RESUMO
Objective: To investigate the inhibitory effects of thalidomide on the expressions of VEGF and bFGF in human lung adenocarcinoma A549 cells and human hepatocellular carcinomas HepG2 cells mediated by cereblon (CRBN). Methods: shRNA technology was used to construct the A549 cell line (A549CRBN) and HepG2 cell line (HepG2CRBN) with stable knockdown of CRBN, which was verified by real-time PCR and Western blot. A549 cells were divided into negative control group (A549luciferase) and CRBN down-regulation group (A549CRBN); HepG2 cells were divided into negative control group (HepG2luciferase) and CRBN down-regulation group (HepG2CRBN). The above cells were seeded into 6-well plates at 3×105 cells/well, and cultured in a 37â, 5% CO2 incubator for 24 h. Then, 1 ml medium containing 100 µmol/L thalidomide (thalidomide group) and 1 ml medium containing 1 DMSO (control group) were added respectively, and the culture was continued for 24 hours before subsequent experiments. Each group was designed with three replicate wells. The effect of thalidomide on the activity of A549 cell line was detected by MTS assay. Real-time PCR was performed to detect mRNA expression levels of VEGF, bFGF and c-jun. ELISA assay was performed to detect protein expressions of VEGF and bFGF. Results: Compared with the control group, thalidomide at the concentrations of 1, 10, 50 and 100 µmol/L had no significant effects on the proliferation of A549 and HepG2 cells (Pï¼0.05). VEGF and bFGF levels in the A549CRBN or HepG2luciferase groups were significantly lower than those in the A549CRBN or HepG2CRBN groups (Pï¼0.05). Compared with the control group of the A549luciferase or HepG2luciferase, thalidomide inhibited the expressions of VEGF and bFGF in A549luciferase and HepG2luciferase cells (Pï¼ 0.05), but did not inhibit the expressions of VEGF and bFGF in A549CRBN and HepG2CRBN cells. Compared with the control group of the HepG2luciferase, thalidomide inhibited c-Jun expression in HepG2luciferase cells (Pï¼0.01), but did not significantly inhibit c-Jun expression in HepG2CRBN cells. Conclusion: The inhibitory effects of thalidomide on VEGF and bFGF expressions may be mediated by CRBN in A549 and HepG2 cells, and c-Jun may be one of the key transcription factors responsible for this inhibition.
Assuntos
Talidomida , Fator A de Crescimento do Endotélio Vascular , Células A549 , Regulação para Baixo , Fator 2 de Crescimento de Fibroblastos , HumanosRESUMO
The SUR2B/Kir6.1 channel openers iptakalim and natakalim reverse cardiac remodeling and ameliorate endothelial dysfunction by re-establishing the balance between the nitric oxide and endothelin systems. In this study, we investigated the microRNAs (miRs) involved in the molecular mechanisms of SUR2B/Kir6.1 channel opening in chronic heart failure. Both iptakalim and natakalim significantly upregulated the expression of miR-1-3p, suggesting that this miR is closely associated with the therapeutic effects against chronic heart failure. Bioinformatic analysis showed that many of the 183 target genes of miR-1-3p are involved in cardiovascular diseases, suggesting that miR-1-3p plays a vital role in such diseases and vascular remodeling. Target gene prediction showed that miR-1-3p combines with the 3' untranslated region (UTR) of endothelin-1 (ET-1) mRNA. Iptakalim and natakalim upregulated miR-1-3p expression and downregulated ET-1 mRNA expression in vitro. The dual luciferase assay confirmed that there is a complementary binding sequence between miR-1-3p and the 3' UTR 158-165 sequence of ET-1 mRNA. To verify the effect of miR-1-3p on ET-1, lentiviral vectors overexpressing or inhibiting miR-1-3p were constructed for the transduction of rat primary cardiac microvascular endothelial cells. The results showed that natakalim enhanced the miR-1-3p level. miR-1-3p overexpression downregulated the expression of ET-1, whereas miR-1-3p inhibition had the opposite effect. Therefore, we verified that SUR2B/Kir6.1 channel openers could correct endothelial imbalance and ameliorate chronic heart failure through the miR-1-3p/ET-1 pathway in endothelial cells. Our study provides comprehensive insights into the molecular mechanisms behind the SUR2B/Kir6.1 channel's activity against chronic heart failure.
