RESUMO
The intron retention (IR) is a phenomenon utilized by cells to allow diverse fates at the same mRNA, leading to a different pattern of synthesis of the same protein. In this study, we analyzed the modulation of phosphoinositide-specific phospholipase C (PI-PLC) enzymes by Harpagophytum procumbens extract (HPE) in synoviocytes from joins of osteoarthritis (OA) patients. In some samples, the PI-PLC γ1 isoform mature mRNA showed the IR and, in these synoviocytes, the HPE treatment increased the phenomenon. Moreover, we highlighted that as a consequence of IR, a lower amount of PI-PLC γ1 was produced. The decrease of PI-PLC γ1 was associated with the decrease of metalloprotease-3 (MMP-3), and MMP-13, and ADAMTS-5 after HPE treatment. The altered expression of MMPs is a hallmark of the onset and progression of OA, thus substances able to decrease their expression are very desirable. The interesting outcomes of this study are that 35% of analyzed synovial tissues showed the IR phenomenon in the PI-PLC γ1 mRNA and that the HPE treatment increased this phenomenon. For the first time, we found that the decrease of PI-PLC γ1 protein in synoviocytes interferes with MMP production, thus affecting the pathways involved in the MMP expression. This finding was validated by the silencing of PI-PLC γ1 in synoviocytes where the IR phenomenon was not present. Our results shed new light on the biochemical mechanisms involved in the degrading enzyme production in the joint of OA patients, suggesting a new therapeutic target and highlighting the importance of personalized medicine.
Assuntos
Fibroblastos , Íntrons , Fosfolipase C gama , RNA Mensageiro , Humanos , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Fibroblastos/metabolismo , Fibroblastos/efeitos dos fármacos , Fosfolipase C gama/metabolismo , Fosfolipase C gama/genética , Células Cultivadas , Osteoartrite/metabolismo , Osteoartrite/patologia , Membrana Sinovial/metabolismo , Membrana Sinovial/citologia , Membrana Sinovial/efeitos dos fármacos , Metaloproteinase 3 da Matriz/metabolismo , Metaloproteinase 3 da Matriz/genética , Proteína ADAMTS5/metabolismo , Proteína ADAMTS5/genética , Sinoviócitos/metabolismo , Sinoviócitos/efeitos dos fármacos , Metaloproteinase 13 da Matriz/metabolismo , Metaloproteinase 13 da Matriz/genéticaRESUMO
PURPOSE: To study the damage and the expression of LC3 and p62 of condylar cartilage in fluorosis mouse. METHODS: Thirty 4-week-old male C57BL/6 mice were randomly divided into control group and the experimental group with 15 animals in each group. The control group received regular drinking water and the experimental group received a fluoride concentration of 75 mg/L drinking water for 8 weeks. The structure of condylar cartilage was observed through modified safranine O-fast green FCF cartilage stain kit. Immunohistochemistry was used to detect the expression of MMP-13, type â ¡ collagen and LC3 and p62. Two-way analysis of variance test was conducted for analysis of semi-quantitative results of immunohistochemistry using SPSS 22.0 software package. RESULTS: Compared with the control group, the fibrocartilage layer of the experimental group became thinner, the condrocytes were smaller, and the staining became deeper.Immunohistochemistry results showed that the expression of MMP-13 and LC3 increased; the expression of type â ¡ collagen and p62 decreased in the experimental group. CONCLUSIONS: There was degeneration of the condylar cartilage and autophagy in mice with drinking water containing 75 mg/L fluoride.
Assuntos
Autofagia , Fluorose Dentária , Metaloproteinase 13 da Matriz , Camundongos Endogâmicos C57BL , Proteínas Associadas aos Microtúbulos , Animais , Camundongos , Autofagia/efeitos dos fármacos , Metaloproteinase 13 da Matriz/metabolismo , Metaloproteinase 13 da Matriz/genética , Masculino , Proteínas Associadas aos Microtúbulos/metabolismo , Fluorose Dentária/metabolismo , Colágeno Tipo II/metabolismo , Côndilo Mandibular/metabolismo , Côndilo Mandibular/patologia , Fluoretos/toxicidade , Cartilagem Articular/metabolismo , Imuno-HistoquímicaRESUMO
An increasing body of evidence supports the involvement of inflammation and immune responses in the occurrence and development of keratoconus (KC). However, the causal relationship between inflammatory factors and KC remains unclear. We employed a 2-way Mendelian randomization (MR) approach to investigate the interaction between KC and inflammatory factors. Instrumental variables for 41 circulating inflammatory regulators and 12 matrix metalloproteinases (MMPs) were selected from genome-wide association studies of European ancestry. Summary statistics for KC were obtained from a genome-wide association study comprising 2116 cases and 24,626 controls of European ancestry. The primary analytical method for assessing causality was the inverse-variance weighted method. Two additional MR methods (MR-Egger and weighted median) were employed to complement the inverse-variance weighted results. In addition, several sensitivity analyses were conducted to evaluate heterogeneity, horizontal pleiotropy, and stability. Our findings indicated that genetically predicted higher levels of macrophage inflammatory protein-1ß (odds ratioâ =â 1.126, 95% confidence interval: 1.029-1.232, Pâ =â .01) and MMP-13 (odds ratioâ =â 1.211, 95% confidence interval: 1.070-1.371, Pâ =â .003) were positively associated with an elevated risk of KC. Conversely, genetically predicted KC was associated with increased levels of interferon-gamma, interleukin-4, and MMP-1. Our current study provided suggestive evidence supporting causal associations of macrophage inflammatory protein-1ß and MMP-13 with the risk of KC. In addition, KC appeared to affect the expression of interferon-gamma, interleukin-4, and MMP-1.
