RESUMEN
Up to date, there is no information on the influence of 2,2,2-tribromoethanol (TBE; Avertin), a commonly used anaesthetic, on mice with impaired antioxidant capacity. We aimed to analyse the effect of a single dose of Avertin on anaesthesia duration time, inflammatory response, oxidative stress and collagen deposition in the large intestine of Nrf2 transcriptional knockout mice (tNrf2-/-). The studies were performed on six-month-old female mice Nrf2+/+ and tNrf2-/- randomly assigned to Avertin (250 mg/kg b.w. single i.p. injection) or vehicle group. We observed a 2-fold increase in anaesthesia time and longer recovery time (p = 0.015) in tNrf2-/- in comparison to Nrf2+/+. However, no hepato- or nephrotoxicity was detected. Interestingly, we found severe changes in colon morphology of untreated tNrf2-/- mice associated with colon shortening (p = 0.02) and thickening (p = 0.015). Avertin treatment caused colon damage manifested with epithelial layer damage and goblet depletion in Nrf2+/+ mice but not in tNrf2-/- individuals. Additionally, Avertin did not induce oxidative stress in colon tissue, but it increased leukocyte infiltration in Nrf2+/+ mice (p = 0.02). Immunofluorescent staining also revealed enhanced deposition of collagen I and collagen III in the colon of untreated tNrf2-/- mice. Avertin contributed to increased deposition of collagen I in Nrf2+/+ mice but reduced deposition of collagen I and III in tNrf2-/- individuals. In conclusion, tNrf2-/- respond to Avertin with prolonged anaesthesia that is not associated with acute toxicity, inflammatory reaction or enhanced oxidative stress. Avertin does not impair intestine morphology in tNrf2-/- mice but can normalise the enhanced fibrosis.
Asunto(s)
Anestésicos/farmacología , Colon/efectos de los fármacos , Estado de Conciencia/efectos de los fármacos , Etanol/análogos & derivados , Factor 2 Relacionado con NF-E2/metabolismo , Periodo de Recuperación de la Anestesia , Anestésicos/toxicidad , Animales , Colágeno/metabolismo , Colon/metabolismo , Colon/patología , Etanol/farmacología , Etanol/toxicidad , Femenino , Fibrosis , Mediadores de Inflamación/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Factor 2 Relacionado con NF-E2/deficiencia , Factor 2 Relacionado con NF-E2/genética , Estrés Oxidativo/efectos de los fármacos , Factores de TiempoRESUMEN
BACKGROUND: Neutrophil gelatinase-associated lipocalin (NGAL), an acute phase protein released by neutrophils, has been described as biomarker of inflammatory states. Type 2 diabetes mellitus (T2DM) is characterized by increased inflammation and an elevated risk for embolization of carotid artery stenosis (CAS). We aimed to explore the role of NGAL systemically and in plaques of diabetics undergoing carotid endarterectomy. Moreover, the potential anti-inflammatory effect of metformin on NGAL was addressed in diabetics. METHODS: Serum NGAL and matrix metalloproteinase (MMP)-9/NGAL levels were measured in 136 patients (67 with T2DM vs. 69 non-diabetics) by specific ELISA. Endarterectomy samples were graded histologically according to the American Heart Association´s classification. NGAL mRNA expression was detected using RealTime-PCR in carotid endarterectomy specimens. RESULTS: Serum NGAL [median 107.4 ng/ml (quartiles: 75.2-145.0) vs. 64.4 (50.4 -81.3), p < 0.0001] and MMP-9/NGAL [41.5 ng/ml (20.8-63.9) vs. 27.6 (16.0-42.4), p = 0.017] were significantly elevated in diabetics compared to non-diabetics, as were leukocytes, neutrophils, C-reactive protein and fibrinogen (all p < 0.05). In patients with symptomatic and asymptomatic CAS diabetics had higher NGAL levels compared to non-diabetics [128.8 ng/ml (100.8-195.6) vs. 64.8 (48.9-82.2] and [101.6 ng/ml (70.1-125.3) vs. 63.8 (51.0-81.3), respectively, both p < 0.0001]. Presence of T2DM and type VI plaques (with surface defect, hemorrhage or thrombus) had a profound impact on NGAL levels (both p < 0.01) in multiple linear regression analysis. NGAL mRNA was detectable in 95% of analyzed carotid artery lesions of diabetics compared to 5% of non-diabetics (p < 0.0001). Accordingly, cerebral embolization was more frequent in diabetics (52.2% vs. 29%, p = 0.006). Metformin treatment was associated with decreased NGAL [60.7 ng/ml (51.9-69.2) vs. 121.7 (103.7-169.9), p < 0.0001] and MMP-9/NGAL [20.8 ng/ml (12.1-26.5) vs. 53.7 (27.4-73.4), p = 0.007] in diabetics and reduced leukocyte infiltration in carotid lesions of diabetics. CONCLUSIONS: Higher NGAL levels in serum and plaques are associated with T2DM in patients with CAS. Metformin significantly reduced the inflammatory burden including NGAL in diabetics. Early treatment of these patients may be recommended, as elevated NGAL levels were linked with vulnerable plaques prone for embolization.