Assuntos
Endotelina-1/metabolismo , Endotélio Vascular/metabolismo , Insuficiência Cardíaca/metabolismo , Canais KATP/metabolismo , MicroRNAs/metabolismo , Receptores de Sulfonilureias/metabolismo , Compostos Alílicos/farmacologia , Compostos Alílicos/uso terapêutico , Animais , Células Cultivadas , Relação Dose-Resposta a Droga , Endotelina-1/antagonistas & inibidores , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/patologia , Células HEK293 , Insuficiência Cardíaca/tratamento farmacológico , Insuficiência Cardíaca/patologia , Humanos , Canais KATP/agonistas , MicroRNAs/antagonistas & inibidores , Propilaminas/farmacologia , Propilaminas/uso terapêutico , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Receptores de Sulfonilureias/agonistasRESUMO
Both iptakalim (Ipt) and natakalim (Nat) activate the SUR2B/Kir6.1 channel, an ATP-sensitive potassium channel (KATP) subtype, with high selectivity. In this study we investigated the therapeutic effects of Ipt and Nat against isoproterenol-induced chronic heart failure (ISO-CHF) in rats, and demonstrated a new therapeutic approach to the treatment of CHF through activation of the SUR2B/Kir6.1 channel in endothelial cells. In ISO-CHF rats, oral administration of Nat (1, 3, 9 mg·kg-1·d-1) or Ipt (3 mg·kg-1·d-1) for 60 days significantly improved cardiac dysfunction, reversed cardiac remodeling, significantly attenuated the pathological increases in BNP levels, and improved endothelial dysfunction by adjusting the balance between endothelin and NO systems. The therapeutic effects of Nat were prevented by the selective KATP blocker glibenclamine (Gli, 50 mg·kg-1·d-1), confirming that these effects were mediated through activation of the SUR2B/Kir6.1 channel in endothelial cells. The molecular mechanisms underlying the therapeutic effects of Nat were further addressed using proteomic methods. We identified 724 proteins in the plasma of ISO-CHF rats; 55 proteins were related to Nat. These differentially expressed proteins were mainly involved in single-organism processes and the regulation of biological quality relative to CHF, including proteasome (Psm) and ATP protein clusters. We screened out PRKAR2ß, GAS6/eNOS/NO and NO/PKG/VASP pathways involved in the amelioration of CHF among the 24 enriched pathways. We further confirmed 6 protein candidates, including PRKAR2ß, GAS6 and VASP, which were involved in the endothelial mechanisms, and ATP, TIMP3 and AGT, which contributed to its cardiovascular actions. This study demonstrates a new pharmacological approach to the treatment of CHF through activation of the SUR2B/Kir6.1 channel in endothelial cells, and that the eNOS/VASP pathways are involved in its signaling mechanisms.
