ABSTRACT
In osteoarthritis (OA), extracellular matrix (ECM) digestion by cartilage-degrading enzymes drives cartilage destruction and generates ECM fragments, such as proteoglycan aggrecan (PG) peptides. PG peptides have been shown to induce immunological functions of chondrocytes. However, the role of PG peptides in stimulating catabolic mediators from chondrocytes has not been investigated. Therefore, we aim to determine the effects and its mechanism by which PG peptides induce chondrocytes to produce catabolic mediators in OA. Human chondrocytes were stimulated with IFNγ and various PG peptides either (i) with or (ii) without TLR2 blockade or (iii) with Lactobacillus species-conditioned medium (LCM), a genus of bacteria with anti-inflammatory properties. Transcriptomic analysis, cartilage-degrading enzyme production and TLR2-intracellular signaling activation were investigated. Chondrocytes treated with PG peptides p16-31 and p263-280 increased expression levels of genes associated with chondrocyte hypertrophy, cartilage degradation and proteolytic enzyme production. TLR2 downstream signaling proteins (STAT3, IkBα and MAPK9) were significantly phosphorylated in p263-280 peptide-stimulated chondrocytes. MMP-1 and ADAMTS-4 were significantly reduced in p263-280 peptides-treated condition with TLR2 blockade or LCM treatment. Phosphorylation levels of IkBa, ERK1/2 and MAPK9 were significantly decreased with TLR2 blockade, but only phosphorylation levels of MAPK9 was significantly decreased with LCM treatment. Our study showed that PG peptide stimulation via TLR2 induced cartilage-degrading enzyme production via activation of MAPK, NFκB and STAT3 pathways.
Subject(s)
Aggrecans , Chondrocytes , Lactobacillus , Toll-Like Receptor 2 , Chondrocytes/metabolism , Chondrocytes/drug effects , Humans , Toll-Like Receptor 2/metabolism , Aggrecans/metabolism , Culture Media, Conditioned/pharmacology , Lactobacillus/metabolism , Signal Transduction/drug effects , Osteoarthritis/metabolism , Osteoarthritis/pathology , Cells, Cultured , ADAMTS4 Protein/metabolism , STAT3 Transcription Factor/metabolism , Peptides/pharmacology , Peptides/metabolism , Proteoglycans/metabolism , Proteoglycans/pharmacology , Matrix Metalloproteinase 1/metabolism , Matrix Metalloproteinase 1/genetics , NF-KappaB Inhibitor alpha/metabolismABSTRACT
BACKGROUND: Osteoarthritis (OA) is a degenerative joint disease characterized by the progressive degeneration of articular cartilage, leading to pain, stiffness, and loss of joint function. The pathogenesis of OA involves multiple factors, including increased intracellular reactive oxygen species (ROS), enhanced chondrocyte apoptosis, and disturbances in cartilage matrix metabolism. These processes contribute to the breakdown of the extracellular matrix (ECM) and the loss of cartilage integrity, ultimately resulting in joint damage and dysfunction. RNA interference (RNAi) therapy has emerged as a promising approach for the treatment of various diseases, including hATTR and acute hepatic porphyria. By harnessing the natural cellular machinery for gene silencing, RNAi allows for the specific inhibition of target genes involved in disease pathogenesis. In the context of OA, targeting key molecules such as matrix metalloproteinase-13 (MMP13), which plays a critical role in cartilage degradation, holds great therapeutic potential. RESULTS: In this study, we developed an innovative therapeutic approach for OA using a combination of liposome-encapsulated siMMP13 and NG-Monomethyl-L-arginine Acetate (L-NMMA) to form an injectable hydrogel. The hydrogel served as a delivery vehicle for the siMMP13, allowing for sustained release and targeted delivery to the affected joint. Experiments conducted on destabilization of the medial meniscus (DMM) model mice demonstrated the therapeutic efficacy of this composite hydrogel. Treatment with the hydrogel significantly inhibited the degradation of cartilage matrix, as evidenced by histological analysis showing preserved cartilage structure and reduced loss of proteoglycans. Moreover, the hydrogel effectively suppressed intracellular ROS accumulation in chondrocytes, indicating its anti-oxidative properties. Furthermore, it attenuated chondrocyte apoptosis, as demonstrated by decreased levels of apoptotic markers. CONCLUSION: In summary, the injectable hydrogel containing siMMP13, endowed with anti-ROS and anti-apoptotic properties, may represent an effective therapeutic strategy for osteoarthritis in the future.
Subject(s)
Apoptosis , Chondrocytes , Hydrogels , Matrix Metalloproteinase 13 , Osteoarthritis , Reactive Oxygen Species , Animals , Osteoarthritis/drug therapy , Osteoarthritis/metabolism , Osteoarthritis/pathology , Reactive Oxygen Species/metabolism , Apoptosis/drug effects , Hydrogels/chemistry , Matrix Metalloproteinase 13/metabolism , Mice , Chondrocytes/metabolism , Chondrocytes/drug effects , Mice, Inbred C57BL , Male , Cartilage, Articular/metabolism , Cartilage, Articular/drug effects , Cartilage, Articular/pathology , Liposomes/chemistry , HumansABSTRACT
Eriodictyol, a flavonoid distributed in citrus fruits, has been known to exhibit anti-inflammatory activity. In this study, destabilized medial meniscus (DMM)-induced OA model was used to investigate the protective role of eriodictyol on OA. Meanwhile, we used an IL-1ß-stimulated human osteoarthritis chondrocytes model to investigate the anti-inflammatory mechanism of eriodictyol on OA. The production of nitric oxide was detected by Griess reaction. The productions of MMP1, MMP3, and PGE2 were detected by ELISA. The expression of LXRα, ABCA1, PI3K, AKT, and NF-κB were measured by western blot analysis. The results demonstrated that eriodictyol could alleviate DMM-induced OA in mice. In vitro, eriodictyol inhibited IL-1ß-induced NO, PGE2, MMP1, and MMP3 production in human osteoarthritis chondrocytes. Eriodictyol also suppressed the phosphorylation of PI3K, AKT, NF-κB p65, and IκBα induced by IL-1ß. Meanwhile, eriodictyol significantly increased the expression of LXRα and ABCA1. Furthermore, eriodictyol disrupted lipid rafts formation through reducing the cholesterol content. And cholesterol replenishment experiment showed that adding water-soluble cholesterol could reverse the anti-inflammatory effect of eriodictyol. In conclusion, the results indicated eriodictyol inhibited IL-1ß-induced inflammation in human osteoarthritis chondrocytes through suppressing lipid rafts formation, which subsequently inhibiting PI3K/AKT/NF-κB signaling pathway.
