RESUMO
BACKGROUND: Guizhou Plateau, as one of the original centers of tea plant, has a profound multi-ethnic cultural heritage and abundant tea germplasm resources. However, the impact of indigenous community factors on the genetic diversity, population structure and geographical distribution of tea plant is still unclear. RESULTS: Using the genotyping-by-sequencing (GBS) approach, we collected 415 tea plant accessions from the study sites, estimated genetic diversity, developed a core collection, and conducted a genome-wide association study (GWAS) based on 99,363 high-quality single-nucleotide polymorphisms (SNPs). A total of 415 tea accessions were clustered into six populations (GP01, GP02, GP03, GP04, GP05 and GP06), and the results showed that GP04 and GP05 had the highest and lowest genetic diversity (Pi = 0.214 and Pi = 0.145, respectively). Moreover, 136 tea accessions (33%) were selected to construct the core set that can represent the genetic diversity of the whole collection. By analyzing seven significant SNP markers associated with the traits such as the germination period of one bud and two leaves (OTL) and the germination period of one bud and three leaves (OtL), four candidate genes possibly related to OTL and OtL were identified. CONCLUSIONS: This study revealed the impact of indigenous communities on the population structure of 415 tea accessions, indicating the importance of cultural practices for protection and utilization of tea plant genetic resources. Four potential candidate genes associated with the OTL and OtL of tea plant were also identified, which will facilitate genetic research, germplasm conservation, and breeding.
Assuntos
Variação Genética , Estudo de Associação Genômica Ampla , Melhoramento Vegetal , Fenótipo , Chá , Polimorfismo de Nucleotídeo ÚnicoRESUMO
BACKGROUND: S1P (sphingosine-1-phosphate) has been reported to possess vasodilatory properties, but the underlying pathways are largely unknown. METHODS: Isolated mouse mesenteric artery and endothelial cell models were used to determine S1P-induced vasodilation, intracellular calcium, membrane potentials, and calcium-activated potassium channels (KCa2.3 and KCa3.1 [endothelial small- and intermediate-conductance calcium-activated potassium channels]). Effect of deletion of endothelial S1PR1 (type 1 S1P receptor) on vasodilation and blood pressure was evaluated. RESULTS: Mesenteric arteries subjected to acute S1P stimulation displayed a dose-dependent vasodilation response, which was attenuated by blocking endothelial KCa2.3 or KCa3.1 channels. In cultured human umbilical vein endothelial cells, S1P stimulated immediate membrane potential hyperpolarization following activation of KCa2.3/KCa3.1 with elevated cytosolic Ca2+. Further, chronic S1P stimulation enhanced expression of KCa2.3 and KCa3.1 in human umbilical vein endothelial cells in dose- and time-dependent manners, which was abolished by disrupting either S1PR1-Ca2+ signaling or downstream Ca2+-activated calcineurin/NFAT (nuclear factor of activated T-cells) signaling. By combination of bioinformatics-based binding site prediction and chromatin immunoprecipitation assay, we revealed in human umbilical vein endothelial cells that chronic activation of S1P/S1PR1 promoted NFATc2 nuclear translocation and binding to promoter regions of KCa2.3 and KCa3.1 genes thus to upregulate transcription of these channels. Deletion of endothelial S1PR1 reduced expression of KCa2.3 and KCa3.1 in mesenteric arteries and exacerbated hypertension in mice with angiotensin II infusion. CONCLUSIONS: This study provides evidence for the mechanistic role of KCa2.3/KCa3.1-activated endothelium-dependent hyperpolarization in vasodilation and blood pressure homeostasis in response to S1P. This mechanistic demonstration would facilitate the development of new therapies for cardiovascular diseases associated with hypertension.
Assuntos
Hipertensão , Vasodilatação , Camundongos , Humanos , Animais , Pressão Sanguínea , Endotélio/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/genética , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Homeostase , Canais de Potássio Ativados por Cálcio de Condutância Baixa/genética , Canais de Potássio Ativados por Cálcio de Condutância Baixa/metabolismoRESUMO
BACKGROUND: Camellia tachangensis F. C. Zhang is a five-compartment species in the ovary of tea group plants, which represents the original germline of early differentiation of some tea group plants. METHODS AND RESULTS: In this study, we analyzed single-nucleotide polymorphisms (SNPs) at the genome level, constructed a phylogenetic tree, analyzed the genetic diversity, and further investigated the population structure of 100 C. tachangensis accessions using the genotyping-by-sequencing (GBS) method. A total of 91,959 high-quality SNPs were obtained. Population structure analysis showed that the 100 C. tachangensis accessions clustered into three groups: YQ-1 (Village Group), YQ-2 (Forest Group) and YQ-3 (Transition Group), which was further consistent with the results of phylogenetic analysis and principal component analyses (PCA). In addition, a comparative analysis of the genetic diversity among the three populations (Forest, Village, and Transition Groups) detected the highest genetic diversity in the Transition Group and the highest differentiation between Forest and Village Groups. CONCLUSIONS: C. tachangensis plants growing in the forest had different genetic backgrounds from those growing in villages. This study provides a basis for the effective protection and utilization of C. tachangensis populations and lays a foundation for future C. tachangensis breeding.
