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1.
Article in English | WPRIM | ID: wpr-881038

ABSTRACT

Due to the poor repair ability of cartilage tissue, regenerative medicine still faces great challenges in the repair of large articular cartilage defects. Quercetin is widely applied as a traditional Chinese medicine in tissue regeneration including liver, bone and skin tissues. However, the evidence for its effects and internal mechanisms for cartilage regeneration are limited. In the present study, the effects of quercetin on chondrocyte function were systematically evaluated by CCK8 assay, PCR assay, cartilaginous matrix staining assays, immunofluorescence assay, and western blotting. The results showed that quercetin significantly up-regulated the expression of chondrogenesis genes and stimulated the secretion of GAG (glycosaminoglycan) through activating the ERK, P38 and AKT signalling pathways in a dose-dependent manner. Furthermore, in vivo experiments revealed that quercetin-loaded silk protein scaffolds dramatically stimulated the formation of new cartilage-like tissue with higher histological scores in rat femoral cartilage defects. These data suggest that quercetin can effectively stimulate chondrogenesis in vitro and in vivo, demonstrating the potential application of quercetin in the regeneration of cartilage defects.


Subject(s)
Animals , Cartilage/cytology , Chondrocytes/drug effects , Chondrogenesis/drug effects , Extracellular Matrix/metabolism , Quercetin/pharmacology , Rats , Signal Transduction/drug effects , Tissue Scaffolds
2.
Arq. bras. med. vet. zootec. (Online) ; 71(2): 509-520, mar.-abr. 2019. graf, ilus
Article in English | LILACS, VETINDEX | ID: biblio-1011276

ABSTRACT

The aim of this study was to evaluate the effect of concentrations of caffeine on the viability, synthesis activity and gene expression in cultures of chondrocytes. Extracted articular cartilage from the femurs and tibias of 15 Wistar rats at three days old to isolate chondrocytes. Chondrocytes were cultured in chondrogenic medium (control) or supplemented with caffeine (0.5, 1.0, 2.0mM). Cell viability, alkaline phosphatase activity and collagen synthesis were assessed using colorimetric assays at 7, 14, 21 days. The chondrocyte cultures of all groups grown under coverslips were stained with hematoxylin-eosin to determine the percentage of cells/field and with PAS, safranin O, alcian blue to determine the percentage of matrix chondrogenic/field at 21 days. The expressions of gene transcripts for aggrecan, collagen-II, Sox-9, Runx-2 and alkaline phosphatase were also evaluated by RT-PCR at 21 days. The means were compared using Student-Newman-Keuls. Caffeine significantly reduced the conversion of MTT to formazan, percentage of cells/field, collagen synthesis, alkaline phosphatase activity, synthesis of PAS+, safranin O+ and alcian blue+ chondrogenic matrix, and the expression of aggrecan, Sox-9 and II collagen. It is concluded that caffeine at concentrations of 0.5, 1.0, 2.0mM has a direct inhibitory effect on chondrogenesis in cultures of chondrocytes from rats.(AU)


O objetivo deste estudo foi avaliar o efeito direto de concentrações de cafeína sobre a viabilidade, atividade de síntese e expressão gênica em culturas de condrócitos de ratos. As cartilagens dos fêmures e tíbias de 15 ratos Wistar com três dias foram extraídas para isolamento de condrócitos. Os condrócitos foram cultivados em meio condrogênico (controle) ou em meio acrescido de diferentes concentrações de cafeína (0,5, 1,0, 2,0mM). Foram avaliadas a viabilidade celular, a atividade da fosfatase alcalina e a síntese de colágeno por ensaios colorimétricos aos sete, 14 e 21 dias. Condrócitos cultivados sob lamínulas foram corados pela hematoxilina e eosina, para se determinar a porcentagem de células/campo, e pelo PAS, safranina O, alcian Blue, para se determinar a porcentagem de matriz condrogênica/campo aos 21 dias. Foi avaliada a expressão de transcriptos gênicos para Sox-9, Runx-2, agrecano, colágeno-II e fosfatase alcalina por qRT-PCR, aos 21 dias. As médias foram comparadas pelo Student-Newman-Keuls. A cafeína reduziu significativamente o MTT em cristais de formazan, a porcentagem de células/campo, a síntese de colágeno, a atividade da fosfatase alcalina e a síntese de matriz condrogênica PAS+, safranina O+, alcian blue+ e expressão de Sox-9 e colágeno-II. Conclui-se que a cafeína, nas concentrações de 0,5, 1,0, 2,0mM, apresenta efeito inibidor direto sobre a condrogênese em culturas de condrócitos de ratos.(AU)


Subject(s)
Animals , Female , Rats , Caffeine , Cartilage, Articular/drug effects , Chondrocytes/drug effects , Chondrogenesis/drug effects
3.
Braz. j. med. biol. res ; 52(9): e8525, 2019. tab, graf
Article in English | LILACS | ID: biblio-1011614

ABSTRACT

Many compounds of ginsenosides show anti-inflammatory properties. However, their anti-inflammatory effects in intervertebral chondrocytes in the presence of inflammatory factors have never been shown. Increased levels of pro-inflammatory cytokines are generally associated with the degradation and death of chondrocytes; therefore, finding an effective and nontoxic substance that attenuates the inflammation is worthwhile. In this study, chondrocytes were isolated from the nucleus pulposus tissues, and the cells were treated with ginsenoside compounds and IL-1β, alone and in combination. Cell viability and death rate were assessed by CCK-8 and flow cytometry methods, respectively. PCR, western blot, and immunoprecipitation assays were performed to determine the mRNA and protein expression, and the interactions between proteins, respectively. Monomeric component of ginsenoside Rd had no toxicity at the tested range of concentrations. Furthermore, Rd suppressed the inflammatory response of chondrocytes to interleukin (IL)-1β by suppressing the increase in IL-1β, tumor necrosis factor (TNF)-α, IL-6, COX-2, and inducible nitric oxide synthase (iNOS) expression, and retarding IL-1β-induced degradation of chondrocytes by improving cell proliferation characteristics and expression of aggrecan and COL2A1. These protective effects of Rd were associated with ubiquitination of IL-1 receptor accessory protein (IL1RAP), blocking the stimulation of IL-1β to NF-κB. Bioinformatics analysis showed that NEDD4, CBL, CBLB, CBLC, and ITCH most likely target IL1RAP. Rd increased intracellular ITCH level and the amount of ITCH attaching to IL1RAP. Thus, IL1RAP ubiquitination promoted by Rd is likely to occur by up-regulation of ITCH. In summary, Rd inhibited IL-1β-induced inflammation and degradation of intervertebral disc chondrocytes by increasing IL1RAP ubiquitination.