Subject(s)
Chondrocytes , Flavanones , NF-kappa B , Osteoarthritis , Phosphatidylinositol 3-Kinases , Proto-Oncogene Proteins c-akt , Signal Transduction , Flavanones/pharmacology , Animals , Humans , Signal Transduction/drug effects , NF-kappa B/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Chondrocytes/drug effects , Chondrocytes/metabolism , Mice , Osteoarthritis/drug therapy , Osteoarthritis/metabolism , Osteoarthritis/pathology , Interleukin-1beta/metabolism , Liver X Receptors/metabolism , Male , Inflammation/drug therapy , Inflammation/metabolism , Inflammation/pathology , ATP Binding Cassette Transporter 1/metabolism , ATP Binding Cassette Transporter 1/genetics , Disease Progression , Disease Models, Animal , Anti-Inflammatory Agents/pharmacology , Nitric Oxide/metabolism , Mice, Inbred C57BLABSTRACT
Chondrocyte ferroptosis induces the occurrence of osteoarthritis (OA). As a key gene of OA, C5a receptor 1 (C5AR1) is related to ferroptosis. Here, we investigated whether C5AR1 interferes with chondrocyte ferroptosis during OA occurrence. C5AR1 was downregulated in PA-treated chondrocytes. Overexpression of C5AR1 increased the cell viability and decreased ferroptosis in chondrocytes. Moreover, Tumor necrosis factor superfamily member 13B (TNFSF13B) was downregulated in PA-treated chondrocytes, and knockdown of TNFSF13B eliminated the inhibitory effect of C5AR1 on ferroptosis in chondrocytes. More importantly, the PI3K/Akt/GSK3ß/Nrf2/HO-1 pathway inhibitor LY294002 reversed the inhibition of C5AR1 or TNFSF13B on ferroptosis in chondrocytes. Finally, we found that C5AR1 alleviated joint tissue lesions and ferroptosis in rats and inhibited the progression of OA in the rat OA model constructed by anterior cruciate ligament transection (ACLT), which was reversed by interfering with TNFSF13B. This study shows that C5AR1 reduces the progression of OA by upregulating TNFSF13B to activate the PI3K/Akt/GSK3ß/Nrf2/HO-1 pathway and thereby inhibiting chondrocyte sensitivity to ferroptosis, indicating that C5AR1 may be a potential therapeutic target for ferroptosis-related diseases.
Subject(s)
Chondrocytes , Ferroptosis , Glycogen Synthase Kinase 3 beta , NF-E2-Related Factor 2 , Osteoarthritis , Phosphatidylinositol 3-Kinases , Proto-Oncogene Proteins c-akt , Rats, Sprague-Dawley , Receptor, Anaphylatoxin C5a , Animals , Ferroptosis/drug effects , Proto-Oncogene Proteins c-akt/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Chondrocytes/metabolism , Chondrocytes/pathology , Chondrocytes/drug effects , NF-E2-Related Factor 2/metabolism , NF-E2-Related Factor 2/genetics , Rats , Osteoarthritis/metabolism , Osteoarthritis/pathology , Osteoarthritis/genetics , Glycogen Synthase Kinase 3 beta/metabolism , Glycogen Synthase Kinase 3 beta/genetics , Male , Receptor, Anaphylatoxin C5a/metabolism , Receptor, Anaphylatoxin C5a/genetics , Signal Transduction , Heme Oxygenase-1/metabolism , Heme Oxygenase-1/genetics , Heme Oxygenase (Decyclizing)ABSTRACT
BACKGROUND: Excessive fluoride exposure induces skeletal fluorosis, but the specific mechanism responsible is still unclear. Therefore, this study aimed to identify the pathogenesis of fluoride-induced bone injuries. METHODS: We systematically searched fluoride-induced bone injury-related genes from five databases. Then, these genes were subjected to enrichment analyses. A TF (transcription factor)-mRNA-miRNA network and protein-protein interaction (PPI) network were constructed using Cytoscape, and the Human Protein Atlas (HPA) database was used to screen the expression of key proteins. The candidate pharmacological targets were predicted using the Drug Signature Database. RESULTS: A total of 85 studies were included in this study, and 112 osteoblast-, 35 osteoclast-, and 41 chondrocyte-related differential expression genes (DEGs) were identified. Functional enrichment analyses showed that the Atf4, Bcl2, Col1a1, Fgf21, Fgfr1 and Il6 genes were significantly enriched in the PI3K-Akt signaling pathway of osteoblasts, Mmp9 and Mmp13 genes were enriched in the IL-17 signaling pathway of osteoclasts, and Bmp2 and Bmp7 genes were enriched in the TGF-beta signaling pathway of chondrocytes. With the use of the TF-mRNA-miRNA network, the Col1a1, Bcl2, Fgfr1, Mmp9, Mmp13, Bmp2, and Bmp7 genes were identified as the key regulatory factors. Selenium methyl cysteine, CGS-27023A, and calcium phosphate were predicted to be the potential drugs for skeletal fluorosis. CONCLUSIONS: These results suggested that the PI3K-Akt signaling pathway being involved in the apoptosis of osteoblasts, with the IL-17 and the TGF-beta signaling pathways being involved in the inflammation of osteoclasts and chondrocytes in fluoride-induced bone injuries.
