RESUMEN
Two eudesmane-guaiane/lindenane heterocoupled sesquiterpenoid dimers, horienoids A (1) and B (2) with new carbon skeletons, from Hedyosmum orientale were characterized by a combined method. Compound 1 featured a unique 2,10-dioxabicyclo[6.2.1]undecane core moiety with an anti-Bredt bridgehead double bond. Their biogenetic pathways were proposed to involve Diels-Alder and cascade rearrangement reactions as the key steps. Compound 2 exhibited a potent anti-inflammatory effect on LPS-induced BV-2 microglial cells.
Asunto(s)
Sesquiterpenos , Sesquiterpenos/farmacologíaRESUMEN
Euphorhelipanes A (1) and B (2), two Euphorbia diterpenoids with a new 4-(5,5-dimethylheptan-2-yl)-2,7-dimethylbicyclo[4.3.0]nonane skeleton, were isolated from a 95% ethanol extract of the whole plants of Euphorbia helioscopia. Their structures were elucidated by spectroscopic data analysis, quantum chemical calculations, and single-crystal X-ray diffraction data. Compounds 1 and 2 represent the first examples of Euphorbia diterpenoids with a 5/6 fused carbon ring system, and their plausible biosynthetic pathways originating from jatrophanes are proposed. Compounds 1 and 2 showed a triglyceride-lowering effect in oleic-acid-stimulated HuH7 cells at concentrations of 1-50 µM.
Asunto(s)
Diterpenos/aislamiento & purificación , Euphorbia/química , Hipolipemiantes/aislamiento & purificación , Triglicéridos/sangre , Línea Celular Tumoral , Diterpenos/química , Diterpenos/farmacología , Humanos , Hipolipemiantes/química , Hipolipemiantes/farmacología , Ácido Oléico/farmacologíaRESUMEN
Four ring-A seco-cucurbitane triterpenoids, colocynthenins A-D (1-4), together with seven known cucurbitane triterpenoids (5-11), were isolated from the fruits of Citrullus colocynthis. Their structures and absolute configurations were elucidated based on spectroscopic analysis and quantum chemical ECD calculations. Compound 1 possesses an unprecedented 2,11-lactone moiety, while compound 2 is the first reported cucurbitane triterpenoid with an unusual cyano group. Compounds 1 and 3 showed acetylcholinesterase inhibitory activities in a standard in vitro assay, with IC50 values of 2.6 and 3.1 µM, respectively.
Asunto(s)
Citrullus colocynthis/química , Triterpenos/aislamiento & purificación , Inhibidores de la Colinesterasa/farmacología , Frutas/química , Triterpenos/química , Triterpenos/farmacologíaRESUMEN
OBJECTIVE: To investigate the expression of macrophage migration inhibition factor (MIF) in pulmonary tissues of the smokers with and without chronic obstructive pulmonary disease (COPD). METHODS: The subjects were assigned into three groups: non-smokers without COPD (control group, n = 12), smokers without COPD (smoker group, n = 13) and smokers with COPD (COPD group, n = 16). The specimens were obtained from lung tissues as far away from cancer focus as possible (> 5 cm). Real-time quantitative PCR and immunohistochemistry were used to investigate the expression and distribution of MIF in pulmonary tissues. The relationship between the severity of airflow obstruction and the differential expressions of MIF in lung tissues of the smokers with or without COPD was analyzed. RESULTS: (1) MIF mRNA expression in COPD group (4.87 ± 1.79) was higher than that in the smoker group (2.16 ± 0.72; P < 0.01), which was higher than that in the control group (1.09 ± 0.48; P < 0.01). (2) Immunohistochemistry analysis showed that MIF protein expression in lung tissues of the COPD group (0.277 ± 0.025) was higher than that in the smokers group (0.199 ± 0.034; P < 0.01), which was significantly higher than that in control group (0.130 ± 0.021; P < 0.01). (3) Correlation analysis of MIF mRNA expression in the lung tissues and pulmonary function parameters of forced expired volume in one second (FEV(1)) percentage of predicted (FEV(1) pred)and ratio of FEV(1) to forced vital capacity (FEV(1)/FVC) suggested that MIF mRNA expression in the lung tissues was negatively related with FEV(1) pred (r = -0.578, P < 0.01) and FEV(1)/FVC (r = -0.607, P < 0.01). CONCLUSIONS: MIF expression significantly increases in the smokers with COPD, and MIF level in the lung is positively correlated with airflow limitation. The results suggest that MIF may play an important role in the pathogenesis of smoking-induced COPD.