Subject(s)
Humans , Male , Female , Adult , Middle Aged , Aged , Chondrocytes/drug effects , Ginsenosides/pharmacology , Interleukin-1beta/drug effects , Interleukin-1 Receptor Accessory Protein/metabolism , Intervertebral Disc Degeneration/metabolism , Dinoprostone/metabolism , Cell Survival/drug effects , Tumor Necrosis Factor-alpha/metabolism , Low Back Pain/metabolism , Nitric Oxide Synthase/metabolism , Chondrocytes/cytology , Chondrocytes/metabolism , Ginsenosides/metabolism , Cyclooxygenase 2/metabolism , Aggrecans/metabolism , Interleukin-1beta/metabolism , Ubiquitination , Nucleus Pulposus/cytology , Nucleus Pulposus/drug effects , Nucleus Pulposus/metabolism , Inflammation/metabolism
4.
Braz. J. Pharm. Sci. (Online) ; 54(4): e17534, 2018. tab, graf
Article in English | LILACS | ID: biblio-1001576

ABSTRACT

Diacerein (DCN) was obtained by diacetylation of an anthraquinone derivative rhein and was approved by FDA in 2008, in the treatment of osteoarthritis due to its inhibitory effect on proinflammatory cytokines, including IL-6 and IL-1ß. It was synthesized in 1980s and marketed as a tablet in some European Union and Asian countries from 1994. Along with its great potential in the treatment of osteoarthritis, its other applications are also being explored day by day, such as in the treatment of psoriasis, epidermolysis bullosa, breast cancer, type 2 diabetes and periodontitis. The main aim of this review is to explore mechanism of action, various applications and side effects associated with DCN. This has been reviewed that apart from the risk of diarrhea on long-term administration of DCN, various clinical studies has also shown its modest benefits in treatment of various pathological conditions. Hence, DCN is emerging as a new and potentially safe derivative with maximum therapeutic efficacies and minimum side effects which can results in improving the living status of patients suffering from various inflammatory diseases


Subject(s)
Osteoarthritis/drug therapy , Pharmaceutical Preparations/analysis , Pharmacologic Actions , Chondrocytes/drug effects
5.
Biol. Res ; 51: 41, 2018. graf
Article in English | LILACS | ID: biblio-983943

ABSTRACT

BACKGROUND: Osteoarthritis (OA) can be defined as degradation of articular cartilage of the joint, and is the most common degenerative disease. To regenerate the damaged cartilage, different experimental approaches including stem cell therapy have been tried. One of the major limitations of stem cell therapy is the poor post-transplantation survival of the stem cells. Anoikis, where insufficient matrix support and adhesion to extracellular matrix causes apoptotic cell death, is one of the main causes of the low post-transplantation survival rate of stem cells. Therefore, enhancing the initial interaction of the transplanted stem cells with chondrocytes could improve the therapeutic efficacy of stem cell therapy for OA. Previously, protein kinase C activator phorbol 12-myristate 13-acetate (PMA)- induced increase of mesenchymal stem cell adhesion via activation of focal adhesion kinase (FAK) has been reported. In the present study, we examine the effect PMA on the adipose-derived stem cells (ADSCs) adhesion and spreading to culture substrates, and further on the initial interaction between ADSC and chondrocytes. RESULTS: PMA treatment increased the initial adhesion of ADSC to culture substrate and cellular spreading with increased expression of adhesion molecules, such as FAK, vinculin, talin, and paxillin, at both RNA and protein level. Priming of ADSC with PMA increased the number of ADSCs attached to confluent layer of cultured chondrocytes compared to that of untreated ADSCs at early time point (4 h after seeding). CONCLUSION: Taken together, the results of this study suggest that priming ADSCs with PMA can increase the initial interaction with chondrocytes, and this proof of concept can be used to develop a non-invasive therapeutic approach for treating OA. It may also accelerate the regeneration process so that it can relieve the accompanied pain faster in OA patients. Further in vivo studies examining the therapeutic effect of PMA pretreatment of ADSCs for articular cartilage damage are required.


Subject(s)
Humans , Stem Cells/drug effects , Protein Kinase C/pharmacology , Cartilage, Articular/cytology , Chondrocytes/cytology , Cell Adhesion , Cell Communication , Cell Differentiation , Cell Survival , Blotting, Western , Cell Culture Techniques , Chondrocytes/drug effects , Reverse Transcriptase Polymerase Chain Reaction
6.
Braz. j. med. biol. res ; 50(4): e5714, 2017. tab, graf
Article in English | LILACS | ID: biblio-839285

