RESUMEN
Immunofluorescence staining is a widely used and powerful tool for the visualization and colocalization of two or more proteins and/or cellular organelles. For colocalization studies in fixed cells, one target protein/organelle is immunostained and visualized by one fluorophore and the other target protein/organelle is immunostained and visualized by a different fluorophore whose excitation emission spectra does not overlap with the first fluorophore. Parkin (PARK2) is an E3 ubiquitin ligase which performs ubiquitination of surface proteins of dysfunctional mitochondria to mark them for autolysosomal degradation. Here we describe the immunofluorescence staining of parkin protein and immunofluorescence or dye-based methods to visualize mitochondria and study the colocalization of parkin and mitochondria in primary human or mouse chondrocytes or cell lines.
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Condrocitos/metabolismo , Técnica del Anticuerpo Fluorescente , Mitocondrias/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Biomarcadores , Células Cultivadas , Técnica del Anticuerpo Fluorescente/métodos , Humanos , Microscopía Confocal/métodos , Mitocondrias/genética , Osteoartritis/genética , Osteoartritis/metabolismo , Osteoartritis/patología , Permeabilidad , Transporte de Proteínas , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
PURPOSE: Transposable elements are known to remodel gene structure and provide a known source of genetic variation. Retrotransposon gag-like-3 (RTL3) is a mammalian retrotransposon-derived transcript (MART) whose function in the skeletal tissue is unknown. This study aimed to elucidate the biological significance of RTL3 in chondrogenesis and type-II collagen (COL2A1) gene expression in chondrocytes. MATERIALS AND METHODS: Expression of RTL3, SOX-9 and COL2A1 mRNAs was determined by TaqMan assays and the protein expression by immunoblotting. RTL3 and Sox-9 depletion in human chondrocytes was achieved using validated siRNAs. An RTL3 mutant (∆RTL3) lacking the zinc finger domain was created using in vitro mutagenesis. Forced expression of RTL3, ∆RTL3, and SOX-9 was achieved using CMV promoter containing expression plasmids. CRISPR-Cas9 was utilized to delete Rtl3 and create a stable ATDC5Rlt3-/- cell line. Matrix deposition and Col2a1 quantification during chondrogenesis were determined by Alcian blue staining and Sircol™ Soluble Collagen Assay, respectively. RESULTS: RTL3 is not ubiquitously expressed but showed strong expression in cartilage, chondrocytes and synoviocytes but not in muscle, brain, or other tissues analyzed. Loss-of-function and gain-of-function studies demonstrated a critical role of RTL3 in the regulation of SOX-9 and COL2A1 expression and matrix synthesis during chondrogenesis. Both RTL3 and SOX-9 displayed co-regulated expression in chondrocytes. Gene regulatory activity of RTL3 requires the c-terminal CCHC zinc-finger binding domain. CONCLUSIONS: Our results identify a novel regulatory mechanism of COL2A1 expression in chondrocytes that may help to further understand the skeletal development and the pathogenesis of diseases with altered COL2A1 expression.
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Condrocitos , Retroelementos , Animales , Diferenciación Celular , Condrocitos/metabolismo , Condrogénesis/genética , Colágeno/metabolismo , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Humanos , Mamíferos/genética , Mamíferos/metabolismo , Factor de Transcripción SOX9/genética , Factor de Transcripción SOX9/metabolismoRESUMEN
Mitochondrial function is impaired in osteoarthritis (OA) but its impact on cartilage catabolism is not fully understood. Here, we investigated the molecular mechanism of mitochondrial dysfunction-induced activation of the catabolic response in chondrocytes. Using cartilage slices from normal and OA cartilage, we showed that mitochondrial membrane potential was lower in OA cartilage, and that this was associated with increased production of mitochondrial superoxide and catabolic genes [interleukin 6 (IL-6), COX-2 (also known as PTGS2), MMP-3, -9, -13 and ADAMTS5]. Pharmacological induction of mitochondrial dysfunction in chondrocytes and cartilage explants using carbonyl cyanide 3-chlorophenylhydrazone increased mitochondrial superoxide production and the expression of IL-6, COX-2, MMP-3, -9, -13 and ADAMTS5, and cartilage matrix degradation. Mitochondrial dysfunction-induced expression of catabolic genes was dependent on the JNK (herein referring to the JNK family)/activator protein 1 (AP1) pathway but not the NFκB pathway. Scavenging of mitochondrial superoxide with MitoTEMPO, or pharmacological inhibition of JNK or cFos and cJun, blocked the mitochondrial dysfunction-induced expression of the catabolic genes in chondrocytes. We demonstrate here that mitochondrial dysfunction contributes to OA pathogenesis via JNK/AP1-mediated expression of catabolic genes. Our data shows that AP1 could be used as a therapeutic target for OA management.This article has an associated First Person interview with the first author of the paper.