Assuntos
Estudo de Associação Genômica Ampla , Inflamação , Ceratocone , Análise da Randomização Mendeliana , Humanos , Análise da Randomização Mendeliana/métodos , Ceratocone/genética , Ceratocone/epidemiologia , Inflamação/genética , Polimorfismo de Nucleotídeo Único , Interleucina-4/genética , Interleucina-4/sangue , Metaloproteinase 13 da Matriz/genética , Predisposição Genética para DoençaRESUMO
BACKGROUND: Osteoarthritis (OA) is a prevalent age-related disease characterized by the gradual deterioration of cartilage. The involvement of chondrocyte senescence is crucial in the pathogenesis of OA. Desferoxamine (DFO) is an iron chelator with therapeutic potential in various diseases. However, the relationship of chondrocyte senescence and iron homeostasis is largely unknown. METHODS: Chondrocyte senescence was induced using tert-butyl hydroperoxide (TBHP), and the impact of DFO on chondrocyte senescence and iron metabolism was assessed through techniques such as western blotting, qRT-PCR, and ß-Galactosidase staining. To assess the impact of DFO on chondrocyte senescence and the progression of osteoarthritis (OA), the surgical destabilization of the medial meniscus model was established. RESULTS: In chondrocytes, TBHP administration resulted in elevated expression of P16, P21, and P53, as well as alterations in SA-ß-gal staining. Nevertheless, DFO effectively mitigated chondrocyte senescence induced by TBHP, and reversed the decrease in collagen II expression and increase in MMP13 expression caused by TBHP. Mechanismly, TBHP induced NCOA4 expression and iron release in chondrocytes. Excessive iron could induce chondrocyte senescence, whereas, DFO could inhibit NCOA4 expression and restore ferritin level, and chelate excessive iron. Importantly, intra-articular injection of DFO enhanced collagen II expression and reduced expression of P16, P21, and MMP13 of cartilage in OA mice, and delayed cartilage degeneration. CONCLUSIONS: Overall, this study provides evidence that DFO has the potential to alleviate chondrocyte senescence induced by TBHP and slow down the progression of osteoarthritis (OA) by effectively chelating excessive iron. These findings suggest that iron chelation could be a promising therapeutic strategy for treating OA.
Assuntos
Senescência Celular , Condrócitos , Desferroxamina , Homeostase , Ferro , Osteoartrite , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Desferroxamina/farmacologia , Desferroxamina/uso terapêutico , Animais , Osteoartrite/tratamento farmacológico , Osteoartrite/patologia , Osteoartrite/metabolismo , Ferro/metabolismo , Senescência Celular/efeitos dos fármacos , Homeostase/efeitos dos fármacos , Camundongos , Células Cultivadas , Masculino , Camundongos Endogâmicos C57BL , Progressão da Doença , terc-Butil Hidroperóxido , Cartilagem Articular/efeitos dos fármacos , Cartilagem Articular/patologia , Cartilagem Articular/metabolismo , Metaloproteinase 13 da Matriz/metabolismo , Metaloproteinase 13 da Matriz/genética , Humanos , Quelantes de Ferro/farmacologia , Quelantes de Ferro/uso terapêutico , Modelos Animais de DoençasRESUMO
Haemophilic arthropathy (HA), a common comorbidity in haemophilic patients leads to joint pain, deformity and reduced quality of life. We have recently demonstrated that a long non-coding RNA, Neat1 as a primary regulator of matrix metalloproteinase (MMP) 3 and MMP13 activity, and its induction in the target joint has a deteriorating effect on articular cartilage. In the present study, we administered an Adeno-associated virus (AAV) 5 vector carrying an short hairpin (sh)RNA to Neat1 via intra-articular injection alone or in conjunction with systemic administration of a capsid-modified AAV8 (K31Q) vector carrying F8 gene (F8-BDD-V3) to study its impact on HA. AAV8K31Q-F8 vector administration at low dose, led to an increase in FVIII activity (16%-28%) in treated mice. We further observed a significant knockdown of Neat1 (~40 fold vs. untreated injured joint, p = 0.005) in joint tissue of treated mice and a downregulation of chondrodegenerative enzymes, MMP3, MMP13 and the inflammatory mediator- cPLA2, in mice receiving combination therapy. These data demonstrate that AAV mediated Neat1 knockdown in combination with F8 gene augmentation can potentially impact mediators of haemophilic joint disease.
