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Combinatorial molecular therapy in pancreatic ductal adenocarcinoma (PDAC) has yielded largely disappointing results in clinical testing to-date as a multitude of adaptive resistance mechanisms is making selection of patients via molecular markers that capture essential, intersecting signaling routes challenging. Here, we report the scaffolding protein connector enhancer of kinase suppressor of Ras 1 (CNKSR1) as mediator of resistance to MAPK (MEK) inhibition. MEK inhibition in CNKSR1high cancer cells induces translocation of CNKSR1 to the plasma membrane where the scaffolding protein interacts with and stabilizes the phosphorylated form of AKT. CNKSR1-mediated AKT activation following MEK inhibition was associated with increased cellular p-PRAS40 levels and reduced nuclear translocation and cellular levels of FoxO1, a negative regulator of AKT signaling. In clinical PDAC specimens, high cytoplasmatic CNKSR1 levels correlated with increased cellular phospho-AKT and mTOR levels. Pharmacological co-blockade of AKT and MEK ranked top in induced synergies with MEK inhibition in CNKSR1high pancreas cancer cells among other inhibitor combinations targeting known CNKSR1 signaling. In vivo, CNKSR1high pancreatic tumors treated with AKT and MEK inhibitors showed improved outcome in the combination arm compared with single-agent treatment, an effect not observed in CNKSR1low models.Our results identify CNKSR1 as regulator of adaptive resistance to MEK inhibition by promoting crosstalk to AKT signaling via a scaffolding function for the phosphorylated form of AKT. CNSKR1 expression might be a possible molecular marker to enrich patients for future AKT-MEK inhibitor precision medicine studies. IMPLICATIONS: The CNKSR1 scaffold, identified within an RNAi screen as a novel mediator of resistance to MEK inhibition in pancreas cancer, connects the MAPK pathway and AKT signaling and may be adopted as a biomarker to select patients for combined MEK AKT blockade.
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Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Carcinoma Ductal Pancreático/tratamiento farmacológico , Carcinoma Ductal Pancreático/genética , Quinasas de Proteína Quinasa Activadas por Mitógenos , Línea Celular Tumoral , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Neoplasias PancreáticasRESUMEN
OBJECTIVES: One of the most prevalent autoimmune disease globally, rheumatoid arthritis (RA) is caused by interplay of multiple inflammatory mediators in specific joints. Altered redox balance is one of the key factors in pathophysiology of RA. This study aims to find whether oxidative stress in peripheral blood neutrophil correlates with the disease activity and disability associated with it. METHODS: Ten healthy controls and 29 RA patients with moderate to severe disease activity (DAS28 score >3.2) were recruited and reactive oxygen species (ROS) level in peripheral blood neutrophil was measured using flow cytometry at baseline visit and after 6 months follow-up. Functional status of RA patients was measured using Health Assessment Questionnaire Disability Index (HAQ-DI). RESULTS: RA patients showed significantly higher level of ROS in compared to healthy control. DAS28 correlated well with ROS at baseline visit (Pearson's r = +0.63) as well as follow-up visit (Pearson's r = +0.75). HAQ-DI showed weak positive correlation at baseline visit (Pearson's r = 0.1) but it was negative at follow-up visit (Pearson's r = -0.19). CONCLUSIONS: Oxidative stress mirrors the disease activity in RA and can be considered as a biomarker, but it is not related with functional ability of the patients.
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INTRODUCTION: The prerequisite for any experimental model in animals is similarity with the human disease, uniformity in disease severity and incidence. In antigen-induced arthritis models it is generally recognized that the major limitation is inconsistency in terms of incidence and severity. As access to strains like DBA/1 mice or Lewis rats is difficult for resource restrained laboratories, this study aimed to establish a robust and reproducible animal model of rheumatoid arthritis (RA). METHODS: Multiple approaches were undertaken for inducing arthritis in Sprague Dawley (SD) and Wistar rats using complete Freund's adjuvant (CFA), collagen type II (CII) emulsion, or different combinations of CII with low dose CFA along with lipopolysaccharide (LPS). The development of arthritis was evaluated by measuring paw edema, arthritis score, body weight, splenic index, histopathology and radiography of paw tissues. RESULTS: The combination of CII with low dose CFA and one injection of LPS resulted in 100% incidence of arthritis with disease severity ranging from moderate to severe and results were corroborated by histopathology and radiography. DISCUSSION: In a head-to-head comparison between SD and Wistar rats, the disease profile was better sustained and consistent in SD rats, and the use of CII with low dose CFA and LPS induced features akin to human RA. Taken together, a reproducible model of arthritis was established which can be replicated in any laboratory with limited resources.