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Cartílago Articular , Factor de Transcripción AP-1 , Cartílago Articular/metabolismo , Células Cultivadas , Condrocitos/metabolismo , Humanos , Interleucina-1beta/metabolismo , Sistema de Señalización de MAP Quinasas , Mitocondrias , Especies Reactivas de Oxígeno/metabolismo , Factor de Transcripción AP-1/genética , Factor de Transcripción AP-1/metabolismoRESUMEN
Ageing is a leading risk factor predisposing cartilage to osteoarthritis. However, little research has been conducted on the effect of ageing on the expression of small non-coding RNAs (sncRNAs). RNA from young and old chondrocytes from macroscopically normal equine metacarpophalangeal joints was extracted and subjected to small RNA sequencing (RNA-seq). Differential expression analysis was performed in R using package DESeq2. For transfer RNA (tRNA) fragment analysis, tRNA reads were aligned to horse tRNA sequences using Bowtie2 version 2.2.5. Selected microRNA (miRNAs or miRs) and small nucleolar RNA (snoRNA) findings were validated using real-time quantitative Polymerase Chain Reaction (qRT-PCR) in an extended cohort of equine chondrocytes. tRNA fragments were further investigated in low- and high-grade OA human cartilage tissue. In total, 83 sncRNAs were differentially expressed between young and old equine chondrocytes, including miRNAs, snoRNAs, small nuclear RNAs (snRNAs), and tRNAs. qRT-PCR analysis confirmed findings. tRNA fragment analysis revealed that tRNA halves (tiRNAs), tiRNA-5035-GluCTC and tiRNA-5031-GluCTC-1 were reduced in both high grade OA human cartilage and old equine chondrocytes. For the first time, we have measured the effect of ageing on the expression of sncRNAs in equine chondrocytes. Changes were detected in a number of different sncRNA species. This study supports a role for sncRNAs in ageing cartilage and their potential involvement in age-related cartilage diseases.
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Senescencia Celular/genética , Condrocitos/metabolismo , ARN Pequeño no Traducido/metabolismo , Envejecimiento/genética , Animales , Cartílago Articular/patología , Condrocitos/patología , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Caballos/genética , Humanos , MicroARNs/genética , MicroARNs/metabolismo , Osteoartritis/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN de Transferencia/genética , ARN de Transferencia/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Reproducibilidad de los Resultados , Análisis de Secuencia de ARNRESUMEN
Osteoarthritis (OA) is the most prevalent joint degenerative disease leading to irreversible structural and functional changes in the joint and is a major cause of disability and reduced life expectancy in ageing population. Despite the high prevalence of OA, there is no disease modifying drug available for the management of OA. Oxidative stress, a result of an imbalance between the production of reactive oxygen species (ROS) and their clearance by antioxidant defense system, is high in OA cartilage and is a major cause of chronic inflammation. Inflammatory mediators, such as interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) are highly upregulated in OA joints and induce ROS production and expression of matrix degrading proteases leading to cartilage extracellular matrix degradation and joint dysfunction. ROS and inflammation are interdependent, each being the target of other and represent ideal target/s for the treatment of OA. Plant polyphenols possess potent antioxidant and anti-inflammatory properties and can inhibit ROS production and inflammation in chondrocytes, cartilage explants and in animal models of OA. The aim of this review is to discuss the chondroprotective effects of polyphenols and modulation of different molecular pathways associated with OA pathogenesis and limitations and future prospects of polyphenols in OA treatment.
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Antiinfecciosos/farmacología , Antioxidantes/farmacología , Antirreumáticos/uso terapéutico , Mediadores de Inflamación/antagonistas & inhibidores , Articulaciones/efectos de los fármacos , Osteoartritis/tratamiento farmacológico , Estrés Oxidativo/efectos de los fármacos , Polifenoles/farmacología , Animales , Humanos , Mediadores de Inflamación/metabolismo , Articulaciones/metabolismo , Articulaciones/patología , Osteoartritis/metabolismo , Osteoartritis/patología , Especies Reactivas de Oxígeno/metabolismo , Transducción de SeñalRESUMEN
Joint inflammation is a key player in the pathogenesis of osteoarthritis (OA). Imperatorin, a plant-derived small molecule has been reported to have anti-inflammatory properties; however, its effect on chondrocytes is not known. Here, we investigated the effects of Imperatorin on interleukin-1ß (IL-1ß) induced expression of inducible nitric oxide synthase (iNOS) and nitric oxide production in primary human OA chondrocytes and cartilage explants culture under pathological conditions and explored the associated signaling pathways. We pretreated chondrocytes or explants with Imperatorin (50 µM) followed by IL-1ß (1 ng/ml), and the culture supernatant was used to determine the levels of nitrite production by Griess assay and chondrocytes were harvested to prepare cell lysate or RNA for gene expression analysis of iNOS by Western blot or qPCR and in explants by immunohistochemistry (IHC). Pretreatment of primary chondrocytes and cartilage explants with Imperatorin suppressed IL-1ß induced expression of iNOS and NO production. Imperatorin blocked the IL-1ß-induced phosphorylation of ERK-MAPK/AP1 signaling pathway to suppress iNOS expression. The role of ERK in the regulation of iNOS expression was verified by using ERK inhibitor. Interestingly, we also found that Imperatorin binds to iNOS protein and inhibits its activity in vitro. Our data demonstrated that Imperatorin possess strong anti-inflammatory activity and may be developed as a therapeutic agent for the management of OA.