Assuntos
Dependovirus , Fator VIII , Vetores Genéticos , Hemofilia A , Metaloproteinase 13 da Matriz , Metaloproteinase 3 da Matriz , RNA Longo não Codificante , Animais , Hemofilia A/genética , Hemofilia A/terapia , Hemofilia A/complicações , Dependovirus/genética , RNA Longo não Codificante/genética , Metaloproteinase 13 da Matriz/metabolismo , Metaloproteinase 13 da Matriz/genética , Camundongos , Metaloproteinase 3 da Matriz/genética , Metaloproteinase 3 da Matriz/metabolismo , Vetores Genéticos/genética , Vetores Genéticos/administração & dosagem , Fator VIII/genética , Fator VIII/metabolismo , Artropatias/terapia , Artropatias/genética , Artropatias/etiologia , Humanos , Terapia Genética/métodos , Camundongos Endogâmicos C57BL , Cartilagem Articular/metabolismo , Cartilagem Articular/patologia , Modelos Animais de Doenças , MasculinoRESUMO
BACKGROUND/AIM: Matrix metalloproteinase 13 (MMP13) has been reported to be involved in tumor development and progression, including of colorectal cancer (CRC). This study aimed at evaluating whether the MMP13 rs2252070 gene polymorphism is associated with clinicopathological factors and its influence on long-term survival in Swedish patients with CRC. PATIENTS AND METHODS: A total of 723 patients with CRC were genotyped using TaqMan single nucleotide polymorphism assays based on polymerase chain reaction. RESULTS: Assessing clinicopathological factors, we demonstrated that having the G/G genotype for MMP13 rs2252070 was significantly associated with poor differentiation, higher serum level of carcinoembryonic antigen and higher lymph node status. Moreover, the presence of a G allele was significantly related to larger tumor size in rectal cancer but had a significantly protective role against mucinous cancer, perineural invasion and lymphovascular invasion. Kaplan-Meier analysis showed no difference between genotypes regarding cancer-specific survival. CONCLUSION: Our findings highlight the potential of MMP13 rs2252070 polymorphism as a useful predictor of poor differentiation, serum level of carcinoembryonic antigen, lymph node status, tumor size, mucinous cancer, perineural invasion and lymphovascular invasion in patients with CRC.
Assuntos
Neoplasias Colorretais , Genótipo , Metaloproteinase 13 da Matriz , Polimorfismo de Nucleotídeo Único , Humanos , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Neoplasias Colorretais/mortalidade , Masculino , Feminino , Suécia/epidemiologia , Metaloproteinase 13 da Matriz/genética , Idoso , Pessoa de Meia-Idade , Predisposição Genética para Doença , Prognóstico , Alelos , Estimativa de Kaplan-Meier , Idoso de 80 Anos ou mais , Adulto , Antígeno Carcinoembrionário/sangue , Estadiamento de Neoplasias , Biomarcadores Tumorais/genética , Estudos de Associação GenéticaRESUMO
BACKGROUND: Articular cartilage defects (ACD) are injuries with a diameter greater than 3 mm, resulting from wear and tear on joints. When the diameter of the defect exceeds 6 mm, it can further damage the surrounding joint cartilage, causing osteoarthritis (OA). Try to explain why OA is an irreversible disease, we hypothesize that damaged articular chondrocytes (DAC) may have reduced capacities to repair cartilage because its extracellular vesicle (EVs) that might directly contribute to OA formation. METHODS: In this study, DAC-EVs and AC-EVs were isolated using ultracentrifugation. Next-generation sequencing was employed to screen for a pathogenic long non-coding RNA (lncRNA). After verifying its function in vitro, the corresponding small interfering RNA (siRNA) was constructed and loaded into extracellular vesicles, which were then injected into the knee joint cavities of rats. RESULTS: The results revealed that DAC-EVs packaged lncRNA LOC102546541 acts as a competitive endogenous RNA (ceRNA) of MMP13, down-regulating miR-632. Consequently, the function of MMP13 in degrading the extracellular matrix is enhanced, promoting the development of osteoarthritis. CONCLUSIONS: This study uncovered a novel mode of OA pathogenesis using rat models, which DAC deliver pathogenic LOC102546541 packaged EVs to normal articular chondrocytes, amplifying the degradation of the extracellular matrix. Nonetheless, the functions of highly homologous human gene of LOC102546541 need to be verified in the future.
Assuntos
Cartilagem Articular , Condrócitos , Modelos Animais de Doenças , Vesículas Extracelulares , Metaloproteinase 13 da Matriz , MicroRNAs , Osteoartrite , RNA Longo não Codificante , Animais , Vesículas Extracelulares/metabolismo , Cartilagem Articular/metabolismo , Cartilagem Articular/patologia , Condrócitos/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Ratos , Osteoartrite/metabolismo , Osteoartrite/patologia , Metaloproteinase 13 da Matriz/metabolismo , Metaloproteinase 13 da Matriz/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Ratos Sprague-Dawley , Masculino , Humanos , Células Cultivadas , RNA Interferente Pequeno/genéticaRESUMO
Extracellular vesicles (EVs) have been found to have the characteristics of their parent cells. Based on the characteristics of these EVs, various studies on disease treatment using mesenchymal stem cell (MSC)-derived EVs with regenerative activity have been actively conducted. The therapeutic nature of MSC-derived EVs has been shown in several studies, but in recent years, there have been many efforts to functionalize EVs to give them more potent therapeutic effects. Strategies for functionalizing EVs include endogenous and exogenous methods. In this study, human umbilical cord MSC (UCMSC)-derived EVs were selected for optimum OA treatments with expectation via bioinformatics analysis based on antibody array. And we created a novel nanovesicle system called the IGF-si-EV, which has the properties of both cartilage regeneration and long-term retention in the lesion site, attaching positively charged insulin-like growth factor-1 (IGF-1) to the surface of the UCMSC-derived Evs carrying siRNA, which inhibits MMP13. The downregulation of inflammation-related cytokine (MMP13, NF-kB, and IL-6) and the upregulation of cartilage-regeneration-related factors (Col2, Acan) were achieved with IGF-si-EV. Moreover, the ability of IGF-si-EV to remain in the lesion site for a long time has been proven through an ex vivo system. Collectively, the final constructed IGF-si-EV can be proposed as an effective OA treatment through its successful MMP13 inhibition, chondroprotective effect, and cartilage adhesion ability. We also believe that this EV-based nanoparticle-manufacturing technology can be applied as a platform technology for various diseases.