Subject(s)
Apoptosis , Fluorides , Inflammation , Osteoblasts , Signal Transduction , Humans , Fluorides/adverse effects , Apoptosis/drug effects , Osteoblasts/drug effects , Osteoblasts/metabolism , Inflammation/chemically induced , Signal Transduction/drug effects , MicroRNAs/metabolism , MicroRNAs/genetics , Osteoclasts/drug effects , Osteoclasts/metabolism , Chondrocytes/drug effects , Chondrocytes/metabolism , Protein Interaction Maps , RNA, Messenger/metabolism , RNA, Messenger/genetics , Gene Regulatory Networks , Gene Expression Regulation/drug effects , Bone Diseases/chemically induced , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
Nicotine is developmentally toxic. Prenatal nicotine exposure (PNE) affects the development of multiple fetal organs and causes susceptibility to a variety of diseases in offspring. In this study, we aimed to investigate the effect of PNE on cartilage development and osteoarthritis susceptibility in female offspring rats. Wistar rats were orally gavaged with nicotine on days 9-20 of pregnancy. The articular cartilage was obtained at gestational day (GD) 20 and postnatal week (PW) 24, respectively. Further, the effect of nicotine on chondrogenic differentiation was explored by the chondrogenic differentiation model in human Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs). The PNE group showed significantly shallower Safranin O staining and lower Collagen 2a1 content of articular cartilage in female offspring rats. Further, we found that PNE activated pyroptosis in the articular cartilage at GD20 and PW24. In vitro experiments revealed that nicotine inhibited chondrogenic differentiation and activated pyroptosis. After interfering with nod-like receptors3 (NLRP3) expression by SiRNA, it was found that pyroptosis mediated the chondrogenic differentiation inhibition of WJ-MSCs induced by nicotine. In addition, we found that α7-nAChR antagonist α-BTX reversed nicotine-induced NLRP3 and P300 high expression. And, P300 SiRNA reversed the increase of NLRP3 mRNA expression and histone acetylation level in its promoter region induced by nicotine. In conclusion, PNE caused chondrodysplasia and poor articular cartilage quality in female offspring rats. PNE increased the histone acetylation level of NLRP3 promoter region by α7-nAChR/P300, which resulting in the high expression of NLRP3. Further, NLRP3 mediated the inhibition of chondrogenic differentiation by activating pyroptosis.
Subject(s)
Cartilage, Articular , Mesenchymal Stem Cells , NLR Family, Pyrin Domain-Containing 3 Protein , Nicotine , Prenatal Exposure Delayed Effects , Pyroptosis , Rats, Wistar , alpha7 Nicotinic Acetylcholine Receptor , Animals , Nicotine/pharmacology , Nicotine/toxicity , Female , Cartilage, Articular/drug effects , Cartilage, Articular/metabolism , Cartilage, Articular/pathology , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , Pregnancy , Pyroptosis/drug effects , Rats , alpha7 Nicotinic Acetylcholine Receptor/metabolism , Mesenchymal Stem Cells/drug effects , Mesenchymal Stem Cells/metabolism , Mesenchymal Stem Cells/cytology , Prenatal Exposure Delayed Effects/metabolism , Prenatal Exposure Delayed Effects/chemically induced , Prenatal Exposure Delayed Effects/pathology , Chondrogenesis/drug effects , Cell Differentiation/drug effects , Humans , Chondrocytes/drug effects , Chondrocytes/metabolism , Chondrocytes/cytologyABSTRACT
Regeneration of hyaline cartilage in human-sized joints remains a clinical challenge, and it is a critical unmet need that would contribute to longer healthspans. Injectable scaffolds for cartilage repair that integrate both bioactivity and sufficiently robust physical properties to withstand joint stresses offer a promising strategy. We report here on a hybrid biomaterial that combines a bioactive peptide amphiphile supramolecular polymer that specifically binds the chondrogenic cytokine transforming growth factor ß-1 (TGFß-1) and crosslinked hyaluronic acid microgels that drive formation of filament bundles, a hierarchical motif common in natural musculoskeletal tissues. The scaffold is an injectable slurry that generates a porous rubbery material when exposed to calcium ions once placed in cartilage defects. The hybrid material was found to support in vitro chondrogenic differentiation of encapsulated stem cells in response to sustained delivery of TGFß-1. Using a sheep model, we implanted the scaffold in shallow osteochondral defects and found it can remain localized in mechanically active joints. Evaluation of resected joints showed significantly improved repair of hyaline cartilage in osteochondral defects injected with the scaffold relative to defects injected with the growth factor alone, including implantation in the load-bearing femoral condyle. These results demonstrate the potential of the hybrid biomimetic scaffold as a niche to favor cartilage repair in mechanically active joints using a clinically relevant large-animal model.