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Antiinflamatorios/farmacología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Furocumarinas/farmacología , Óxido Nítrico Sintasa de Tipo II/metabolismo , Osteoartritis/prevención & control , Factor de Transcripción AP-1/metabolismo , Antiinflamatorios/uso terapéutico , Cartílago/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Condrocitos/citología , Condrocitos/efectos de los fármacos , Condrocitos/ultraestructura , Furocumarinas/uso terapéutico , Humanos , Interleucina-1beta/antagonistas & inhibidores , Interleucina-1beta/toxicidad , Simulación del Acoplamiento Molecular , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo II/química , Óxido Nítrico Sintasa de Tipo II/genética , Nitritos/análisis , Cultivo Primario de Células , Proteoma/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Regulación hacia ArribaRESUMEN
Accumulating evidence suggests that inflammation has a key role in the pathogenesis of osteoarthritis (OA). Nitric oxide (NO) has been established as one of the major inflammatory mediators in OA and drives many pathological changes during the development and progression of OA. Excessive production of NO in chondrocytes promotes cartilage destruction and cellular injury. The synthesis of NO in chondrocytes is catalyzed by inducible NO synthase (iNOS), which is thereby an attractive therapeutic target for the treatment of OA. A number of direct and indirect iNOS inhibitors, bioactive compounds, and plant-derived small molecules have been shown to exhibit chondroprotective effects by suppressing the expression of iNOS. Many of these iNOS inhibitors hold promise for the development of new, disease-modifying therapies for OA; however, attempts to demonstrate their success in clinical trials are not yet successful. Many plant extracts and plant-derived small molecules have also shown promise in animal models of OA, though further studies are needed in human clinical trials to confirm their therapeutic potential. In this review, we discuss the role of iNOS in OA pathology and the effects of various iNOS inhibitors in OA.
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Óxido Nítrico Sintasa de Tipo II/metabolismo , Osteoartritis/metabolismo , Osteoartritis/patología , Animales , Humanos , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Inflamación/patología , Mediadores de Inflamación/metabolismo , Osteoartritis/tratamiento farmacológico , Extractos Vegetales/farmacologíaRESUMEN
OBJECTIVE: Cytokine expression is tightly regulated posttranscriptionally, but high levels of interleukin-6 (IL-6) in patients with osteoarthritis (OA) indicate that regulatory mechanisms are disrupted in this disorder. The enzyme ZCCHC6 (zinc-finger CCHC domain-containing protein 6; TUT-7) has been implicated in posttranscriptional regulation of inflammatory cytokine expression, but its role in OA pathogenesis is unknown. The present study was undertaken to investigate whether ZCCHC6 directs the expression of IL-6 and influences OA pathogenesis in vivo. METHODS: Human and mouse chondrocytes were stimulated with recombinant IL-1ß. Expression of ZCCHC6 in human chondrocytes was knocked down using small interfering RNAs. IL-6 transcript stability was determined by actinomycin D chase, and 3'-uridylation of microRNAs was determined by deep sequencing. Zcchc6-/- mice were produced by gene targeting. OA was surgically induced in the knee joints of mice, and disease severity was scored using a semiquantitative grading system. RESULTS: ZCCHC6 was markedly up-regulated in damaged cartilage from human OA patients and from wild-type mice with surgically induced OA. Overexpression of ZCCHC6 induced the expression of IL-6, and its knockdown reduced IL-6 transcript stability and IL-1ß-induced IL-6 expression in chondrocytes. Reintroduction of Zcchc6 in Zcchc6-/- mouse chondrocytes rescued the IL-1ß-induced IL-6 expression. Knockdown of ZCCHC6 reduced the population of micro-RNA 26b (miR-26b) with 3'-uridylation by 60%. Zcchc6-/- mice with surgically induced OA produced low levels of IL-6 and exhibited reduced cartilage damage and synovitis in the joints. CONCLUSION: These findings indicate that ZCCHC6 enhances IL-6 expression in chondrocytes through transcript stabilization and by uridylating miR-26b, which abrogates repression of IL-6. Inhibition of IL-6 expression and significantly reduced OA severity in Zcchc6-/- mice identify ZCCHC6 as a novel therapeutic target to inhibit disease pathogenesis.