Assuntos
Camellia , Variação Genética , Filogenia , Polimorfismo de Nucleotídeo Único , Camellia/genética , Polimorfismo de Nucleotídeo Único/genética , China , Variação Genética/genética , Genética Populacional/métodos , Genótipo , Análise de Componente Principal , Genoma de PlantaRESUMO
BACKGROUND: Vascular endothelial dysfunction is regarded as an early event of hypertension. Galectin-3 (Gal-3) is known to participate in various pathological processes. Whilst previous studies showed that inhibition of Gal-3 effectively ameliorates angiotensin II (Ang II)-induced atherosclerosis or hypertension, it remains unclear whether Ang II regulates Gal-3 expression and actions in vascular endothelium. METHODS: Using techniques of molecular biology and myograph, we investigated Ang II-mediated changes in Gal-3 expression and activity in thoracic aortas and mesenteric arteries from wild-type and Gal-3 gene deleted (Gal-3-/-) mice and cultured endothelial cells. RESULTS: The serum level of Gal-3 was significantly higher in hypertensive patients or in mice with chronic Ang II-infusion. Ang II infusion to wild-type mice enhanced Gal-3 expression in the aortic and mesenteric arteries, elevated systolic blood pressure and impaired endothelium-dependent relaxation of the thoracic aortas and mesenteric arteries, changes that were abolished in Gal-3-/- mice. In human umbilical vein endothelial cells, Ang II significantly upregulated Gal-3 expression by promoting nuclear localization of Yes-associated protein (YAP) and its interaction with transcription factor Tead1 with enhanced YAP/Tead1 binding to Gal-3 gene promoter region. Furthermore, Gal-3 deletion augmented the bioavailability of nitric oxide, suppressed oxidative stress, and alleviated inflammation in the thoracic aorta of Ang II-infused mice or endothelial cells exposed to Ang II. CONCLUSIONS: Our results demonstrate for the first time that Ang II upregulates Gal-3 expression via increment in YAP nuclear localization in vascular endothelium, and that Gal-3 mediates endothelial dysfunction contributing to the development of hypertension.
Assuntos
Angiotensina II , Hipertensão , Camundongos , Humanos , Animais , Angiotensina II/farmacologia , Angiotensina II/metabolismo , Galectina 3/genética , Galectina 3/metabolismo , Hipertensão/metabolismo , Transdução de Sinais , Células Endoteliais da Veia Umbilical Humana/metabolismo , Endotélio Vascular/metabolismo , Pressão SanguíneaRESUMO
Takotsubo syndrome (TTS) is characterized by short-term contractile dysfunction with its mechanism undefined. We showed that activation of cardiac Hippo pathway mediates mitochondrial dysfunction and that stimulation of ß-adrenoceptors (ßAR) activates Hippo pathway. Here, we investigated the role of ßAR-Hippo signaling in mediating mitochondrial dysfunction in isoproterenol (Iso)-induced TTS-like mouse model. Elderly postmenopausal female mice were administered with Iso (1.25 mg/kg/h for 23 h). Cardiac function was determined by serially echocardiography. At days 1 and 7 post-Iso exposure, mitochondrial ultrastructure and function were examined by electron microscopy and various assays. Alterations in cardiac Hippo pathway and effects of genetic inactivation of Hippo kinase (Mst1) on mitochondrial damage and dysfunction in the acute phase of TTS were investigated. Isoproterenol exposure induced acute increase in biomarkers of cardiac damage and ventricular contractile dysfunction and dilation. At day 1 post-Iso, we observed extensive abnormalities in mitochondrial ultrastructure, downregulation of mitochondrial marker proteins, and mitochondrial dysfunction evidenced by lower ATP content, increased lipid droplets, higher contents of lactate, and augmented reactive oxygen species (ROS). All changes were reversed by day 7. ßAR stimulation led to activation of cardiac Hippo pathway with enhanced expression of Hippo kinase Mst1 and inhibitory YAP phosphorylation, as well as reduced nuclear YAP-TEAD1 interaction. In mice with cardiac expression of inactive mutant Mst1 gene, acute mitochondrial damage and dysfunction were mitigated. Stimulation of cardiac ßAR activates Hippo pathway that mediates mitochondrial dysfunction with energy insufficiency and enhanced ROS, promoting acute but short-term ventricular dysfunction.NEW & NOTEWORTHY Takotsubo syndrome (TTS) is featured by activation of sympatho-ß-adrenoceptor (ßAR) system leading to acute loss of ventricular contractile performance. However, the molecular mechanism remains undefined. We demonstrated, in an isoproterenol-induced murine TTS-like model, extensive mitochondrial damage, metabolic dysfunction, and downregulated mitochondrial marker proteins, changes temporarily associated with cardiac dysfunction. Mechanistically, stimulation of ßAR activated Hippo signaling pathway and genetic inactivation of Mst1 kinase ameliorated mitochondrial damage and metabolic dysfunction at the acute phase of TTS.