Subject(s)
Chondrogenesis , Tissue Scaffolds , Transforming Growth Factor beta1 , Animals , Tissue Scaffolds/chemistry , Sheep , Transforming Growth Factor beta1/metabolism , Chondrogenesis/drug effects , Polymers/chemistry , Hyaluronic Acid/chemistry , Hyaluronic Acid/pharmacology , Cartilage, Articular/drug effects , Regeneration/drug effects , Cell Differentiation/drug effects , Tissue Engineering/methods , Humans , Biocompatible Materials/chemistry , Chondrocytes/drug effects , Hyaline Cartilage/metabolismABSTRACT
This study aimed to investigate whether the beneficial effects of PCA on chondrocyte senescence are mediated through the regulation of mitophagy. Chondrocyte senescence plays a significant role in the development and progression of knee osteoarthritis (OA). The compound protocatechuic aldehyde (PCA), which is abundant in the roots of Salvia miltiorrhiza, has been reported to have antioxidant properties and the ability to protect against cellular senescence. To achieve this goal, a destabilization of the medial meniscus (DMM)-induced mouse OA model and a lipopolysaccharide (LPS)-induced chondrocyte senescence model were used, in combination with PINK1 gene knockdown or overexpression. After treatment with PCA, cellular senescence was assessed using Senescence-Associated ß-Galactosidase (SA-ß-Gal) staining, DNA damage was evaluated using Hosphorylation of the Ser-139 (γH2AX) staining, reactive oxygen species (ROS) levels were measured using Dichlorodihydrofluorescein diacetate (DCFH-DA) staining, mitochondrial membrane potential was determined using a 5,5',6,6'-TETRACHLORO-1,1',3,3'-*. TETRAETHYBENZIMIDA (JC-1) kit, and mitochondrial autophagy was examined using Mitophagy staining. Western blot analysis was also performed to detect changes in senescence-related proteins, PINK1/Parkin pathway proteins, and mitophagy-related proteins. Our results demonstrated that PCA effectively reduced chondrocyte senescence, increased the mitochondrial membrane potential, facilitated mitochondrial autophagy, and upregulated the PINK1/Parkin pathway. Furthermore, silencing PINK1 weakened the protective effects of PCA, whereas PINK1 overexpression enhanced the effects of PCA on LPS-induced chondrocytes. PCA attenuates chondrocyte senescence by regulating PINK1/Parkin-mediated mitochondrial autophagy, ultimately reducing cartilage degeneration.
Subject(s)
Benzaldehydes , Catechols , Cellular Senescence , Chondrocytes , Mitophagy , Protein Kinases , Ubiquitin-Protein Ligases , Chondrocytes/drug effects , Chondrocytes/metabolism , Chondrocytes/pathology , Animals , Cellular Senescence/drug effects , Ubiquitin-Protein Ligases/metabolism , Ubiquitin-Protein Ligases/genetics , Mitophagy/drug effects , Protein Kinases/metabolism , Mice , Catechols/pharmacology , Benzaldehydes/pharmacology , Reactive Oxygen Species/metabolism , Mitochondria/drug effects , Mitochondria/metabolism , Mitochondria/pathology , Male , Mice, Inbred C57BL , Autophagy/drug effects , Membrane Potential, Mitochondrial/drug effects , Osteoarthritis, Knee/pathology , Osteoarthritis, Knee/metabolism , Osteoarthritis, Knee/drug therapyABSTRACT
This study aims to investigate the mechanism of Huangqin Qingre Chubi Capsules(HQC) in delaying chondrocyte senescence of osteoarthritic(OA) rats by regulating the p53/p21 signaling pathway. Rheumatic fever paralysis models of OA rats were induced based on monosodiun iodoacetate(MIA) combined with external rheumatic fever environmental stimuli and divided into normal(Con) group, OA model(MIA) group, OA model+rheumatic fever stimulation model(MIA-M) group, MIA-M+HQC low-dose(MIA-M+HQC-L) group, medium-dose(MIA-M+HQC-M) group, and high-dose(MIA-M+HQC-H) group, and MIA-M+glucosamine(MIA-M+GS) group. The models were successfully prepared and administered by gavage for 30 d. The pathological changes of cartilage were observed by hematoxylin-eosin(HE) and Senna O solid green(SO) staining. The expression of interleukin(IL)-1ß and IL-6 was detected by enzyme-linked immunosorbent assay(ELISA). Flow cytometry(FCM) was used to detect apoptosis and cell cycle. The mRNA expression of MMP13, ADAMTS-5, COLâ ¡, and TGF-ß was detected by RT-qPCR. The protein expression of p53/p21, p16, Bax, and Bcl-2 was detected by Western blot. The articular cartilage surface of rats in the Con group was smooth, and the tide line was smooth. The cartilage layer of MIA and MIA-M groups was obviously damaged, and the cartilage matrix was reduced. The above conditions were more severe in the MIA-M group. The cartilage surface of the HQC high-dose group and MIA-M+GS group was basically intact with clear delamination. Compared with the MIA-M+HQC-H group, Mankin's score was higher in the HQC low-dose and medium-dose groups, and the change was not obvious in the MIA-M+GS group. Compared with the Con group, the proportion of chondrocytes G_1 was elevated in the MIA and MIA-M groups, and the proportion of the S phase and G_2 phase was significantly decreased. In addition, the apoptosis rate was increased. Compared with MIA-M, HQC groups inhibited apoptosis and promoted cell proliferation in a concentration-dependent manner. Compared with the MIA-M+HQC-H group, the effect was more significant in the HQC high-dose group than in the HQC medium-low dose, while it was not significant in the MIA-M+GS group. Compared with the Con group, IL-1ß and IL-6 were elevated in the MIA and MIA-M groups, and mRNA levels of MMP13 and ADAMTS-5 were elevated. p53, p21, p16, and Bax protein were elevated, and mRNA levels of COLâ ¡ and TGF-ß were decreased. Compared with the MIA-M group, IL-1ß and IL-6 decreased after drug interventions of HQC and GS, and mRNA levels of MMP13 and ADAMTS-5, as well as protein levels of p53, p21, Bax, and p16 decreased. In addition, Bcl-2 increased. The improvement of these indexes was significantly better in the MIA-M+HQC-H group than in the HQC low-dose and medium-dose groups, and the difference with the MIA-M+GS group was not significant. HQC delayed MIA-induced chondrocyte senescence in OA rats, inhibited inflammatory response and extracellular matrix(ECM) degradation, and its mechanism may be related to the inhibition of the p53/p21 pathway.