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Silenciador del Gen , Interleucina-6/metabolismo , Nucleotidiltransferasas/metabolismo , Osteoartritis/genética , Índice de Severidad de la Enfermedad , Animales , Condrocitos/metabolismo , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Humanos , Articulación de la Rodilla/metabolismo , Ratones , ARN Nucleotidiltransferasas , ARN Interferente Pequeño/metabolismo , Regulación hacia ArribaRESUMEN
BACKGROUND/AIMS: Butein (2',3,4,4'-Tetrahydroxychalcone), a polyphenol produced by several plants including Butea monoserpma, has been reported to exert potent anti-inflammatory activity but the mechanism remains unknown. In the present work we investigated the mechanism of Butein-mediated suppression of IL-6 expression in normal and human osteoarthritis (OA) chondrocytes under pathological conditions. METHODS: Expression level of interleukin-6 (IL-6) protein in OA cartilage was analyzed by immunohistochemistry using a validated antibody. Chondrocytes derived from normal or OA cartilage by enzymatic digestion were pretreated with Butein followed by stimulation with interleukin-1ß (IL-1ß) and the levels of IL-6 mRNA were quantified by TaqMan assay and the protein levels were measured by Western immunoblotting. Autophagy activation was determined by Western blotting and confocal microscopy. Autophagy was inhibited by siRNA mediated knockdown of ATG5. RESULTS: Expression of IL-6 protein was high in the OA cartilage compared to smooth cartilage from the same patient. OA chondrocytes and cartilage explants stimulated with IL-1ß showed high level expression of IL-6 mRNA and protein. Butein increased the phosphorylation of AMPKαThr-172, TSC2Ser-1387 and ULK1Ser-317 and inhibited the phosphorylation of mTORSer-2448 and its downstream target p70S6K and increased autophagy flux that correlated with the suppression of the IL-1ß mediated expression of IL-6 in normal and OA chondrocytes. In OA chondrocytes with siRNA-mediated knockdown of ATG5 expression, treatment with Butein failed to activate autophagy and abrogated the suppression of IL-1ß induced IL-6 expression. CONCLUSION: Our findings demonstrate for the first time that Butein activate autophagy in OA chondrocytes via AMPK/TSC2/ULK1/mTOR pathway. Additionally, activation of autophagy was essential to block the IL-1ß-induced expression of IL-6 in OA chondrocytes. These data support further studies to evaluate the use of Butein or compounds derived from it for the management of OA.
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Autofagia/efectos de los fármacos , Chalconas/farmacología , Interleucina-1beta/farmacología , Interleucina-6/metabolismo , Transducción de Señal/efectos de los fármacos , Proteínas Quinasas Activadas por AMP/metabolismo , Proteína 5 Relacionada con la Autofagia/antagonistas & inhibidores , Proteína 5 Relacionada con la Autofagia/genética , Proteína 5 Relacionada con la Autofagia/metabolismo , Homólogo de la Proteína 1 Relacionada con la Autofagia/metabolismo , Cartílago Articular/citología , Cartílago Articular/patología , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Condrocitos/citología , Condrocitos/efectos de los fármacos , Condrocitos/metabolismo , Humanos , Interleucina-6/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Osteoartritis/metabolismo , Osteoartritis/patología , Fosforilación/efectos de los fármacos , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Proteína 2 del Complejo de la Esclerosis Tuberosa , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Nrf2, a redox regulated transcription factor, has recently been shown to play a role in cartilage integrity but the mechanism remains largely unknown. Osteoarthritis (OA) is a multifactorial disease in which focal degradation of cartilage occurs. Here, we studied whether Nrf2 exerts chondroprotective effects by suppressing the oxidative stress and apoptosis in IL-1ß stimulated human OA chondrocytes. Expression of Nrf2 and its target genes HO-1, NQO1 and SOD2 was significantly high in OA cartilage compared to normal cartilage and was also higher in damaged area compared to smooth area of OA cartilage of the same patient. Human chondrocytes treated with IL-1ß resulted in robust Nrf2/ARE reporter activity, which was inhibited by pretreatment with antioxidants indicating that Nrf2 activity was due to IL-1ß-induced ROS generation. Ectopic expression of Nrf2 significantly suppressed the IL-1ß-induced generation of ROS while Nrf2 knockdown significantly increased the basal as well as IL-1ß-induced ROS levels in OA chondrocytes. Further, Nrf2 activation significantly inhibited the IL-1ß-induced activation of extrinsic and intrinsic apoptotic pathways as determined by inhibition of DNA fragmentation, activation of Caspase-3,-8,-9, cleavage of PARP, release of cytochrome-c, suppression of mitochondrial dysfunction and mitochondrial ROS production in OA chondrocytes. Nrf2 over-expression in OA chondrocytes increased the expression of anti-apoptotic proteins while pro-apoptotic proteins were suppressed. Importantly, Nrf2 over-expression activated ERK1/2 and its downstream targets-ELK1, P70S6K and P90RSK and suppressed the IL-1ß-induced apoptosis whereas inhibition of ERK1/2 activation abrogated the protective effects of Nrf2 in OA chondrocytes. Taken together, our data demonstrate that Nrf2 is a stress response protein in OA chondrocytes with anti-oxidative and anti-apoptotic function and acts via activation of ERK1/2/ELK1-P70S6K-P90RSK signaling axis. These activities of Nrf2 make it a promising candidate for the development of novel therapies for the management of OA.
