Bixa orellana ethyl acetate fraction and its isolated compound ellagic acid attenuate the progression of MIA-induced osteoarthritis in rat knees.

Osteoarthritis (OA) is a pathology that is characterized by progressive erosion of articular cartilage. In this context, medicinal plants have become relevant tools regarding their potential role in the prevention and treatment of OA, being safe and effective. The aim of this work was investigate the therapeutic efficacy of the ethyl acetate fraction of Bixa orellana leaves (BoEA) and ellagic acid (ElAc) for the therapeutic treatment of OA induced by monosodium iodoacetate (MIA) in rats. The plant material was extracted via maceration with 70 % hydroalcoholic solvent (BoHE). The ethyl acetate (BoEA) fraction was by solvents in increasing order of polarity. The ElAc was identified and isolated in BoEA using high performance liquid chromatography (HPLC-DAD) and analytical curve. The OA was induced using MIA in the right knee at the knee joint. Doses of BoEA and ElAc were administered daily (every 24 h, orally) at concentrations of 50, 100 and 50 mg/kg, respectively, for 28 days after induced OA. We evaluated the animals through clinical and radiological examinations every 7 days and, on the 29th day, the animals were euthanized, the joints being removed for histopathological analysis and the serum for cytokine analysis. BoEA and ElAc compounds reduced inflammation and nociception in OA and were as effective as indomethacin in clinical parameters of joint discomfort and allodynia in rats, in addition to showing improvements in radiological and histopathological images, acting on the progress of cartilage deterioration, proving properties related to anti-inflammatory and analgesic processes, being important allies for new therapeutic interventions for the treatment of OA.

Response of Bovine Cumulus-Oocytes Complexes to Energy Pathway Inhibition during In Vitro Maturation.

Glucose or fatty acids (FAs) metabolisms may alter the ovarian follicle environment and thus determine oocyte and the nascent embryo quality. The aim of the experiment was to investigate the effect of selective inhibition of glucose (iodoacetate + DHEA) or FA (etomoxir) metabolism on in vitro maturation (IVM) of bovine COCs (cumulus-oocyte complexes) to investigate oocyte's development, quality, and energy metabolism. After in vitro fertilization, embryos were cultured to the blastocyst stage. Lipid droplets, metabolome, and lipidome were analyzed in oocytes and cumulus cells. mRNA expression of the selected genes was measured in the cumulus cells. ATP and glutathione relative levels were measured in oocytes. Changes in FA content in the maturation medium were evaluated by mass spectrometry. Our results indicate that only glucose metabolism is substantial to the oocyte during IVM since only glucose inhibition decreased embryo culture efficiency. The most noteworthy differences in the reaction to the applied inhibition systems were observed in cumulus cells. The upregulation of ketone body metabolism in the cumulus cells of the glucose inhibition group suggest possibly failed attempts of cells to switch into lipid consumption. On the contrary, etomoxir treatment of the oocytes did not affect embryo development, probably due to undisturbed metabolism in cumulus cells. Therefore, we suggest that the energy pathways analyzed in this experiment are not interchangeable alternatives in bovine COCs.

Quantitation of Gait and Stance Alterations Due to Monosodium Iodoacetate-induced Knee Osteoarthritis in Yucatan Swine.

Knee osteoarthritis is one of the most common causes of chronic pain worldwide, and several animal models have been developed to investigate disease mechanisms and treatments to combat associated morbidities. Here we describe a novel method for assessment of locomotor pain behavior in Yucatan swine. We used monosodium iodoacetate (MIA) to induce osteoarthritis in the hindlimb knee, and then conducted live observation, quantitative gait analysis, and quantitative weight-bearing stance analysis. We used these methods to test the hypothesis that locomotor pain behaviors after osteoarthritis induction would be detected by multiparameter quantitation for at least 12 wk in a novel large animal model of osteoarthritis. MIA-induced knee osteoarthritis produced lameness quantifiable by all measurement techniques, with onset at 2 to 4 wk and persistence until the conclusion of the study at 12 wk. Both live observation and gait analysis of kinetic parameters identified mild and moderate osteoarthritis phenotypes corresponding to a binary dose relationship. Quantitative stance analysis demonstrated the greatest sensitivity, discriminating between mild osteoarthritis states induced by 1.2 and 4.0 mg MIA, with stability of expression for as long as 12 wk. The multiparameter quantitation used in our study allowed rejection of the null hypothesis. This large animal model of quantitative locomotor pain resulting from MIA-induced osteoarthritis may support the assessment of new analgesic strategies for human knee osteoarthritis.