Assuntos
Via de Sinalização Hippo , Cardiomiopatia de Takotsubo , Feminino , Camundongos , Animais , Cardiomiopatia de Takotsubo/induzido quimicamente , Isoproterenol , Espécies Reativas de Oxigênio , Modelos Animais de Doenças , Receptores Adrenérgicos betaRESUMO
ABSTRACT: Mitochondrial dysfunction plays a key role in the development of heart failure, but targeted therapeutic interventions remain elusive. Previous studies have shown coenzyme Q10 (CoQ10) insufficiency in patients with heart disease with undefined mechanism and modest effectiveness of CoQ10 supplement therapy. Using 2 transgenic mouse models of cardiomyopathy owing to cardiac overexpression of Mst1 (Mst1-TG) or ß 2 -adrenoceptor (ß 2 AR-TG), we studied changes in cardiac CoQ10 content and alterations in CoQ10 biosynthesis genes. We also studied in Mst1-TG mice effects of CoQ10, delivered by oral or injection regimens, on both cardiac CoQ10 content and cardiomyopathy phenotypes. High performance liquid chromatography and RNA sequencing revealed in both models significant reduction in cardiac content of CoQ10 and downregulation of most genes encoding CoQ10 biosynthesis enzymes. Mst1-TG mice with 70% reduction in cardiac CoQ10 were treated with CoQ10 either by oral gavage or i.p. injection for 4-8 weeks. Oral regimens failed in increasing cardiac CoQ10 content, whereas injection regimen effectively restored the cardiac CoQ10 level in a time-dependent manner. However, CoQ10 restoration in Mst1-TG mice did not correct mitochondrial dysfunction measured by energy metabolism, downregulated expression of marker proteins, and oxidative stress nor to preserve cardiac contractile function. In conclusion, mouse models of cardiomyopathy exhibited myocardial CoQ10 deficiency likely due to suppressed endogenous synthesis of CoQ10. In contrast to ineffectiveness of oral administration, CoQ10 administration by injection regimen in cardiomyopathy mice restored cardiac CoQ10 content, which, however, failed in achieving detectable efficacy at molecular and global functional levels.
Assuntos
Cardiomiopatias , Ubiquinona , Camundongos , Animais , Ubiquinona/metabolismo , Ubiquinona/uso terapêutico , Cardiomiopatias/tratamento farmacológico , Cardiomiopatias/genética , Coração , Camundongos TransgênicosRESUMO
Activation of the sympatho-ß-adrenergic receptors (ß-ARs) system is a hallmark of heart failure, leading to fibrosis and arrhythmias. Connexin 43 (Cx43) is the most abundant gap junctional protein in the myocardium. Current knowledge is limited regarding Cx43 remodelling in diverse cell types in the diseased myocardium and the underlying mechanism. We studied cell type-dependent changes in Cx43 remodelling due to ß-AR overactivation and molecular mechanisms involved. Mouse models of isoproterenol stimulation or transgenic cardiomyocyte overexpression of ß2 -AR were used, which exhibited cardiac fibrosis and up-regulated total Cx43 abundance. In both models, whereas Cx43 expression in cardiomyocytes was reduced and more laterally distributed, fibroblasts exhibited elevated Cx43 expression and enhanced gap junction communication. Mechanistically, activation of ß2 -AR in fibroblasts in vitro elevated Cx43 expression, which was abolished by the ß2 -antagonist ICI-118551 or protein kinase A inhibitor H-89, but simulated by the adenylyl cyclase activator forskolin. Our in vitro and in vivo data showed that ß-AR activation-induced production of IL-18 sequentially stimulated Cx43 expression in fibroblasts in a paracrine fashion. In summary, our findings demonstrate a pivotal role of ß-AR in mediating distinct and cell type-dependent changes in the expression and distribution of Cx43, leading to pathological gap junction remodelling in the myocardium.
Assuntos
Conexina 43/metabolismo , Receptores Adrenérgicos beta/metabolismo , Animais , Arritmias Cardíacas/tratamento farmacológico , Arritmias Cardíacas/metabolismo , Células Cultivadas , Conexinas/metabolismo , Fibroblastos/metabolismo , Junções Comunicantes/efeitos dos fármacos , Junções Comunicantes/metabolismo , Isoproterenol/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Miocárdio/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Propanolaminas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacosRESUMO
Chronic islet inflammation is associated with development of type 2 diabetes mellitus (T2DM). Intermediate-conductance calcium-activated K+ (KCa3.1) channel plays an important role in inflammatory diseases. However, the role and regulation of KCa3.1 in pancreatic ß cells in progression of T2DM remain unclarified. In the present study, we evaluated the effect of the specific KCa3.1 channel blocker 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34) on diabetic phenotype in the db/db model. In diabetic mice, blockade of KCa3.1 significantly improved glucose tolerance, enhanced secretion of postprandial insulin level, and reduced loss of ß-cell mass through attenuating the expression and secretion of inflammatory mediators. Furthermore, in cultured pancreatic ß cells, exposure to high levels of glucose or palmitic acid significantly increased expression and current density of the KCa3.1 channel as well as secretion of proinflammatory chemokines, and the effects were similarly reversed by preincubation with TRAM-34 or a NF-κB inhibitor pyrrolidinedithiocarbamate. Additionally, expression of KCa3.1 in pancreas islet cells was up-regulated by activation of NF-κB with IL-1ß stimulation. In summary, up-regulated KCa3.1 due to activation of NF-κB pathway leads to pancreatic inflammation via expression and secretion of chemokines and cytokines by pancreatic ß cells, thereby facilitating progression of T2DM.-Pang, Z.-D., Wang, Y., Wang, X.-J., She, G., Ma, X.-Z., Song, Z., Zhao, L.-M., Wang, H.-F., Lai, B.-C., Gou, W., Du, X.-J., Deng, X.-L. KCa3.1 channel mediates inflammatory signaling of pancreatic ß cells and progression of type 2 diabetes mellitus.
Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Células Secretoras de Insulina/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Transdução de Sinais , Animais , Glicemia/metabolismo , Linhagem Celular , Células Cultivadas , Diabetes Mellitus Tipo 2/prevenção & controle , Insulina/sangue , Células Secretoras de Insulina/efeitos dos fármacos , Interleucina-1beta/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Pirazóis/farmacologia , Pirazóis/uso terapêuticoRESUMO
Elevated plasma free fatty acids level has been implicated in the development of insulin resistance, inflammation, and endothelial dysfunction in diabetic and nondiabetic individuals. However, the underlying mechanisms still remain to be defined. Herein, we investigated the effect of palmitic acid (PA), the most abundant saturated fatty acid in the human body, on small-conductance Ca2+-activated potassium channels (KCa2.3)-mediated relaxation in rodent resistance arteries and the underlying molecular mechanism. The effect of PA on KCa2.3 in endothelium was evaluated using real-time PCR, Western blotting, whole-cell patch voltage-clamp, wire and pressure myograph system, and reactive oxygen species (ROS) were measured by using dihydroethidium and 2', 7'-dichlorofluorescein diacetate. KCa2.3-mediated vasodilatation responses to acetylcholine and NS309 (agonist of KCa2.3 and KCa3.1) were impaired by incubation of normal mesenteric arteries with 100⯵Mâ¯PA for 24â¯h. In cultured human umbilical vein endothelial cells (HUVECs), PA decreased KCa2.3 current and expression at mRNA and protein levels. Incubation with the NADPH oxidase (Nox) inhibitor dibenziodolium (DPI) partly inhibited the PA-induced ROS production and restored KCa2.3 expression. Inhibition of either p38-MAPK or NF-κB using specific inhibitors (SB203580, SB202190 or Bay11-7082, pyrrolidinedithiocarbamate) attenuated PA-induced downregulation of KCa2.3 and inhibition of p38-MAPK also attenuated PA-induced phosphorylation of NF-κB p65. Furthermore, DPI reversed the increment of phospho-p38-MAPK by PA. These results demonstrated that PA downregulated KCa2.3 expressions via Nox/ROS/p38-MAPK/NF-κB signaling leading to endothelial vasodilatory dysfunction.
Assuntos
Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Ácido Palmítico/farmacologia , Canais de Potássio Ativados por Cálcio de Condutância Baixa/metabolismo , Animais , Células Cultivadas , Células Endoteliais da Veia Umbilical Humana , Humanos , Masculino , Artérias Mesentéricas/efeitos dos fármacos , Artérias Mesentéricas/metabolismo , Artérias Mesentéricas/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , NADPH Oxidases/metabolismo , NF-kappa B/metabolismo , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Vasodilatação/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Monocyte migration into diseased tissues contributes to the pathogenesis of diseases. Intermediate-conductance Ca2+-activated K+ (KCa3.1) channels play an important role in cell migration. However, the role of KCa3.1 channels in mediating monocyte migration induced by palmitic acid (PA) is still unclear. Using cultured THP-1 cells and peripheral blood mononuclear cells from healthy subjects, we investigated the role and signaling mechanisms of KCa3.1 channels in mediating the migration induced by PA. Using methods of Western blotting analysis, RNA interference, cell migration assay and ELISA, we found that PA-treated monocytes exhibited increment of the protein levels of KCa3.1 channel and monocyte chemoattractant protein-1 (MCP-1), and the effects were reversed by co-incubation of PA with anti-TLR2/4 antibodies or by specific inhibitors of p38-MAPK, or NF-κB. In addition, PA increased monocyte migration, which was abolished by a specific KCa3.1 channel blocker, TRAM-34, or KCa3.1 small interfering RNA (siRNA). The expression and secretion of MCP-1 induced by PA was also similarly prevented by TRAM-34 and KCa3.1 siRNA. These results demonstrate for the first time that PA upregulates KCa3.1 channels through TLR2/4, p38-MAPK and NF-κB pathway to promote the expression of MCP-1, and then induce the trans-endothelial migration of monocytes.
Assuntos
Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Monócitos/efeitos dos fármacos , Monócitos/fisiologia , Ácido Palmítico/farmacologia , Quimiocina CCL2/metabolismo , Humanos , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/antagonistas & inibidores , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/genética , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , NF-kappa B/metabolismo , Bloqueadores dos Canais de Potássio/farmacologia , Pirazóis/farmacologia , RNA Interferente Pequeno/genética , Transdução de Sinais/efeitos dos fármacos , Células THP-1 , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
BACKGROUND Lymphocyte-to-monocyte ratio (LMR) is an independent predictive factor of clinical outcome of acute ischemic stroke and cancer, but the predictive effect of LMR in spontaneous intracerebral hemorrhage (ICH) is unknown. Thus, the aim of this study was to explore the impact of peripheral LMR on the neurological deterioration (ND) during the initial week after spontaneous ICH onset, as well as 90-day mortality. MATERIAL AND METHODS The clinical data of 558 consecutive patients with ICH were retrospectively analyzed. LMR is calculated by absolute lymphocyte count divided by absolute monocyte count. RESULTS Of these patients, 166 patients experienced ND during the first week after admission and 72 patients died within 90 days. Multivariate analysis indicated that white blood cells (WBC), absolute neutrophil count (ANC), absolute lymphocyte count (ALC), neutrophil-to-lymphocyte ratio (NLR), LMR were significantly associated with ND during the initial week after ICH onset and also were associated with 90-day mortality. Moreover, NLR and LMR showed a higher predictive ability in ND during the initial week after ICH onset than 90-day mortality in receiver operating characteristic analysis. The best cut-off points of NLR and LMR in predicting ND and 90-day mortality were 10.24 and 2.21 and 16.81 and 2.19, respectively. CONCLUSIONS Our results suggest that LMR on admission is a predictive factor for ND during the initial week after ICH onset, as well as 90-day mortality.