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Condrocitos/fisiología , Factor 2 Relacionado con NF-E2/metabolismo , Osteoartritis/metabolismo , Apoptosis , Hidrolasas de Éster Carboxílico/metabolismo , Caspasas/metabolismo , Células Cultivadas , Humanos , Interleucina-1beta/inmunología , Sistema de Señalización de MAP Quinasas , Factor 2 Relacionado con NF-E2/genética , Estrés Oxidativo , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Proteínas Quinasas S6 Ribosómicas 90-kDa/metabolismo , Regulación hacia Arriba , Proteína Elk-1 con Dominio ets/metabolismoRESUMEN
Osteoarthritis (OA) is the most common joint disease and the leading cause of chronic disability in middle-aged and older populations worldwide. The development of disease modifying therapy for OA is in its infancy largely because the regulatory mechanisms for the molecular effectors of OA pathogenesis are poorly understood. Recent studies identified epigenetic events as a critical regulator of molecular players involved in the induction and development of OA. Epigenetic mechanisms include DNA methylation, non-coding RNA and histone modifications. The aim of this review is to briefly highlight the recent advances in the epigenetics of cartilage and potential of HDACs (Histone deacetylases) inhibitors in the therapeutic management of OA. We summarize the recent studies utilizing HDAC inhibitors as potential therapeutics for inhibiting disease progression and preventing the cartilage destruction in OA. HDACs control normal cartilage development and homeostasis and understanding the impact of HDACs inhibitors on the disease pathogenesis is of interest because of its importance in affecting overall cartilage health and homeostasis. These findings also shed new light on cartilage disease pathophysiology and provide substantial evidence that HDACs may be potential novel therapeutic targets in OA.
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Inhibidores de Histona Desacetilasas/uso terapéutico , Osteoartritis/tratamiento farmacológico , Osteoartritis/genética , Animales , Epigénesis Genética , HumanosRESUMEN
MicroRNAs, a group of small, noncoding RNAs that post-transcriptionally regulate gene expression, play important roles in chondrocyte function and in the development of osteoarthritis. We characterized the dynamic repertoire of the chondrocyte miRNome and miRISC-associated miRNome by deep sequencing analysis of primary human chondrocytes. IL-1ß treatment showed a modest effect on the expression profile of miRNAs in normal and osteoarthritis (OA) chondrocytes. We found a number of miRNAs that showed a wide range of sequence modifications including nucleotide additions and deletions at 5' and 3' ends; and nucleotide substitutions. miR-27b-3p showed the highest expression and miR-140-3p showed the highest number of sequence variations. AGO2 RIP-Seq analysis revealed the differential recruitment of a subset of expressed miRNAs and isoforms of miRNAs (isomiRs) to the miRISC in response to IL-1ß, including miR-146a-5p, miR-155-5p and miR-27b-3p. Together, these results reveal a complex repertoire of miRNAs and isomiRs in primary human chondrocytes. Here, we also show the changes in miRNA composition of the miRISC in primary human chondrocytes in response to IL-1ß treatment. These findings will provide an insight to the miRNA-mediated control of gene expression in the pathogenesis of OA.
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Condrocitos/química , Condrocitos/efectos de los fármacos , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , MicroARNs/análisis , Complejo Silenciador Inducido por ARN/análisis , Células Cultivadas , Variación Genética , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Interleucina-1beta/metabolismo , MicroARNs/genéticaRESUMEN
BACKGROUND/OBJECTIVE: Osteoarthritis (OA) is a leading cause of joint dysfunction, disability and poor quality of life in the affected population. The underlying mechanism of joint dysfunction involves increased oxidative stress, inflammation, high levels of cartilage extracellular matrix degrading proteases and decline in autophagy-a mechanism of cellular defense. There is no disease modifying therapies currently available for OA. Different parts of the Butea monosperma (Lam.) plant have widely been used in the traditional Indian Ayurvedic medicine system for the treatment of various human diseases including inflammatory conditions. Here we studied the chondroprotective effect of hydromethanolic extract of Butea monosperma (Lam.) flowers (BME) standardized to the concentration of Butein on human OA chondrocytes stimulated with IL-1ß. METHODS: The hydromethanolic extract of Butea monosperma (Lam.) (BME) was prepared with 70% methanol-water mixer using Soxhlet. Chondrocytes viability after BME treatment was measured by MTT assay. Gene expression levels were determined by quantitative polymerase chain reaction (qPCR) using TaqMan assays and immunoblotting with specific antibodies. Autophagy activation was determined by measuring the levels of microtubule associated protein 1 light chain 3-II (LC3-II) by immunoblotting and visualization of autophagosomes by transmission electron and confocal microscopy. RESULTS: BME was non-toxic to the OA chondrocytes at the doses employed and suppressed the IL-1ß induced expression of inerleukin-6 (IL-6) and matrix metalloprotease-3 (MMP-3), MMP-9 and MMP-13. BME enhanced autophagy in chondrocytes as determined by measuring the levels of LC3-II by immunoblotting and increased number of autophagosomes in BME treated chondrocytes by transmission electron microscopy and confocal microscopy. BME upregulated the expression of several autophagy related genes and increased the autophagy flux in human OA chondrocytes under pathological conditions. Further analysis revealed that BME activated autophagy in chondrocytes via inhibition of mammalian target of rapamycin (mTOR) pathway. Of importance is our finding that BME-mediated suppression of IL-1ß induced expression of IL-6, MMP-3, -9, and -13 was autophagy dependent and was abrogated by inhibition of autophagy. CONCLUSION: The above results show that the Butea monosperma (Lam.) extract has strong potential to activate autophagy and suppress IL-1ß induced expression of IL-6 and MMP-3, -9 and -13 in human OA chondrocytes. This study shows that BME or compounds derived from BME can be developed as safe and effective chondroprotective agent(s) that function by activating autophagy to suppress the expression of inflammatory and catabolic factors associated with OA pathogenesis.