Asunto(s)
Oxidorreductasas Intramoleculares/metabolismo , Pulmón/metabolismo , Factores Inhibidores de la Migración de Macrófagos/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Fumar/metabolismo , Anciano , Estudios de Casos y Controles , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
OBJECTIVE: To investigate the expression of ß-catenin in pulmonary tissues of smokers with and without chronic obstructive pulmonary disease (COPD). METHODS: Pulmonary tissues were obtained from patients who had underwent pneumonectomy in Tongji Hospital. The subjects were assigned into non-smokers without COPD (control group), smokers without COPD (smoker group) and smokers with COPD (smoker + COPD group) based on their pulmonary functions and smoking history, with 12 subjects each group. The specimens were obtained as far from the tumor focus (> 5 cm) as possible. Immunofluorescence staining, Western blot and real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR) were used to investigate the expression and localization of ß-catenin in pulmonary tissues. Numerical data were expressed as the mean ± standard deviation, and were assessed for significance by one-way analysis of variance followed by a Student-Newman-Keuls test for multiple comparisons. The difference of enumeration data was detected by Chi-Square test. Relationship was estimated by Pearson correlation. RESULTS: Immunofluorescence analysis revealed that ß-catenin mainly expressed in the cell membrane of epithelial cells. There was also a positive expression in the cytoplasm and the nuclei of the epithelial cells. The number of alveolar epithelial cells with ß-catenin expressed in the cytoplasm and(or) nucleus was (1.2 ± 0.6)/HP in smokers + COPD group. And the protein and mRNA expression of ß-catenin in pulmonary tissues in smokers + COPD group were 0.26 ± 0.11 and 0.351 ± 0.129, respectively, which were significantly less than those of the smoker group and the control group [(5.0 ± 2.5)/HP and (8.4 ± 3.5)/HP, 0.62 ± 0.23 and 1.00 ± 0.50, 0.60 ± 0.14 and 1.03 ± 0.27]. The differences among the 3 groups were significant (F = 12.809 - 38.776, P < 0.05). Correlation analysis between ß-catenin expression and pulmonary function suggested that the protein and mRNA expression of ß-catenin positively related with FEV(1)%pred (P < 0.05) and FEV(1)/FVC (r = 0.402 - 0.558, P < 0.05). CONCLUSION: ß-catenin expression significantly was decreased in smokers with COPD, and ß-catenin level in the lungs was positively correlated with pulmonary function.
Asunto(s)
Pulmón/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , beta Catenina/metabolismo , Adulto , Anciano , Estudios de Casos y Controles , Femenino , Humanos , Pulmón/fisiopatología , Masculino , Persona de Mediana Edad , Enfermedad Pulmonar Obstructiva Crónica/fisiopatología , Pruebas de Función Respiratoria , Fumar/metabolismo , beta Catenina/genéticaRESUMEN
OBJECTIVE: To study the effect of electroacupuncture on nitric oxide synthase (NOS) in rats with cerebral ischemia-reperfusion injury. METHODS: Focal cerebral ischemia-reperfusion model was established using modified intravascular suture technique. The NO content in the brain tissue was detected by nitrite reduction and the expressions of nNOS and iNOS were detected by immunohistochemistry. Eighty rats in this experiment were divided into the normal group, the cerebral ischemia-reperfusion injury model group (as the model group), the cerebral ischemia-reperfusion injury + electroacupuncture group (as the acupuncture group), and the cerebral ischemia-reperfusion injury + phosphatidylinositol 3 kinase (PI3-K) inhibitor group (as the inhibitor group). Each group consisted of twenty rats. Five microL PI3-K inhibitor LY294002 (400 microL) was slowly injected at the lateral cerebral ventricle of rats in the inhibitor group at a constant speed using microinjector according to Konig Klippel atlas of the stereotaxis instrument. Shuigou (DU26) and Chengjiang (RN24) were selected to determine levels of NO and NOS. RESULTS: After 24-h ischemia-reperfusion, the NO levels of the hippocampus and the cerebral cortex increased abnormally, and the expressions of nNOS and iNOS increased, showing significant difference when compared with those of the normal group (P<0.05). By electroacupuncture at Shuigou (DU26) and Chengjiang (RN24), the ischemic cerebral ischemia-reperfusion injury neuron loss was inhibited. Meanwhile, the high levels of NO, nNOS and iNOS in the cerebral cortex and the hippocampus were significantly inhibited (P<0.05). The abnormally increased expressions of nNOS and iNOS were reversed, showing significant difference when compared with the model group (P<0.05). But when compared with the normal group, there was no significant difference (P>0.05). The effects of electroacupuncture reversed the abnormally increased NO levels of the hippocampus and the cerebral cortex and expressions of nNOS and iNOS after LY294002 oppressed anti-PI3K to block the TrkA acceptor circuit. The NO levels of the hippocampus and the cerebral cortex and expressions of nNOS and iNOS increased again, showing significant difference when compared with the acupuncture group (P<0.05). CONCLUSIONS: Acupuncture fought against cerebral ischemia and reperfusion in the loss of neurons, at the same time, the abnormal regulation of NOS had reverse effect partly through TrkA/PI3K mediated signal transduction pathway.