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Estenosis Carotídea/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Lipocalina 2/metabolismo , Metformina/uso terapéutico , Anciano , Biomarcadores/sangre , Arterias Carótidas/metabolismo , Enfermedades de las Arterias Carótidas/metabolismo , Estenosis Carotídea/complicaciones , Diabetes Mellitus Tipo 2/complicaciones , Femenino , Humanos , Masculino , Persona de Mediana Edad , Proteínas Proto-Oncogénicas/sangreRESUMEN
The aim of this study was to assess the immune response to parainfluenza virus type 3 (PIV3), rhinovirus 1B (RV1B) and intracellular Toll-like receptors (TLR) agonists in nasal epithelial cells (NECs) from patients with allergic rhinitis and healthy controls. NECs were obtained from eight patients with allergic rhinitis (AR) and 11 non-atopic healthy controls (HC) by nasal scraping, grown to confluence and exposed to PIV3, RV1B infection or TLR-3 and TLR-7/8 agonists. Interferon (IFN)-λ1, IFN-α, IFN-ß and regulated on activation, normal T expressed and secreted (RANTES) release into the cell culture supernatants was assessed at 8, 24 and 48 h upon infection or 8 and 24 h after stimulation with poly(I:C) and R848. mRNA levels of IFNs, RANTES, interferon regulatory transcription factor (IRF)3, IRF7 and viral gene copy number were determined using real-time polymerase chain reaction (RT-PCR). PIV3 but not RV1B replication 48 h after infection was significantly lower (P < 0·01) in NECs from AR patients compared to HC. PIV3 infection induced significantly less IFN-λ1 (both protein and mRNA) in NECs from AR compared to HC. IFN-ß mRNA expression and RANTES protein release and mRNA expression tended to be smaller in AR compared HC cells in response to both viruses. Stimulation with TLR-3 agonist [poly (I:C)] induced similar IFN-λ1 and RANTES generation in AR and HC subjects. Viral infections in NECs induced IRF7 expression, which correlated with IFN and RANTES expression. These data suggest that virus proliferation rates and the immune response profile are different in nasal epithelial cells from patients with allergic rhinitis compared to healthy individuals.
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Resfriado Común/inmunología , Células Epiteliales/inmunología , Inmunidad Innata , Virus de la Parainfluenza 3 Humana/fisiología , Infecciones por Respirovirus/inmunología , Rinitis Alérgica/inmunología , Rhinovirus/fisiología , Replicación Viral , Adulto , Células Cultivadas , Quimiocina CCL5/genética , Quimiocina CCL5/metabolismo , Células Epiteliales/efectos de los fármacos , Células Epiteliales/virología , Femenino , Humanos , Imidazoles/farmacología , Factor 7 Regulador del Interferón/genética , Factor 7 Regulador del Interferón/metabolismo , Interferones/genética , Interferones/metabolismo , Masculino , Persona de Mediana Edad , Nariz/patología , Poli I-C/farmacología , Rinitis Alérgica/virología , Receptor Toll-Like 3/agonistas , Receptor Toll-Like 7/agonistas , Adulto JovenRESUMEN
OBJECTIVE: Neutrophil gelatinase-associated lipocalin (NGAL), a protein found in activated neutrophils, is expressed in kidney tubule cells in response to noxious stimuli, and is thus recognized as a marker of acute kidney injury. Recent studies have suggested that NGAL could also have pathophysiological importance in cardiovascular diseases. The aim of the present study was to examine NGAL expression in human carotid endarterectomy tissues ex vivo as well as the effects of NGAL in the main cell types involved in atherogenesis, namely in human macrophages, endothelial cells, and smooth muscle cells in vitro. METHODS: NGAL