Assuntos
Compostos Alílicos/uso terapêutico , Células Endoteliais/efeitos dos fármacos , Insuficiência Cardíaca/tratamento farmacológico , Canais KATP/agonistas , Propilaminas/uso terapêutico , Receptores de Sulfonilureias/agonistas , Compostos Alílicos/antagonistas & inibidores , Compostos Alílicos/farmacologia , Animais , Relação Dose-Resposta a Droga , Células Endoteliais/metabolismo , Glibureto/análogos & derivados , Glibureto/farmacologia , Insuficiência Cardíaca/induzido quimicamente , Isoproterenol , Peptídeo Natriurético Encefálico/metabolismo , Propilaminas/antagonistas & inibidores , Propilaminas/farmacologia , Proteômica , Ratos , Remodelação Ventricular/efeitos dos fármacosRESUMO
OBJECTIVE: To construct T vectors based on Xcm I recognition site and optimize the PCR fragments for its ligation. METHODS: We firstly cloned the human histone H4 cDNA containing one Xcm I recognition site at both its 5' and 3' end into pCDNA 3.0 vector and then generated T vector with pCDNA 3.0 backbone by cutting the recombinant plasmid with Xcm I. To increase the ligation efficiency, the primers were firstly phosphorylated before DNA fragments amplification and then the PCR products were treated with Taq DNA polymerase and dATP after PCR amplification. Two DNA fragments with the length of 312 bp and 1 329 bp were ligated to it and the ligation mixture was transformed into E. coli DH5α competent cells and the positive rates of the transformants were evaluated by PCR and DNA agarose gel electrophoresis. RESULTS: Our results showed that the T vector produced by our method could ligate to the target DNA fragments with high efficiency. Besides, the phosphorylation state of the primers used for PCR amplification is also an important factor determining the cloning efficiency. What's more, as for longer DNA fragments, retreatment with Taq DNA polymerase and dATP after PCR amplification and purification could improve the ligation efficiency significantly. CONCLUSION: Our protocol may overcome the dependence on blue/white screening to get positive clones and provide a potent way to generate T vectors and ligate them to the target PCR fragment.
Assuntos
Escherichia coli/genética , Vetores Genéticos , Histonas/genética , Reação em Cadeia da Polimerase/métodos , Clonagem Molecular , DNA Complementar/genética , HumanosRESUMO
OBJECTIVE: To study the dilatation characteristics of ATP-sensitive potassium channel (KATP) SUR2B/Kir6.1 subtype opener iptakalim (Ipt) in pulmonary arterioles, and to explore its possible mechanism. METHODS: Vessels pressure-diameter monitoring perfusion technique was used to observe the dilatation effects of Ipt in rats fourth pulmonary arterioles (n=6~8). After the pulmonary arterioles were pre-treated with removing endothelium or pre-incubated with KATP channel blocker glibenclamide (Gli), cyclo-oxygenase (COX) inhibitor indomethacin (Indo) and nitric oxide synthase (NOS) inhibitor L-Nω-Nitro-arginine methyl ester(L-NAME), the dilatation effects of Ipt were observed. RESULTS: Pulmonary arterioles could be relaxed by Ipt, the maximal relaxation rate was (60.53±2.08)%. Compaired with control group, the effects of Ipt in endothelium denuded arterioles were significantly decreased, the maximal relaxation rate was (9.47±1.56)% (P<0.01). The maximal relaxation rate were decreased to(17.49±1.47)%,(37.00±3.88)% and(24.91±2.30)% respectively after Gli,Indo,L-NAME were pre-incubated (P<0.01). CONCLUSIONS: Pulmonary arterioles can be relaxed by Ipt. The selective activation of KATP SUR2B/Kir6.1 subtype by Ipt was involved in its mechanisms. The endothelium-dependently dilatation of Ipt was related to nitric oxide (NO) and prostacyclin (PGI2) released by endothelial cells.