ABSTRACT

Inflammation of cartilage is a primary symptom for knee-joint osteoarthritis. Matrix metalloproteinases (MMPs) are known to play an important role in the articular cartilage destruction related to osteoarthritis. Naringenin is a plant-derived flavonoid known for its anti-inflammatory properties. We studied the effect of naringenin on the transcriptional expression, secretion and enzymatic activity of MMP-3 in vivo in the murine monosodium iodoacetate (MIA) osteoarthritis model. The assessment of pain behavior was also performed in the MIA rats. The destruction of knee-joint tissues was analyzed microscopically. Moreover, the effect of naringenin was also studied in vitro in IL-1β activated articular chondrocytes. The transcriptional expression of MMP-3, MMP-1, MMP-13, thrombospondin motifs (ADAMTS-4) and ADAMTS-5 was also studied in primary cultured chondrocytes of rats. Naringenin caused significant reduction in pain behavior and showed marked improvement in the tissue morphology of MIA rats. Moreover, a significant inhibition of MMP-3 expression in MIA rats was observed upon treatment with naringenin. In the in vitro tests, naringenin caused a significant reduction in the transcriptional expression, secretion and enzymatic activity of the studied degradative enzymes. The NF-κB pathway was also found to be inhibited upon treatment with naringenin in vitro. Overall, the study suggests that naringenin alleviated pain and regulated the production of matrix-metalloproteinases via regulation of NF-κB pathway. Thus, naringenin could be a potent therapeutic option for the treatment of osteoarthritis.


Subject(s)
Animals , Male , Anti-Inflammatory Agents/pharmacology , Arthralgia/enzymology , Chondrocytes/enzymology , Flavanones/pharmacology , Knee Joint/enzymology , Matrix Metalloproteinase 3/biosynthesis , Osteoarthritis, Knee/enzymology , Arthralgia/drug therapy , Blotting, Western , Cell Proliferation/drug effects , Cells, Cultured , Chondrocytes/drug effects , Disease Models, Animal , Gene Expression , Interleukin-1beta/analysis , Interleukin-1beta/drug effects , Interleukin-1beta/metabolism , Knee Joint/pathology , Matrix Metalloproteinase 3/analysis , NF-kappa B/analysis , NF-kappa B/drug effects , NF-KappaB Inhibitor alpha/analysis , NF-KappaB Inhibitor alpha/drug effects , Osteoarthritis, Knee/drug therapy , Osteoarthritis, Knee/pathology , Random Allocation , Rats, Wistar , Reproducibility of Results , Reverse Transcriptase Polymerase Chain Reaction , Time Factors , Treatment Outcome
7.
Rev. bras. anestesiol ; 66(1): 29-36, Jan.-Feb. 2016. graf
Article in Portuguese | LILACS | ID: lil-773481

ABSTRACT

BACKGROUND: Intra-articular injections of local anesthetics are commonly used to enhance post-operative analgesia following orthopedic surgery as arthroscopic surgeries. Nevertheless, recent reports of severe complications due to the use of intra-articular local anesthetic have raised concerns. OBJECTIVES: The study aims to assess use of vitamin C in reducing adverse effects of the most commonly employed anesthetics - ropivacaine, bupivacaine and lidocaine - on human chondrocytes. METHODS: The chondrocyte viability following exposure to 0.5% bupivacaine or 0.75% ropivacaine or 1.0% lidocaine and/or vitamin C at doses 125, 250 and 500 µM was determined by LIVE/DEAD assay and annexin V staining. Expression levels of caspases 3 and 9 were assessed using antibodies by Western blotting. Flow cytometry was performed to analyze the generation of reactive oxygen species. RESULTS: On exposure to the local anesthetics, chondrotoxicity was found in the order ropivacaine < bupivacaine < lidocaine. Vitamin C effectively improved the reduced chondrocyte viability and decreased the raised apoptosis levels following exposure to anesthesia. At higher doses, vitamin C was found efficient in reducing the generation of reactive oxygen species and as well down-regulate the expressions of caspases 3 and 9. CONCLUSIONS: Vitamin C was observed to effectively protect chondrocytes against the toxic insult of local anesthetics ropivacaine, bupivacaine and lidocaine.


JUSTIFICATIVA: Injeções de anestésicos locais por via intra-articular são comumente usadas para melhorar a analgesia no período pós-operatório de cirurgia ortopédica como artroscopia. No entanto, relatos recentes de complicações graves devido ao uso de anestésico local por via intra-articular causou preocupações. OBJETIVOS: O objetivo do estudo foi avaliar o uso de vitamina C para reduzir os efeitos adversos dos anestésicos mais comumente usados (ropivocaína, bupivacaína e lidocaína) sobre condrócitos humanos. MÉTODOS: A viabilidade dos condrócitos após a exposição à bupivacaína a 0,5% ou ropivacaína a 0,75% ou lidocaína a 1,0% e/ou vitamina C em doses de 125, 250 e 500 µM foi determinada pelo ensaio Vivo/Morto e coloração com anexina V. Os níveis de expressão das caspases 3 e 9 foram avaliados com o uso de anticorpos pela técnica Western blotting. Citometria de fluxo foi feita para analisar a geração de espécies reativas ao oxigênio. RESULTADOS: Na exposição aos anestésicos locais, condrotoxicidade foi encontrada na seguinte ordem: ropivacaína < bupivacaína < lidocaína. A vitamina C efetivamente melhorou a redução da viabilidade dos condrócitos e diminuiu os níveis elevados de apoptose após a exposição à anestesia. Em doses mais altas, a vitamina C foi eficiente para reduzir a geração de espécies reativas ao oxigênio e assim regular negativamente a expressão das caspases 3 e 9. CONCLUSÕES: Observamos que a vitamina C foi eficaz na proteção dos condrócitos contra a agressão tóxica dos anestésicos locais ropivacaína, bupivacaína e lidocaína.