protein was analyzed in human endarterectomy samples from patients with asymptomatic and symptomatic carotid stenosis by immunofluorescence, and NGAL mRNA expression was detected using RealTime-PCR. Human monocyte derived macrophages (MDM), human coronary artery smooth muscle cells (HCASMC), and human umbilical vein endothelial cells (HUVEC) were treated with recombinant human (rh) NGAL at different concentrations. Interleukin (IL)-6, IL-8, and monocyte chemo-attractant protein-1 (MCP-1) were determined by specific enzyme linked immunosorbent assays (ELISAs) in culture supernatants of such treated cells. RESULTS: Expression of NGAL protein was demonstrated by macrophages, smooth muscle cells, and endothelial cells in human carotid atherosclerotic tissue. NGAL mRNA expression was detected at a higher rate in atherosclerotic tissue of patients with symptomatic carotid stenosis (in 70%; n = 19) compared with asymptomatic patients (in 37%; n = 20, p < .001). Treatment of MDM, HCASMC, and HUVEC with rhNGAL led to a significant (p < 0.05) and concentration dependent increase of pro-inflammatory cytokines IL-6, IL-8, and MCP-1 in all cell types analyzed. CONCLUSION: By induction of pro-inflammatory mediators in human macrophages, smooth muscle cells and endothelial cells, NGAL, which is predominantly expressed in atherosclerotic plaques of symptomatic patients, could be involved in creating the local and systemic pro-inflammatory environment characteristic for atherosclerosis.
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Enfermedades de las Arterias Carótidas/metabolismo , Inflamación/metabolismo , Lipocalina 2/metabolismo , Quimiocina CCL2/metabolismo , Relación Dosis-Respuesta a Droga , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Técnica del Anticuerpo Fluorescente , Humanos , Técnicas In Vitro , Inflamación/etiología , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Lipocalina 2/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
The α7 pentamer nicotinic acetylcholine receptors (nAChRs) are a target in transduction of anti-inflammatory signals from the central nervous system to the gastrointestinal (GI) tract. The aim of this study was to investigate the anti-inflammatory action of the novel α7 nAChR partial agonist encenicline and to determine the mechanism underlying its activity. Anti-inflammatory activity of encenicline was evaluated using trinitrobenzenesulfonic acid (TNBS)- and dextran sulfate sodium (DSS)-induced models of colitis. Macroscopic score, ulcer score, colon length and thickness, as well as myeloperoxidase (MPO) activity were recorded. Immunohistochemistry (IHC) was used to measure the infiltration of immune cells in the colon. Furthermore, we employed flow cytometry to determine the effect of encenicline on frequencies of FoxP3(+) and interleukin (IL)-17A(+) T cells in the mouse colon. Encenicline attenuated TNBS- and DSS-induced colitis in mice via α7 nAChRs, as indicated by significantly reduced macroscopic parameters and MPO activity. Treatment with encenicline significantly reduced the infiltration of macrophages, neutrophils, and B cells in the colon of TNBS-treated animals, as indicated by IHC. In the TNBS model encenicline reduced the frequency of FoxP3(+) IL-17A(+) T cells in the colon. In the DSS-model treatment encenicline increased the frequency of FoxP3(+) T cells and reduced IL-17A(+) T cells. Stimulation of α7 nAChR with partial agonist encenicline alleviates colitis via alteration of the number and/or activation status of the immune cells in the gut, emphasizing a potential role of α7 nAChRs as a target for anticolitic drugs.