Assuntos
Arteríolas/fisiologia , Canais KATP/fisiologia , Receptores de Sulfonilureias/fisiologia , Animais , Arteríolas/efeitos dos fármacos , Dilatação , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/fisiologia , Epoprostenol , Glibureto/farmacologia , Indometacina/farmacologia , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico , RatosRESUMO
AIM: To determine whether administration of choline could attenuate brain injury in a rat model of ischemic stroke and the underlying mechanisms. METHODS: A rat model of ischemic stroke was established through permanent middle cerebral artery occlusion (pMCAO). After the surgery, the rats were treated with choline or choline plus the specific α7 nAChR antagonist methyllycaconitine (MLA), or with the control drug nimodipine for 10 days. The neurological deficits, brain-infarct volume, pial vessel density and the number of microvessels in the cortex were assessed. Rat brain microvascular endothelial cells (rBMECs) cultured under hypoxic conditions were used in in vitro experiments. RESULTS: Oral administration of choline (100 or 200 mg·kg(-1)·d(-1)) or nimodipine (20 mg·kg(-1)·d(-1)) significantly improved neurological deficits, and reduced infarct volume and nerve cell loss in the ischemic cerebral cortices in pMCAO rats. Furthermore, oral administration of choline, but not nimodipine, promoted the pial arteriogenesis and cerebral-cortical capillary angiogenesis in the ischemic regions. Moreover, oral administration of choline significantly augmented pMCAO-induced increases in the expression levels of α7 nAChR, HIF-1α and VEGF in the ischemic cerebral cortices as well as in the serum levels of VEGF. Choline-induced protective effects were prevented by co-treatment with MLA (1 mg·kg(-1)·d(-1), ip). Treatment of rBMECs cultured under hypoxic conditions in vitro with choline (1, 10 and 100 µmol/L) dose-dependently promoted the endothelial-cell proliferation, migration and tube formation, as well as VEGF secretion, which were prevented by co-treatment with MLA (1 µmol/L) or by transfection with HIF-1α siRNA. CONCLUSION: Choline effectively attenuates brain ischemic injury in pMCAO rats, possibly by facilitating pial arteriogenesis and cerebral-cortical capillary angiogenesis via upregulating α7 nAChR levels and inducing the expression of HIF-1α and VEGF.
Assuntos
Indutores da Angiogênese/uso terapêutico , Encéfalo/irrigação sanguínea , Encéfalo/efeitos dos fármacos , Colina/uso terapêutico , Infarto da Artéria Cerebral Média/tratamento farmacológico , Nootrópicos/uso terapêutico , Animais , Encéfalo/patologia , Infarto da Artéria Cerebral Média/patologia , Masculino , Neovascularização Fisiológica/efeitos dos fármacos , Ratos Sprague-DawleyRESUMO
The primary object of this fundamental research was to survey the synergistic cardiovascular effects of iptakalim, a novel ATP-sensitive potassium channel (K(ATP)) opener, and clinical first-line antihypertensive drugs, such as calcium antagonists, thiazide diuretics and ß receptor blockers by a 2 x 2 factorial-design experiment. It would provide a theoretical basis for the development of new combined antihypertensive therapy program after iptakalim is applied to the clinic. Amlodipine besylate, hydrochlorothiazide and propranolol were chosen as clinical first-line antihypertensive drugs. Blood pressure, heart rate (HR) and cardiac functions were observed in anesthetized normal rats by an eight-channel physiological recorder. The results showed that iptakalim monotherapy in a low dose could produce significant antihypertensive effect. There was no interaction between iptakalim and amlodipine on the maximal changes of systolic blood pressure (SBP), diastolic blood pressure (DBP), mean arterial blood pressure (MABP), the left ventricular systolic pressure (LVSP), and the left ventricular end-diastolic pressure (LVEDP) (P > 0.05). However, the effects of combination iptakalim/amlodipine on the maximal changes of SBP, DBP, MABP, LVSP and LVEDP were more obvious than those of iptakalim or amlodipine monotherapy. And there was strong positive interaction between iptakalim and amlodipine on the maximal changes of HR (P>0.05). According to the maximal changes of DBP, MABP, LVSP and LVEDP (P < 0.05) of combination iptakalim with hydrochlorothiazide, there was strong positive interaction between them. But there was no interaction between iptakalim and hydrochlorothiazide on the maximal drop of SBP and HR (P > 0.05). According to the maximal drops of DBP, MABP of combination iptakalim with propranolol, there was strong positive interaction between them (P < 0.05). But there was no interaction between iptakalim and propranolol on the maximal changes of SBP, LVSP, LVEDP and HR (P > 0.05). In conclusion, it was the first time to study the effects of amlodipine, hydrochlorothiazide or propranolol, which had different mechanisms of action from iptakalim, on cardiovascular effects of iptakalim in anesthetized normal rats. This study proved that the combination of iptakalim with hydrochlorothiazide or propranolol respectively had significant synergism on lowering blood pressure, while the combination of iptakalim/amlodipine had additive action on lowering blood pressure. Meanwhile the antihypertensive effect was explicit, stable and long-lasting. Iptakalim thus appears suitable for the clinical treatment of hypertensive people who need two or more kinds of antihypertensive agents.