Subject(s)
Humans , Ascorbic Acid/pharmacology , Reactive Oxygen Species/metabolism , Chondrocytes/drug effects , Anesthetics, Local/toxicity , Ascorbic Acid/administration & dosage , Bupivacaine/toxicity , Down-Regulation/drug effects , Cells, Cultured , Apoptosis/drug effects , Chondrocytes/pathology , Dose-Response Relationship, Drug , Caspase 3/genetics , Caspase 9/genetics , Flow Cytometry , Ropivacaine , Amides/toxicity , Lidocaine/toxicity , Antioxidants/administration & dosage , Antioxidants/pharmacology
8.
Pakistan Journal of Pharmaceutical Sciences. 2015; 28 (2 Supp.): 667-670
in English | IMEMR | ID: emr-168095

ABSTRACT

Discuss the internal mechanism of delaying degeneration of lumber intervertebral disc. The cartilage of lumbar intervertebral disc of SD rats was selected in vitro, then cultured by tissue explant method, and identified by HE staining, toluidine blue staining and immunofluorescence. The optimal concentration of psoralen was screened by cell proliferation assay and RT-PCR method. The cells in third generation with good growth situation is selected and placed in 6-well plate at concentration of 1×10[5]/well and its expression was tested. Compared to concentration of 0, the mRNA expression of Col2al [Collagen a] secreted by was up regulated chondrocyte of lumbar intervertebral disc at the concentration of 12.5 and 25 microM [P<0.0 or P<0.01]. The aggrecan mRNA of psoralen group was higher than blank control group [P<0.01]; compared with IL-1beta induced group, the mRNA expression of Col2al was significantly increased but the mRNA expression of ADAMTS-5 was significantly decreased in psoralen group [P<0.01]. These findings suggest that, psoralen can remit the degeneration of lumbar intervertebral disc induced by IL-1beta to some extent, and affect the related factors of IL-1beta signaling pathway


Subject(s)
Animals, Laboratory , Chondrocytes/drug effects , Intervertebral Disc Degeneration , Intervertebral Disc , Lumbar Vertebrae , Interleukin-1beta , Rats
9.
Braz. j. med. biol. res ; 47(8): 637-645, 08/2014. tab, graf
Article in English | LILACS | ID: lil-716279

ABSTRACT

Tissue engineering encapsulated cells such as chondrocytes in the carrier matrix have been widely used to repair cartilage defects. However, chondrocyte phenotype is easily lost when chondrocytes are expanded in vitro by a process defined as “dedifferentiation”. To ensure successful therapy, an effective pro-chondrogenic agent is necessary to overcome the obstacle of limited cell numbers in the restoration process, and dedifferentiation is a prerequisite. Gallic acid (GA) has been used in the treatment of arthritis, but its biocompatibility is inferior to that of other compounds. In this study, we modified GA by incorporating sulfamonomethoxine sodium and synthesized a sulfonamido-based gallate, JJYMD-C, and evaluated its effect on chondrocyte metabolism. Our results showed that JJYMD-C could effectively increase the levels of the collagen II, Sox9, and aggrecan genes, promote chondrocyte growth, and enhance secretion and synthesis of cartilage extracellular matrix. On the other hand, expression of the collagen I gene was effectively down-regulated, demonstrating inhibition of chondrocyte dedifferentiation by JJYMD-C. Hypertrophy, as a characteristic of chondrocyte ossification, was undetectable in the JJYMD-C groups. We used JJYMD-C at doses of 0.125, 0.25, and 0.5 µg/mL, and the strongest response was observed with 0.25 µg/mL. This study provides a basis for further studies on a novel agent in the treatment of articular cartilage defects.


Subject(s)
Animals , Rabbits , Benzamides/chemical synthesis , Cell Dedifferentiation/drug effects , Cell Proliferation/drug effects , Chondrocytes/drug effects , Phenotype , Pyrimidines/chemical synthesis , Aggrecans/genetics , Aggrecans/metabolism , Anti-Infective Agents/chemistry , Anti-Infective Agents/pharmacology , Benzamides/pharmacology , Cell Survival , Cell Dedifferentiation/immunology , Chondrocytes/cytology , Chondrocytes/metabolism , Chondrogenesis/drug effects , Collagen Type I/genetics , Collagen Type I/metabolism , Collagen Type II/genetics , Collagen Type II/metabolism , Glycosaminoglycans/analysis , Immunohistochemistry , Laser Scanning Cytometry , Primary Cell Culture , Pyrimidines/pharmacology , Real-Time Polymerase Chain Reaction , SOX9 Transcription Factor/genetics , SOX9 Transcription Factor/metabolism , Tissue Engineering
10.
Article in English | WPRIM | ID: wpr-108336

ABSTRACT

BACKGROUND/AIMS: SKI306X, a mixed extract of three herbs, Clematis mandshurica (CM), Prunella vulgaris (PV), and Trichosanthes kirilowii (TK), is chondroprotective in animal models of osteoarthritis (OA). The objectives of this study were to investigate its effect on interleukin (IL)-1beta-induced degradation of glycosaminoglycan (GAG) and the basis of its action in human OA cartilage, as well as to screen for the presence of inhibitors of matrix metalloproteinase (MMP)-13 and a disintegrin and metalloprotease with thrombospondin motifs (ADAMTS)-4 in SKI306X and its component herbs, as well as in fractions from SKI306X. METHODS: Human OA chondrocytes and cartilage explants were obtained during total knee replacements and incubated with IL-1beta +/- oncostatin M with or without SKI306X or its component herb extracts. GAG degradation was assayed in cartilage explants using a commercial kit. Expression of genes involved in cartilage destruction was measured by real-time polymerase chain reaction using chondrocyte RNA. SKI306X was fractionated by preparative liquid chromatography to test for the presence of inhibitors of MMP-13 and ADAMTS-4. RESULTS: SKI306X and PV inhibited IL-1beta-induced GAG release from cartilage explants, and SKI306X, CM, PV, and TK inhibited IL-1beta-induced MMP gene expression. Unexpectedly, SKI306X greatly stimulated IL-1beta + oncostatin M-induced ADAMTS-4 gene expression, probably due to its TK component. Some fractions of SKI306X also inhibited ADAMTS-4 activity. CONCLUSIONS: SKI306X and its herbal components inhibit GAG degradation and catabolic gene expression in human OA chondrocytes and cartilage explants. SKI306X likely also contains one or more ADAMTS-4 inhibitor.