Assuntos
Hemorragia Cerebral/fisiopatologia , Linfócitos/citologia , Monócitos/citologia , Adulto , Idoso , Hemorragia Cerebral/mortalidade , China , Feminino , Humanos , Contagem de Leucócitos/métodos , Contagem de Linfócitos/métodos , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Doenças Neurodegenerativas , Razão de Chances , Prognóstico , Curva ROC , Estudos Retrospectivos , Fatores de Risco , Acidente Vascular Cerebral/mortalidade , Acidente Vascular Cerebral/fisiopatologiaRESUMO
PURPOSE: This aim of this study was to investigate the key genes and pathways involved in the response to pain in goat and sheep by transcriptome sequencing. METHODS: Chronic pain was induced with the injection of the complete Freund's adjuvant (CFA) in sheep and goats. The animals were divided into four groups: CFA-treated sheep, control sheep, CFA-treated goat, and control goat groups (n = 3 in each group). The dorsal root ganglions of these animals were isolated and used for the construction of a cDNA library and transcriptome sequencing. Differentially expressed genes (DEGs) were identified in CFA-induced sheep and goats and gene ontology (GO) enrichment analysis was performed. RESULTS: In total, 1748 and 2441 DEGs were identified in CFA-treated goat and sheep, respectively. The DEGs identified in CFA-treated goats, such as C-C motif chemokine ligand 27 (CCL27), glutamate receptor 2 (GRIA2), and sodium voltage-gated channel alpha subunit 3 (SCN3A), were mainly enriched in GO functions associated with N-methyl-D-aspartate (NMDA) receptor, inflammatory response, and immune response. The DEGs identified in CFA-treated sheep, such as gamma-aminobutyric acid (GABA)-related DEGs (gamma-aminobutyric acid type A receptor gamma 3 subunit [GABRG3], GABRB2, and GABRB1), SCN9A, and transient receptor potential cation channel subfamily V member 1 (TRPV1), were mainly enriched in GO functions related to neuroactive ligand-receptor interaction, NMDA receptor, and defense response. CONCLUSIONS: Our data indicate that NMDA receptor, inflammatory response, and immune response as well as key DEGs such as CCL27, GRIA2, and SCN3A may regulate the process of pain response during chronic pain in goats. Neuroactive ligand-receptor interaction and NMDA receptor as well as GABA-related DEGs, SCN9A, and TRPV1 may modulate the process of response to pain in sheep. These DEGs may serve as drug targets for preventing chronic pain.
Assuntos
Dor Crônica/genética , Gânglios Espinais/fisiopatologia , Transdução de Sinais/genética , Transcriptoma/genética , Adjuvantes Imunológicos , Animais , Dor Crônica/induzido quimicamente , Dor Crônica/fisiopatologia , Modelos Animais de Doenças , Adjuvante de Freund , Perfilação da Expressão Gênica , Biblioteca Gênica , Ontologia Genética , Cabras , Limiar da Dor/fisiologia , Ovinos , Transdução de Sinais/fisiologia , Transcriptoma/fisiologiaRESUMO
The present study was designed to investigate whether large conductance Ca2+ -activated K+ (BK) channels were regulated by epidermal growth factor (EGF) receptor (EGFR) tyrosine kinase. BK current and channel tyrosine phosphorylation level were measured in BK-HEK 293 cells expressing both functional α-subunits and the auxiliary ß1-subunits using electrophysiology, immunoprecipitation and Western blotting approaches, respectively, and the function of rat cerebral basilar arteries was determined with a wire myography system. We found that BK current in BK-HEK 293 cells was increased by the broad spectrum protein tyrosine kinase (PTK) inhibitor genistein and the selective EGFR tyrosine kinase inhibitor AG556, one of the known tyrphostin. The effect of genistein or AG556 was antagonized by the protein tyrosine phosphatase (PTP) inhibitor orthovanadate. On the other hand, orthovanadate or EGF decreased BK current, and the effect was counteracted by AG556. The tyrosine phosphorylation level of BK channels (α- and ß1-subunits) was increased by EGF and orthovanadate, while decreased by genistein and AG556, and the reduced tyrosine phosphorylation of BK channels by genistein or AG556 was reversed by orthovanadate. Interestingly, AG556 induced a remarkable enhancement of BK current in rat cerebral artery smooth muscle cells and relaxation of pre-contracted rat cerebral basilar arteries with denuded endothelium, and these effects were antagonized by the BK channel blocker paxilline or orthovanadate. These results demonstrate that tyrosine phosphorylation of BK channels by EGFR kinase decreases the channel activity, and inhibition of EGFR kinase by AG556 enhances the channel activity and dilates rat cerebral basilar arteries.