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Colitis Ulcerosa/tratamiento farmacológico , Colitis Ulcerosa/patología , Colon/patología , Agonistas Nicotínicos/uso terapéutico , Quinuclidinas/uso terapéutico , Tiofenos/uso terapéutico , Receptor Nicotínico de Acetilcolina alfa 7/agonistas , Animales , Colitis Ulcerosa/inducido químicamente , Sulfato de Dextran , Citometría de Flujo , Factores de Transcripción Forkhead/metabolismo , Hexametonio/farmacología , Interleucina-17/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Antagonistas Nicotínicos/farmacología , Peroxidasa/metabolismo , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismo , Ácido TrinitrobencenosulfónicoRESUMEN
OBJECTIVE/BACKGROUND: Matrix metalloproteinases (MMPs) play a pivotal role in the development and progression of abdominal aortic aneurysms (AAAs). The action of MMPs depends on a balance between tissue inhibitors of MMPs (TIMPs) and compounds that may prolong protease activity, such as neutrophil gelatinase-associated lipocalin (NGAL). METHODS: The study was designed to analyse gene expression and protein concentration of MMPs, TIMPs, and NGAL in AAA walls and intraluminal thrombi (ILTs) of patients on simvastatin (n = 10) and not on statins (n = 10). The patients were matched by age, sex, and AAA diameter. Expression of MMP2, MMP9, TIMP1, TIMP2, and NGAL was investigated by real time polymerase chain reaction, and MMP2, MMP9, MMP9/TIMP1, MMP9/TIMP2, and MMP9/NGAL protein levels by enzyme-linked immunosorbent assay. RESULTS: MMP2 and MMP9 protein and mRNA levels were comparable in the simvastatin and non-statin groups (p > .05); however, there was a significant decrease in TIMP1 mRNA in AAA tissue (p = .04). Moreover, a significant increase in MMP9/TIMP2 complex concentration in ILTs of patients on simvastatin was noted (median 94.71 ng/mL in the simvastatin group vs. 36.80 ng/mL in the non-statin group; p = .01). No significant difference was observed for NGAL mRNA or protein content in AAA and ILT. CONCLUSION: Simvastatin treatment in patients with AAAs may influence the concentration of proteases and their inhibitors (TIMPs) in aneurysmal wall tissue and ILTs. Thus, further studies should be undertaken to understand the different influence of statin therapy on the components of the MMP/TIMP system in AAAs and ILTs.
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Proteínas de Fase Aguda/metabolismo , Aneurisma de la Aorta Abdominal/tratamiento farmacológico , Lipocalinas/metabolismo , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Simvastatina/farmacología , Trombosis/tratamiento farmacológico , Inhibidor Tisular de Metaloproteinasa-1/metabolismo , Inhibidor Tisular de Metaloproteinasa-2/metabolismo , Anciano , Aneurisma de la Aorta Abdominal/metabolismo , Femenino , Humanos , Lipocalina 2 , Masculino , Persona de Mediana Edad , ARN Mensajero/metabolismo , Trombosis/metabolismoRESUMEN
Oenothera paradoxa (EP) preparations are commonly used in folk medicine to treat skin diseases, neuralgia, and gastrointestinal (GI) disorders. Several reports suggested that EP preparations exhibit potent anti-inflammatory and antioxidant activities both in vitro and in vivo. Here, we aimed to characterize the action of EP pomace polyphenol extract in mouse model of colitis. We analyzed the composition of EP pomace polyphenol extract using reversed phase HPLC system and ultra-performance liquid chromatography (UPLC) system coupled with a quadrupole-time of flight (Q-TOF) MS instrument. Then, we used a well-established animal model of 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis to determine the anti-inflammatory action of EP pomace polyphenol extract. We also investigated the effect of the EP pomace polyphenol extract on pro-inflammatory (IL-1ß and TNF-α) cytokine mRNA levels and hydrogen peroxide concentration in the inflamed colon. Administration of EP pomace polyphenol extract significantly improved macroscopic and microscopic damage scores, as well as myeloperoxidase (MPO) activity in TNBS-treated mice. The anti-inflammatory effect of the extract was observed after intracolonic and oral administration and was dose-dependent. Significant reduction of tissue hydrogen peroxide level after treatment with EP pomace polyphenol extract suggests that its therapeutic effect is a result of free radical scavenging. This novel finding indicates that the application of the EP pomace polyphenol extract in patients with inflammatory bowel diseases (IBDs) may become an attractive supplementary treatment for conventional anti-inflammatory therapy.