Does cartilage ERα overexpression correlate with osteoarthritic chondrosenescence? Indications from Labisia pumila OA mitigation.

Chondrosenescence (chondrocyte senescence) and subchondral bone deterioration in osteoarthritic rats were analyzed after treatment with the estrogenic herb Labisia pumila (LP) or diclofenac. Osteoarthritis (OA) was induced in bilaterally ovariectomized (OVX) rats by injecting mono-iodoacetate into the right knee joints. Rats were grouped (n = 8) into nontreated OVX+OA control, OVX+OA + diclofenac (5 mg/kg) (positive control), OVX+OA + LP leaf extract (150 and 300 mg/kg) and healthy sham control. After 8 weeks' treatment, their conditions were evaluated via serum biomarkers, knee joint histology, bone histomorphometry, protein and mRNA expressions. The LP significantly reduced cartilage erosion, femur bone surface alteration, bone loss and porosity and increased trabecular bone thickness better than diclofenac and the non-treated OA. The cartilage catabolic markers' (matrix metalloproteinase (MMP)-13, RUNX2, COL10a, ERa, CASP3 and HIF-2 alpha) mRNA expressions were down-regulated and serum bone formation marker, PINP, was increased by LP in a dose-dependent manner. The LP (containing myricetin and gallic acid) showed protection against chondrosenescence, chondrocyte death, hypoxia-induced cartilage catabolism and subchondral bone deterioration. The bone and cartilage protective effects were by suppressing proteases (collagen break-down), bone resorption and upregulating subchondral bone restoration. The cartilage ER alpha over-expression showed a strong positive correlation with MMP-13, COL10 alpha1, histological, micro-computed tomography evidence for cartilage degradation and chondrosenescence.

Isorhamnetin ameliorates inflammatory responses and articular cartilage damage in the rats of monosodium iodoacetate-induced osteoarthritis.

Context: Osteoarthritis (OA) is a degenerative joint disease with damage to the articular cartilage. Active production of inflammatory cytokine/chemokine and matrix metalloproteinases may be found during the progression of OA. Isorhamnetin had the effects of anti-inflammatory, antioxidant, anti-ischemia, anti-atherosclerotic hepatoprotective and anticancer activities. Objective: Our study was focused on the effects of isorhamnetin treatment in OA. Materials and methods: We used monosodium iodoacetate (MIA)-induced OA rats to evaluate the effects of isorhamnetin related anti-inflammatory process. The rats in all groups were sacrificed on four weeks post-MIA injection. The measurements of knee joint swelling, histological analysis, serum inflammatory biomarkers and western blot were evaluated. Results: We found that isorhamnetin may reduce MIA-induced knee swelling by significantly reduction of articular cartilage damage.in rats. Suppression of pro-inflammatory cytokines production was found after isohamnetin treatment. Isorhamnetin inhibited the production of NO and PGE2, and the expression of iNOS and COX-2. The production of COMP, CTX-II and osteopontin (OPN) were also inhibited in MIA-induced OA rats. Discussion and conclusions: Isorhamnetin may modulate the inflammatory progression of OA in MIA-induced OA rats. The prevention of cartilage damage was found in OA after adequate isorhamnetin treatment. Isorhamnetin may serve as a potential agent for the management of OA.

Joint unloading inhibits articular cartilage degeneration in knee joints of a monosodium iodoacetate-induced rat model of osteoarthritis.