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Butea , Condrocitos/metabolismo , Interleucina-1beta/farmacología , Interleucina-6/biosíntesis , Metaloproteinasas de la Matriz/biosíntesis , Osteoartritis/metabolismo , Anciano , Autofagia/efectos de los fármacos , Autofagia/fisiología , Condrocitos/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Flores , Expresión Génica , Humanos , Interleucina-6/genética , Metaloproteinasa 13 de la Matriz/biosíntesis , Metaloproteinasa 13 de la Matriz/genética , Metaloproteinasa 3 de la Matriz/biosíntesis , Metaloproteinasa 3 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/biosíntesis , Metaloproteinasa 9 de la Matriz/genética , Metaloproteinasas de la Matriz/genética , Persona de Mediana Edad , Extractos Vegetales/aislamiento & purificación , Extractos Vegetales/farmacologíaRESUMEN
Wogonin has recently been shown to possess anti-inflammatory and chondroprotective properties and is of considerable interest due to its broad pharmacological activities. The present study highlights that Wogonin binds DNA and exerts chondroprotective effects in vitro. Wogonin showed strong binding with chondrocytes genomic DNA in vitro. The mode of binding of Wogonin to genomic-DNA was assessed by competing Wogonin with EtBr or DAPI, known DNA intercalator and a minor groove binder, respectively. EtBr fluorescence reduced significantly with increase in Wogonin concentration suggesting possible DNA intercalation of Wogonin. Further, in silico molecular docking of Wogonin on mammalian DNA also indicated possible intercalation of Wogonin with DNA. The denaturation and FRET studies revealed that Wogonin prevents denaturation of DNA strands and provide stability to genomic DNA against a variety of chemical denaturants. The cellular uptake study showed that Wogonin enters osteoarthritis chondrocytes and was mainly localized in the nucleus. Wogonin treatment to OA chondrocytes protects the fragmentation of genomic DNA in response to IL-1ß as evaluated by DNA ladder and TUNEL assay. Treatment of chondrocytes with Wogonin resulted in significant suppression of IL-1ß-mediated induction of ROS. Further, Wogonin exhibited protective potential through potent suppression of extrinsic and intrinsic apoptotic pathways and induction of anti-apoptotic proteins in IL-1ß-stimulated osteoarthritis chondrocytes. Our data thus suggest that DNA intercalation by Wogonin may result in the stabilization of genomic DNA leading to protective activity.
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Condrocitos/efectos de los fármacos , ADN/metabolismo , Flavanonas/farmacología , Sustancias Intercalantes/farmacología , Interleucina-1beta/farmacología , Sustancias Protectoras/farmacología , Apoptosis/efectos de los fármacos , Sitios de Unión , Células Cultivadas , Condrocitos/citología , Condrocitos/metabolismo , ADN/química , Flavanonas/química , Flavanonas/metabolismo , Flavonoides/química , Flavonoides/farmacología , Transferencia Resonante de Energía de Fluorescencia , Humanos , Sustancias Intercalantes/química , Sustancias Intercalantes/metabolismo , Simulación del Acoplamiento Molecular , Conformación de Ácido Nucleico , Osteoartritis/metabolismo , Osteoartritis/patología , Sustancias Protectoras/química , Sustancias Protectoras/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
This article contains data related to the article "Wogonin, a plant derived small molecule exerts potent anti-inflammatory and chondroprotective effects through activation of ROS/ERK/Nrf2 signaling pathways in human Osteoarthritis chondrocytes" (Khan et al. 2017) [1]. The data are related to effects of Wogonin on the viability and IL-1ß-stimulated activation of NF-κB and ERK1/2, JNK1/2 and p38 MAPKs in human OA chondrocytes. Gene expression data representing the chondrogenic phenotype and the efficiency of Nrf2 knockdown in monolayer culture of human OA chondrocytes were shown. Moreover, mass spectrometric calibration curve of Wogonin used to quantify the intracellular uptake were also presented. The data are presented in the form of figures and significance of these has been given in the research article (Khan et al. 2017) [1].
RESUMEN
Human chondrocytes accumulate an ECM-rich matrix by secreting matrix macromolecules during monolayer culture, which makes them difficult to transfect efficiently. Here we report a non-viral based protocol to transfect the primary human chondrocytes with high efficiency in suspension. Chondrocyte cultures were digested using Pronase and Collagenase and transfected in suspension. Transfection efficiencies of more than 80% were achieved routinely using the protocol described. The viability of siRNA transfected or un-transfected chondrocytes was not affected and resulted in 80-90% knockdown of the target mRNA levels. This protocol may be useful in gene knockdown, and ectopic overexpression studies in chondrocytes.