Asunto(s)
Isquemia Encefálica/metabolismo , Electroacupuntura , Óxido Nítrico Sintasa de Tipo II/metabolismo , Óxido Nítrico Sintasa de Tipo I/metabolismo , Daño por Reperfusión/metabolismo , Animales , Óxido Nítrico/metabolismo , Ratas , Ratas Sprague-Dawley , Transducción de SeñalRESUMEN
Aim: Coronavirus disease 2019 (COVID-19) is a form of disease triggered by a new strain of coronavirus. This paper proposes a novel model termed "deep fractional max pooling neural network (DFMPNN)" to diagnose COVID-19 more efficiently. Methods: This 12-layer DFMPNN replaces max pooling (MP) and average pooling (AP) in ordinary neural networks with the help of a novel pooling method called "fractional max-pooling" (FMP). In addition, multiple-way data augmentation (DA) is employed to reduce overfitting. Model averaging (MA) is used to reduce randomness. Results: We ran our algorithm on a four-category dataset that contained COVID-19, community-acquired pneumonia, secondary pulmonary tuberculosis (SPT), and healthy control (HC). The 10 runs on the test set show that the micro-averaged F1 (MAF) score of our DFMPNN is 95.88%. Discussions: This proposed DFMPNN is superior to 10 state-of-the-art models. Besides, FMP outperforms traditional MP, AP, and L2-norm pooling (L2P).
Asunto(s)
COVID-19 , Neumonía , Algoritmos , Humanos , Redes Neurales de la Computación , SARS-CoV-2RESUMEN
Accumulating evidence indicated that smoking might directly induce pulmonary vascular remodeling at the initial stage of chronic obstructive pulmonary disease (COPD). However, the molecular mechanism underlying this process remains poorly understood. To investigate the role of cyclin D1 in pulmonary vascular remodeling, we constructed a plasmid-based short hairpin RNA (shRNA) to knock down the expression of cyclin D1 in smoking rats. Specific shRNA against cyclin D1 significantly prevented the cyclin D1 expression and the cell proliferation in rat pulmonary artery smooth muscle cells (rPASMCs). Furthermore, the plasmid-based shRNA successfully decreased the cyclin D1 protein in intra-pulmonary arteries of smoking rats and subsequently decreased the wall thickness of pulmonary vessels and the percentage of muscularized vessels. We conclude that the plasmid-based shRNA against cyclin D1 gene attenuated pulmonary vascular remodeling in smoking rats. Cyclin D1 might play a critical role in cigarette smoke-induced pulmonary vascular remodeling via regulating rPASMCs proliferation.
Asunto(s)
Ciclina D1/genética , Hiperplasia/prevención & control , Pulmón/irrigación sanguínea , Músculo Liso Vascular/patología , ARN Interferente Pequeño/uso terapéutico , Fumar/patología , Animales , Arterias/metabolismo , Arterias/patología , Presión Sanguínea/fisiología , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Ciclina D1/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Hiperplasia/inducido químicamente , Hiperplasia/patología , Pulmón/metabolismo , Pulmón/patología , Pulmón/fisiopatología , Masculino , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/efectos de los fármacos , Plásmidos/genética , Arteria Pulmonar/fisiopatología , ARN Interferente Pequeño/farmacología , Ratas , Ratas Wistar , Humo/efectos adversos , Fumar/efectos adversos , Fumar/metabolismo , Fumar/fisiopatología , Nicotiana , TransfecciónRESUMEN
Novel antilipid peroxidative carbazole alkaloids, antiostatin A5 (1), antiostatin A6 (2), and (±)-morindolestatin (3), were isolated from a new soil-derived Streptomyces sp. Compound 2 possesses an unusual cyclohexene side chain. Compound 3 was a pair of enantiomers featuring an unprecedented [1,4]oxazino[2,3-c]carbazole ring system. The absolute configuration of 3 was determined by online HPLC-ECD and ECD calculation. A racemization mechanism and putative biosynthetic pathway are discussed.
