenin. The co-expression plasmid pcDNA3.1 myc-siRNA-MDM2-beta-catenin transfection did not affect the proliferation of OA chondrocytes. In general, the high expression of MDM2 in OA rats restricts the proliferation of chondrocytes, which may be related to the main pathogenesis of the occurrence and development of OA in vivo, and the regulation of MDM2 on the proliferation of chondrocytes may be achieved through the Wnt/ β-catenin pathway.

Identification of thrombin as a key regulator of chondrocyte catabolic activity through RNA-Seq and experimental verification

The present study was designed to explore the relationship between thrombin and catabolic activity in chondrocytes. Primary rat chondrocytes were cultured for 24 h with rat serum (RS), rat plasma (RP), or rat plasma supplemented with thrombin (RPT). RNA-sequencing was then performed. Cell proliferation was analyzed by EdU uptake, CCK-8 assays and protein–protein interaction (PPI) network of proliferation-related genes. Heatmaps were used to visualize differences in gene expression. Gene Ontology (GO) enrichment analyses of up- and down-regulated differentially expressed genes were conducted. Molecular probes were used to label the endoplasmic reticulum in chondrocytes from three treatment groups. Immunofluorescence and Safranin O staining were used to assess type II collagen (Col2a1) expression and proteoglycan synthesis, whereas Lox expression was assessed by immunocytochemistry. The expression of enzymes involved in the synthesis and maturation of extracellular matrix (ECM) components and chemokines were measured by qPCR while matrix metalloproteinases (MMPs) levels were evaluated by Western blotting. Relevant nodules were selected through further PPI network analyses. A total of 727 and 1162 genes were up- and down-regulated based on the Venn diagrams comparison among groups. Thrombin was thus able to promote chondrocyte proliferation and a shift towards fibrotic morphology, while upregulating MMPs and chemokines linked to ECM degradation. In addition, thrombin decreased the enzyme expression involved in the synthesis and maturation of ECM.

Similarities and differences between rat and mouse chondrocyte gene expression induced by IL-1\u03b2

BackgroundOsteoarthritis (OA) is the most prevalent degenerative joint disease. In vitro experiments are an intuitive method used to investigate its early pathogenesis. Chondrocyte inflammation models in rats and mice are often used as in vitro models of OA. However, similarities and differences between them in the early stages of inflammation have not been reported.ObjectiveThis paper seeks to compare the chondrocyte phenotype of rats and mice in the early inflammatory state and identify chondrocytes suitable for the study of early OA.MethodsUnder similar conditions, chondrocytes from rats and mice were stimulated using the same IL-1β concentration for a short period of time. The phenotypic changes of chondrocytes were observed under a microscope. The treated chondrocytes were subjected to RNA-seq to identify similarities and differences in gene expression. Chondrocytes were labelled with EdU for proliferation analysis. Cell proliferation-associated proteins, including minichromosome maintenance 2 (MCM2), minichromosome maintenance 5 (MCM5), Lamin B1, proliferating cell nuclear antigen (PCNA), and Cyclin D1, were analysed by immunocytochemical staining, cell immunofluorescence, and Western blots to verify the RNA-seq results.ResultsRNA-seq revealed that the expression patterns of cytokines, chemokines, matrix metalloproteinases, and collagen were similar between the rat and mouse chondrocyte inflammation models. Nonetheless, the expression of proliferation-related genes showed the opposite pattern. The RNA-seq results were further verified by subsequent experiments. The expression levels of MCM2, MCM5, Lamin B1, PCNA, and Cyclin D1 were significantly upregulated in rat chondrocytes (P < 0.05) and mouse chondrocytes (P < 0.05).ConclusionsBased on the findings, the rat chondrocyte inflammation model may help in the study of the early pathological mechanism of OA.

CF101 alleviates OA progression and inhibits the inflammatory process via the AMP/ATP/AMPK/mTOR axis.