Assuntos
Anlodipino/farmacologia , Anti-Hipertensivos/farmacologia , Pressão Sanguínea/efeitos dos fármacos , Hidroclorotiazida/farmacologia , Propranolol/farmacologia , Propilaminas/farmacologia , Animais , Sinergismo Farmacológico , Frequência Cardíaca , Hipertensão , RatosRESUMO
OBJECTIVE: To establish a method for real-time recording the oxygen consumption of mice under normobaric hypoxia. METHODS: The experimental apparatus was made up of animal container, filling water control system, electronic balance, hose, a computer with weight recording software, etc. The working principle was that the oxygen consumed by animal was replaced by water filling which was controlled by the pneumatic and hydraulic actuator. The water was weighted by an electronic balance and the weight signal was recorded into excel file at the same time. The accuracy and precision of the apparatus were detected by a 10 ml syringe. The oxygen consumption characteristics of 6 acute repetitive hypoxia mice and 6 normal mice were observed. RESULTS: The P value for the paired t test was 1 and the CV value was 4%. The survival time and total oxygen consumption of acute repetitive hypoxia mice were both significantly increased compared to normal mice (P < 0.05), which were (58.8 +/- 6.8) min and (46.0 +/- 8.7) min respectively for the survival time and (85.1 +/- 8.5) ml and (73.6 +/- 5.4) ml respectively for total oxygen consumption. CONCLUSION: The hypoxia tolerance of the acute repetitive hypoxia mice can significantly improved by taking more oxygen in the animal cabin. The accuracy and precision of the apparatus are high and it can be used for the determination of oxygen consumption in hypoxia research.
Assuntos
Hipóxia/fisiopatologia , Monitorização Fisiológica/instrumentação , Consumo de Oxigênio/fisiologia , Animais , CamundongosRESUMO
Endothelial dysfunction can lead to congestive heart failure and the activation of endothelial ATP-sensitive potassium (K(ATP)) channels may contribute to endothelial protection. Therefore, the present study was carried out to investigate the hypothesis that natakalim, a novel K(ATP) channel opener, ameliorates post-infarction left ventricular remodeling and failure by correcting endothelial dysfunction. The effects of myocardial infarction were assessed 8 weeks following left anterior descending coronary artery occlusion in male Wistar rats. Depressed blood pressure, cardiac dysfunction, evidence of left ventricular remodeling and congestive heart failure were observed in the rats with myocardial infarction. Treatment with natakalim at daily oral doses of 1, 3 or 9 mg/kg/day for 8 weeks prevented these changes. Natakalim also prevented the progression to cardiac failure, which was demonstrated by the increase in right ventricular weight/body weight (RVW/BW) and relative lung weight, signs of cardiac dysfunction, as well as the overexpression of atrial and brain natriuretic peptide mRNAs. Our results also demonstrated that natakalim enhanced the downregulation of endothelium-derived nitric oxide, attenuated the upregulation of inducible nitric oxide synthase-derived nitric oxide (NO), inhibited the upregulated endothelin system and corrected the imbalance between prostacyclin and thromboxane A(2). Overall, our findings suggest that natakalim prevents post-infarction hypertrophy and cardiac failure by restoring the coordinated balance between endothelial function and cardiac hypertrophy.