Asunto(s)
Alcaloides/aislamiento & purificación , Productos Biológicos/aislamiento & purificación , Carbazoles/aislamiento & purificación , Streptomyces/química , Alcaloides/química , Alcaloides/metabolismo , Productos Biológicos/química , Productos Biológicos/metabolismo , Carbazoles/química , Estructura Molecular , Estereoisomerismo , Streptomyces/metabolismoRESUMEN
T-cell immunoglobulin- and mucin-domain-containing molecule-3 (Tim-3) is preferentially expressed on Th1-helper type T-cells and functions to repress the Th1-mediated immune response. However, the role of Tim-3 during the inflammatory pathogenesis of asthma remains unclear. This study determines the expression level of Tim-3 in CD4+ T-cells within the peripheral blood and bronchoalveolar lavage fluid (BALF) isolated from a murine model of atopic asthma and explores the potential role of Tim-3 during the inflammatory response. Mice were randomly divided into normal control, asthma day 1, and asthma day 7 groups, and peripheral blood T lymphocytes and BALF cells were collected. The ratio of Tim-3+/CD4+ cells among the total CD4+cell populations from peripheral blood and BALF was determined by flow cytometry, and the expression of the Tim-3 mRNA was determined by Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR). In contrast with the normal control group, the ratio of Tim-3+/CD4+:CD4+ cells and the level of Tim-3 mRNA in both the peripheral blood T lymphocytes and BALF cells among the asthma day 1 and asthma day 7 groups were significantly increased (p < 0.01), and those in the asthma day 7 group were higher than the asthma day 1 group (p < 0.05). There was also a positive correlation between the ratio of Tim-3+/CD4+:CD4+ detected in BALF and that the ratio detected in peripheral blood T lymphocytes (r = 0.84, p < 0.01). Therefore, the expression of Tim-3 is increased in CD4+ T-cells following airway challenge and likely affects asthma-induced inflammation by repressing the Th1-mediated immune response.
Asunto(s)
Asma/inmunología , Receptores Virales/metabolismo , Células TH1/metabolismo , Animales , Asma/inducido químicamente , Líquido del Lavado Bronquioalveolar/citología , Líquido del Lavado Bronquioalveolar/inmunología , Recuento de Linfocito CD4 , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Recuento de Células , Eosinófilos/citología , Expresión Génica/genética , Receptor 2 Celular del Virus de la Hepatitis A , Leucocitos/citología , Masculino , Ratones , Ratones Endogámicos , Ovalbúmina/inmunología , Receptores Virales/genética , Subgrupos de Linfocitos T/citología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Células TH1/inmunologíaRESUMEN
OBJECTIVE: To investigate the gene mutation types and distribution features of α- and ß-thalassemia in reproductive population of Xing bin district of Guangxi Lai bin city so as to provide the scientific basis for formulating the preventive and control measures. METHODS: The high risk population with thalassemia in 6 498 people of child-bearing age admited in department of antenatal care of our hospital from January 2017 to December 2017 were screened by blood cell test and hemoglobin electrophoresis. The gene mutation types and mutation frequency in αandßthalassemia positive cases were diagnosed and analyzied by Gap-PCR and PCR-RDB. RESULTS: The inital screening showed that there were 1 432 cases of thalassemia positive accounting for 22.04%; the gene diagnoses showed that there were 920 cases of thalassemia gene positive accounting for 14.16%. Among 920 cases, 593 cases were α-thalassemia accounting for 64.45% (593/920); the gene mutation types were 19 kinds. The α-deletion type gene was mainly --SEA (47.22%), the α-mutatin type gene was mainly -αcsα(13.66%); 260 cases were the ß-thalassemia accounting for 28.26%, (260/920), the gene mutation types were 9 kinds, out of which the ß41-42 ßN was main (50.38%), followed by ß17/ßN (38.08%)ï¼there were 2 kinds of gene mutation types accounted for 88.46%; the αß-thalassemia numbered 67 cases (7.28%), the mutation types were mainly --SEA/ß41-42 (17.91%) and -α3.7/ß41-42 (17.91%). CONCLUSION: The α-and ß-thalassemia mostly observed in the childbearing population of Laibin city Xinbin district possess the gene comblexity and diversity as well as the significant genetic heterogeneily.The results of this study provide the reference basis for the prevention of thalassemia and eugenic works.
Asunto(s)
Mutación , Niño , China , Femenino , Genotipo , Humanos , Embarazo , Talasemia alfa , Talasemia betaRESUMEN
Euphorkanlide A (1), a highly modified ingenane diterpenoid with a C24 appendage forming an additional hexahydroisobenzofuran-fused 19-membered macrocyclic bis-lactone ring system was isolated from the roots of Euphorbia kansuensis. Its structure was determined by extensive spectroscopic analysis and quantum-chemical calculations. Compound 1 showed significant cytotoxicities against a panel of cancer cell lines (IC50s < 5 µM). Mechanistic study revealed that 1 could induce the generation of ROS, leading to cell cycle arrest and cell apoptosis in drug-resistant cancer cell line HCT-15/5-FU.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Diterpenos/farmacología , Euphorbia/química , Antineoplásicos Fitogénicos/química , Antineoplásicos Fitogénicos/aislamiento & purificación , Apoptosis/efectos de los fármacos , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Diterpenos/química , Diterpenos/aislamiento & purificación , Relación Dosis-Respuesta a Droga , Resistencia a Antineoplásicos/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Conformación Molecular , Relación Estructura-ActividadRESUMEN
Five undescribed cycloartane triterpenoids, including two cycloartane trinor-triterpenoids, were isolated from a 70% ethanol extract of the whole plant of Actaea vaginata (Ranunculaceae), together with thirteen known cycloartane triterpenoids. Their structures were determined by spectroscopic techniques and quantum chemical calculations for intramolecular noncovalent interactions with reduced density gradient method. All compounds were evaluated for their anti-inflammatory effects by a lipopolysaccharide (LPS)-stimulated nitric oxide (NO) production model in RAW264.7 macrophage cells, and some showed potent inhibitory effects with IC50 values ranging from 5.0 to 24.4⯵M. Further mechanism studies showed that one compound dose-dependently suppressed LPS-induced NO production and pro-inflammatory cytokines secretion, and decreased the expression of iNOS, through inhibiting NF-κB activation.