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Artritis Experimental/fisiopatología , Artritis Reumatoide/fisiopatología , Modelos Animales de Enfermedad , Animales , Colágeno Tipo II/toxicidad , Femenino , Adyuvante de Freund/toxicidad , Lipopolisacáridos/toxicidad , Masculino , Ratas , Ratas Sprague-Dawley , Ratas Wistar , Reproducibilidad de los Resultados , Índice de Severidad de la Enfermedad , Especificidad de la EspecieRESUMEN
Rheumatoid arthritis (RA) is a systemic inflammatory autoimmune disorder wherein the contributory role of oxidative stress has been established in the synovial fluid. As availability of synovial fluid is limited, this study aimed to evaluate in the peripheral blood of patients with RA, the relationship if any, between the extent of oxidative stress in terms of generation of reactive oxygen species (ROS) in neutrophils, plasma NADPH oxidase and myeloperoxidase activity with markers of oxidative damage, circulating cytokines and disease activity score (DAS28). In patients with RA, neutrophils in peripheral blood demonstrated an enhanced generation of ROS, coupled with depletion of free radical scavenging activity. Furthermore, the NADPH oxidase and myeloperoxidase activity was enhanced as were markers of damage. There was a positive correlation between the DAS 28 and generation of ROS, NADPH oxidase and myeloperoxidase activity as also with oxidative stress mediated protein carbonylation. Patients with RA demonstrated an increase in proinflammatory (IL-17, IL-23, and IFN-γ) and some anti-inflammatory (IL-4, IL-5, and TGF-ß) cytokines. Although the levels of IL-17 correlated positively with generation of ROS, myeloperoxidase, markers of protein damage and DAS28, IL-23 correlated positively only with protein damage, and negatively with free radical scavenging activity. Importantly, incubation of neutrophils from healthy donors with plasma or SF from patients with RA translated into an enhanced generation of ROS, along with an elevation of intracellular proinflammatory cytokines. Taken together, in patients with RA, circulating neutrophils mediated a shift in the oxidant/antioxidant balance favouring the former, which translated into protein damage and contributed towards disease progression.
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Artritis Reumatoide/sangre , Citocinas/metabolismo , Adulto , Progresión de la Enfermedad , Femenino , Humanos , Masculino , Persona de Mediana Edad , Oxidación-Reducción , Estrés Oxidativo , Adulto JovenRESUMEN
The cornerstone of treatment for rheumatoid arthritis is low dose methotrexate (MTX), but its use is limited by concerns regarding its potential for hepatotoxicity. Allylpyrocatechol (APC), a phytoconstituent sourced from leaves of Piper betle demonstrated antioxidant, anti-inflammatory, and antiarthritic properties. The present study aimed to evaluate the combined effect of APC and MTX on limiting progression of lipopolysaccharide accelerated collagen-induced arthritis, along with reduction of MTX-induced hepatic damage. A collagen-induced arthritis (CIA) model was established by immunising Sprague-Dawley rats with bovine collagen type II (CII) and lipopolysaccharide, followed by a booster dose of CII on day 15. Rats from days 11-27 were administered APC (20 mg/kg), methotrexate (1.5 mg/kg), or a combination of MTX and APC. The combinatorial therapy of APC and MTX significantly improved the parameters of arthritis as evident from the reduction in paw oedema and arthritic score and was endorsed by radiological and histopathological changes. This combination prevented the rise in levels of proinflammatory cytokines, tumour necrosis factor (TNF-α), and interleukin 6 (IL-6). Furthermore, unlike MTX-monotherapy, the APC-MTX combination decreased the associated cachexia, splenomegaly, and oxidative stress. Importantly, the hepatic damage mediated by MTX monotherapy was effectively attenuated by the inclusion of APC. Taken together, antioxidants such as APC when combined with MTX not only potentiated the antiarthritic effect but importantly alleviated the MTX-induced hepatic damage, thus endorsing its effectiveness in preventing progression of articular diseases such as rheumatoid arthritis.