Subject(s)
ADAM Proteins/antagonists & inhibitors , Cartilage, Articular/drug effects , Cells, Cultured , Chondrocytes/drug effects , Down-Regulation/drug effects , Drugs, Chinese Herbal/pharmacology , Glycosaminoglycans/metabolism , Humans , Interleukin-1beta/metabolism , Matrix Metalloproteinase 13/metabolism , Matrix Metalloproteinase Inhibitors/pharmacology , Oncostatin M/metabolism , Osteoarthritis, Knee/drug therapy , Procollagen N-Endopeptidase/antagonists & inhibitors
11.
Article in English | WPRIM | ID: wpr-208229

ABSTRACT

Withaferin A (WFA) is known as a constituent of Ayurvedic medicinal plant, Withania somnifera, and has been used for thousands of years. Although WFA has been used for the treatment of osteoarthritis (OA) and has a wide range of biochemical and pharmacologic activities, there are no findings suggesting its properties on chondrocytes or cartilage. The aim of the present study is to investigate the effects of WFA on apoptosis with focus on generation of intracellular reactive oxygen species (ROS). Here we showed that WFA significantly increased the generation of intracellular ROS in a dose-dependent manner. We also determined that WFA markedly leads to apoptosis as evidenced by accumulation of p53 by Western blot analysis. N-Acetyl-L-Cystein (NAC), an antioxidant, prevented WFA-caused expression of p53 and inhibited apoptosis of chondrocytes. We also found that WFA causes the activation of PI3K/Akt and JNKinase. Inhibition of PI3K/Akt and JNKinase with LY294002 (LY)/triciribine (TB) or SP600125 (SP) in WFA-treated cells reduced accumulation of p53 and inhibited fragmented DNA. Our findings suggested that apoptosis caused by WFA-induced intracellular ROS generation is regulated through PI3K/Akt and JNKinase in rabbit articular chondrocytes.


Subject(s)
Animals , Anti-Inflammatory Agents/administration & dosage , Apoptosis/drug effects , Cartilage, Articular/cytology , Cells, Cultured , Chondrocytes/drug effects , Dose-Response Relationship, Drug , MAP Kinase Kinase 4/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Rabbits , Reactive Oxygen Species/metabolism , Withanolides/administration & dosage
12.
Cell Journal [Yakhteh]. 2013; 15 (1): 19-28
in English | IMEMR | ID: emr-143263

ABSTRACT

A study of the histological events under interleukin-1 alpha [IL-1alpha] induction of bovine nasal cartilage [BNC] could result in useful data to better understand the mechanisms involved in tissue breakdown in joint diseases. The aim of this study was to investigate the effects of IL-1alpha on chondrocyte phenotype and extracellular matrix [ECM] changes in BNC explants. In this experimental study, samples were divided into two groups. Group I [control group] BNC explants were cultured only in Dulbecco's modified Eagle's medium [DMEM]. In group II, BNC explants were treated with IL-1alpha [10 ng/ml] for 28 days. Then, samples were harvested on culture days 3, 7, 14, 21 and 28 and chondrocyte morphology and ECM alterations were assessed by invert microscopy and histology by hematoxylin and eosin [H and E] and Alcian blue. Cell viability was evaluated by the lactate dehydrogenase [LDH] assay test. Data were analyzed by the t test and p<0.05 was considered significant. IL-1alpha induced significant morphological changes in cartilage. In the presence of IL-1alpha, most chondrocytes transformed into a fibroblast-like morphology with a granular black point appearance. An increase in the cell: matrix ratio was observed and there were decreased numbers of chondrocytes.IL-1alpha induced breakdown of ECM. We observed partial degradation of ECM between days 7-14 and complete degradation occurred between days 21-28 of culture. The LDH levels increased. IL-1alpha induced morphological changes in chondrocytes and increased destruction of cartilage ECM. There was a parallel correlation between proteoglycan degradation and changes in chondrocyte morpholgy


Subject(s)
Animals , Chondrocytes/drug effects , Nasal Cartilages/drug effects , Extracellular Matrix , L-Lactate Dehydrogenase
13.
Clinics ; 67(1): 35-40, 2012. ilus
Article in English | LILACS | ID: lil-610621

ABSTRACT

OBJECTIVE: Hypoxia-inducible factor 1 alpha regulates genes related to cellular survival under hypoxia. This factor is present in osteroarthritic chondrocytes, and cytokines, such as interleukin-1 beta, participate in the pathogenesis of osteoarthritis, thereby increasing the activities of proteolytic enzymes, such as matrix metalloproteinases, and accelerating cartilage destruction. We hypothesize that Hypoxia Inducible Factor-1 alpha (HIF-1α) can regulate cytokines (catabolic action) and/or growth factors (anabolic action) in osteoarthritis. The purpose of this study was to investigate the modulation of HIF-1α in human osteoarthritic chondrocytes by interleukin-1 beta (IL-1β) and insulin-like growth factors I (IGF-I) and II (IGF-II) and to determine the involvement of the phosphatidylinositol-3kinase (PI-3K) pathway in this process. METHODS: Human osteroarthritic chondrocytes were stimulated with IL-1β, IGF-I and IGF-II and LY294002, a specific inhibitor of PI-3K. Nuclear protein levels and gene expression were analyzed by western blot and quantitative reverse transcription-polymerase chain reaction analyses, respectively. RESULTS: HIF-1α expression was upregulated by IL-1β at the protein level but not at the gene level. IGF-I treatment resulted in increases in both the protein and mRNA levels of HIF-1α , whereas IGF-II had no effect on its expression. However, all of these stimuli exploited the PI-3K pathway. CONCLUSION: IL-1β upregulated the levels of HIF-1α protein post-transcriptionally, whereas IGF-I increased HIF-1α at the transcript level. In contrast, IGF-II did not affect the protein or gene expression levels of HIF-1α . Furthermore, all of the tested stimuli exploited the PI-3K pathway to some degree. Based on these findings, we are able to suggest that Hypoxia inducible Factor-1 exhibits protective activity in chondrocytes during osteoarthritis.