Asunto(s)
Actaea/química , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Triterpenos/química , Triterpenos/farmacología , Animales , Antiinflamatorios no Esteroideos/química , Antiinflamatorios no Esteroideos/farmacología , Macrófagos/citología , Macrófagos/metabolismo , Ratones , Modelos Moleculares , Conformación Molecular , FN-kappa B/metabolismo , Óxido Nítrico/biosíntesis , Células RAW 264.7 , Transducción de Señal/efectos de los fármacosRESUMEN
Fourteen acetylbenzofuran derivatives, including three undescribed carbon skeletons with a newly formed hexane or benzene ring on the other side of the benzofuran ring, (±)-eupatonin A (1), (±)-eupatonin B (2), and eupatonin C (3), two new benzofurans (-)-12ß-hydroxygynunone (4) and (+)-12-hydroxyl-13-noreuparin (5), as well as 9 known ones (6-14), were isolated from 95% ethanol extract of the roots of Eupatorium chinense. Their structures were determined by spectroscopic methods and quantum chemical DFT and TDDFT calculations of the NMR chemical shifts and ECD spectra, which helped in the determination of the relative configurations of 1 and 2 and the absolute configurations of 4 and 5, respectively. 1 and 2 were further identified to be racemic mixtures by chiral HPLC analysis. All compounds were evaluated for insulin-stimulated glucose uptake in differentiated C2C12 myotubes. Compounds 1, 3, 4, 5, 11, 12, and 13 markedly enhanced insulin-mediated glucose uptake. (±)-Eupatonin A (1) activated the IRS-1/Akt/GSK-3ß signaling pathway and enhanced insulin stimulated GLUT4 membrane translocation in C2C12 myotubes. On LPS stimulated RAW264.7 macrophages, several compounds exhibited significant inhibitory effect on NO production with IC50 values ranging from 4.94 to 9.70 µΜ. (±)-Eupatonin A (1) again dose-dependently suppressed LPS-induced NO production and decreased the expression of inducible NO synthase (iNOS), through inhibiting NF-κB activity.
Asunto(s)
Benzofuranos/farmacología , Eupatorium/química , Macrófagos/efectos de los fármacos , Fibras Musculares Esqueléticas/efectos de los fármacos , Animales , China , Proteínas de Unión al ADN/metabolismo , Glucosa/metabolismo , Insulina , Ratones , Estructura Molecular , Óxido Nítrico Sintasa de Tipo II/metabolismo , Fitoquímicos/aislamiento & purificación , Fitoquímicos/farmacología , Raíces de Plantas/química , Células RAW 264.7 , Transducción de Señal/efectos de los fármacos , Factores de Transcripción/metabolismoRESUMEN
AIM: To determine whether protein kinase C (PKC) has any effect on the expression of cyclinD1, a key regulator of growth control and G1/S transition, and to investigate the underlying molecular mechanisms of PKC involving the remodeling of the asthmatic airway smooth muscle (ASM). METHODS: The treatment of synchronized ASM cells from asthmatic rats with PKC-specific agonist phorbol 12-myristate 13-acetate (PMA) and antagonist 2-{1-[3-(amidinothio) propyl]-1Hindol-3-yl}-3-(1-methylindol-3-yl) maleimide methanesulfonate salt (Ro31-8220) was followed by the proliferation assay. PKCalpha and cyclinD1 expressions in ASM cells (ASMC) were detected by RT-PCR and Western blotting. The relation between PKCalpha and cyclinD1 was assessed by linear regression analysis. The effect of the construct recombinant plasmid pcDNA3.1-antisense cyclinD1 (pcDNA3.1-ascyclinD1) on the proliferation of ASMC was found to be induced by PMA. RESULTS: The data showed phorbol ester-dependent PKCalpha promoted the proliferation of ASMC. The closely-positive correlation existed between the expression of PKCalpha and cyclinD1 at the transcriptional (r=0.821, P<0.01) and translational (r=0.940, P<0.01) levels. pcDNA3.1-ascyclinD1 could inhibit the proliferation of ASMC. pcDNA3.1-ascyclinD1 almost completely attenuated the PMA-induced proliferation effect as Ro31-8220+pcDNA3.1. CONCLUSION: The proliferation of ASMC by PKC might by regulated by the cyclinD1 expression in asthmatic rats.