CF101 (IB-MECA) is an adenosine A3 receptor agonist that has anti-inflammatory and pain-relieving properties. Adenosine A3 receptor activation can delay the process of Osteoarthritis(OA) and prevent the occurrence of OA. However, the mechanism of CF101 on OA is still unknown. This study aimed to investigate the effect of CF101 on rats induced by anterior cruciate ligament-transection (ACLT) and rat chondrocytes induced by IL-1ß. ACLT-induced OA rats were administered CF101, and autophagy levels were measured to determine whether CF101 had an autophagy-mediated protective effect on articular cartilage. Furthermore, the mechanism by which CF101 protected articular cartilage in IL-1ß-induced chondrocytes mimicking OA was investigated. In rats treated with ACLT, CF101 was able to delay the progression of OA, as well as reduce inflammation and type II collagen degradation factors. In addition, in vitro experiments revealed that CF101 reduced type II collagen degradation factors in OA chondrocytes. In rats treated with ACLT and OA chondrocytes, CF101 enhanced autophagy and increased the ratio of AMP/ATP and AMPK protein levels while decreasing mTOR expression. Treatment of OA chondrocytes with 3-MA prior to treatment with CF101 resulted in inhibition of autophagy factor levels, as well as increased levels of inflammatory factors and type II collagen degradation compared to the CF101 group. These findings demonstrated that CF101 could protect articular cartilage against OA by enhancing the ratio of ATP/AMP and altering the AMPK/mTOR pathway to enhance autophagy and reduce inflammation. In addition, inhibition of autophagy resulted in a reduced CF101 effect.

Timosaponin B-II alleviates osteoarthritis-related inflammation and extracellular matrix degradation through inhibition of mitogen-activated protein kinases and nuclear factor-κB pathways in vitro

ABSTRACT Osteoarthritis (OA), an inflammatory response in chondrocytes, leads to extracellular matrix (ECM) degradation and cartilage destruction. Timosaponin B-II (TB-II) is the main bioactive component of Rhizoma Anemarrhenae with reported antioxidant and anti-inflammatory effects. This study investigated the anti-OA function and mechanism of TB-II on IL-1β-stimulated SW1353 cells and primary rat chondrocytes. We firstly screened the concentration of TB-II in SW1353 cells and primary rat chondrocytes using CCK-8 assay. Thereafter, SW1353 cells and chondrocytes were, respectively, pretreated with TB-II (20 and 40 μg/mL) and TB-II (10 and 30 μg/mL) for 24 h and then stimulated with interleukin 1β (IL-1β, 10 ng/mL) for another 24 hours. Results showed that TB-II suppressed the production of reactive oxygen species, the protein levels of inducible nitric oxide synthase and cyclooxygenase-2 in IL-1β-stimulated SW1353 cells and chondrocytes. IL-1β-induced high secretion levels of nitric oxide and prostaglandin 2, TNF-α, IL-6 and MCP-1 were down-regulated by TB-II treatment, indicating an anti-inflammatory effect of TB-II on OA in vitro condition. Moreover, TB-II weakened the mRNA and protein expression of (matrix metalloproteinase) MMPs including MMP-1, MMP-3, and MMP-13, indicating the protection of TB-II against ECM degradation. Mechanically, TB-II suppressed MAPKs and NF-κB pathways under IL-1β stimulation evidenced by the down-regulated protein expression of p-ERK, p-p38, p-JNK, p-p65 and the reduced translocation of p65 subunit to the nucleus. The present study demonstrated that TB-II might become a novel therapeutic agent for OA treatment through repressing IL-1β-stimulated inflammation, oxidative stress and ECM degradation via suppressing the MAPKs and NF-κB pathways.

Ononin ameliorates inflammation and cartilage degradation in rat chondrocytes with IL-1\u03b2-induced osteoarthritis by downregulating the MAPK and NF-\u03baB pathways

BackgroundOsteoarthritis (OA) treatment aims to improve inflammation and delay cartilage degeneration. However, there is no effective strategy presently available. Ononin, a representative isoflavone glycoside component extracted from natural Chinese herbs, exerts anti-inflammatory and proliferative effects. However, the therapeutic effect of ononin on chondrocyte inflammation remains unclear.MethodsIn this study, we explored the therapeutic effect and potential mechanism of ononin in OA by establishing an interleukin-1 beta (IL-1β)-induced chondrocyte inflammation model.ResultsOur results verified that ononin alleviated the IL-1β-induced decrease in chondrocyte viability, attenuated the overexpression of the inflammatory factors tumour necrosis factor α (TNF-α) and interleukin 6 (IL-6), and simultaneously inhibited the expression of cartilage extracellular matrix (ECM)-degrading enzymes such as matrix metalloproteinase-13 (MMP-13). Furthermore, the decomposition of Collagen II protein could be alleviated in the OA model by ononin. Finally, ononin improved chondrocyte inflammation by downregulating the mitogen-activated protein kinase (MAPK) and nuclear factor kappa-B (NF-κB) signalling pathways.ConclusionOur findings suggested that ononin could inhibit the IL-1β-induced proinflammatory response and ECM degradation in chondrocytes by interfering with the abnormal activation of the MAPK and NF-κB pathways, indicating its protective effect against OA.