Subject(s)
Chondrocytes , Drugs, Chinese Herbal , Osteoarthritis , Rats, Sprague-Dawley , Signal Transduction , Tumor Suppressor Protein p53 , Animals , Chondrocytes/drug effects , Chondrocytes/metabolism , Drugs, Chinese Herbal/administration & dosage , Drugs, Chinese Herbal/pharmacology , Osteoarthritis/metabolism , Osteoarthritis/drug therapy , Osteoarthritis/genetics , Rats , Signal Transduction/drug effects , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , Male , Cellular Senescence/drug effects , Cyclin-Dependent Kinase Inhibitor p21/genetics , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Capsules , Humans , Apoptosis/drug effectsABSTRACT
Tissue engineering is theoretically considered a promising approach for repairing osteochondral defects. Nevertheless, the insufficient osseous support and integration of the cartilage layer and the subchondral bone frequently lead to the failure of osteochondral repair. Drawing from this, it was proposed that incorporating glycine-modified attapulgite (GATP) into poly(1,8-octanediol-co-citrate) (POC) scaffolds via the one-step chemical cross-linking is proposed to enhance cartilage and subchondral bone defect repair simultaneously. The effects of the GATP incorporation ratio on the physicochemical properties, chondrocyte and MC3T3-E1 behavior, and osteochondral defect repair of the POC scaffold were also evaluated. In vitro studies indicated that the POC/10% GATP scaffold improved cell proliferation and adhesion, maintained cell phenotype, and upregulated chondrogenesis and osteogenesis gene expression. Animal studies suggested that the POC/10% GATP scaffold has significant repair effects on both cartilage and subchondral bone defects. Therefore, the GATP-incorporated scaffold system with dual-lineage bioactivity showed potential application in osteochondral regeneration.
Subject(s)
Tissue Engineering , Tissue Scaffolds , Animals , Tissue Scaffolds/chemistry , Mice , Chondrocytes/cytology , Chondrocytes/drug effects , Chondrocytes/metabolism , Bone Regeneration/drug effects , Chondrogenesis/drug effects , Osteogenesis/drug effects , Cell Proliferation/drug effects , Rabbits , Bone and Bones/drug effects , Regeneration/drug effectsABSTRACT
Objectives: Osteochondral defects (OCDs) are localized areas of damaged cartilage and underlying subchondral bone that can produce pain and seriously impair joint function. Literature reports indicated that icariin (ICA) has the effect of promoting cartilage repair. However, its mechanism remains unclear. Here, we explored the effects of icariin and extracellular vesicles (EVs) from rabbit synovial-derived mesenchymal stem cells (rSMSCs) on repairing of OCDs. Materials and Methods: Rabbit primary genicular chondrocytes (rPGCs), knee skeletal muscle cells (rSMCKs), and rSMSCs, and extracellular vesicles derived from the latter two cells (rSMCK-EVs and rSMSC-EVs) were isolated and identified. The rPGCs were stimulated with ICA, rSMSC-EVs either separately or in combination. The rSMCK-EVs were used as a control. After stimulation, chondrogenic-related markers were analyzed by quantitative RT-PCR and western blotting. Cell proliferation was determined by the CCK-8 assay. The preventative effects of ICA and SMSC-EVs in vivo were determined by H&E and toluidine blue staining. Immunohistochemical analyses were performed to evaluate the levels of COL2A1 and ß-catenin in vivo. Results. In vitro, the proliferation of rPGCs was markedly increased by ICA treatment in a dose-dependent manner. When compared with ICA or rSMSC-EVs treatment alone, combined treatment with ICA and SMSC-EVs produced stronger stimulative effects on cell proliferation. Moreover, combined treatment with ICA and rSMSC-EVs promoted the expression of chondrogenic-related gene, including COL2A1, SOX-9, and RUNX2, which may be via the activation of the Wnt/ß-catenin pathway. In vivo, combined treatment with rSMSC-EVs and ICA promoted cartilage repair in joint bone defects. Results also showed that ICA or rSMSC-EVs both promoted the COL2A1 and ß-catenin protein accumulation in articular cartilage, and that was further enhanced by combined treatment with rSMSC-EVs and ICA. Conclusion: Our findings highlight the promising potential of using combined treatment with ICA and rSMSC-EVs for promoting osteochondral repair.
Subject(s)
Chondrocytes , Chondrogenesis , Extracellular Vesicles , Flavonoids , Mesenchymal Stem Cells , Synovial Membrane , Wnt Signaling Pathway , Animals , Rabbits , Flavonoids/pharmacology , Mesenchymal Stem Cells/metabolism , Mesenchymal Stem Cells/cytology , Wnt Signaling Pathway/drug effects , Extracellular Vesicles/metabolism , Chondrocytes/metabolism , Chondrocytes/drug effects , Synovial Membrane/metabolism , Synovial Membrane/cytology , Chondrogenesis/drug effects , Cell Proliferation/drug effects , beta Catenin/metabolism , Cartilage, Articular/metabolism , Cartilage, Articular/drug effectsABSTRACT
Osteoarthritis (OA) is a degenerative joint disorder that is distinguished by inflammation and chronic cartilage damage. Interleukin-1ß (IL-1ß) is a proinflammatory cytokine that plays an important role in the catabolic processes that underlie the pathogenesis of OA. In this study, we investigate the therapeutic efficacy of exosomes derived from untreated bone-marrow-derived mesenchymal stem cells (BMMSC-Exo) and those treated with cinnamaldehyde (BMMSC-CA-Exo) for preventing the in vitro catabolic effects of IL-1ß on chondrocytes. We stimulated chondrocytes with IL-1ß to mimic the inflammatory microenvironment of OA. We then treated these chondrocytes with BMMSC-Exo and BMMSC-CA-Exo isolated via an aqueous two-phase system and evaluated their effects on the key cellular processes using molecular techniques. Our findings revealed that treatment with BMMSC-Exo reduces the catabolic effects of IL-1ß on chondrocytes and alleviates inflammation. However, further studies directly comparing treatments with BMMSC-Exo and BMMSC-CA-Exo are needed to determine if CA preconditioning can provide additional anti-inflammatory benefits to the exosomes beyond those of CA preconditioning or treatment with regular BMMSC-Exo. Through a comprehensive molecular analysis, we elucidated the regulatory mechanisms underlying this protective effect. We found a significant downregulation of proinflammatory signaling pathways in exosome-infected chondrocytes, suggesting the potential modulation of the NF-κB and MAPK signaling cascades. Furthermore, our study identified the molecular cargo of BMMSC-Exo and BMMSC-CA-Exo, determining the key molecules, such as anti-inflammatory cytokines and cartilage-associated factors, that may contribute to their acquisition of chondroprotective properties. In summary, BMMSC-Exo and BMMSC-CA-Exo exhibit the potential as therapeutic agents for OA by antagonizing the in vitro catabolic effects of IL-1ß on chondrocytes. The regulation of the proinflammatory signaling pathways and bioactive molecules delivered by the exosomes suggests a multifaceted mechanism of action. These findings highlight the need for further investigation into exosome-based therapies for OA and joint-related diseases.