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Condrocitos/metabolismo , Plásmidos/genética , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Transfección/métodos , Agrecanos/genética , Cartílago Articular/metabolismo , Cartílago Articular/patología , Células Cultivadas , Condrocitos/citología , Colágeno Tipo II/genética , Humanos , Nucleotidiltransferasas/antagonistas & inhibidores , Nucleotidiltransferasas/genética , Polinucleotido Adenililtransferasa , Factores de Escisión y Poliadenilación de ARNm/antagonistas & inhibidores , Factores de Escisión y Poliadenilación de ARNm/genéticaRESUMEN
Osteoarthritis (OA) is a common joint disorder with varying degrees of inflammation and sustained oxidative stress. The root extract of Scutellaria baicalensis (SBE) has been used for the treatment of inflammatory and other diseases. Here, we performed activity-guided HPLC-fractionation of SBE, identified the active ingredient(s) and investigated its chondroprotective potential. We found that the Wogonin containing fraction-4 (F4) was the most potent fraction based on its ability to inhibit ROS production and the suppression of catabolic markers including IL-6, COX-2, iNOS, MMP-3, MMP-9, MMP-13 and ADAMTS-4 in IL-1ß-treated OA chondrocytes. OA chondrocytes treated with F4 in the presence of IL-1ß showed significantly enhanced expression of anabolic genes ACAN and COL2A1. In an in vitro model of cartilage degradation treatment with F4 inhibited s-GAG release from IL-1ß-treated human cartilage explants. The inhibitory effect of F4 was not mediated through the inhibition of MAPKs and NF-κB activation but was mediated through the suppression of c-Fos/AP-1 activity at transcriptional and post transcriptional levels in OA chondrocytes. Purified Wogonin mimicked the effects of F4 in IL-1ß-stimulated OA chondrocytes. Our data demonstrates that a Wogonin-rich fraction of SBE exert chondroprotective effects through the suppression of c-Fos/AP-1 expression and activity in OA chondrocytes under pathological conditions.
Asunto(s)
Condrocitos/efectos de los fármacos , Flavanonas/farmacología , Extractos Vegetales/farmacología , Raíces de Plantas/química , Scutellaria baicalensis/química , Factor de Transcripción AP-1/metabolismo , Células Cultivadas , Fraccionamiento Químico/métodos , Condrocitos/metabolismo , Ciclooxigenasa 2/genética , Ciclooxigenasa 2/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Interleucina-1beta/farmacología , Interleucina-6/genética , Interleucina-6/metabolismo , Osteoartritis/patología , Sustancias Protectoras/farmacología , Especies Reactivas de Oxígeno/metabolismo , Factor de Transcripción AP-1/genéticaRESUMEN
Pomegranate fruit extract (PE) rich in polyphenols has been shown to exert chondroprotective effects, but the mechanism is not established. Here, we used an in vitro model of inflammation in osteoarthritis (OA) to investigate the potential of PE to suppress interleukin 1 beta (IL-1ß)-stimulated expression of inflammatory cytokine IL-6, generation of reactive oxygen species (ROS) levels, and investigated the mechanism of NF-κB inhibition by analyzing the activation of the kinases upstream of IκBα in primary human chondrocytes. Total and phosphorylated forms of kinases and expression of IL-6 were determined at protein and mRNA levels by western immunoblotting and Taqman assay, respectively. Dihydrorhodamine 123 staining estimated ROS generation. Pomegranate fruit extract inhibited the mRNA and protein expression of IL-6, generation of ROS, and inhibited the IL-1ß-mediated phosphorylation of inhibitor of nuclear factor kappa-B kinase subunit beta (IKKß), expression of IKKß mRNA, degradation of IκBα, and activation and nuclear translocation of NF-κB/p65 in human chondrocytes. Importantly, phosphorylation of NF-κB-inducing kinase was blocked by PE in IL-1ß-treated human OA chondrocytes. Taken together, these data suggest that PE exerts the chondroprotective effect(s) by suppressing the production of IL-6 and ROS levels. Inhibition of NF-κB activation by PE was blocked via modulation of activation of upstream kinases in human OA chondrocytes. Copyright © 2017 John Wiley & Sons, Ltd.