Assuntos
Vesículas Extracelulares , Fator de Crescimento Insulin-Like I , Células-Tronco Mesenquimais , Osteoartrite , RNA Interferente Pequeno , Fator de Crescimento Insulin-Like I/metabolismo , Vesículas Extracelulares/metabolismo , Humanos , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/citologia , Osteoartrite/terapia , Osteoartrite/metabolismo , RNA Interferente Pequeno/genética , Animais , Metaloproteinase 13 da Matriz/metabolismo , Metaloproteinase 13 da Matriz/genéticaRESUMO
Osteoarthritis is the most prevalent type of degenerative arthritis. It is characterized by persistent pain, joint dysfunction, and physical disability. Pain relief and inflammation control are prioritised during osteoarthritis treatment Mume Fructus (Omae), a fumigated product of the Prunus mume fruit, is used as a traditional medicine in several Asian countries. However, its therapeutic mechanism of action and effects on osteoarthritis and articular chondrocytes remain unknown. In this study, we analyzed the anti-osteoarthritis and articular regenerative effects of Mume Fructus extract on rat chondrocytes. Mume Fructus treatment reduced the interleukin-1ß-induced expression of matrix metalloproteinase 3, matrix metalloproteinase 13, and a disintegrin and metalloproteinase with thrombospondin type 1 motifs 5. Additionally, it enhanced collagen type II alpha 1 chain and aggrecan accumulation in rat chondrocytes. Furthermore, Mume Fructus treatment regulated the inflammatory cytokine levels, mitogen-activated protein kinase phosphorylation, and nuclear factor-kappa B activation. Overall, our results demonstrated that Mume Fructus inhibits osteoarthritis progression by inhibiting the nuclear factor-kappa B and mitogen-activated protein kinase pathways to reduce the levels of inflammatory cytokines and prevent cartilage degeneration. Therefore, Mume Fructus may be a potential therapeutic option for osteoarthritis.
Assuntos
Cartilagem Articular , Condrócitos , Interleucina-1beta , Osteoartrite , Extratos Vegetais , Prunus , Animais , Masculino , Ratos , Proteína ADAMTS5/metabolismo , Proteína ADAMTS5/genética , Agrecanas/metabolismo , Cartilagem Articular/efeitos dos fármacos , Cartilagem Articular/metabolismo , Células Cultivadas , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Colágeno Tipo II/metabolismo , Regulação para Baixo/efeitos dos fármacos , Frutas/química , Interleucina-1beta/efeitos dos fármacos , Interleucina-1beta/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Metaloproteinase 13 da Matriz/metabolismo , Metaloproteinase 13 da Matriz/genética , Metaloproteinase 3 da Matriz/metabolismo , Metaloproteinase 3 da Matriz/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/efeitos dos fármacos , NF-kappa B/metabolismo , Osteoartrite/tratamento farmacológico , Osteoartrite/metabolismo , Osteoartrite/patologia , Extratos Vegetais/farmacologia , Prunus/química , Ratos Sprague-DawleyRESUMO
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.
Assuntos
Proteínas de Ciclo Celular , Progressão da Doença , Metaloproteinase 13 da Matriz , Osteoartrite , Regulação para Cima , Humanos , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Condrócitos/metabolismo , Condrócitos/patologia , Biologia Computacional , Lipopolissacarídeos/farmacologia , Metaloproteinase 13 da Matriz/metabolismo , Metaloproteinase 13 da Matriz/genética , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Osteoartrite/induzido quimicamente , Osteoartrite/genética , Osteoartrite/patologia , Mapas de Interação de ProteínasRESUMO
Our objective in this study is to determine whether intra-articular injection of miRNA-1 can attenuate the progression of OA in rats by down regulating Ihh. Knee chondrocytes were isolated from male Sprague-Dawley rats aged 2-3 days. Second-generation chondrocytes were transfected with miR-1 mimic and empty vector with lipo3000 for 6 h and then stimulated with 10 ng/mL IL-1ß for 24 h. OA-related and cartilage matrix genes were quantified using real-time quantitative polymerase chain reaction (RT-qPCR). Two-month-old male Sprague-Dawley rats were divided into three groups (n = 30?): sham operation group + 50 µL saline, anterior cruciate ligament transection (ACLT) group + 50 µL miR-1 agomir (concentration), and control group ACLT + 50 µL miR-1 agomir. Treatment was started one week after the operation. All animals were euthanized eight weeks after the operation. X-rays and micro-CT were used to detect imaging changes in the knee joints. FMT was used to monitor joint inflammation in vivo. Safranin O staining was used to detect morphological changes in articular cartilage. Immunohistochemistry was used to detect Col2, Col10, metalloproteinase-13 (MMP-13). RT-qPCR was used to detect gene changes includingmiR-1, Col2, Col10, MMP-13, Ihh, Smo, Gli1, Gli2, and Gli3. Overexpression of miR-1 in IL-1ß-stimulated chondrocytes reduced the levels of Ihh, MMP-13, and Col10 but increased the levels of Col2 and aggrecan. Intra-articular injection of miR-1 agomir reduced osteophyte formation, inflammation, and prevented cartilage damage. RT-qPCR results indicated that the miR-1 agomir increased articular cartilage anabolism and inhibited cartilage catabonism. miR-1 can attenuate the progression of OA by downregulating Ihh.