Subject(s)
Acrolein , Chondrocytes , Exosomes , Inflammation , Interleukin-1beta , Mesenchymal Stem Cells , Signal Transduction , Exosomes/metabolism , Interleukin-1beta/metabolism , Acrolein/analogs & derivatives , Acrolein/pharmacology , Mesenchymal Stem Cells/metabolism , Mesenchymal Stem Cells/drug effects , Chondrocytes/metabolism , Chondrocytes/drug effects , Signal Transduction/drug effects , Inflammation/metabolism , Animals , Osteoarthritis/metabolism , Osteoarthritis/drug therapy , Humans , Cells, CulturedABSTRACT
The most widely used synthetic glucocorticoid, dexamethasone (DEX), causes stunted growth in children when used excessively or for long periods of time; however, there are still plenty of pediatric patients require long-term treatment with DEX. As an alternative, growth hormone is used in combination, but it has side effects, a high cost, and psychological factors, and it is not satisfactory in terms of effectiveness. It is necessary to develop a safe and affordable treatment that can replace it. The Korean Food and Drug Administration approved HT042, a standardized functional food ingredient, with the claim that it can help height growth of children. In this study, it was found that HT042 activated the Indian hedgehog/parathyroid hormone-related protein signaling pathway and enhanced the number of growth hormone receptors and insulin-like growth factor-1 receptors on the growth plate surface, which were reduced by DEX treatment, and restored growth retardation. In metatarsal bone and primary chondrocyte models, it was found that HT042 can promote the length of growth plate and recover DEX-induced growth retardation. It was also found that HT042 promotes cell proliferation using bromodeoxyuridine and terminal deoxynucleotidyl transferase dUTP nick end labeling assays; moreover, we verified increased expression of GHR/IGF-1R and Ihh/PTHrP pathway activity using qRT-PCR, western blotting, and siRNA analyses to verify its direct action on the growth plate. The anti-apoptotic effect of HT042 was identified by regulating the expression of apoptotic factors such as caspase-3, Bcl2, Bclx, and Bax. These results were identified using both ex vivo and in vitro models. Our study verified that co-administration of HT042 could recover the DEX induced growth retardation.
Subject(s)
Cell Proliferation , Dexamethasone , Growth Plate , Metatarsal Bones , Plant Extracts , Signal Transduction , Animals , Dexamethasone/pharmacology , Metatarsal Bones/drug effects , Growth Plate/drug effects , Rats , Cell Proliferation/drug effects , Signal Transduction/drug effects , Plant Extracts/pharmacology , Male , Hedgehog Proteins/metabolism , Chondrocytes/drug effects , Chondrocytes/metabolism , Parathyroid Hormone-Related Protein/metabolism , Rats, Sprague-Dawley , Apoptosis/drug effects , Receptors, Somatotropin/metabolism , Receptors, Somatotropin/genetics , Receptor, IGF Type 1/metabolism , Receptor, IGF Type 1/genetics , Bone Development/drug effects , Growth Disorders/chemically inducedABSTRACT
The actions of the retinoic acid nuclear receptor gamma (RARγ) agonist, palovarotene, on pre-existing osteochondromas were investigated using a mouse multiple osteochondroma model. This approach was based on the knowledge that patients often present to the clinic after realizing the existence of osteochondroma masses, and the findings from preclinical investigations are the effects of drugs on the initial formation of osteochondromas. Systemic administration of palovarotene, with increased doses (from 1.76 to 4.0 mg/kg) over time, fully inhibited tumor growth, keeping the tumor size (0.31 ± 0.049 mm3) similar to the initial size (0.27 ± 0.031 mm3, p = 0.66) while the control group tumor grew (1.03 ± 0.23 mm3, p = 0.023 to the drug-treated group). Nanoparticle (NP)-based local delivery of the RARγ agonist also inhibited the growth of osteochondromas at an early stage (Control: 0.52 ± 0.11 mm3; NP: 0.26 ± 0.10, p = 0.008). Transcriptome analysis revealed that the osteoarthritis pathway was activated in cultured chondrocytes treated with palovarotene (Z-score = 2.29), with the upregulation of matrix catabolic genes and the downregulation of matrix anabolic genes, consistent with the histology of palovarotene-treated osteochondromas. A reporter assay performed in cultured chondrocytes demonstrated that the Stat3 pathway, but not the Stat1/2 pathway, was stimulated by RARγ agonists. The activation of Stat3 by palovarotene was confirmed using immunoblotting and immunohistochemistry. These findings suggest that palovarotene treatment is effective against pre-existing osteochondromas and that the Stat3 pathway is involved in the antitumor actions of palovarotene.