Asunto(s)
Asma/patología , Proliferación Celular/efectos de los fármacos , Ciclina D1/biosíntesis , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/patología , Proteína Quinasa C/fisiología , Animales , Células Cultivadas , Ciclina D1/genética , Dermatoglifia del ADN , Regulación de la Expresión Génica , Masculino , Plásmidos/genética , Antígeno Nuclear de Célula en Proliferación/metabolismo , Ratas , Ratas Endogámicas BNRESUMEN
BACKGROUND: Although it is recognized that bronchial smooth muscle cells (BSMCs) play a key role in airway remodeling during chronic asthma, it is not well understood how BSMCs exert their inflammatory functions. The extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway is an important signaling pathway in chronic asthma, but its influence on secretion by BSMCs has not been well-studied. We investigated the impact of ERK1/2 signaling pathway on secretion by BSMCs in a rat model of chronic asthma in this study. METHODS: To create a rat model of chronic asthma, Wistar rats underwent ovalbumim (OVA) injection and eight weeks of inhalation. BSMCs were isolated and cultured in vitro. Epidermal growth factor, PD98059 and ERK1/2 antisense oligonucleotide were used to explore the role of ERK1/2 signaling pathway. The expression of P-ERK1/2 (phospho-ERK1/2) in BSMCs was analyzed by Western blot and reverse transcriptase-polymerase chain reaction (RT-PCR). Secretion of BSMCs was detected by enzyme-linked immunosorbent assay (ELISA). RESULTS: Phospho-ERK1/2 expression was increased in BSMCs of chronic asthmatic rats compared with the controls. PD98059 inhibited expression of phospho-ERK1/2 protein, while treatment with an antisense oligonucleotide inhibited the expression of P-ERK1/2 mRNA and protein. BSMCs obtained from the chronic asthma group secreted significantly greater quantities of growth factors (transforming growth factor (TGF)-beta(1), vascular endothelial growth factor (VEGF) and connective tissue growth factor (CTGF)), cytokines (regulated upon activation, normal T cell-expressed and secreted (RANTES) and eotaxin), and extracellular matrix (fibronectin and collagen I) compared with normal controls. Epidermal growth factor stimulated secretion in both groups, but the response of the chronic asthma group was more intense. Both PD98059 and antisense oligonucleotide suppressed secretion by BSMCs in chronic ashmatic rats. Antisense oligonucleotide reduced the level of RANTES nearly to that of normal controls, while PD98059 could not. CONCLUSION: These results suggest that ERK1/2 signaling pathway may play an important role in the augmented secretion of BSMCs in chronic asthmatic rats, and ERK1/2 antisense oligonucleotide effectively inhibits the process.
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Asma/metabolismo , Bronquios/metabolismo , Sistema de Señalización de MAP Quinasas/fisiología , Proteína Quinasa 1 Activada por Mitógenos/fisiología , Proteína Quinasa 3 Activada por Mitógenos/fisiología , Miocitos del Músculo Liso/metabolismo , Animales , Quimiocina CCL5/metabolismo , Enfermedad Crónica , Modelos Animales de Enfermedad , Masculino , Ratas , Ratas Wistar , Factor de Crecimiento Transformador beta1/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
BACKGROUND: Airway smooth muscle (ASM) is suspected to be a determining factor in the structural change of asthma. However, the role of protein kinase C alpha (PKCalpha) and cyclin D1 involved in the dysfunction of ASM leading to asthmatic symptoms is not clear. In this study, the central role of PKCalpha and cyclin D1 in ASM proliferation in asthmatic rats was explored. METHODS: Thirty-six pathogen-free male Brown Norway (BN) rats were randomly divided into 2 groups: control groups (group N1, N2 and N3) and asthmatic groups (group A1, A2, and A3). Groups A1, A2 and A3 were challenged with ovalbumin (OA) for 2 weeks, 4 weeks and 8 weeks respectively. Control animals were exposed to an aerosolized sterile phosphate buffered saline (PBS). The ASM mass and nucleus numbers were studied to estimate the degree of airway remodeling by the hematoxylin-eosin staining method. PKCalpha and cyclin D1 expression in the ASM cells was detected by reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry. The relation between PKCalpha and cyclin D1 was assessed by linear regression analysis. PKC agonist phorbol 12-myristate 13-acetate (PMA), PKC inhibitor Ro31-8220 and an antisense oligonucleotide against cyclin D1 (ASOND) were used to treat ASM cells (ASMCs) obtained from the 2 weeks asthmatic rats. The cyclin D1 protein expression level was detected by Western blotting. RESULTS: Compared with the control group, the PKCalpha and cyclin D1 mRNA levels were increased in the asthmatic group. Similar to RT-PCR results, immunohistochemistry analysis for PKCalpha and cyclin D1 expression revealed an increased production in ASMCs after allergen treatment for 2, 4 and 8 weeks compared with the respective control groups. No difference in expression of PKCalpha and cyclin D1 in ASM were found in the 2, 4 or 8 weeks asthmatic rats. There were significant positive correlations between PKCalpha and cyclin D1 expression, both transcriptionally (r = 0.944, P < 0.01) and translationally (r = 0.826, P < 0.01), in ASM. The content of cyclin D1 in asthmatic ASMCs increased after being stimulated by PMA, and decreased when induced by Ro31-8220. ASOND targeting for cyclin D1 lowered the expression of cyclin D1 induced by PMA. CONCLUSIONS: Increased expression of PKCalpha and cyclin D1 in ASM along with smooth muscle structure changes might implicate PKCalpha and cyclin D1 participation in the proliferation of ASM and contribute to the pathogenesis of asthma after repeated allergen exposure in rats. The results suggested that cyclin D1 might be downstream of PKC signal transduction pathway.