Assuntos
Cartilagem Articular , MicroRNAs , Osteoartrite , Ratos , Masculino , Animais , Proteínas Hedgehog , MicroRNAs/genética , MicroRNAs/uso terapêutico , Ratos Sprague-Dawley , Metaloproteinase 13 da Matriz/genética , Osteoartrite/tratamento farmacológico , Osteoartrite/genética , Condrócitos , Injeções Intra-Articulares , Inflamação , Modelos Animais de DoençasRESUMO
Osteoarthritis (OA) is the most prevalent degenerative cartilage disease, but no effective treatment is currently available to ameliorate the dysregulation of cartilage catabolism. Cartilage degeneration is closely related to the change in the physiology of chondrocytes: for example, chondrocytes of the OA patients overexpress matrix metallopeptidase 13 (MMP13), a.k.a. collagenase 3, which damages the extracellular matrix (ECM) of the cartilage and deteriorate the disease progression. Inhibiting MMP13 has shown to be beneficial for OA treatments, but delivering therapeutics to the chondrocytes embedded in the dense cartilage is a challenge. Here, we engineered the exosome surface with the cartilage affinity peptide (CAP) through lipid insertion to give chondrocyte-targeting exosomes, CAP-Exo, which was then loaded with siRNA against MMP13 (siMMP13) in the interior to give CAP-Exo/siMMP13. Intra-articular administration of CAP-Exo/siMMP13 reduced the MMP13 level and increased collagen COL2A1 and proteoglycan in cartilage in a rat model of anterior cruciate ligament transection (ACLT)-induced OA. Proteomic analysis showed that CAP-Exo/siMMP13 treatment restored the altered protein levels in the IL-1ß-treated chondrocytes. Taken together, a facile exosome engineering method enabled targeted delivery of siRNA to chondrocytes and chondrocyte-specific silencing of MMP13 to attenuate cartilage degeneration.
Assuntos
Condrócitos , Exossomos , Metaloproteinase 13 da Matriz , Osteoartrite , RNA Interferente Pequeno , Ratos Sprague-Dawley , Regeneração , Exossomos/metabolismo , Animais , Condrócitos/metabolismo , Metaloproteinase 13 da Matriz/metabolismo , Metaloproteinase 13 da Matriz/genética , RNA Interferente Pequeno/administração & dosagem , Osteoartrite/terapia , Masculino , Cartilagem Articular/metabolismo , Peptídeos/administração & dosagem , Peptídeos/química , Células Cultivadas , Humanos , Ratos , Cartilagem/metabolismoRESUMO
Type 2 diabetes is linked with increased incidence and severity of osteoarthritis. The purpose of this study was to determine the effect of extracellular glucose within the normal blood glucose and hyperglycemic range on catabolic enzyme production by chondrocytes isolated from osteoarthritic (OA) and macroscopically normal (MN) human cartilage under oxygenated (18.9% oxygen) and hypoxic (1% oxygen) conditions. OA and MN chondrocytes were maintained in 4, 6, 8, or 10 mM glucose for 24 h. Glucose consumption, GLUT1 glucose transporter levels, MMP13 and ADAMTS5 production, and levels of RUNX2, a transcriptional regulator of MMP13, ADAMTS5, and GLUT1, were assessed by enzyme-linked assays, RT-qPCR and/or western blot. Under oxygenated conditions, glucose consumption and GLUT1 protein levels were higher in OA but not MN chondrocytes in 10 mM glucose compared to 4 mM. Both RNA and protein levels of MMP13 and ADAMTS5 were also higher in OA but not MN chondrocytes in 10 mM compared to 4 mM glucose under oxygenated conditions. Expression of RUNX2 was overall lower in MN than OA chondrocytes and there was no consistent effect of extracellular glucose concentration on RUNX2 levels in MN chondrocytes. However, protein (but not RNA) levels of RUNX2 were elevated in OA chondrocytes maintained in 10 mM versus 4 mM glucose under oxygenated conditions. In contrast, neither RUNX2 levels or MMP13 or ADAMTS5 expression were increased in OA chondrocytes maintained in 10 mM compared to 4 mM glucose in hypoxia. Elevated extracellular glucose leads to increased glucose consumption and increased RUNX2 protein levels, promoting production of MMP13 and ADAMTS5 by OA chondrocytes in oxygenated but not hypoxic conditions. These findings suggest that hyperglycaemia may exacerbate chondrocyte-mediated cartilage catabolism in the oxygenated superficial zone of cartilage in vivo in patients with undertreated type 2 diabetes, contributing to increased OA severity.