Subject(s)
Humans , Chondrocytes/drug effects , Gene Expression Regulation/drug effects , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Insulin-Like Growth Factor I/pharmacology , Insulin-Like Growth Factor II/pharmacology , Interleukin-1beta/pharmacology , Osteoarthritis/metabolism , Chondrocytes/metabolism , Enzyme Inhibitors/pharmacology , Gene Expression Regulation/genetics , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Osteoarthritis/genetics , /antagonists & inhibitors , /metabolism , RNA, Messenger/analysis , Statistics, Nonparametric , Signal Transduction/drug effects , Signal Transduction/genetics
14.
Article in English | WPRIM | ID: wpr-193635

ABSTRACT

The study investigated the effects of adenovirus-mediated gene transfection of basic fibroblast growth factor (bFGF), bFGF combined with interleukin-1 receptor antagonist protein (IL-Ra) and/or insulin-like growth factor-1 (IGF-1) both in human osteoarthritis (OA) chondrocytes and rabbits OA model. Human OA chondrocytes were delivered by adenovirus-mediated bFGF, IL-Ra and IGF-1 vectors, respectively. Chondrocyte proliferation, glycosaminoglycan (GAG) content, expression of type II collagen, ADAMTS-5, MMP-13, MMP-3 and TIMP-1 were determined. Rabbit OA model was induced by anterior cruciate ligament transaction (ACLT) in knees. Adenoviral vectors encoding human bFGF, IL-Ra and IGF-1 were injected intraarticularly into the knee joints after ACLT. The effects of adenovirus- mediated gene transfection on rabbit OA were evaluated. In vitro, the transfected genes were expressed in cell supernatant of human OA chondrocytes. AdbFGF group significantly promoted chondrocyte proliferation, and increased GAG and type II collagen synthesis than in the OA group. As two or three genes were transfected in different combinations, there was significant enhancement on the GAG content, type II collagen synthesis, and TIMP-1 levels, while ADAMTS-5, MMP-13, and MMP-3 levels were reduced. In vivo, the transfected genes were expressed in synovial fluid of rabbits. Intraarticular delivery of bFGF enhanced the expression of type II collagen in cartilage and decreased cartilage Mankin score compared with the OA control group (P = 0.047; P < 0.01, respectively). Multiple-gene transfection in different combinations showed better results than bFGF transfection alone. This study suggests that bFGF gene transfection is effective in treating experimental OA. Multiple gene transfection has better biologic effects on OA.


Subject(s)
Adenoviridae/genetics , Animals , Chondrocytes/drug effects , Collagen Type II/genetics , Fibroblast Growth Factor 2/genetics , Genetic Therapy/methods , Genetic Vectors/administration & dosage , Humans , Insulin-Like Growth Factor I/genetics , Interleukin 1 Receptor Antagonist Protein/genetics , Interleukin-1/genetics , Matrix Metalloproteinase 13/genetics , Matrix Metalloproteinase 3/genetics , Osteoarthritis/therapy , Rabbits , Tissue Inhibitor of Metalloproteinase-1/genetics , Transfection
15.
Article in English | WPRIM | ID: wpr-122638

ABSTRACT

Endoplasmic reticulum (ER) stress regulates a wide range of cellular responses including apoptosis, proliferation, inflammation, and differentiation in mammalian cells. In this study, we observed the role of 2-deoxy-D-glucose (2DG) on inflammation of chondrocytes. 2DG is well known as an inducer of ER stress, via inhibition of glycolysis and glycosylation. Treatment of 2DG in chondrocytes considerably induced ER stress in a dose- and time-dependent manner, which was demonstrated by a reduction of glucose regulated protein of 94 kDa (grp94), an ER stress-inducible protein, as determined by a Western blot analysis. In addition, induction of ER stress by 2DG led to the expression of COX-2 protein with an apparent molecular mass of 66-70kDa as compared with the normally expressed 72-74 kDa protein. The suppression of ER stress with salubrinal (Salub), a selective inhibitor of eif2-alpha dephosphorylation, successfully prevented grp94 induction and efficiently recovered 2DG-modified COX-2 molecular mass and COX-2 activity might be associated with COX-2 N-glycosylation. Also, treatment of 2DG increased phosphorylation of Src in chondrocytes. The inhibition of the Src signaling pathway with PP2 (Src tyrosine kinase inhibitor) suppressed grp94 expression and restored COX-2 expression, N-glycosylation, and PGE2 production, as determined by a Western blot analysis and PGE2 assay. Taken together, our results indicate that the ER stress induced by 2DG results in a decrease of the transcription level, the molecular mass, and the activity of COX-2 in rabbit articular chondrocytes via a Src kinase-dependent pathway.


Subject(s)
Animals , Cartilage, Articular/pathology , Cells, Cultured , Chondrocytes/drug effects , Cyclooxygenase 2/genetics , Deoxyglucose/pharmacology , Down-Regulation , Endoplasmic Reticulum/drug effects , Glycosylation/drug effects , Inflammation , Rabbits , Signal Transduction/drug effects , Stress, Physiological/drug effects , src-Family Kinases/metabolism
16.
JAMC-Journal of Ayub Medical College-Abbotabad-Pakistan. 2008; 20 (4): 77-81
in English | IMEMR | ID: emr-101899