Asunto(s)
Asma/patología , Ciclina D1/fisiología , Pulmón/patología , Miocitos del Músculo Liso/patología , Proteína Quinasa C-alfa/fisiología , Animales , Proliferación Celular , Ciclina D1/genética , Masculino , Proteína Quinasa C-alfa/genética , ARN Mensajero/análisis , Ratas , Ratas Endogámicas BNRESUMEN
This study is to investigate the expression of CyclinD1 in asthmatic rats and construct expression plasmids of sense and antisense CyclinD1 gene and transfect them to asthmatic airway smooth muscle cell to study the effects of CyclinD1 on the proliferation of airway smooth muscle cells in asthmatic rats. CyclinD1 cDNA was obtained by RT-PCR of total RNA extracted from the airway smooth muscle in asthmatic rats. The sequence was inserted into eukaryotic expression vector pcDNA3.1 (+) to recombinate the sense and antisense pcDNA3.1-CyclinD1 eukaryotic expression vector. The two recombinations and vector were then separately transfected into airway smooth muscle cell in asthmatic rats by using liposome. The expression level of CyclinD1 was certificated by Western blotting analysis. The proliferations of ASMCs isolated from asthmatic rats were examined with cell cycle analysis, MTT colorimetric assay and proliferating cell nuclear antigen (PCNA) immunocytochemical staining. Results showed (1) Compared with control group, the content of CyclinD1 was significantly increased; (2) It was comformed by restriction endonucleasa digestion and DNA sequence analysis that the expression plasmid of sense and antisense CyclinD1 were successfully recombinated. There was significant change of CyclinD1 expression between vector and sense CyclinD1 transfected cells, and the expression level of CyclinD1 in ASMC transfected with antisense CyclinD1 was lower than that in vector transfected cells (P <0.01); (3) In the asthmatic groups, compared with the vecter group, the percentage of S + G2M phase, absorbance A value of MTT and the expression rate of PCNA protein in ASMC transfected with pcDNA3. 1-CyclinD1 vector significantly increased. The values decreased remarkably in the pcDNA3,1-as CyclinD1 group. Statistical analysis revealed that there were significant differences in these indicators of cell proliferation in three groups (P <0.01). In the normal groups, statistical analysis revealed that there were significant differences in the percentage of S + G2M phase, a value of MTT and the expression rate of PCNA protein in three groups (P <0.01). Sense CyclinD1 eukaryotic expression vectors could have a positive effect on the proliferation of ASMC, however the antisence one have a negative effect, which implicated that CyclinD1 might contribute to the process of airway smooth muscle cell proliferation.