OBJECTIVE: The aim of the study was to examine how mechanical unloading affects articular cartilage degeneration in the patellofemoral (PF) and tibiofemoral (TF) joints of a monosodium iodoacetate (MIA)-induced rat model of osteoarthritis (OA). DESIGN: The study involved 60 male rats. OA was induced by intra-articular injecting MIA into both knee joints. All animals were equally divided into two groups: sedentary (SE) and hindlimb unloading (HU) groups. Histopathological changes in the articular cartilage of the PF and TF joints were evaluated using the Osteoarthritis Research Society International (OARSI) score and modified Mankin score at 2 and 4 weeks after MIA injection. RESULTS: In the SE and HU groups, representative histopathological changes in OA were detected in the PF and TF joints. The OARSI and modified Mankin scores for the PF and TF joints tended to increase over time after the injection of 0.2 mg or 1.0 mg of MIA in the SE and HU groups. Both the scores for the HU group were significantly lower than those for the SE group [OARSI score: P < 0.0001 (1.0-mg injection at 4 weeks); modified Mankin score: P = 0.0116 (0.2-mg injection at 4 weeks); P = 0.0004 and < 0.0001 (1.0-mg injection at 2 and 4 weeks, respectively)]. CONCLUSION: This study revealed new histological evidence that indicates that unloading condition suppresses articular cartilage degeneration and is beneficial in many areas of basal and clinical research involving OA.

Protective effects of dioscin against cartilage destruction in a monosodium iodoacetate (MIA)-indcued osteoarthritis rat model.

BACKGROUND: Osteoarthritis (OA) is a disabling disease of joint with no clear treatment. The finding of medicine be of benefit to joint is an important topic for osteoarthritis prevention and treatment. The present study was designed to explore the therapeutic effects and possible underlying mechanism of dioscin, a natural steroidal saponin, on osteoarthritis. METHODS: OA models were created via intra-joint injection of monosodium iodoacetate (MIA) in rats. After the administration of dioscin, the effects of dioscin were estimated with western blotting, qRT-PCR and histologic staining. RESULTS: The results showed that dioscin exerted cartilage and extracelluar matrix (ECM) protective effects via suppressing ER-stress, oxidative stress, apoptosis and inflammation. More significantly, it also ameliorated the progress of OA via inhibiting Wnt/ß-catenin pathway and up-regulating PPAR-γ expression. CONCLUSION: Our work showed the good protective effects of dioscin on MIA-induced OA for the first time. Dioscin is a promising drug on OA treatment although further researches are needed in the future.

Differential killing and radio-modifying effects of iodoacetate in mammalian normal and cancer cells.

To explore possible applications of iodoacetate (IA), a glycolytic inhibitor, in cancer treatment, we screened its cytotoxicity and radioprotective/sensitizing efficacy in three different mammalian cell lines; A549 (human lung carcinoma), MCF7 (human mammary cancer), a non-cancerous CHO (Chinese hamster ovary) cells and human lymphocytes. Experiments were carried out using IA concentrations ranging from 0.01 to 2.5 µg/ml, with or without 60Coγ-radiation. In the outcomes, IA was found to exhibit higher toxicity in the cancer cells, whereas it was non-toxic/marginally toxic to the non-cancerous cells. Considerably higher glucose uptake in both cancer cells lines was observed indicating higher rates of glycolysis. IA significantly inhibited glycolysis as reflected by GAPDH activity inhibition. Radiomodifying effects of IA were found to be concentration dependent in both cancerous and non-cancerous cells. The response in non-cancerous was found to be biphasic: at lower concentrations, it offered significant radioprotection; however, the protection decreased with increasing concentration. Moreover, at the highest tested concentration, marginal radiosensitization was also observed (as indicated by clonogenic assay). In both cancer cells, IA offered significant amount of radiosensitization which was considerably high at higher concentrations. Further experiments were carried out to estimate the Dose Modification Factor (DMF) to quantify and compare relative radiosensitization by IA in cancer and normal cell lines. The DMF was calculated for three different concentrations of IA, 0.5, 1, and 1.5 µg/ml, and corresponding values were found to be 1.26, 1.43, and 1.89 for A549 cancer cells, whereas for normal CHO cells, it was 1.13, 1.13, and 1.24. In conclusion, differential killing and radiosensitizing effects of IA suggest that it may have potential use as a anticancer agent and radiosensitizer in cancer therapy.

ADX71943 and ADX71441, novel positive allosteric modulators of the GABAB receptor with distinct central/peripheral profiles, show efficacy in the monosodium iodoacetate model of chronic osteoarthritis pain in the rat.