ABSTRACT

To assess the preventive role of zinc chloride on toxicity of ciprofloxacin administration in wistar albino rat litter. It was a Prospective experimental study. The study was carried out in the department of Anatomy, Basic Medical Sciences Institute, Jinnah Postgraduate Medical Centre, Karachi, Pakistan during March 2002 to February 2003 one year study. Ciprofloxacin and zinc chloride were administered to newly born albino rat litters separately and simultaneously at a dose of 20 mg/kg body weight and 1200 micro g/Kg body weight respectively, intraperitonealy twice daily from 1-14 day after birth. The animals were sacrificed by deep ether anaesthesia. The fore and hind limbs were dis-articulated from the axial skeleton, soft tissue was removed and bones were fixed in 10% buffered formalin. Decalcification was done in 10% nitric acid and 10% formic acid changes. After paraplast embedding, 4 micro m thick longitudinal sections of proximal and distal ends of long bones were cut by a rotary microtome. Routine staining with haemotoxylin and eosin was performed. Histomorphometery was done to measure the thickness of epiphyseal cartilage and was compared with similar values of the control animals. The results were statistically analyzed to evaluate the significance. Our study revealed that ciprofloxacin administration in new born albino rat litter decreased the width of epiphyseal growth plate cartilage by 13.7 +/- 0.42 micro m, 10.43% in humerus and 6.6 +/- 1.2 micro m 4.72% in femur as compared to control, whereas, simultaneous zinc chloride administration restricted the decrease to 1.27 micro m +/- SD in humerus and 2.05 micro m +/- SD in femur. Simultanous zinc chloride administration minimized the epiphseal cartilage damage induced by ciprofloxacin in Wistar albino rat litter


Subject(s)
Male , Female , Animals, Laboratory , Chlorides , Zinc Compounds , Cartilage/drug effects , Growth Plate/drug effects , Chondrocytes/drug effects , Cartilage/growth & development , Rats, Wistar , Prospective Studies , Litter Size , Animals, Newborn
17.
Braz. j. med. biol. res ; 40(8): 1101-1109, Aug. 2007. tab, ilus
Article in English | LILACS | ID: lil-456807

ABSTRACT

Children with chronic renal failure in general present growth retardation that is aggravated by corticosteroids. We describe here the effects of methylprednisolone (MP) and recombinant human growth hormone (rhGH) on the growth plate (GP) of uremic rats. Uremia was induced by subtotal nephrectomy in 30-day-old rats, followed by 20 IU kg-1 day-1 rhGH (N = 7) or 3 mg kg-1 day-1 MP (N = 7) or 20 IU kg-1 day-1 rhGH + 3 mg kg-1 day-1 MP (N = 7) treatment for 10 days. Control rats with intact renal function were sham-operated and treated with 3 mg kg-1 day-1 MP (N = 7) or vehicle (N = 7). Uremic rats (N = 7) were used as untreated control animals. Structural alterations in the GP and the expression of anti-proliferating cell nuclear antigen (PCNA) and anti-insulin-like growth factor I (IGF-I) by epiphyseal chondrocytes were evaluated. Uremic MP rats displayed a reduction in the proliferative zone height (59.08 ± 4.54 vs 68.07 ± 7.5 æm, P < 0.05) and modifications in the microarchitecture of the GP. MP and uremia had an additive inhibitory effect on the proliferative activity of GP chondrocytes, lowering the expression of PCNA (19.48 ± 11.13 vs 68.64 ± 7.9 percent in control, P < 0.0005) and IGF-I (58.53 ± 0.96 vs 84.78 ± 2.93 percent in control, P < 0.0001), that was counteracted by rhGH. These findings suggest that in uremic rats rhGH therapy improves longitudinal growth by increasing IGF-I synthesis in the GP and by stimulating chondrocyte proliferation.


Subject(s)
Animals , Female , Humans , Rats , Glucocorticoids/pharmacology , Growth Plate/drug effects , Human Growth Hormone/pharmacology , Methylprednisolone/pharmacology , Uremia/metabolism , Autoantibodies/metabolism , Cell Proliferation , Chondrocytes/drug effects , Growth Plate/metabolism , Growth Plate/pathology , Insulin-Like Growth Factor I/metabolism , Proliferating Cell Nuclear Antigen/metabolism , Rats, Wistar , Tibia/drug effects , Tibia/pathology , Uremia/pathology
18.
Rev. Fac. Cienc. Méd. (Córdoba) ; 64(2): 9-15, 2007. ilus
Article in Spanish | LILACS | ID: lil-511492

ABSTRACT

La osteoartritis (OA) es una enfermedad articular crónica, progresiva que se instala como consecuencia de un proceso complejo que involcra alteraciones mecánicas y biológicas del sistema músculo-esquelético, siendo resultante de múltiples interacciones entre factores genéticos e injurias extrínsecas. La patogenia de esta enfermedad se ralaciona con alta y desviada producción de citokinas flogógenas y de enzimas proteolíticas, que degradan y destruyen la matriz extracelular en tejidos articulares y peri-articulares. Se estudiaron 20 casos con OA, de los cuales se obtuvo cartílago durante intervenciones quirúrgicas programadas. El cartílago se cultivó en medio Dulbecco-Eagle, con o sin agregado de AINEs o condromodulares. En los sobrenadantes se determinaron óxido nítrico por reacción de Giess y la medición espectrofotométrica; y colagenasa por ELISA doble sándwich en presencia de anticuerpos monoclonales. En ausencia de AINEs, los cultivos de condrocitos produjeron 1950 ± 665ng/ml de MMP-1, La adición de Diclofenac redujo esa cifra a 1140 ± 155 ng/ml, aunque esta diferencia no fue estadisticamente significativa, (p<0.60). Por el contrario, Celecoxib redujo el nivel de la enzima a 760 ± 75ng/ml (p<0,01) y la Glucosamina también provocó un descenso (950 ± 89 ng/ml) significativo (p<0.05). Los niveles de ON en ausencia de AINEs llegaron a 47,3 ± 4,9 µM. Su producción no varió significativamente con la adición de Diclofenac, Ceecoxib o Glucosamina (p=ns). Los resultados indicarían la incapacidad de Diclofenac para modificar la generación de enzimas proteolíticas, mientras que Celecoxib y Glucosamina disminuyen su producción significativamente. Ninguno de los fármacos utilizados en nuestro trabajo ha logrado alterar la concentración de ON. Muchos integrantes quedan aún sin resolver y todavía se carece de fármacos de eficacia comprobada para alterar el curso natural de la enfermedad.