Assuntos
Receptores ErbB/antagonistas & inibidores , Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Tirfostinas/farmacologia , Animais , Artéria Basilar/citologia , Separação Celular , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/metabolismo , Genisteína/farmacologia , Células HEK293 , Humanos , Indóis/farmacologia , Ativação do Canal Iônico/efeitos dos fármacos , Masculino , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Fosforilação/efeitos dos fármacos , Fosfotirosina/metabolismo , Subunidades Proteicas/metabolismo , Ratos Sprague-Dawley , Vanadatos/farmacologia , Vasodilatação/efeitos dos fármacosRESUMO
PLA2G6 or GVIA calcium-independent PLA2 (iPLA2ß) is identified as one of the NAFLD modifier genes in humans, and thought to be a target for NAFLD therapy. iPLA2ß is known to play a house-keeping role in phospholipid metabolism and remodeling. However, its role in NAFLD pathogenesis has not been supported by results obtained from high-fat feeding of iPLA2ß-null (PKO) mice. Unlike livers of human NAFLD and genetically obese rodents, fatty liver induced by high-fat diet is not associated with depletion of hepatic phospholipids. We therefore tested whether iPLA2ß could regulate obesity and hepatic steatosis in leptin-deficient mice by cross-breeding PKO with ob/ob mice to generate ob/ob-PKO mice. Here we observed an improvement in ob/ob-PKO mice with significant reduction in serum enzymes, lipids, glucose, insulin as well as improved glucose tolerance, and reduction in islet hyperplasia. The improvement in hepatic steatosis measured by liver triglycerides, fatty acids and cholesterol esters was associated with decreased expression of PPARγ and de novo lipogenesis genes, and the reversal of ß-oxidation gene expression. Notably, ob/ob livers contained depleted levels of lysophospholipids and phospholipids, and iPLA2ß deficiency in ob/ob-PKO livers lowers the former, but replenished the latter particularly phosphatidylethanolamine (PE) and phosphatidylcholine (PC) that contained arachidonic (AA) and docosahexaenoic (DHA) acids. Compared with WT livers, PKO livers also contained increased PE and PC containing AA and DHA. Thus, iPLA2ß deficiency protected against obesity and ob/ob fatty liver which was associated with hepatic fatty-acyl phospholipid remodeling. Our results support the deleterious role of iPLA2ß in severe obesity associated NAFLD.
Assuntos
Ácidos Graxos/sangue , Fosfolipases A2 do Grupo VI/deficiência , Fígado/enzimologia , Hepatopatia Gordurosa não Alcoólica/prevenção & controle , Obesidade/prevenção & controle , Fosfolipídeos/sangue , Animais , Apoptose , Ácido Araquidônico/sangue , Glicemia/metabolismo , Ésteres do Colesterol/sangue , Modelos Animais de Doenças , Ácidos Docosa-Hexaenoicos/sangue , Regulação da Expressão Gênica , Genótipo , Fosfolipases A2 do Grupo VI/genética , Insulina/sangue , Resistência à Insulina , Fígado/patologia , Lisofosfolipídeos/sangue , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Hepatopatia Gordurosa não Alcoólica/sangue , Hepatopatia Gordurosa não Alcoólica/enzimologia , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/patologia , Obesidade/sangue , Obesidade/enzimologia , Obesidade/genética , Obesidade/patologia , Oxirredução , PPAR gama/genética , PPAR gama/metabolismo , Fenótipo , Fosfatidilcolinas/sangue , Fosfatidiletanolaminas/sangue , Triglicerídeos/sangueRESUMO
The present study was designed to investigate the effect of equol on cerebral blood flow and the underlying molecular mechanisms. The regional cerebral blood flow in parietal lobe of rats was measured by using a laser Doppler flowmetry. Isolated cerebral basilar artery and mesenteric artery rings from rats were used for vascular reactivity measurement with a multi wire myography system. Outward K(+) current in smooth muscle cells of cerebral basilar artery, large-conductance Ca(2+)-activated K(+) (BK) channel current in BK-HEK 293 cells stably expressing both human α (hSlo)- and ß1-subunits, and hSlo channel current in hSlo-HEK 293 cells expressing only the α-subunit of BK channels were recorded with whole cell patch-clamp technique. The results showed that equol significantly increased regional cerebral blood flow in rats, and produced a concentration-dependent but endothelium-independent relaxation in rat cerebral basilar arteries. Both paxilline and iberiotoxin, two selective BK channel blockers, significantly inhibited equol-induced vasodilation in cerebral arteries. Outward K(+) currents in smooth muscle cells of cerebral basilar artery were increased by equol and fully reversed by washout or blockade of BK channels with iberiotoxin. Equol remarkably enhanced human BK current in BK-HEK 293 cells, but not hSlo current in hSlo-HEK 293 cells, and the increase was completely abolished by co-application of paxilline. Our findings provide the first information that equol selectively stimulates BK channel current by acting on its ß1 subunit, which may in turn contribute to the equol-mediated vasodilation and cerebral blood flow increase.