We tested novel positive allosteric modulators (PAMs) of the γ-aminobutyric acid receptor B (GABAB), ADX71943 and ADX71441in the monosodium iodoacetate model of chronic osteoarthritis pain in rats with the objective to delineate the role of peripheral versus central GABAB receptor populations in modulation of chronic pain. Anesthetized Sprague-Dawley rats received an injection of monosodium iodoacetate into the knee and were tested for hyperalgesia starting post-MIA day 14. Effects of compounds on ipsilateral joint compression threshold were evaluated on post-MIA day 14 (after acute treatment), as well as after repeated, daily treatment on days 21 and 28 (ADX71943 only) and were compared to those of celecoxib (30mg/kg, p.o.). The PAMs were also tested in the rat rotarod test for potential muscle-relaxant effects. Acutely, ADX71943 (1-30mg/kg, p.o.), the peripherally restricted PAM, resulted in similar increases in pain threshold across the doses on day 14, while showing reduced efficacy on day 21 and no efficacy on day 28. A clear reduction in the efficacy of celecoxib across testing was also noted in this experiment. Acutely ADX71441 (0.3-15mg/kg, p.o.), the central-peripheral PAM, resulted in over 2-fold increases in pain threshold at 15mg/kg (but not at lower doses) on day 14, while causing more modest effects on day 21. Celecoxib increased pain threshold after both acute and daily treatment, showing overall similar efficacy. Thus, early, presumably more inflammatory phase of osteoarthritis pain in more sensitive to GABAB PAMs with peripherally restricted profile, while later, presumably more neuropathic phase is more sensitive to PAMs with central-peripheral profile.

2-Deoxy-d-glucose enhances tonic inhibition through the neurosteroid-mediated activation of extrasynaptic GABAA receptors.

OBJECTIVE: The inhibition of glycolysis exerts potent antiseizure effects, as demonstrated by the efficacy of ketogenic and low-glucose/nonketogenic diets in the treatment of drug-resistant epilepsy. ATP-sensitive potassium (KATP ) channels have been initially identified as the main determinant of the reduction of neuronal hyperexcitability. However, a plethora of other mechanisms have been proposed. Herein, we report the ability of 2-deoxy-d-glucose (2-DG), a glucose analog that inhibits glycolytic enzymes, of potentiating γ-aminobutyric acid (GABA)ergic tonic inhibition via neurosteroid-mediated activation of extrasynaptic GABAA receptors. METHODS: Acute effects of 2-DG on the ATP-sensitive potassium currents, GABAergic tonic inhibition, firing activity, and interictal events were assessed in hippocampal slices by whole-cell patch-clamp and local field potential recordings of dentate gyrus granule cells. RESULTS: Acute application of 2-DG activates two distinct outward conductances: a KATP channel-mediated current and a bicuculline-sensitive tonic current. The effect of 2-DG on such GABAergic tonic currents was fully prevented by either finasteride or PK11195, which are specific inhibitors of the neurosteroidogenesis pathway acting via different mechanisms. Moreover, the oxidized form of vitamin C, dehydroascorbic acid, known for its ability to induce neurosteroidogenesis, also activated a bicuculline-sensitive tonic current in a manner indistinguishable from that of 2-DG. Finally, we found that the enhancement of KATP current by 2-DG primarily regulates intrinsic firing rate of granule cells, whereas the increase of the GABAergic tonic current plays a key role in reducing the frequency of interictal events evoked by treatment of hippocampal slices with the convulsive agent 4-aminopyridine. SIGNIFICANCE: We demonstrated, for the first time, that 2-DG potentiates the extrasynaptic tonic GABAergic current through activation of neurosteroidogenesis. Such tonic inhibition represents the main conductance responsible for the antiseizure action of this glycolytic inhibitor.

Neural correlates of hyperalgesia in the monosodium iodoacetate model of osteoarthritis pain.