Assuntos
Proteína ADAMTS5 , Hipóxia Celular , Condrócitos , Glucose , Metaloproteinase 13 da Matriz , Osteoartrite , Humanos , Condrócitos/metabolismo , Condrócitos/patologia , Proteína ADAMTS5/metabolismo , Proteína ADAMTS5/genética , Metaloproteinase 13 da Matriz/metabolismo , Metaloproteinase 13 da Matriz/genética , Glucose/metabolismo , Glucose/farmacologia , Osteoartrite/metabolismo , Osteoartrite/patologia , Osteoartrite/genética , Idoso , Feminino , Oxigênio/metabolismo , Oxigênio/farmacologia , Masculino , Pessoa de Meia-Idade , Células Cultivadas , Cartilagem Articular/metabolismo , Cartilagem Articular/patologia , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/genéticaRESUMO
OBJECTIVE: Matrix metalloproteinase 13 (MMP13) is an extracellular matrix protease that affects the progression of atherosclerotic plaques and arterial thrombi by degrading collagens, modifying protein structures and regulating inflammatory responses, but its role in deep vein thrombosis (DVT) has not been determined. The purpose of this study was to investigate the potential effects of MMP13 and MMP13-related genes on the formation of DVT. METHODS: We altered the expression level of MMP13 in vivo and conducted a transcriptome study to examine the expression and relationship between MMP13 and MMP13-related genes in a mouse model of DVT. After screening genes possibly related to MMP13 in DVT mice, the expression levels of candidate genes in human umbilical vein endothelial cells (HUVECs) and the venous wall were evaluated. The effect of MMP13 on platelet aggregation in HUVECs was investigated in vitro. RESULTS: Among the differentially expressed genes, interleukin 1 beta, podoplanin (Pdpn), and factor VIII von Willebrand factor (F8VWF) were selected for analysis in mice. When MMP13 was inhibited, the expression level of PDPN decreased significantly in vitro. In HUVECs, overexpression of MMP13 led to an increase in the expression level of PDPN and induced platelet aggregation, while transfection of PDPN-siRNA weakened the ability of MMP13 to increase platelet aggregation. CONCLUSIONS: Inhibiting the expression of MMP13 could reduce the burden of DVT in mice. The mechanism involves downregulating the expression of Pdpn through MMP13, which could provide a novel gene target for DVT diagnosis and treatment.
Assuntos
Trombose Venosa , Animais , Humanos , Camundongos , Modelos Animais de Doenças , Células Endoteliais da Veia Umbilical Humana/metabolismo , Metaloproteinase 13 da Matriz/genética , Agregação Plaquetária , Trombose Venosa/genéticaRESUMO
Intervertebral disc degeneration (IVDD) is a progressive degenerative disease influenced by various factors. Genkwanin, a known anti-inflammatory flavonoid, has not been explored for its potential in IVDD management. This study aims to investigate the effects and mechanisms of genkwanin on IVDD. In vitro, cell experiments revealed that genkwanin dose-dependently inhibited Interleukin-1ß-induced expression levels of inflammatory factors (Interleukin-6, inducible nitric oxide synthase, cyclooxygenase-2) and degradation metabolic protein (matrix metalloproteinase-13). Concurrently, genkwanin upregulated the expression of synthetic metabolism genes (type II collagen, aggrecan). Moreover, genkwanin effectively reduced the phosphorylation of phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin, mitogen-activated protein kinase (MAPK), and nuclear factor-κB (NF-κB) pathways. Transcriptome sequencing analysis identified integrin α2 (ITGA2) as a potential target of genkwanin, and silencing ITGA2 reversed the activation of PI3K/AKT pathway induced by Interleukin-1ß. Furthermore, genkwanin alleviated Interleukin-1ß-induced senescence and apoptosis in nucleus pulposus cells. In vivo animal experiments demonstrated that genkwanin mitigated the progression of IVDD in the rat model through imaging and histological examinations. In conclusion, This study suggest that genkwanin inhibits inflammation in nucleus pulposus cells, promotes extracellular matrix remodeling, suppresses cellular senescence and apoptosis, through the ITGA2/PI3K/AKT, NF-κB and MAPK signaling pathways. These findings indicate that genkwanin may be a promising therapeutic candidate for IVDD.
Assuntos
Apoptose , Senescência Celular , Flavonoides , Degeneração do Disco Intervertebral , Transdução de Sinais , Animais , Humanos , Masculino , Ratos , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Apoptose/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Flavonoides/farmacologia , Flavonoides/uso terapêutico , Integrina alfa2/metabolismo , Integrina alfa2/genética , Interleucina-1beta/metabolismo , Degeneração do Disco Intervertebral/tratamento farmacológico , Degeneração do Disco Intervertebral/patologia , Degeneração do Disco Intervertebral/metabolismo , Metaloproteinase 13 da Matriz/metabolismo , Metaloproteinase 13 da Matriz/genética , Núcleo Pulposo/efeitos dos fármacos , Núcleo Pulposo/patologia , Núcleo Pulposo/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacosRESUMO
BACKGROUND: Osteoarthritis (OA) is a joint disease characterized by inflammation and progressive cartilage degradation. Chondrocyte apoptosis is the most common pathological feature of OA. Interleukin-1ß (IL-1ß), a major inflammatory cytokine that promotes cartilage degradation in OA, often stimulates primary human chondrocytes in vitro to establish an in vitro OA model. Moreover, IL-1ß is involved in OA pathogenesis by stimulating the phosphoinositide-3-kinase (PI3K)/Akt and mitogen-activated protein kinases pathways. The G-protein-coupled receptor, cc chemokine receptor 10 (CCR10), plays a vital role in the occurrence and development of various malignant tumors. However, the mechanism underlying the role of CCR10 in the pathogenesis of OA remains unclear. We aimed to evaluate the protective effect of CCR10 on IL-1ß-stimulated CHON-001 cells and elucidate the underlying mechanism. METHODS: The CHON-001 cells were transfected with a control small interfering RNA (siRNA) or CCR10-siRNA for 24 h, and stimulated with 10 ng/mL IL-1ß for 12 h to construct an OA model in vitro. The levels of CCR10, cleaved-caspase-3, MMP-3, MMP-13, Collagen II, Aggrecan, p-PI3K, PI3K, p-Akt, Akt, phosphorylated-mammalian target of rapamycin (p-mTOR), and mTOR were detected using quantitative reverse transcription polymerase chain reaction and western blotting. Viability, cytotoxicity, and apoptosis of CHON-001 cells were assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, lactate dehydrogenase assay (LDH), and flow cytometry analysis, respectively. Inflammatory cytokines (TNF-α, IL-6, and IL-8) were assessed using enzyme-linked immunosorbent assay. RESULTS: Level of CCR10 was substantially higher in the IL-1ß-stimulated CHON-001 cells than that in the control group, whereas CCR10 was down-regulated in the CCR10-siRNA transfected CHON-001 cells compared to that in the control-siRNA group. Notably, CCR10 inhibition alleviated IL-1ß-induced inflammatory injury in the CHON-001 cells, as verified by enhanced cell viability, inhibited LDH release, reduced apoptotic cells, and cleaved-caspase-3 expression. Meanwhile, IL-1ß induced the release of tumor necrosis factor alpha, IL-6, and IL-8, increase of MMP-3 and MMP-13, and decrease of Collagen II and Aggrecan in the CHON-001 cells, which were reversed by CCR10-siRNA. However, these effects were reversed upon PI3K agonist 740Y-P treatment. Further, IL-1ß-induced PI3K/Akt/mTOR signaling pathway activation was inhibited by CCR10-siRNA, which was increased by 740Y-P treatment. CONCLUSION: Inhibition of CCR10 alleviates IL-1ß-induced chondrocytes injury via PI3K/Akt/mTOR pathway inhibition, suggesting that CCR10 might be a promising target for novel OA therapeutic strategies.
Assuntos
Osteoartrite , Fragmentos de Peptídeos , Fosfatidilinositol 3-Quinase , Receptores do Fator de Crescimento Derivado de Plaquetas , Humanos , Agrecanas , Caspase 3 , Colágeno , Citocinas , Interleucina-6 , Interleucina-8 , Metaloproteinase 13 da Matriz/genética , Metaloproteinase 3 da Matriz , Osteoartrite/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatidilinositóis , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores CCR10 , RNA Interferente Pequeno , Serina-Treonina Quinases TORRESUMO
AIM: To investigate the linkage of matrix metalloproteinase (MMP) gene polymorphisms with the pathogenesis of knee osteoarthritis (OA). METHODS: This meta-analysis study systematically retrieved relevant studies from PubMed, Embase, the Cochrane Central, Wanfang Data, CNKI, and SinoMed up to November 2020. Odds ratios (ORs) and 95% confidence intervals (CIs) were used to estimate the association between MMP gene polymorphisms and OA. RESULTS: A total of nine case-control studies comprising 1719 knee OA patients and 1904 controls were included in this meta-analysis. The results revealed that MMP-1-1607 (rs1799750) 1G/2G polymorphism was not significantly associated with knee OA risk in four genetic models (OR (95% CI): allele model: 0.89 (0.57, 1.40), p = .615); dominant mode: 0.82 (0.47, 1.44), p = .486; recessive model: 0.88 (0.49, 1.57), p = .659; homozygote model: 0.79 (0.34, 1.82), p = .576. The association was significant for dominant model of MMP-3 C/T: 1.54 (1.10-2.15), p = .013, especially in Asian ethnicity (1.63 (1.11, 2.39), p = .013). Variants of MMP-13 C/T polymorphism were associated with increased risk of knee OA development based on dominant model: 1.56 (1.19, 2.06), p = .001 and homozygote model: 2.12 (1.44, 3.13), p < .001, and there were significant associations between MMP-13 C/T polymorphism and knee OA risk in Asian ethnicity under different genetic models (all p > .05). CONCLUSIONS: Present evidence suggested that the gene polymorphisms of MMP-1-1607 1G/2G may not be associated with the risk of OA. But, the dominant model of MMP-3 and MMP-13 polymorphisms in Asian ethnicity was significantly correlated with knee OA.
Assuntos
Osteoartrite do Joelho , Humanos , Osteoartrite do Joelho/diagnóstico , Osteoartrite do Joelho/genética , Predisposição Genética para Doença , Metaloproteinase 1 da Matriz/genética , Metaloproteinase 13 da Matriz/genética , Metaloproteinase 3 da Matriz/genética , Genótipo , Polimorfismo de Nucleotídeo Único , Estudos de Casos e ControlesRESUMO
Targeted drug delivery and the reduction of off-target effects are crucial for the promising clinical application of nucleic acid drugs. To address this challenge, a new approach for treating osteoarthritis (OA) that accurately delivers antisense oligonucleotides (ASO) targeting matrix metalloproteinase-13 (ASO-MMP13) to chondrocytes, is developed. Small extracellular vesicles (exos) are ligated with chondrocyte affinity peptide (CAP) using Sortase A and subsequently incubated with cholesterol-modified ASO-MMP13 to construct a chondrocyte-targeted drug delivery exo (CAP-exoASO). Compared with exos without CAP (ExoASO), CAP-exoASOs attenuate IL-1ß-induced chondrocyte damage and prolong the retention time of ASO-MMP13 in the joint without distribution in major organs following intra-articular injection. Notably, CAP-exoASOs decrease MMP13 expression (P < 0.001) and upregulate COL2A1 expression (P = 0.006), resulting in reorganization of the cartilage matrix and alleviation of progression in the OA model. Furthermore, the Osteoarthritis Research Society International (OARSI) score of articular cartilage tissues treated with CAP-exoASO is comparable with that of healthy rats (P = 0.148). A mechanistic study demonstrates that CAP-exoASO may reduce inflammation by suppressing the IL-17 and TNF signaling pathways. Based on the targeted delivery effect, CAP-exoASOs successfully accomplish cartilage repair and have considerable potential for development as a promising therapeutic modality for satisfactory OA therapy.