Assuntos
Circulação Cerebrovascular/efeitos dos fármacos , Equol/farmacologia , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/fisiologia , Miócitos de Músculo Liso/efeitos dos fármacos , Vasodilatadores/farmacologia , Animais , Artérias Cerebrais/efeitos dos fármacos , Artérias Cerebrais/fisiologia , Células HEK293 , Humanos , Técnicas In Vitro , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/genética , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/fisiologia , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/genética , Masculino , Artérias Mesentéricas/efeitos dos fármacos , Artérias Mesentéricas/fisiologia , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/fisiologia , Subunidades Proteicas/genética , Subunidades Proteicas/fisiologia , Ratos , Ratos Sprague-Dawley , Vasodilatação/efeitos dos fármacosRESUMO
The intermediate-conductance Ca(2+)-activated K(+) (KCa3.1) channels play a pivotal role in the proliferation and collagen secretion of cardiac fibroblasts. However, their contribution in cardiac fibrosis remains unknown. This study was designed to investigate whether KCa3.1 channels mediate the development of cardiac fibrosis. Pressure-overloaded rats were induced by abdominal aortic constriction and treated without or with KCa3.1 blocker (TRAM-34) or angiotensin type 1 receptor blocker (losartan) for 2 weeks. Besides the increase of blood pressure, angiotensin (Ang) II level in the plasma and myocardium, left ventricle mass and hydroxyproline concentration, myocardial hypertrophy, as well as significant collagen deposition in the perivascular regions and interstitium of the myocardium were observed in pressure-overloaded rats. The expression of leukocyte differentiation antigens (CD45 and CD3), macrophage surface marker (F4/80), tumor necrosis factor alpha, and monocyte chemotactic protein-1 (MCP-1) also significantly increased. All these alterations were prevented by losartan and TRAM-34. TRAM-34 also reduced the increase of renin and angiotensinogen in the plasma and myocardium of pressure-overloaded rats. Ang II promoted the migration of monocytes through endothelial cells and the secretion of MCP-1 from human umbilical vein endothelial cells in vitro, which was inhibited by TRAM-34. In conclusion, the present study demonstrates that TRAM-34 alleviates cardiac fibrosis induced by pressure overload, which is related to its inhibitory action on KCa3.1 channels and Ang II level. Our findings indicate that the inhibition of KCa3.1 channels may represent a novel approach of preventing the progression of cardiac fibrosis, and also add to the already developing literature of promising targets for TRAM-34.
Assuntos
Miocárdio/metabolismo , Miocárdio/patologia , Canais de Potássio Shaw/metabolismo , Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Angiotensinogênio/sangue , Angiotensinogênio/metabolismo , Animais , Aorta Abdominal/efeitos dos fármacos , Pressão Sanguínea , Cardiomegalia/patologia , Citocinas/metabolismo , Fibrose , Hidroxiprolina/metabolismo , Losartan/farmacologia , Masculino , Bloqueadores dos Canais de Potássio/farmacologia , Pirazóis/farmacologia , Ratos , Ratos Sprague-Dawley , Renina/sangue , Renina/metabolismo , Canais de Potássio Shaw/antagonistas & inibidoresRESUMO
The present study was designed to investigate the effect of metformin on the impairment of intermediate-conductance and small-conductance Ca(2+)-activated potassium channels (IKCa and SKCa)-mediated relaxation in diabetes and the underlying mechanism. The endothelial vasodilatation function of mesenteric arteries was assessed with the use of wire myography. Expression levels of IKCa and SKCa and phosphorylated Thr(172) of AMP-activated protein kinase (AMPK) were measured using Western blot technology. The channel activity was observed using a whole-cell patch voltage clamp. Reactive oxygen species (ROS) were measured using dihydroethidium and 2',7'-dichlorofluorescein diacetate. Metformin restored the impairment of IKCa- and SKCa-mediated vasodilatation in mesenteric arteries from streptozotocin-induced type 2 diabetic rats and that from normal rats incubated with advanced glycation end products (AGEs) for 3 hours. In cultured human umbilical vein endothelial cells (HUVECs), 1 µM metformin reversed AGE-induced increase of ROS and attenuated AGE- and H2O2- induced downregulation of IKCa and SKCa after long-term incubation (>24 hours). Short-term treatment (3 hours) with 1 µM metformin reversed the decrease of IKCa and SKCa currents induced by AGE incubation for 3 hours without changing the channel expression or the AMPK activation in HUVECs. These results are the first to demonstrate that metformin restored IKCa- and SKCa-mediated vasodilatation impaired by AGEs in rat mesenteric artery, in which the upregulation of channel activity and protein expression is likely involved.
Assuntos
Produtos Finais de Glicação Avançada/metabolismo , Metformina/farmacologia , Canais de Potássio Cálcio-Ativados/metabolismo , Vasodilatação/efeitos dos fármacos , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Linhagem Celular , Diabetes Mellitus Experimental/metabolismo , Regulação para Baixo/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Masculino , Artérias Mesentéricas/efeitos dos fármacos , Artérias Mesentéricas/metabolismo , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
The present study was designed to investigate the role of advanced glycation end products (AGEs) in intermediate-conductance and small-conductance Ca(2+)-activated potassium channels (KCa3.1 and KCa2.3)-mediated relaxation in rat resistance arteries and the underlying mechanism. The endothelial function of mesenteric arteries was assessed with the use of wire myography. Expression levels of KCa3.1 and KCa2.3 were measured by using Western blot. Reactive oxygen species (ROS) were measured by using dihydroethidium and 2', 7'-dichlorofluorescein diacetate. KCa3.1 and KCa2.3-mediated vasodilatation responses to acetylcholine and NS309 (opener of KCa3.1 and KCa2.3) were impaired by incubation of the third-order mesenteric arteries from normal rats with AGEs (200 µg ml(-1) for 3 h). In cultured human umbilical vein endothelial cells (HUVECs), AGEs increased ROS level and decreased the protein expression of KCa3.1 and KCa2.3. Antioxidant alpha lipoic acid restored the impairment in both vasodilatation function and expression of KCa3.1 and KCa2.3. H2O2 could mimic the effect of AGEs on the protein expression of KCa3.1 and KCa2.3 in cultured HUVECs. These results demonstrate for the first time that AGEs impaired KCa3.1 and KCa2.3-mediated vasodilatation in rat mesenteric arteries via downregulation of both KCa3.1 and KCa2.3, in which the enhanced oxidative stress was involved.