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Colitis/tratamiento farmacológico , Oenothera biennis/química , Extractos Vegetales/farmacología , Polifenoles/farmacología , Administración Oral , Animales , Antiinflamatorios/administración & dosificación , Antiinflamatorios/aislamiento & purificación , Antiinflamatorios/farmacología , Cromatografía Líquida de Alta Presión , Colitis/fisiopatología , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Depuradores de Radicales Libres/administración & dosificación , Depuradores de Radicales Libres/aislamiento & purificación , Peróxido de Hidrógeno/metabolismo , Interleucina-1beta/genética , Masculino , Espectrometría de Masas , Ratones , Ratones Endogámicos C57BL , Extractos Vegetales/administración & dosificación , Polifenoles/administración & dosificación , Polifenoles/aislamiento & purificación , Ácido Trinitrobencenosulfónico/toxicidad , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
BACKGROUND: Cyclophilin A (CyPA), a cyclosporine A-binding protein, influences abdominal aortic aneurysm (AAA) formation and the ERK1/2 signalling pathway in animal and in vitro studies. Statins decrease CyPA in smooth muscle cells although their influence on CyPA in human AAA is unknown. MATERIAL AND METHODS: The study was performed on AAA wall-tissue samples obtained from 30 simvastatin-treated and 15 non-statin patients (2:1 case to control). The patients were matched by age, sex and AAA diameter. We investigated the gene expression of CyPA, its receptor extracellular matrix metalloproteinase inducer (EMMPRIN) by real-time RT-PCR. CyPA and EMMPRIN protein level and phosphorylated extracellular signal-regulated kinases 1 and 2 (ERK1/2) were measured by Western blot. RESULTS: The AAA wall tissue from simvastatin-treated patients had significantly lower CyPA gene expression and protein levels (P = 0.0018, P = 0.0083, respectively). Furthermore, phosphorylation of ERK1 and ERK2 was markedly suppressed in the simvastatin group (P = 0.0002, P = 0.0027, respectively). However, simvastatin did not influence EMMPRIN gene and protein expression. CONCLUSION: Simvastatin-treated patients with AAA exert lower CyPA messenger RNA (mRNA), as well as CyPA intracellular protein levels and a decreased amount of phospho-ERK1/2. Thus, the interference with signalling pathways leading to CyPA formation and ERK1/2 activation reveals a new anti-inflammatory role of statins in AAA.
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Antiinflamatorios/uso terapéutico , Aorta Abdominal/efectos de los fármacos , Aneurisma de la Aorta Abdominal/tratamiento farmacológico , Ciclofilina A/análisis , Inhibidores de Hidroximetilglutaril-CoA Reductasas/uso terapéutico , Proteína Quinasa 1 Activada por Mitógenos/análisis , Proteína Quinasa 3 Activada por Mitógenos/análisis , Simvastatina/uso terapéutico , Anciano , Anciano de 80 o más Años , Aorta Abdominal/enzimología , Aneurisma de la Aorta Abdominal/enzimología , Aneurisma de la Aorta Abdominal/genética , Basigina/análisis , Basigina/genética , Western Blotting , Estudios de Casos y Controles , Ciclofilina A/genética , Regulación hacia Abajo , Femenino , Humanos , Modelos Lineales , Masculino , Persona de Mediana Edad , Fosforilación , ARN Mensajero/análisis , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
OBJECTIVES: Statins have been reported to suppress the progression of abdominal aortic aneurysm (AAA). However, the effects of statins on inflammatory processes and free radicals generation are poorly understood. METHODS: Wall samples from 51 patients (simvastatin patients, n = 34; non-statin patients, n = 17; matched by sex, age and aneurysm size) subjected to elective open AAA repair were analysed. We examined the effects of simvastatin on lipid peroxidation (4-hydroxy-trans-2-nonenal (4-HNE)), hydrogen peroxide (H(2)O(2)), tumour necrosis factor alpha (TNF-α) concentration, superoxide dismutase (SOD) and catalase (CAT) activity as well as nuclear factor kappa B (NF-κB) pathway activation in human AAA wall samples. RESULTS: Treatment with simvastatin resulted in a decrease in 4-HNE and TNF-α concentration (median 4.18 µg/mg protein vs. 4.75, p = 0.012; median 10.33 pg/ml vs. 11.81, p = 0.026, respectively). CAT activity was higher in the simvastatin group (median 3.98 U ml vs. 3.19, p = 0.023). NF-κB expression was lower (p = 0.018) in the simvastatin group. However, simvastatin had little effect on H(2)O(2) concentration (p = 0.832) and SOD activity (p = 0.401). CONCLUSION: Simvastatin inhibits free radicals and TNF-α generation and improves antioxidant capacity of human AAA wall tissue, possibly through the suppression of NF-κB activity. This may be one possible explanation how statins can inhibit AAA oxidative stress.