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Antiinflamatorios/farmacología , Antioxidantes/farmacología , Antirreumáticos/farmacología , Artritis Experimental/tratamiento farmacológico , Catecoles/farmacología , Enfermedad Hepática Inducida por Sustancias y Drogas/prevención & control , Metotrexato/farmacología , Animales , Artritis Experimental/inducido químicamente , Artritis Experimental/genética , Artritis Experimental/inmunología , Caquexia/inducido químicamente , Caquexia/genética , Caquexia/inmunología , Caquexia/prevención & control , Enfermedad Hepática Inducida por Sustancias y Drogas/genética , Enfermedad Hepática Inducida por Sustancias y Drogas/inmunología , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Colágeno Tipo II/administración & dosificación , Sinergismo Farmacológico , Edema/inducido químicamente , Edema/genética , Edema/inmunología , Edema/prevención & control , Femenino , Regulación de la Expresión Génica , Interleucina-6/antagonistas & inhibidores , Interleucina-6/genética , Interleucina-6/inmunología , Lipopolisacáridos/antagonistas & inhibidores , Lipopolisacáridos/farmacología , Hígado/efectos de los fármacos , Hígado/inmunología , Hígado/patología , Masculino , Estrés Oxidativo/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Esplenomegalia/inducido químicamente , Esplenomegalia/genética , Esplenomegalia/inmunología , Esplenomegalia/prevención & control , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
Rheumatoid arthritis (RA), an inflammatory autoimmune disorder, is characterized by synovial hyperplasia and bony destruction. The pathogenesis of RA includes redox dysregulation, concomitant with increased levels of proinflammatory mediators. As the ability of allylpyrocatechol (APC), a phytoconstituent of Piper betle leaves, to alleviate oxidative stress has been demonstrated in patients with RA, its antiarthritic activity was evaluated in an animal model of arthritis, and the underlying mechanism(s) of action clarified. The animal model was established by immunizing rats with bovine collagen type II (CII) followed by lipopolysaccharide, along with a booster dose of CII on day 15. Rats were treated with APC or methotrexate (MTX) from days 11 to 27, when paw edema, radiography, histopathology, and markers of inflammation were evaluated. The pro/antiinflammatory signaling pathways were studied in a RAW264.7 macrophage cell line. Allylpyrocatechol (APC) prevented the progression of arthritis as was evident from the reduction in paw edema, and attenuation of damage to bones and cartilage shown by radiography and histopathology. Additionally, there was reduction in the levels of proinflammatory cytokines [tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6)] and restoration of the redox balance. Importantly, MTX ameliorated the features of arthritis but not the associated oxidative stress. In RAW264.7, APC inhibited generation of nitric oxide and proinflammatory cytokines (TNF-α, IL-6, and IL-12p40), and modulated the phosphorylation of proinflammatory (extracellular signal-regulated kinase 1/2, stress-activated protein kinase/c-Jun N-terminal protein kinase, and Janus kinase/signal transducers and activators of transcription) and cytoprotective (nuclear factor erythroid 2-related factor 2, heme oxygenase-1) signaling pathways. Taken together, APC controlled the development of arthritis, possibly via modulation of signaling pathways, and deserves further consideration as a therapy for RA.