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Quinasa I-kappa B/metabolismo , Interleucina-6/metabolismo , Lythraceae/química , FN-kappa B/metabolismo , Extractos Vegetales/farmacología , Proteínas Serina-Treonina Quinasas/metabolismo , Condrocitos/efectos de los fármacos , Frutas/química , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Quinasa I-kappa B/genética , Interleucina-1beta/metabolismo , Interleucina-6/genética , FN-kappa B/genética , Fosforilación/efectos de los fármacos , Extractos Vegetales/química , Polifenoles/farmacología , Proteínas Serina-Treonina Quinasas/genética , Transducción de Señal/efectos de los fármacos , Factor de Transcripción ReIA/metabolismo , Quinasa de Factor Nuclear kappa BRESUMEN
Osteoarthritis (OA), characterized by progressive destruction of articular cartilage, is the most common form of human arthritis. Here, we evaluated the potential chondroprotective and anti-inflammatory effects of Wogonin, a naturally occurring flavonoid, in IL-1ß-stimulated human OA chondrocytes and cartilage explants. Wogonin completely suppressed the expression and production of inflammatory mediators including IL-6, COX-2, PGE2, iNOS and NO in IL-1ß-stimulated OA chondrocytes. Further, Wogonin exhibits potent chondroprotective potential by switching the signaling axis of matrix degradation from catabolic towards anabolic ends and inhibited the expression, production and activities of matrix degrading proteases including MMP-13, MMP-3, MMP-9, and ADAMTS-4 in OA chondrocytes, and blocked the release of s-GAG and COL2A1 in IL-1ß-stimulated OA cartilage explants. Wogonin also elevated the expression of cartilage anabolic factors COL2A1 and ACAN in chondrocytes and inhibited the IL-1ß-mediated depletion of COL2A1 and proteoglycan content in the matrix of cartilage explants. The suppressive effect of Wogonin was not mediated through the inhibition of MAPKs or NF-κB activation. Instead, Wogonin induced mild oxidative stress through the generation of ROS and depletion of cellular GSH, thereby modulating the cellular redox leading to the induction of Nrf2/ARE pathways through activation of ROS/ERK/Nrf2/HO-1-SOD2-NQO1-GCLC signaling axis in OA chondrocytes. Molecular docking studies revealed that Wogonin can disrupt KEAP-1/Nrf-2 interaction by directly blocking the binding site of Nrf-2 in the KEAP-1 protein. Genetic ablation of Nrf2 using specific siRNA, significantly abrogated the anti-inflammatory and chondroprotective potential of Wogonin in IL-1ß-stimulated OA chondrocytes. Our data indicates that Wogonin exerts chondroprotective effects through the suppression of molecular events involved in oxidative stress, inflammation and matrix degradation in OA chondrocytes and cartilage explants. The study provides novel insights into the development of Nrf2 as a promising candidate and Wogonin as a therapeutic agent for the management of OA.
Asunto(s)
Flavanonas/administración & dosificación , Inflamación/tratamiento farmacológico , Proteína 1 Asociada A ECH Tipo Kelch/genética , Factor 2 Relacionado con NF-E2/genética , Osteoartritis/tratamiento farmacológico , Antiinflamatorios/administración & dosificación , Condrocitos/efectos de los fármacos , Condrocitos/patología , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Inflamación/metabolismo , Inflamación/patología , Proteína 1 Asociada A ECH Tipo Kelch/química , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Simulación del Acoplamiento Molecular , Factor 2 Relacionado con NF-E2/antagonistas & inhibidores , Factor 2 Relacionado con NF-E2/química , Factor 2 Relacionado con NF-E2/metabolismo , Osteoartritis/metabolismo , Osteoartritis/patología , Unión Proteica , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
There is growing evidence in support of the involvement of inflammatory response in the pathogenesis of osteoarthritis (OA). Harpagoside, one of the bioactive components of Harpagophytum procumbens (Hp), has been shown to possess anti-inflammatory properties. Here we used an in vitro model of inflammation in OA to investigate the potential of harpagoside to suppress the production of inflammatory cytokines/chemokines such as IL-6 and matrix degrading proteases. We further investigated the likely targets of harpagoside in primary human OA chondrocytes. OA chondrocytes were pre-treated with harpagoside before stimulation with IL-1ß. mRNA expression profile of 92 cytokines/chemokines was determined using TaqMan Human Chemokine PCR Array. Expression levels of selected mRNAs were confirmed using TaqMan assays. Protein levels of IL-6 and MMP-13 were assayed by ELISA and immunoblotting. Total protein levels and phosphorylation of signaling proteins were determined by immunoblotting. Cellular localization of IL-6 and c-Fos was performed by immunofluorescence and confocal microscopy. DNA binding activity of c-FOS/AP-1 was determined by ELISA. Harpagoside significantly altered the global chemokine expression profile in IL-1ß-stimulated OA chondrocytes. Expression of IL-6 was highly induced by IL-1ß, which was significantly inhibited by pre-treatment of OA chondrocytes with harpagoside. Harpagoside did not inhibit the IL-1ß-induced activation of NF-κB and C/EBPß transcription factors but suppressed the IL-1ß-triggered induction, phosphorylation, and DNA binding activity of c-FOS, one of the main components of AP-1 transcription factors. Further, harpagoside significantly inhibited the expression of MMP-13 in OA chondrocytes under pathological conditions. siRNA-mediated knockdown of IL-6 resulted in suppressed expression and secretion of MMP-13 directly linking the role of IL-6 with MMP-13 expression. Taken together, the present study suggests that harpagoside exerts a significant anti-inflammatory effect by inhibiting the inflammatory stimuli mediated by suppressing c-FOS/AP-1 activity in OA chondrocytes under pathological conditions. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:311-320, 2017.