Osteoarthritis is a chronic and progressive joint disease. It is established by a complex process involving mechanical and biological alterations of the musculoskeletal system, which are generated by a great variety of interactions between genetic factors and extrinsic injuries. The pathogenesis of this disease is related to an increased and divergent production of inflammatory markers and proteolytic enzymes that promote the degradation and destruction of the extracellular matrix of articular and periarticular tissues. Cartilage samples were taken from 20 osteoarthritic patients during programmed surgical interventions. The cartilage samples were cultured in Dulbecco-Eagle medium, with or without the addition of NSAIDs or modulators of chondrocyte metabolism. The content of nitric oxide in the supernatant was quantified using the Griess reaction; the concentration of MMP-1 was quantified via double-sandwich ELISA. Untreated chondrocyte cultures produced 1950 +/- 665 ng/ml MMP-1. With the addition of Diclofenac this value decreased to 1140 +/- 155 ng/ml, although this difference was not statistically significant (p < 0.06). However, in the presence of Celecoxib the level significantly dropped to 760 +/- 75 ng/ml (p < 0.01). Although the addition ofglucosamine did not produce such a noticeable reduction in the level of MMP-1 (950 +/- 89 ng/ml), it was statistically significant (p < 0.05). On the contrary, none of the drugs (Diclofenac, Celecoxib, Glucosamine) modified the level of nitric oxide which had a mean value of 47.3 +/- 4.9 microM in the control samples. This investigation evidenced the inability of Diclofenac to significantly modify the production of proteolytic enzymes in osteoarthritic chondrocyte cultures. However, both Celecoxib and Glucosamine significantly reduced the production of MMP-1. On the contrary, none of the drugs used in this study managed to modify the concentration of nitric oxide. To the present day, no drugs have been found to be...


Subject(s)
Humans , Aged , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Cartilage, Articular/drug effects , Chondrocytes/drug effects , Cyclooxygenase Inhibitors/pharmacology , Matrix Metalloproteinases/metabolism , Osteoarthritis/drug therapy , Analysis of Variance , Biomarkers/metabolism , Chondrocytes/metabolism , Diclofenac/pharmacology , Enzyme-Linked Immunosorbent Assay , Glucosamine/pharmacology , Matrix Metalloproteinases/drug effects , Nitric Oxide/metabolism , Osteoarthritis/enzymology , Pyrazoles/pharmacology , Sulfonamides/pharmacology
19.
JAMC-Journal of Ayub Medical College-Abbotabad-Pakistan. 2006; 18 (3): 50-54
in English | IMEMR | ID: emr-77349

ABSTRACT

Administration of quinolone therapy is controversial during growing age as stated by earlier worker. The flroquinolones are currently not indicated for young children, because of arthropathy and adverse effect on growing cartilage shown by studies. However the effects of ciprofloxacin on epiphyseal growth plate has remained undocumented. This study is therefore, undertaken to determine the risk of ciprofloxacin administration an growing cartilage by prospective experimental animal study model using Wistar albino rat pups. Ciprofloxacin was administered to newly born Wistar albino rat pups with a doze of 20mg/kg body weight intraperitonealy twice a day from day-1 to day-14 after birth. The animals were sacrificed by deep ether anesthesia. The limbs were disarticulated from axial skeleton, soft tissue was removed. The intact bone mean length in millimeter of right and left humerus and femur was measured with the help of electronic vernier caliper and bones were fixed in 10% buffered farmalin. Decalcification was done in 10% nitric acid and 10% formic acid changes. After paraplast embeding, 4 mm thick longitudinal sections of the proximal long bones were cut by a rotary microtome. Routine staining with haemotoxylin and eosin was performed. Histomorphometry was done measuring the thickness of epiphyseal cartilage and was compared with similar value of control animals. The results were statistically analysed to find out the significance. The ciprofloxacin induces a mordanting effect as abviated by increased basophilia. Our study reveales that cirprofloxacin administration in the newly born pups decreased the width of epiphyseal growth plate cartilage by 10.43% in humerus and 4.72% in femur as compared to the growth of control cartilage. The decrease in the width was brought about mainly by the reduced count of the proliferative cells in the proliferative zone and the diminuation in the average size of the hypertrophic condryocytes in the hypertrophic zone. The reserve zone has become markedly reduced in thickness. The ciprofloxacin post-natal administration effected growth plate retardation by inhibiting the mitosis in the proliferative zone and also effected the mean length of humora and femora leading to reduction in limb length of rat pups


Subject(s)
Animals , Growth Plate/drug effects , Growth Plate/growth & development , Growth Plate/pathology , Cartilage/drug effects , Cartilage/growth & development , Osteogenesis/drug effects , Chondrocytes/drug effects , Rats
20.
Article in English | WPRIM | ID: wpr-53150

ABSTRACT

Although the apoptosis of chondrocytes plays an important role in endochondral ossification, its mechanism has not been elucidated. In this study, we show that guanosine induces chondrocyte apoptosis based on the results of acridine orange/ ethidium bromide staining, caspase-3 activation, and sub-G1 fraction analysis. The potent inhibitory effect of dipyridamole, a nucleoside transporter blocker, indicates that extracellular guanosine must enter the chondrocytes to induce apoptosis. We found that guanosine promotes Fas-Fas ligand interaction which, in turn, leads to chondrocyte apoptosis. These findings indicate a novel mechanism for endochondral ossification via metabolic regulation.


Subject(s)
Tumor Necrosis Factors/metabolism , Signal Transduction/drug effects , Receptors, Tumor Necrosis Factor/metabolism , Rats, Sprague-Dawley , Rats , Nucleoside Transport Proteins/metabolism , Membrane Glycoproteins/metabolism , Guanosine/pharmacology , Fas Ligand Protein , Chondrocytes/drug effects , Apoptosis/drug effects , fas Receptor , Animals
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