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Artritis Experimental/inducido químicamente , Artritis Experimental/tratamiento farmacológico , Catecoles/farmacología , Colágeno/efectos adversos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Animales , Artritis Experimental/metabolismo , Artritis Experimental/patología , Artritis Reumatoide/inducido químicamente , Artritis Reumatoide/tratamiento farmacológico , Artritis Reumatoide/metabolismo , Artritis Reumatoide/patología , Catecoles/uso terapéutico , Bovinos , Progresión de la Enfermedad , Femenino , Hemo-Oxigenasa 1/metabolismo , Mediadores de Inflamación/metabolismo , Quinasas Janus/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Macrófagos/patología , Masculino , Proteínas de la Membrana/metabolismo , Ratones , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Oxidación-Reducción/efectos de los fármacos , Células RAW 264.7 , Ratas , Proteínas Recombinantes de Fusión/efectos de los fármacos , Factores de Transcripción STAT/metabolismoRESUMEN
Intrinsically cancer cells have higher basal levels of reactive oxygen species (ROS), which when augmented by pro-oxidants such as Malabaricone-A (MAL-A) triggers apoptotic cell death, secondary to 'turning on' of the apoptosis related cell signaling pathways. The effects of MAL-A upon key inflammation related signaling molecules were evaluated by western blotting in U937, a histiocytic lymphoma derived cell line. The impact of inhibitors of the pro-apoptotic MAPK and anti-apoptotic PI3K/AKT signaling pathways upon MAL-A induced cytotoxicity and generation of ROS was evaluated by a cell viability assay and flow cytometry respectively in two hematopoietic cell lines, U937 and MOLT3. MAL-A enhanced phosphorylation of the components of the pro-apoptotic pathway, namely ASK1, p38 and JNK. Alongside, MAL-A decreased the phosphorylation of AKT and mTOR. The cytotoxicity of MAL-A was attenuated by inhibitors of p38 and JNK, whereas its cytotoxic potential was enhanced in the presence of a PI3K/AKT inhibitor. Similarly, MAL-A mediated generation of ROS was decreased by inhibitors of p38MAPK and JNK, whereas the PI3K/AKT inhibitor potentiated its generation of ROS. Taken together, MAL-A mediated its cytotoxicity by enhanced generation of ROS via modulation of the apoptosis related cellular signaling pathways and tilting the balance towards a pro-apoptotic scenario. This was achieved via an up-regulation of MAPK (p38 and JNK) along with down-regulation of the PI3K/AKT/mTOR pathway indicating that manipulation of these pathways by compounds such as MAL-A are promising therapeutic targets, worthy of future pharmacological consideration.
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Apoptosis , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Oxidantes/farmacología , Especies Reactivas de Oxígeno/metabolismo , Resorcinoles/farmacología , Apoptosis/efectos de los fármacos , Proteínas de Ciclo Celular/metabolismo , Inhibidores Enzimáticos/farmacología , Humanos , MAP Quinasa Quinasa 4/metabolismo , Proteína Oncogénica v-akt/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR/metabolismo , Células U937 , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Methotrexate (MTX), a folate antagonist, is currently used as first line therapy for autoimmune diseases like rheumatoid arthritis and psoriasis, but its use is limited by the associated hepatotoxicity. As leaves of Piper betle, belonging to family Piperaceae, have antioxidant and anti-inflammatory properties, the present study was undertaken to investigate the potential of Piper betle leaf extract (PB) in attenuating MTX-induced hepatotoxicity. Rats pre-treated with PB (50 or 100 mg kg(-1) b.w., p.o.) were administered with a single dose of MTX (20 mg kg(-1), b.w., i.p.) and its hepatoprotective efficacy was compared with folic acid (1 mg kg(-1) b.w., i.p.), conventionally used to minimize MTX-induced toxicity. MTX-induced hepatotoxicity was confirmed by increased activities of marker enzymes, alanine transaminase, aspartate transaminase, and alkaline phosphatase which were remitted by pre-treatment with PB and corroborated with histopathology. Additionally, MTX-induced hepatic oxidative stress which included increased generation of reactive oxygen species, enhanced lipid peroxidation, depleted levels of glutathione and decreased activities of antioxidant enzymes was effectively mitigated by PB, indicative that its promising antioxidant-mediated hepatoprotective activity was worthy of future pharmacological consideration.