Subject(s)
Chondrocytes , Disease Models, Animal , Osteochondroma , Receptors, Retinoic Acid , Retinoic Acid Receptor gamma , Animals , Mice , Receptors, Retinoic Acid/agonists , Receptors, Retinoic Acid/metabolism , Osteochondroma/drug therapy , Osteochondroma/pathology , Osteochondroma/metabolism , Chondrocytes/metabolism , Chondrocytes/drug effects , Chondrocytes/pathology , STAT3 Transcription Factor/metabolism , Cell Proliferation/drug effects , Bone Neoplasms/drug therapy , Bone Neoplasms/pathology , Bone Neoplasms/metabolism , MaleABSTRACT
BACKGROUND: This study aimed to investigate functions of GLP-1R agonist by liraglutide (LIRA) and revealing the mechanism related to AGEs/RAGE in chondrocytes. METHODS: To illustrate potential effect of GLP-1R agonist on AGEs induced chondrocytes, chondrocytes were administrated by AGEs with LIRA and GLP-1R inhibitor exendin. Inflammatory factors were assessed using ELISA. Real-time PCR was used to evaluate the catabolic activity MMPs and ADAMTS mRNA level, as well as anabolic activity (aggrecan and collagen II). RAGE expression was investigated by Western blotting. TUNEL, caspase3 activity and immunofluorescence were performed to test the apoptotic activity. RESULTS: Our results showed that treatment with LIRA at > 100 nM attenuated the AGE-induced chondrocyte viability. Western bolt demonstrated that GLP-1R activation by LIRA treatment reduced RAGE protein expression compared with the AGEs groups. ELISA showed that LIRA hindered the AGEs-induced production of inflammatory cytokines (IL-6, IL-12 and TNF-α) in primary chondrocytes. AGEs induced catabolism levels (MMP-1, -3, -13 and ADAMTS-4, 5) are also attenuated by LIRA, causing the retention of more extracellular matrix (Aggrecan and Collagen II). TUNEL, caspase3 activity and immunofluorescence results indicated that LIRA inhibited the AGEs-induced production of inflammatory cytokines in primary chondrocytes and attenuated the caspase 3 level, leading to the reduced apoptotic activity. All the protective effects are reversed by exendin (GLP-1R blockers). CONCLUSIONS: The present study demonstrates for the first time that LIRA, an agonist for GLP-1R which is commonly used in type 2 diabetes reverses AGEs induced chondrocyte inflammation and apoptosis through suppressing RAGE signaling, contributing to reduced catabolism and retention of more extracellular matrix. The above results indicate the possible effect of GLP-1R agonist on treating OA.
Subject(s)
Apoptosis , Chondrocytes , Glucagon-Like Peptide-1 Receptor , Glycation End Products, Advanced , Inflammation , Liraglutide , Receptor for Advanced Glycation End Products , Signal Transduction , Chondrocytes/drug effects , Chondrocytes/metabolism , Chondrocytes/pathology , Liraglutide/pharmacology , Glycation End Products, Advanced/metabolism , Glucagon-Like Peptide-1 Receptor/agonists , Glucagon-Like Peptide-1 Receptor/metabolism , Receptor for Advanced Glycation End Products/metabolism , Apoptosis/drug effects , Animals , Signal Transduction/drug effects , Inflammation/metabolism , Inflammation/drug therapy , Inflammation/pathology , Cells, CulturedABSTRACT
Chondrocyte apoptosis is recognized as one of the pathological features involved in cartilage degeneration driving the onset and progression of knee osteoarthritis (OA). This study aimed to determine the molecular mechanism underlying the effect of clusterin (CLU), anti-apoptotic molecule, in human knee OA chondrocytes. Primary knee OA chondrocytes were isolated from the cartilage of knee OA patients and divided into five groups: (1) the cells treated with interleukin (IL)-1ß, (2) CLU alone, (3) a combination of IL-1ß and CLU, (4) LY294002 (PI3K inhibitor) along with IL-1ß and CLU, and (5) the untreated cells. Production of apoptotic, inflammatory, anabolic, and catabolic mediators in knee OA chondrocytes was determined after treatment for 24 h. Our in vitro study uncovered that CLU significantly suppressed the production of inflammatory mediators [nitric oxide (NO), IL6, and tumor necrosis factor (TNF)-α] and apoptotic molecule (caspase-3, CASP3). CLU significantly upregulated messenger ribonucleic acid (mRNA) expressions of anabolic factors [SRY-box transcription factor-9 (SOX9) and aggrecan (ACAN)], but significantly downregulated mRNA expressions of IL6, nuclear factor kappa-B (NF-κB), CASP3, and matrix metalloproteinase-13 (MMP13). Anti-apoptotic and anti-inflammatory effects of CLU were mediated through activating PI3K/Akt signaling pathway. The findings suggest that CLU might have beneficial effects on knee OA chondrocytes by exerting anti-apoptotic and anti-inflammatory functions via PI3K/Akt pathway, making CLU a promising target for potential therapeutic interventions in knee OA.
Subject(s)
Apoptosis , Chondrocytes , Clusterin , Interleukin-1beta , Osteoarthritis, Knee , Humans , Chondrocytes/drug effects , Chondrocytes/metabolism , Chondrocytes/pathology , Osteoarthritis, Knee/pathology , Osteoarthritis, Knee/metabolism , Apoptosis/drug effects , Clusterin/metabolism , Clusterin/genetics , Interleukin-1beta/metabolism , Signal Transduction/drug effects , Cells, Cultured , Male , Middle Aged , Aged , Inflammation/metabolism , Inflammation/pathology , Proto-Oncogene Proteins c-akt/metabolism , Female , Phosphatidylinositol 3-Kinases/metabolism , Morpholines/pharmacology , Chromones/pharmacology , SOX9 Transcription Factor/metabolism , SOX9 Transcription Factor/genetics , Matrix Metalloproteinase 13/metabolism , Inflammation Mediators/metabolism , Nitric Oxide/metabolismABSTRACT
Medicine intervention is the major clinical treatment used to relieve the symptoms and delay the progression of rheumatoid arthritis (RA), but is limited by its poor targeted delivery and short therapeutic duration. Herein, we developed an injectable and bioadhesive gelatin-based (Gel) hydrogel as a local depot of leonurine (Leon)-loaded and folate-functionalized polydopamine (FA-PDA@Leon) nanoparticles for anti-inflammation and chondroprotection in RA. The nanoparticles could protect Leon and facilitate its entry into the M1 phenotype macrophage for intracellular delivery of Leon, while the hydrogel tightly adhered to the tissues in the joint cavity and prolonged the retention of FA-PDA@Leon nanoparticles, thus achieving higher availability and therapeutic efficiency of Leon. In vitro and in vivo experiments demonstrated that the Gel/FA-PDA@Leon hydrogel could strongly suppress the inflammatory response by down-regulating the JAK2/STAT3 signaling pathway in macrophages and protect the chondrocytes from ferritinophagy/ferroptosis. This contributed to maintaining the structural integrity of articular cartilage and accelerating the joint functional recovery. This work provides an effective and convenient strategy to achieve higher bioavailability and long-lasting therapeutic duration of medicine intervention in arthritis diseases.