Assuntos
Compostos Alílicos/farmacologia , Cardiomegalia/tratamento farmacológico , Endotélio Vascular/efeitos dos fármacos , Infarto do Miocárdio/tratamento farmacológico , Propilaminas/farmacologia , Remodelação Ventricular/efeitos dos fármacos , Administração Oral , Animais , Pressão Sanguínea/efeitos dos fármacos , Relação Dose-Resposta a Droga , Endotelinas/sangue , Endotélio Vascular/fisiopatologia , Epoprostenol/sangue , Insuficiência Cardíaca/tratamento farmacológico , Insuficiência Cardíaca/etiologia , Insuficiência Cardíaca/prevenção & controle , Ventrículos do Coração/efeitos dos fármacos , Ventrículos do Coração/metabolismo , Hidroxiprolina/sangue , Imuno-Histoquímica , Masculino , Microscopia Eletrônica de Transmissão , Infarto do Miocárdio/complicações , Miocárdio/metabolismo , Peptídeo Natriurético Encefálico/metabolismo , Óxido Nítrico/sangue , Ratos , Ratos Wistar , Tromboxano A2/sangueRESUMO
AIM: The non-neuronal acetylcholine system (NNAS) in endothelial cells participates in modulating endothelial function, vascular tone, angiogenesis and inflammation, thus plays a critical role in cardiovascular diseases. In this study, we used a proteomic approach to study potential downstream receptor-effectors of NNAS that were involved in regulating cellular function in endothelial cells. METHODS: Human umbilical vein endothelial cells were incubated in the presence of acetylcholine, oxotremorine, pilocarpine or nicotine at the concentration of 10 µmol/L for 12 h, and the expressed proteins in the cells were separated and identified with two-dimensional electrophoresis (2-DE) and LC-MS. The protein spots with the largest changes were identified by LC-MS. Biowork software was used for database search of the peptide mass fingerprints. RESULTS: Over 1200 polypeptides were reproducibly detected in 2-DE with a pH range of 3-10. Acetylcholine, oxotremorine, pilocarpine and nicotine treatment caused 16, 9, 8 and 9 protein spots, respectively, expressed differentially. Four protein spots were identified as destrin, FK506 binding protein 1A (FKBP1A), macrophage migration inhibitory factor (MIF) and profilin-1. Western blotting analyses showed that treatment of the cells with cholinergic agonists significantly decreased the expression of destrin, FKBP1A and MIF, and increased the expression of profilin-1. CONCLUSION: A set of proteins differentially expressed in endothelial cells in response to cholinergic agonists may have important implications for the downstream biological effects of NNAS.
Assuntos
Células Endoteliais/metabolismo , Proteoma/metabolismo , Receptores Colinérgicos/metabolismo , Células Cultivadas , Agonistas Colinérgicos/farmacologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Proteômica/métodos , SoftwareRESUMO
OBJECTIVE: High altitue pulmonary edema (HAPE) impacts seriously people's health at high altitude. Screening of susceptibility genes for HAPE will be used for the evaluation and protection of susceptible people. METHODS: We performed a genome-wide association study (GWAS) using Affymetrix SNP array 6.0 in 23 HAPE patients and 17 healthy controls. GO and Pathway analysis softwares were used to analyze and draw gene network. RESULTS: Thirty-nine SNPs were found to be significantly different between case and control groups (P < 10(-4)). GO and Pathway analysis of 27 genes around the 39 SNPs indicated that these genes mainly participate in the regulating of cell proliferation, regulation of nitrogen compound metabolic process and G-protein coupled receptor protein signaling pathway and so on. CONCLUSION: It suggests that these SNPs and genes found in this study may be associated with the susceptibility of HAPE.