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Antioxidantes/farmacología , Enfermedad Hepática Inducida por Sustancias y Drogas/prevención & control , Metotrexato/toxicidad , Estrés Oxidativo/efectos de los fármacos , Piper betle/metabolismo , Extractos Vegetales/farmacología , Alanina Transaminasa/metabolismo , Fosfatasa Alcalina/metabolismo , Animales , Aspartato Aminotransferasas/metabolismo , Catalasa/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Femenino , Ácido Fólico/farmacología , Glutatión/metabolismo , Glutatión Peroxidasa/metabolismo , Peroxidación de Lípido/fisiología , Hígado/patología , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismo , Superóxido Dismutasa/metabolismoRESUMEN
PURPOSE: The 'two-faced' character of reactive oxygen species (ROS) plays an important role in cancer biology by acting as secondary messengers in intracellular signaling cascades, enhancing cell proliferation and survival, thereby sustaining the oncogenic phenotype. Conversely, enhanced generation of ROS can trigger an oxidative assault leading to a redox imbalance translating into an apoptotic cell death. Intrinsically, cancer cells have higher basal levels of ROS which if supplemented by additional oxidative insult by pro-oxidants can be cytotoxic, an example being Malabaricone-A (MAL-A). MAL-A is a plant derived diarylnonanoid, purified from fruit rind of the plant Myristica malabarica whose anti-cancer activity has been demonstrated in leukemic cell lines, the modality of cell death being apoptosis. This study aimed to compare the degree of effectiveness of MAL-A in leukemic vs. solid tumor cell lines. METHODS: The cytotoxicity of MAL-A was evaluated by the MTS-PMS cell viability assay in leukemic cell lines (MOLT3, K562 and HL-60) and compared with solid tumor cell lines (MCF7, A549 and HepG2); further studies then proceeded with MOLT3 vs. MCF7 and A549. The contribution of redox imbalance in MAL-A induced cytotoxicity was confirmed by pre-incubating cells with an antioxidant, N-acetyl-L-cysteine (NAC) or a thiol depletor, buthionine sulfoximine (BSO). MAL-A induced redox imbalance was quantitated by flow cytometry, by measuring the generation of ROS and levels of non protein thiols using dichlorofluorescein diacetate (CM-H2DCFDA) and 5-chloromethylfluorescein diacetate (CMFDA) respectively. The activities of glutathione peroxidase (GPx), superoxide dismutase, catalase (CAT), NAD(P)H dehydrogenase (quinone 1) NQO1 and glutathione-S-transferase GST were measured spectrophotometrically. The mitochondrial involvement of MAL-A induced cell death was measured by evaluation of cardiolipin peroxidation using 10-N-nonyl acridine orange (NAO), transition pore activity with calcein-AM, while the mitochondrial transmembrane electrochemical gradient (∆ψ(m)) was measured by JC-1, fluorescence being acquired in a flow cytometer. The apoptotic mode of cell death was evaluated by double staining with annexin V-FITC and propidium iodide (PI), cell cycle analysis by flow cytometry and caspase-3 activity spectrophotometrically. The expression of Nrf2 and HO-1 was examined by western blotting. RESULTS: MAL-A demonstrated a higher degree of cytotoxicity in three leukemic cell lines whose IC50 ranged from 12.70 ± 0.10 to 18.10 ± 0.95 µg/ml, whereas in three solid tumor cell lines, the IC50 ranged from 28.10 ± 0.58 to 55.26 ± 5.90 µg/ml. This higher degree of cytotoxicity in MOLT3, a leukemic cell line was due to a higher induction of redox imbalance, evident by both an increased generation of ROS and concomitant depletion of thiols. This was confirmed by pre-incubation with NAC and BSO, wherein NAC decreased MAL-A induced cytotoxicity by 2.04 fold while BSO enhanced MAL-A cytotoxicity and decreased the IC50 by 5.60 fold. However, in solid tumor cell lines (MCF7 and A549), NAC minimally decreased MAL-A induced cytotoxicity, and BSO increased the IC50 by 1.96 and 2.39 fold respectively. Furthermore, the generation of ROS by MAL-A increased maximally in MOLT3 as the fluorescence increased from 44.28 ± 7.85 to 273.99 ± 32.78, and to a lesser degree in solid tumor cell lines, MCF7 (44.28 ± 14.89 to 207.97 ± 70.64) and A549 (37.87 ± 3.24 to 147.12 ± 38.53). In all three cell lines there was a concomitant depletion of thiols as in MOLT3, the GMFC decreased from 340.65 ± 60.39 to 62.67 ± 11.32, in MCF7 (277.82 ± 50.32 to 100.39 ± 31.93) and in A549 (274.05 ± 59.13 to 83.15 ± 21.43). In MOLT3 as compared to MCF7 and A549, decrease in the activities of GPx, CAT, NQO1 and GST was substantially greater. In all cell lines, the MAL-A induced redox imbalance translated into triggering of initial mitochondrial apoptotic events. Here again, MAL-A