BACKGROUND: The mechanisms driving osteoarthritic pain remain poorly understood, but there is increasing evidence for a role of the central nervous system in the chronification of pain. We used functional magnetic resonance imaging to investigate the influence of a model of unilateral knee osteoarthritis on nociceptive processing. RESULTS: Four to five weeks post intra-articular injection of monosodium iodoacetate (MIA, 1 mg) into the left knee, Sprague Dawley rats were anesthetized for functional magnetic resonance imaging studies to characterize the neural response to a noxious stimulus (intra-articular capsaicin injection). In a two-arm cross-over design, 5 µM/50 µl capsaicin was injected into either the left knee (n = 8, CAPS-MIA) or right control knee (n = 8, CAPS-CON), preceded by contralateral vehicle (SAL) injection. To assess neural correlates of mechanical hyperalgesia, hindpaws were stimulated with von Frey hairs (8 g: MIA; 15 g: control knee, based on behavioral withdrawal responses). The CAPS-MIA group exhibited significant activation of the periaqueductal gray, unilateral thalamus and bilateral mensencephalon, superior-colliculus, and hippocampus, with no significant activation in the other groups/conditions. Capsaicin injection increased functional connectivity in the mid-brain network and mediodorsal thalamic nucleus, hippocampus, and globus pallidus, which was significantly stronger in CAPS-MIA compared to CAPS-CON groups. Mechanical stimulation of the hyperalgesic (ipsilateral to MIA knee) and normalgesic (contralateral) hindpaws evoked qualitatively different brain activation with more widespread brainstem and anterior cingulate (ACC) activation when stimulating the hyperalgesic paw, and clearer frontal sensory activation from the normalgesic paw. CONCLUSIONS: We provide evidence for modulation of nociceptive processing in a chronic knee osteoarthritis pain model with stronger brain activation and alteration of brain networks induced by the pro-nociceptive stimulus. We also report a shift to a medial pain activation pattern following stimulation of the hyperalgesic hindpaw. Taken together, our data support altered neural pain processing as a result of peripheral and central pain sensitization in this model.

Listeria monocytogenes batch culture growth response to metabolic inhibitors.

In certain environments nutrient and energy sources available to microorganisms can be limited. Foodborne pathogens must efficiently adapt in order to be successfully transmitted through the food chain to their hosts. For the intracellular foodborne pathogen Listeria monocytogenes, little is known regarding its response to nutrient/energy-limiting conditions. The alternative stress responsive sigma factor σ(B) has been reported to contribute to survival under specific stresses. Therefore, the effects of several metabolic inhibitors on growth of L. monocytogenes wild-type and a ΔsigB mutant were examined. In the absence of inhibitors, both strains reached stationary phase after 18 h at 23°C and 10 h at 37°C. All of the metabolic inhibitors slowed growth of either strain, with few differences observed among the different inhibitors.

"The group facilitates everything": meanings patients with type 2 diabetes mellitus assigned to health education groups / "O grupo facilita tudo": significados atribuídos por pacientes portadores de diabetes mellitus tipo 2 a grupos de educação em saúde / "El grupo facilita todo": significados atribuidos por pacientes con diabetes mellitus tipo 2 a grupos de educación para la salud

OBJECTIVE: to interpret the meanings patients with type 2 diabetes mellitus assign to health education groups. METHOD: ethnographic study conducted with Hyperdia groups of a healthcare unit with 26 informants, with type 2 diabetes mellitus, and having participated in the groups for at least three years. Participant observation, social characterization, discussion groups and semi-structured interviews were used to collect data. Data were analyzed through the thematic coding technique. RESULTS: four thematic categories emerged: ease of access to the service and healthcare workers; guidance on diabetes; participation in groups and the experience of diabetes; and sharing knowledge and experiences. The most relevant aspect of this study is the social use the informants in relation to the Hyperdia groups under study. CONCLUSION: the studied groups are agents producing senses and meanings concerning the process of becoming ill and the means of social navigation within the official health system. We expect this study to contribute to the actions of healthcare workers coordinating these groups given the observation of the cultural universe of these individuals seeking professional care in the various public health care services. .

OBJETIVO: interpretar os significados atribuídos por pacientes portadores de diabetes mellitus tipo 2 a grupos de educação em saúde. MÉTODO: estudo etnográfico em cinco grupos Hiperdia de um centro de saúde, com 26 informantes portadores de diabetes mellitus tipo 2 que participavam dos grupos há, no mínimo, três anos. Para coligir as informações, utilizaram-se observação participante, caracterização social, grupos de discussão e entrevistas semiestruturadas. Os dados foram analisados por meio da técnica de codificação temática. RESULTADOS: emergiram quatro categorias temáticas - facilidades de acesso ao serviço e profissionais de saúde, orientações sobre o diabetes, participação nos grupos e experiência com o diabetes e compartilhamento de saberes e experiências. O aspecto mais relevante deste estudo diz respeito aos usos sociais que os informantes conferiam aos grupos Hiperdia pesquisados. CONCLUSÃO: os grupos estudados mostraram-se como instâncias produtoras de sentidos e de significados, concernentes ao processo de adoecimento e aos modos de navegação social no interior do sistema oficial de saúde. Almeja-se que este estudo possa contribuir para as ações dos profissionais de saúde que atuam nesses grupos, tendo em vista a observação do universo cultural dos indivíduos que procuram por cuidado profissional, nos diversos serviços públicos de saúde. .