Asunto(s)
Asma/patología , Proliferación Celular/efectos de los fármacos , Ciclina D1/antagonistas & inhibidores , ADN sin Sentido/farmacología , Miocitos del Músculo Liso/efectos de los fármacos , Animales , Ciclo Celular/efectos de los fármacos , Codón/genética , Codón/farmacología , Ciclina D1/agonistas , Ciclina D1/genética , ADN sin Sentido/genética , Modelos Animales de Enfermedad , Expresión Génica , Vectores Genéticos/genética , Masculino , Miocitos del Músculo Liso/patología , Ratas , Ratas Sprague-Dawley , Recombinación Genética/genética , Sistema Respiratorio , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción Genética , TransfecciónRESUMEN
OBJECTIVE: To explore the role of PKCalpha-ERK1/2 cascade in PMA induced up-regulation of cyclinD1 and P21(cip1) in human airway smooth muscle cells (HASMCs) sensitized by sera from atopic asthmatics. METHODS: HASMCs in cultures were passively sensitized by 10% serum from asthmatic patients and were randomly divided into five groups: the control group, PMA treated group, PMA and PKCalpha mismatched Oligodeoxynucleotides (PKCalpha-mmODN) treated group, PMA and PKCalpha antisense Oligodeoxynucleotides (PKCalpha-asODN) treated group, PMA and U0126 (MAP Kinase Kinase inhibitor)treated group. The expression of p-PKCalpha, ERK1/2, p-ERK1/2, cyclinD1 and P21(cip1) protein were determined by western blotting. The proliferation of HASMC was examined by cell cycle analysis and MTT colorimetric assay. RESULTS: Compared with the control group, the expression of p-PKCalpha and ERK1/2, p-ERK1/2 protein increased, the expression of cyclinD1, P21(cip1) protein increased correspondingly (the A value % control was 2.10 +/- 0.29, 1.67 +/- 0.19, 2.20 +/- 0.27, 1.99 +/- 0.22 and 3.11 +/- 0.29 respectively; q value was 9.87, 7.06, 10.57, 11.10 and 20.33 respectively; all P < 0.05) in PMA treated group, and cells proliferation [the percentage of cells in S phase was (30.3 +/- 2.4)%, A(490) value was 0.80 +/- 0.06] enhanced significantly compared with those [the percentage of cells in S phase was (13.9 +/- 2.6)%, A(490) value was 0.41 +/- 0.04] of the control group (q = 6.07, 12.63; all P < 0.05). In PMA and PKCalpha-asODN treated group, the level of p-PKCalpha decreased, the expression of ERK1/2, p-ERK1/2 and the expression of cyclinD1, P21(cip1) decreased correspondingly (the A value % control was 1.23 +/- 0.19, 1.34 +/- 0.18, 1.52 +/- 0.20, 1.45 +/- 0.18 and 1.49 +/- 0.18 respectively; q value was 7.49, 3.58, 5.97, 6.06 and 15.65 respectively; all P < 0.05), and cells proliferation reduced significantly [the percentage of cells in S phase was (21.2 +/- 2.8)%, A(490) value was 0.51 +/- 0.04; q = 6.07, 12.63; all P < 0.05], as compared with those of the PMA treated group. In PMA and U0126 treated group, the level of p-PKCalpha had no significant change (A value was1.99 +/- 0.18, q = 0.94, P > 0.05), but the levels of ERK1/2, p-ERK1/2 decreased, the expression of cyclinD1, P21(cip1) reduced (the A value % control was 0.95 +/- 0.21, 1.15 +/- 0.19, 1.37 +/- 0.15 and 1.96 +/- 0.21 respectively; q value was 7.79, 9.16, 6.92 and 11.16 respectively; all P < 0.05), and cells proliferation reduced significantly [the percentage of cells in S phase was (22.0 +/- 3.2)%, A(490) value was 0.49 +/- 0.03; q = 5.51, 13.45; all P < 0.05], as compared with those of the PMA treated group. CONCLUSION: ERK1/2 is one of the downstream regulators of PKCalpha, and PKCalpha-ERK1/2 cascade is involved in PMA induced up-regulation of cyclinD1 and P21(cip1) and proliferation in HASMC sensitized by sera from atopic asthmatics.
Asunto(s)
Asma/metabolismo , Ciclina D1/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Miocitos del Músculo Liso/metabolismo , Proteína Quinasa C-alfa/metabolismo , Adulto , Asma/sangre , Ciclo Celular , Proliferación Celular , Células Cultivadas , Femenino , Regulación de la Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Miocitos del Músculo Liso/citología , Transducción de Señal , Regulación hacia Arriba , Adulto JovenRESUMEN
BACKGROUND: It has been demonstrated that the phenotypic modulation of airway smooth muscle cells (ASMCs) is important to the pathogenesis of airway remodeling in chronic asthma. The extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway is one of the most important transduction pathways involved in the process of asthma; however, its role in the phenotypic transition of ASMCs remains unclear. OBJECTIVES: To examine the role of ERK1/2 in the phenotypic modulation of ASMCs in the rat model of chronic asthma. METHODS: Bronchial smooth muscle strips were cultured in vitro in the presence of the ERK1/2 agonist epidermal growth factor or/and the MEK inhibitor PD98059. The phenotype of ASMCs was determined by observing these cells under an electron microscope and analyzing expression of phenotypic markers (smooth muscle alpha-actin for the contractile phenotype and osteopontin for the synthetic) by using Western blot and reverse-transcriptase polymerase chain reaction, respectively. RESULTS: The phenotype of the ASMCs from the chronic asthmatic rats changed from the contractile type to the synthetic type with synthetic organelles abundantly gathered around the nucleus and altered expression of phenotypic markers. ERK1/2 was strongly expressed in the ASMCs of the chronic asthmatic rats and its activation by epidermal growth factor excessively promoted the synthetic function of ASMCs; the MEK inhibitor PD98059, however, reversed this phenotypic change in the ASMCs. CONCLUSIONS: Our results reveal a key role of the ERK1/2 signaling pathway in the phenotypic modulation of ASMCs in chronic asthmatic rats, indicating that specific inhibition of ERK1/2 in ASMCs may be therapeutically valuable in the control of airway remodeling in chronic asthma.