Subject(s)
Arthritis, Rheumatoid , Ferroptosis , Hydrogels , Inflammation , Nanoparticles , Polymers , Animals , Arthritis, Rheumatoid/drug therapy , Arthritis, Rheumatoid/pathology , Hydrogels/chemistry , Mice , Nanoparticles/chemistry , Inflammation/drug therapy , Inflammation/pathology , Ferroptosis/drug effects , Polymers/chemistry , RAW 264.7 Cells , Nanomedicine/methods , Indoles/chemistry , Indoles/pharmacology , Indoles/administration & dosage , Male , Macrophages/drug effects , Macrophages/metabolism , Folic Acid/chemistry , Gelatin/chemistry , Humans , Chondrocytes/drug effects , Chondrocytes/metabolismABSTRACT
Temporomandibular joint osteoarthritis (TMJ OA) is characterized by the degeneration of cartilage and subchondral bone. In this study, we observed a significant increase in cell-free DNA (cfDNA) levels during the progression of TMJ OA. Bioinformatics analysis identified TLR9 as a pivotal molecule in TMJ OA pathogenesis. The polyamidoamine (PAMAM) dendrimer characterized by a well-structured, highly branched, and reactive nature, exhibits robust binding and clearance capabilities for cfDNA. However, the abundant amino groups on the surface of PAMAM lead to its inherent toxicity. To mitigate this, PEG-5000 was conjugated to the surface of PAMAM dendrimers, enhancing safety. Our results indicate that PEG-PAMAM effectively inhibits the upregulation of the TLR9 protein in TMJ OA, significantly suppressing the activation of the p-IκBα/p-NF-κB signaling pathway and subsequently decreasing chondrocyte inflammation and apoptosis, as evidenced by both in vivo and in vitro experiments. We conclude that PEG-PAMAM is a safe and effective material for in vivo applications, offering a promising therapeutic strategy for TMJ OA by targeting cfDNA clearance.
Subject(s)
Cell-Free Nucleic Acids , Dendrimers , Osteoarthritis , Polyethylene Glycols , Temporomandibular Joint , Dendrimers/chemistry , Dendrimers/pharmacology , Osteoarthritis/drug therapy , Osteoarthritis/pathology , Osteoarthritis/metabolism , Animals , Polyethylene Glycols/chemistry , Temporomandibular Joint/pathology , Temporomandibular Joint/drug effects , Temporomandibular Joint/metabolism , Adsorption , Humans , Toll-Like Receptor 9/metabolism , Male , Chondrocytes/drug effects , Chondrocytes/metabolism , Nylons/chemistry , Nylons/pharmacology , Apoptosis/drug effects , MiceABSTRACT
Osteoarthritis (OA) is a joint disease caused by inflammation of cartilage and synovial tissue. Suppressing the process of inflammatory reaction and the generation of oxidative stress is an effective strategy to alleviate the progression of OA. Liensinine is one of the main components of lotus seeds, which has anti-hypertensive and anti-arrhythmia activities. In this study, we aimed to determine the anti-inflammatory effect of liensinine in an OA. Here, we found that liensinine significantly inhibited the inflammatory response of SW1353 cells and primary chondrocytes by inhibiting the release of inflammatory cytokines and oxidative stress. Moreover, we showed that liensinine was able to inhibit the activation of the NF-κB signaling pathway in IL-1ß-induced SW1353 cells. Lastly, we found that liensinine significantly ameliorated cartilage damage and inflammatory response in papain-induced rats. Our study demonstrated a significant protective effect of liensinine against OA, which might be by inhibiting the activation of the NF-κB signaling pathway, and provide a new insight for the treatment of OA using liensinine.
Subject(s)
Anti-Inflammatory Agents , Chondrocytes , Interleukin-1beta , NF-kappa B , Osteoarthritis , Animals , Humans , Male , Rats , Anti-Inflammatory Agents/pharmacology , Anti-Inflammatory Agents/therapeutic use , Cells, Cultured , Chondrocytes/drug effects , Cytokines/metabolism , Interleukin-1beta/metabolism , Isoquinolines , NF-kappa B/metabolism , Osteoarthritis/drug therapy , Osteoarthritis/chemically induced , Oxidative Stress/drug effects , Papain , Phenols , Rats, Sprague-Dawley , Signal Transduction/drug effectsABSTRACT
Transforming growth factor (TGF)-ß1 is a multifunctional protein that is essential in many cellular processes that include fibrosis, inflammation, chondrogenesis, and cartilage repair. In particular, cartilage repair is important to avoid physical disability since this tissue does not have the inherent capacity to regenerate beyond full development. We report here on supramolecular coassemblies of two peptide amphiphile molecules, one containing a TGF-ß1 mimetic peptide, and another which is one of two constitutional isomers lacking bioactivity. Using human articular chondrocytes, we investigated the bioactivity of the supramolecular copolymers of each isomer displaying either the previously reported linear form of the mimetic peptide or a novel cyclic analogue. Based on fluorescence depolarization and 1H NMR spin-lattice relaxation times, we found that coassemblies containing the cyclic compound and the most dynamic isomer exhibited the highest intracellular TGF-ß1 signaling and gene expression of cartilage extracellular matrix components. We conclude that control of supramolecular motion is emerging as an important factor in the binding of synthetic molecules to receptors that can be tuned through chemical structure.