OBJETIVO: interpretar los significados atribuidos por pacientes con diabetes mellitus tipo 2 a los grupos de educación para la salud. MÉTODO: estudio etnográfico en cinco grupos Hiperdia de un centro de salud, con 26 informantes con diabetes mellitus tipo 2 que participaban de los grupos hace, por lo menos, tres años. Para recolectar las informaciones se utilizaron la observación participante, la caracterización social, los grupos de discusión y las entrevistas semiestructuradas. Los datos fueron analizados por medio de la técnica de codificación temática. RESULTADOS: surgieron cuatro categorías temáticas: facilidades de acceso al servicio y profesionales de la salud; orientaciones sobre la diabetes; participación en los grupos y experiencia con la diabetes; y, compartir conocimientos y experiencias. El aspecto más relevante de este estudio se refiere a los usos sociales que los informantes daban a los grupos Hiperdia investigados. CONCLUSIÓN: los grupos estudiados se mostraron capaces de producir sentidos y significados concernientes al proceso de enfermarse y a los modos de navegación social en el interior del sistema oficial de salud. El objetivo de este estudio es que pueda contribuir para las acciones de los profesionales de la salud que actúan en esos grupos, considerando la observación del universo cultural de los individuos que buscan cuidados profesionales en los diversos servicios públicos de salud. .

Chemokine expression in peripheral tissues from the monosodium iodoacetate model of chronic joint pain.

BACKGROUND: Chronic pain arising from degenerative diseases of the joint such as osteoarthritis (OA) has a strong peripheral component which is likely to be mediator driven. Current treatments which reduce the production of such mediators i.e. non-steroidal anti-inflammatory drugs (NSAIDs), can help to lessen pain in OA patients. However, this is not always the case and complete pain relief is rarely achieved, suggesting that additional unidentified mediators play a role. Here we have investigated the notion that chemokines might act as such pain mediators in OA. RESULTS: Using the monosodium iodoacetate (MIA) model of chronic joint pain the expression of over 90 different inflammatory mediators, mainly cytokines and chemokines, were measured in tissues taken from the femorotibial joint (cartilage, subchondral bone, fat pad) using custom-made quantitative real-time polymerase chain reaction (qPCR) array cards. At both the day 3 and 14 time points, numerous inflammatory mediators were significantly up-regulated in these tissues, although it was clear that the largest transcriptional dysregulation occurred in the cartilage. Using individual qPCR to measure immune cell markers, a significant infiltration of macrophages was measured in the cartilage and fat pad at day 3. Neutrophil infiltration was also measured in the fat pad at the same time point, but no infiltration was observed at day 14. Combination of mRNA expression data from different time points and tissues identified the chemokines, CCL2, 7 and 9 as being consistently up-regulated. The overall increase in CCL2 expression was also measured at the protein level. CONCLUSION: Chemokines in general and CCL2, 7 and 9 in particular, represent promising targets for further studies into the identification of new pain mediators in chronic joint pain.

Diethylmaleate and iodoacetate in combination caused profound cell death in astrocytes.