Assuntos
Produtos Finais de Glicação Avançada/farmacologia , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/efeitos dos fármacos , Artérias Mesentéricas/fisiologia , Estresse Oxidativo/fisiologia , Canais de Potássio Ativados por Cálcio de Condutância Baixa/efeitos dos fármacos , Vasodilatação/efeitos dos fármacos , Alcanos/farmacologia , Animais , Células Endoteliais da Veia Umbilical Humana , Humanos , Técnicas In Vitro , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/biossíntese , Masculino , Artérias Mesentéricas/efeitos dos fármacos , NG-Nitroarginina Metil Éster/farmacologia , Pirazóis/farmacologia , Compostos de Quinolínio/farmacologia , Ratos , Ratos Sprague-Dawley , Canais de Potássio Ativados por Cálcio de Condutância Baixa/biossínteseRESUMO
SKCa (small-conductance Ca(2+)-activated K(+)) channels are widely distributed in different tissues, including the brain, pancreatic islets and myocardium and play an important role in controlling electrical activity and cellular functions. However, intracellular signal modulation of SKCa channels is not fully understood. The present study was designed to investigate the potential regulation of hSKCa1 (human SKCa1) channels by PTKs (protein tyrosine kinases) in HEK (human embryonic kidney)-293 cells expressing the hSKCa1 (KCNN1) gene using approaches of whole-cell patch voltage-clamp, immunoprecipitation, Western blotting and mutagenesis. We found that the hSKCa1 current was inhibited by the broad-spectrum PTK inhibitor genistein, the selective EGFR (epidermal growth factor receptor) kinase inhibitors T25 (tyrphostin 25) and AG556 (tyrphostin AG 556), but not by the Src-family kinases inhibitor PP2. The inhibitory effect of these PTK inhibitors was significantly antagonized by the PTP (protein tyrosine phosphatase) inhibitor orthovanadate. The tyrosine phosphorylation level of hSKCa1 channels was reduced by genistein, T25 or AG556. The reduced tyrosine phosphorylation was countered by orthovanadate. Interestingly, the Y109F mutant hSKCa1 channel lost the inhibitory response to T25 or AG556, and showed a dramatic reduction in tyrosine phosphorylation levels and a reduced current density. These results demonstrate the novel information that hSKCa1 channels are inhibited by genistein, T25 and AG556 via EGFR tyrosine kinase inhibition, which is related to the phosphorylation of Tyr(109) in the N-terminus. This effect may affect electrical activity and cellular functions in brain, pancreatic islets and myocardium.
Assuntos
Receptores ErbB/antagonistas & inibidores , Receptores ErbB/fisiologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Canais de Potássio Ativados por Cálcio de Condutância Baixa/antagonistas & inibidores , Genisteína/farmacologia , Células HEK293 , Humanos , Inibidores de Proteínas Quinases/farmacologia , Canais de Potássio Ativados por Cálcio de Condutância Baixa/biossíntese , Canais de Potássio Ativados por Cálcio de Condutância Baixa/genética , Tirfostinas/farmacologiaRESUMO
Activation of the sympatho-ß-adrenergic receptor (ßAR) system is the hallmark of heart disease with adverse consequences that facilitate the onset and progression of heart failure (HF). Use of ß-blocking drugs has become the front-line therapy for HF. Last decade has witnessed progress in research demonstrating a pivotal role of Hippo pathway in cardiomyopathy and HF. Clinical studies have revealed myocardial Hippo pathway activation/YAP-TEAD1 inactivation in several types of human cardiomyopathy. Experimental activation of cardiac Hippo signaling or inhibition of YAP-TEAD1 have been shown to leads dilated cardiomyopathy with severe mitochondrial dysfunction and metabolic reprogramming. Studies have also convincingly shown that stimulation of ßAR activates cardiac Hippo pathway with inactivation of the down-stream effector molecules YAP/TAZ. There is strong evidence for the adverse consequences of the ßAR-Hippo signaling leading to HF. In addition to promoting cardiomyocyte death and fibrosis, recent progress is the demonstration of mitochondrial dysfunction and metabolic reprogramming mediated by ßAR-Hippo pathway signaling. Activation of cardiac ßAR-Hippo signaling is potent in downregulating a range of mitochondrial and metabolic genes, whereas expression of pro-inflammatory and pro-fibrotic factors are upregulated. Coupling of ßAR-Hippo pathway signaling is mediated by several kinases, mechanotransduction and/or Ca2+ signaling, and can be blocked by ß-antagonists. Demonstration of the converge of ßAR signaling and Hippo pathway bears implications for a better understanding on the role of enhanced sympathetic nervous activity, efficacy of ß-antagonists, and metabolic therapy targeting this pathway in HF. In this review we summarize the progress and discuss future research directions in this field.