induced a higher degree of cardiolipin peroxidation in MOLT3 (67.01%) than MCF7 and A549 (29.15% and 44.30%), as also down regulated the mitochondrial transition pore activity from baseline to a higher extent, GMFC being 48.05 ± 2.37 to 10.70 ± 3.97 (MOLT3), 43.55 ± 3.36 to 15.36 ± 0.60 (MCF7) and 39.58 ± 0.4 to 12.65 ± 1.56 (A549). Perturbation of mitochondrial membrane potential evident by a decrease in the ratio of red/green (J-aggregates/monomers) was 134 fold (14.73/0.11) in MOLT3, 45 fold in MCF7 (20.72/0.46) and 34 fold in A549 (22.01/0.64). The extent of apoptosis using a similar concentration of MAL-A was maximal in MOLT3, wherein a 105 fold increase in annexin V binding was evident (0.83 ± 0.51 to 87.08 ± 9.85%) whereas it increased by 43.11 fold in MCF7 (0.69 ± 0.30 to 29.75 ± 11.79%) and 47.52 fold in A549 (0.61 ± 0.31 to 28.99 ± 17.21%). MAL-A induced apoptosis was also associated with a higher degree of caspase-3 activity in MOLT3 vs. MCF7 or A549 which translated into halting of cell cycle progression, evident by an increment in the sub-G0/G1 population [19.26 fold in MOLT3 (0.95 ± 0.45 vs. 18.30 ± 1.90%), 11.01 fold in MCF7 (0.97 ± 0.37 vs. 10.68 ± 0.69%) and 8.58 fold in A549 (1.06 ± 0.45 vs. 9.10 ± 1.05%)]. MAL-A effectively inhibited Nrf2 and HO-1, more prominently in MOLT3. Furthermore, the decreased expression of Nrf2 in MOLT3 correlated with the decreased activities of NQO1 and GST, suggesting that targeting of the Nrf2 anti-oxidant pathway could be considered. CONCLUSION: Taken together, MAL-A a pro-oxidant compound is likely to be more effective in leukemias, meriting further pharmacological consideration.
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Antineoplásicos Fitogénicos/farmacología , Especies Reactivas de Oxígeno/metabolismo , Resorcinoles/farmacología , Apoptosis/efectos de los fármacos , Células HL-60/efectos de los fármacos , Células Hep G2/efectos de los fármacos , Humanos , Células K562/efectos de los fármacos , Células MCF-7/efectos de los fármacos , Myristicaceae/química , Oxidación-ReducciónRESUMEN
BACKGROUND: Generalized vitiligo is a disease with unpredictable bursts of activity, goal of treatment during the active phase being to stabilize the lesions. This emphasizes the need for a prospective marker for monitoring disease activity to help decide the duration of therapy. AIMS AND OBJECTIVES: In the present study, we examined whether reactive oxygen species (ROS) generated in erythrocytes can be translated into a marker of activity in vitiligo. MATERIALS AND METHODS: Level of intracellular ROS was measured flow cytometrically in erythrocytes from venous blood of 21 patients with generalized vitiligo and 21 healthy volunteers using the probe dichlorodihydrofluorescein diacetate. RESULTS: The levels of ROS differed significantly between patients and healthy controls, as well as between active versus stable disease groups. In the active disease group, ROS levels were significantly lower in those being treated with systemic steroids than those that were not. ROS levels poorly correlated with disease duration or body surface area involved. CONCLUSION: A long-term study based on these findings can be conducted to further validate the potential role of ROS in monitoring disease activity vitiligo.
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Rheumatoid arthritis (RA) is a debilitating autoimmune disease whose etiology remains unknown, but studies have consistently implicated a plethora of inflammatory mechanisms culminating in chronic symmetric and erosive synovitis. Importantly, reactive oxygen species (ROS) have been attributed to directly contribute towards the destructive, proliferative synovitis evident in RA. Accordingly, this study aimed to establish whether the degree of oxidative stress and disease activity score (DAS28) correlated with the downstream effects of oxidative damage. The redox status of neutrophils sourced from synovial fluid (SF) was measured by flow cytometry in terms of total ROS and hydroxyl radicals. Among the molecular damage markers, protein carbonylation and lipid peroxidation were detected by spectrophotometry and S-nitrosothiols by fluorimetry. Neutrophils constituted the major cellular component of the SF of patients with RA and their levels of ROS and hydroxyl radicals correlated strongly with protein carbonylation and lipid peroxidation. However, all the oxidative damage markers correlated positively with DAS28. Taken together, in patients with RA, the strong correlation between levels of ROS and DAS28 with markers of oxidative damage suggests that measurement of oxidative stress could serve as a biomarker for monitoring disease severity in RA.