Energy failure and oxidative stress have been implicated in the pathogenesis of ischemia. Here, we report a potential link between cytosolic phospholipase A2 (cPLA2) activation and energy failure/oxidative stress-induced astrocyte damage involving reactive oxygen species (ROS), protein kinase C-α (PKC-α), Src, Raf, and extracellular signal-regulated kinase (ERK) signaling and concurrent elevation of endogenous chelatable zinc. Energy failure and oxidative stress were produced by treating astrocytes with glycolytic inhibitor iodoacetate and glutathione chelator diethylmaleate, respectively. Diethylmaleate and iodoacetate in combination caused augmented damage to astrocytes in a time- and concentration-dependent manner. The cell death caused by diethylmaleate/iodoacetate was accompanied by increased ROS generation, PKC-α membrane translocation, Src, Raf, ERK, and cPLA2 phosphorylation. Pharmacological studies revealed that these activations all contributed to diethylmaleate/iodoacetate-induced astrocyte death. Intriguingly, the mobilization of endogenous chelatable zinc was observed in diethylmaleate/iodoacetate-treated astrocytes. Zinc appears to act as a downstream mediator in response to diethylmaleate/iodoacetate treatment because of the attenuating effects of its chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine. These observations indicate that ROS/PKC-α, Src/Raf/ERK signaling and cPLA2 are active participants in diethylmaleate/iodoacetate-induced astrocyte death and contribute to a vicious cycle between the depletion of ATP/glutathione and the mobilization of chelatable zinc as critical upstream effectors in initiating cytotoxic cascades.

CIB1 prevents nuclear GAPDH accumulation and non-apoptotic tumor cell death via AKT and ERK signaling.

CIB1 is a 22-kDa regulatory protein previously implicated in cell survival and proliferation. However, the mechanism by which CIB1 regulates these processes is poorly defined. Here, we report that CIB1 depletion in SK-N-SH neuroblastoma and MDA-MB-468 breast cancer cells promotes non-apoptotic, caspase-independent cell death that is not initiated by increased outer mitochondrial membrane permeability or translocation of apoptosis-inducing factor to the nucleus. Instead, cell death requires nuclear GAPDH accumulation. Furthermore, CIB1 depletion disrupts two commonly dysregulated, oncogenic pathways-PI3K/AKT and Ras/MEK/ERK, resulting in a synergistic mechanism of cell death, which was mimicked by simultaneous pharmacological inhibition of both pathways, but not either pathway alone. In defining each pathway's contributions, we found that AKT inhibition alone maximally induced GAPDH nuclear accumulation, whereas MEK/ERK inhibition alone had no effect on GAPDH localization. Concurrent GAPDH nuclear accumulation and ERK inhibition were required, however, to induce a significant DNA damage response, which was critical to subsequent cell death. Collectively, our results indicate that CIB1 is uniquely positioned to regulate PI3K/AKT and MEK/ERK signaling and that simultaneous disruption of these pathways synergistically induces a nuclear GAPDH-dependent cell death. The mechanistic insights into cell death induced by CIB1 interference suggest novel molecular targets for cancer therapy.

Alteration patterns of trabecular bone microarchitectural characteristics induced by osteoarthritis over time.

Information regarding the alteration of trabecular bone microarchitecture, which is one of the important criteria to estimate bone condition, induced by osteoarthritis (OA) is sparse. The current study therefore aimed to identify and quantify patterns of alterations in trabecular bone microarchitectural characteristics at tibial epiphysis induced by OA using in vivo microcomputed tomography. Fourteen 8-week-old female Sprague Dawley rats were randomly divided into control (n = 7) and OA (n = 7) groups. Rats in the OA group were administered monoiodoacetate into the knee-joint cavity. The tibial joints were scanned by in vivo microcomputed tomography at 0, 4, and 8 weeks after administration. Two-way analysis of variance with Tukey's honestly significant difference post hoc test was carried out for statistical analyses. The results showed that patterns of alterations in the trabecular bone microarchitectural characteristics in the OA group were not different from those in the control group from 0 to 4 weeks (P > 0.05), but differed from 4 to 8 weeks (P < 0.05). In particular, both trabecular bone thickness and trabecular bone separation distributions over time (4-8 weeks) differed significantly (P < 0.05). These findings suggest that the patterns of bone microarchitecture changes brought about by OA should be periodically considered in the diagnosis and management of arthritic symptoms over time. Improved understanding of the alteration pattern on trabecular bone microarchitecture may assist in developing more targeted treatment interventions for OA.

Effect of glycolysis inhibition on mitochondrial function in rat brain.