Assuntos
Aminoaciltransferases , Proteínas de Bactérias , Condrócitos , Cisteína Endopeptidases , Vesículas Extracelulares , Metaloproteinase 13 da Matriz , Osteoartrite , Metaloproteinase 13 da Matriz/metabolismo , Metaloproteinase 13 da Matriz/genética , Animais , Osteoartrite/terapia , Osteoartrite/metabolismo , Osteoartrite/patologia , Osteoartrite/genética , Condrócitos/metabolismo , Ratos , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/química , Aminoaciltransferases/metabolismo , Aminoaciltransferases/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Cisteína Endopeptidases/metabolismo , Cisteína Endopeptidases/genética , Masculino , Sistemas de Liberação de Medicamentos/métodos , Ratos Sprague-Dawley , Oligonucleotídeos Antissenso/farmacologia , Oligonucleotídeos Antissenso/química , Oligonucleotídeos Antissenso/administração & dosagem , Cartilagem Articular/metabolismo , Cartilagem Articular/patologiaRESUMO
BACKGROUND: Matrix metalloproteinase (MMP)1, MMP9, MMP11, and MMP13 are overexpressed in malignant melanoma (MM), being associated with tumor invasive phase, metastases, and more aggressive neoplastic phenotypes. AIM: The main objective of the current study was to correlate the expression of the MMPs with the evolution of MM toward distant metastasis. PATIENTS, MATERIALS AND METHODS: We designed a retrospective cohort study, including 13 patients with metastatic MM. Data concerning age, sex, localization of the primary lesion and metastasis, and histological and immunohistochemical features (intensity of expression and percent of positive cells for MMPs) were statistically processed. RESULTS: The time between the diagnosis of primitive melanoma and the diagnosis of metastasis ranged between 0 and 73 months, with a mean value of 18.3 months. The metastases rich in MMP1- and MMP9-positive cells occurred earlier than the metastases with low levels of positive cells. The mean period until metastasis was shorter for the MMP1-expressing tumors than the ones without MMP1 expression. MMP13 expression in the tumor and its metastasis was significantly linked with the time until the metastasis occurrence. CONCLUSIONS: This study emphasizes the roles of MMP1, MMP9, and MMP13 in the process of metastasis in melanoma and the opportunity to use them as therapeutic targets and surveillance molecules.
Assuntos
Metaloproteinase 13 da Matriz , Metaloproteinase 1 da Matriz , Metaloproteinase 9 da Matriz , Melanoma , Humanos , Metaloproteinase 1 da Matriz/genética , Metaloproteinase 1 da Matriz/metabolismo , Metaloproteinase 11 da Matriz/genética , Metaloproteinase 11 da Matriz/metabolismo , Metaloproteinase 13 da Matriz/genética , Metaloproteinase 13 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Melanoma/genética , Melanoma/metabolismo , Estudos RetrospectivosRESUMO
We investigated the effects of a Tankyrase (TNKS-1/2) inhibitor on mechanical stress-induced gene expression in human chondrocytes and examined TNKS-1/2 expression in human osteoarthritis (OA) cartilage. Cells were seeded onto stretch chambers and incubated with or without a TNKS-1/2 inhibitor (XAV939) for 12 h. Uni-axial cyclic tensile strain (CTS) (0.5 Hz, 8% elongation, 30 min) was applied and the gene expression of type II collagen a1 chain (COL2A1), aggrecan (ACAN), SRY-box9 (SOX9), TNKS-1/2, a disintegrin and metalloproteinase with thrombospondin motifs-5 (ADAMTS-5), and matrix metalloproteinase-13 (MMP-13) were examined by real-time PCR. The expression of ADAMTS-5, MMP-13, nuclear translocation of nuclear factor-κB (NF-κB), and ß-catenin were examined by immunocytochemistry and Western blotting. The concentration of IL-1ß in the supernatant was examined by enzyme-linked immunosorbent assay (ELISA). TNKS-1/2 expression was assessed by immunohistochemistry in human OA cartilage obtained at the total knee arthroplasty. TNKS-1/2 expression was increased after CTS. The expression of anabolic factors were decreased by CTS, however, these declines were abrogated by XAV939. XAV939 suppressed the CTS-induced expression of catabolic factors, the release of IL-1ß, as well as the nuclear translocation of NF-κB and ß-catenin. TNKS-1/2 expression increased in mild and moderate OA cartilage. Our results demonstrated that XAV939 suppressed mechanical stress-induced expression of catabolic proteases by the inhibition of NF-κB and activation of ß-catenin, indicating that TNKS-1/2 expression might be associated with OA pathogenesis.