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Artritis Reumatoide/metabolismo , Estrés Oxidativo/fisiología , Especies Reactivas de Oxígeno/metabolismo , Líquido Sinovial/metabolismo , Sinovitis/metabolismo , Adulto , Artritis Reumatoide/patología , Biomarcadores/metabolismo , Femenino , Humanos , Peroxidación de Lípido/fisiología , Masculino , Persona de Mediana Edad , Neutrófilos/metabolismo , Oxidación-Reducción , Sinovitis/patología , Adulto JovenRESUMEN
OBJECTIVES: To evaluate the effect of vildagliptin alone and in combination with metformin or rosiglitazone on murine hepatic steatosis in diet-induced nonalcoholic fatty liver disease (NAFLD). MATERIALS AND METHODS: Male C57BL/6 mice were fed with high fat diet (60 Kcal %) and fructose (40%) in drinking water for 60 days to induce NAFLD. After the induction period, animals were divided into different groups and treated with vildagliptin (10 mg/kg), metformin (350 mg/kg), rosiglitazone (10 mg/kg), vildagliptin (10 mg/kg) + metformin (350 mg/kg), or vildagliptin (10 mg/kg) + rosiglitazone (10 mg/kg) orally for 28 days. Following parameters were measured: body weight, food intake, plasma glucose, triglyceride (TG), total cholesterol, liver function tests, and liver TG. Liver histopathology was also examined. RESULTS: Oral administration of vildagliptin and rosiglitazone in combination showed a significant reduction in fasting plasma glucose, hepatic steatosis, and liver TGs. While other treatments showed less or no improvement in the measured parameters. CONCLUSIONS: These preliminary results demonstrate that administration of vildagliptin in combination with rosiglitazone could be a promising therapeutic strategy for the treatment of NAFLD.
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Adamantano/análogos & derivados , Hígado Graso/tratamiento farmacológico , Nitrilos/uso terapéutico , Pirrolidinas/uso terapéutico , Tiazolidinedionas/uso terapéutico , Adamantano/administración & dosificación , Adamantano/farmacología , Adamantano/uso terapéutico , Animales , Quimioterapia Combinada , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Nitrilos/administración & dosificación , Nitrilos/farmacología , Enfermedad del Hígado Graso no Alcohólico , Tamaño de los Órganos/efectos de los fármacos , Pirrolidinas/administración & dosificación , Pirrolidinas/farmacología , Rosiglitazona , Tiazolidinedionas/administración & dosificación , Tiazolidinedionas/farmacología , Triglicéridos/metabolismo , VildagliptinaRESUMEN
OBJECTIVES: The objective of this study was to evaluate the peripheral analgesic effect of Piper betle leaf extract (PBE) along with establishing its putative mechanism of action. MATERIALS AND METHODS: Male Swiss albino mice after pre-treatment (1 h) with different doses of PBE were injected 0.8% (v/v) acetic acid i.p.; the onset and number of writhes were noted up to 15 min. To evaluate the mechanism of action, the murine peritoneal exudate was incubated with PBE for 1 h, followed by exposure to arachidonic acid (AA) and generation of reactive oxygen species (ROS) was measured by flow cytometry using 2',7'-dichlorodihydrofluorescein diacetate. RESULTS: PBE in a dose dependent manner significantly reduced acetic acid induced writhing response in mice (P < 0.001). In peritoneal exudates, PBE significantly inhibited AA induced generation of ROS, P < 0.01. CONCLUSIONS: The present study indicates that PBE has promising analgesic activity, worthy of future pharmacological consideration.
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Ácido Araquidónico/farmacología , Nocicepción/efectos de los fármacos , Piper/química , Extractos Vegetales/uso terapéutico , Animales , Citometría de Flujo , Masculino , Ratones , Hojas de la Planta/química , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Allylpyrocatechol (APC) is responsible for the antiinflammatory activity exhibited by the methanolic extract of leaves of Piper betle. As antiinflammatory compounds may display antioxidant properties and vice versa, we investigated the antioxidant effect of APC. APC effectively reduced phorbol-myristate-acetate-induced generation of reactive oxygen species and superoxide in murine peritoneal macrophages as well as inhibited Escherichia-coli-induced phagocytic activity of macrophages. Furthermore, pBluescript SK(+) plasmid DNA damage induced by addition of sodium ascorbate was attenuated by APC as it inhibited transformation of the supercoiled form to a relaxed form. In addition, APC increased the enzymatic (catalase) and nonenzymatic (GSH) antioxidant components of murine macrophages. Taken together, APC exhibited an antioxidant activity which was mediated both via decreased generation of free radicals along with increase in cellular antioxidants.