Inhibition of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase enhances the neural vulnerability to excitotoxicity both in vivo and in vitro through an unknown mechanism possibly related to mitochondrial failure. However, as the effect of glycolysis inhibition on mitochondrial function in brain has not been studied, the aim of the present work was to evaluate the effect of glycolysis inhibition induced by iodoacetate on mitochondrial function and oxidative stress in brain. Mitochondria were isolated from brain cortex, striatum and cerebellum of rats treated systemically with iodoacetate (25 mg/kg/day for 3 days). Oxygen consumption, ATP synthesis, transmembrane potential, reactive oxygen species production, lipoperoxidation, glutathione levels, and aconitase activity were assessed. Oxygen consumption and aconitase activity decreased in the brain cortex and striatum, showing that glycolysis inhibition did not trigger severe mitochondrial impairment, but a slight mitochondrial malfunction and oxidative stress were present.

The contribution of spinal glial cells to chronic pain behaviour in the monosodium iodoacetate model of osteoarthritic pain.

BACKGROUND: Clinical studies of osteoarthritis (OA) suggest central sensitization may contribute to the chronic pain experienced. This preclinical study used the monosodium iodoacetate (MIA) model of OA joint pain to investigate the potential contribution of spinal sensitization, in particular spinal glial cell activation, to pain behaviour in this model. Experimental OA was induced in the rat by the intra-articular injection of MIA and pain behaviour (change in weight bearing and distal allodynia) was assessed. Spinal cord microglia (Iba1 staining) and astrocyte (GFAP immunofluorescence) activation were measured at 7, 14 and 28 days post MIA-treatment. The effects of two known inhibitors of glial activation, nimesulide and minocycline, on pain behaviour and activation of microglia and astrocytes were assessed. RESULTS: Seven days following intra-articular injection of MIA, microglia in the ipsilateral spinal cord were activated (p < 0.05, compared to contralateral levels and compared to saline controls). Levels of activated microglia were significantly elevated at day 14 and 21 post MIA-injection. At day 28, microglia activation was significantly correlated with distal allodynia (p < 0.05). Ipsilateral spinal GFAP immunofluorescence was significantly (p < 0.01) increased at day 28, but not at earlier timepoints, in the MIA model, compared to saline controls. Repeated oral dosing (days 14-20) with nimesulide attenuated pain behaviour and the activation of microglia in the ipsilateral spinal cord at day 21. This dosing regimen also significantly attenuated distal allodynia (p < 0.001) and numbers of activated microglia (p < 0.05) and GFAP immunofluorescence (p < 0.001) one week later in MIA-treated rats, compared to vehicle-treated rats. Repeated administration of minocycline also significantly attenuated pain behaviour and reduced the number of activated microglia and decreased GFAP immunofluorescence in ipsilateral spinal cord of MIA treated rats. CONCLUSIONS: Here we provide evidence for a contribution of spinal glial cells to pain behaviour, in particular distal allodynia, in this model of osteoarthritic pain. Our data suggest there is a potential role of glial cells in the central sensitization associated with OA, which may provide a novel analgesic target for the treatment of OA pain.

The protective role of the pericellular matrix in chondrocyte apoptosis.

INTRODUCTION: This study was designed to quantify the role of the pericellular matrix (PCM) in chondrocyte apoptosis using chondrons, which are a cartilage functional unit including a chondrocyte and its associated PCM. METHODS: Chondrocytes and chondrons were enzymatically isolated from human articular cartilage and exposed to monosodium iodoacetate (MIA) and staurosporine for apoptosis induction. Chondrons were defined by the presence of type VI collagen, a basic component of the PCM. Apoptosis of chondrocytes and chondrons was measured with annexin V binding by flow cytometry and verified with terminal dUTP nick end-labeling staining. In a separate experiment, isolated chondrocytes were treated with soluble type VI collagen, before or after apoptosis induction with MIA, and cell death was measured by the activity of LDH and terminal dUTP nick end-labeling staining. RESULTS: Chondrocytes treated with MIA incurred 27% cell death, compared with 12% in chondrons. On treating with MIA, 9% of chondrocytes underwent apoptosis, compared with only 1.6% of chondrons. Similarly, staurosporine induced 13% apoptosis in chondrocytes, whereas it was 3% in chondrons. Preincubation of type VI collagen effectively prevented chondrocytes from MIA-induced cell death. After apoptosis was induced with MIA, however, treatment with type VI collagen failed to rescue chondrocytes from death. CONCLUSION: The PCM, a native microenvironment of chondrocytes, protects chondrocytes from apoptosis. Type VI collagen is a functional component of the PCM that contributes to the survival of chondrocytes.