Assuntos
Doença da Altitude/genética , Predisposição Genética para Doença , Hipertensão Pulmonar/genética , Polimorfismo de Nucleotídeo Único , Adulto , Povo Asiático/genética , Estudos de Casos e Controles , Estudo de Associação Genômica Ampla , Humanos , Adulto JovemRESUMO
OBJECTIVE: To investigate the effects of non-neuronal muscarinic receptors (NNMR) stimulation on atherosclerosis and endothelial cells activation. METHODS: Atherosclerosis model was established in ApoE-/- mice by a high fat diet for 7 weeks. During the experimental periods, animals were received a low (7 mg/kg/d) or a high (21 mg/kg/d) dose of arecoline by gavage. At the termination of the treatments, serum total cholesterol and NO levels were measured, and the aorta morphology was analyzed by hematoxylin and eosin staining. The gene expression of monocyte chemoattractant protein-1 (MCP-1) and adhesion molecules in the thoracic aortas was determined by RT-PCR, and the MCP-1 protein expression and NF-κB activity were detected by Western blot analysis. NO production, MCP-1 secretion in cultured rat aortic endothelial cells (RAECs), and monocyte-endothelium adhesion assay were also performed after arecoline treatments. RESULTS: Arecoline efficiently decreased atherosclerotic plaque areas, increased serum nitric oxide (NO) content, suppressed the mRNA and protein expression of MCP-1, and modulated the IκB-α degradation and P65 phosphorylation in the aortae of ApoE-/- mice. Furthermore, arecoline promoted NO production and suppressed MCP-1 secretion in cultured RAECs after ox-LDL exposure, and either atropine or NG-nitro-L-arginine methylester could abrogate these effects. Arecoline also significantly inhibited the adherence of U937 monocytes to the ox-LDL injured human umbilical vein endothelial cells, which could be abolished by atropine. CONCLUSION: Our results indicate that arecoline attenuates the progression of atherosclerosis and inhibits endothelial cells activation and adherence by stimulating endothelial NNMR. These effects, at least in part, are due to its modulation on NF-κB activity.
Assuntos
Arecolina/farmacologia , Aterosclerose/prevenção & controle , Células Endoteliais/citologia , Receptores Muscarínicos/fisiologia , Animais , Aorta/citologia , Apolipoproteínas E , Aterosclerose/fisiopatologia , Moléculas de Adesão Celular/metabolismo , Quimiocina CCL2/metabolismo , Colesterol/sangue , Progressão da Doença , Células Endoteliais/efeitos dos fármacos , Endotélio Vascular , Células Endoteliais da Veia Umbilical Humana/citologia , Humanos , Proteínas I-kappa B/metabolismo , Lipoproteínas LDL , Camundongos , Camundongos Knockout , Monócitos/citologia , Inibidor de NF-kappaB alfa , Óxido Nítrico/sangue , Nitroarginina/farmacologia , Ratos , Fator de Transcrição RelA/metabolismoRESUMO
OBJECTIVE: To study the effects of iptakalim (Ipt), an ATP-sensitive potassium channel opener, on cardiac remodeling induced by isoproterenol (ISO) in Wistar rats. METHODS: ISO was given subcutaneously (85 mg/(kg x d), sc, 7 days) to induce cardiac remodeling in rats. The rats in Ipt treated group were administrated with Ipt 3 mg/kg (po) after ISO injection. After treated with Ipt for 6 weeks, the hemodynamic parameters were tested by an eight channel physiological recorder (RM-6000). Then the heart weight was weighed and the cardiac remodeling index was calculated. HE stain and Masson's stain were employed to perform histological analysis, the hydroxyproline(Hyp) content in cardiac tissue was detected by colorimetric method, radioimmunoassay was used to measure the plasma levels of endothelin-1 (ET-1) and prostacyclin (PGI2). RESULTS: Six weeks after ISO injection, the cardiac functions of model group were damaged markedly compared with those of normal group. The characteristics of ventricular remodeling in model group included that the heart weight index, myocyte cross-sectional area, myocardial fibrosis, and the hydroxyproline content in cardiac tissue were all increased significantly. The plasma level of ET-1 was increased, while the plasma level of PGI2 was decreased significantly. These changes could be reversed by Ipt treatment (3 mg/(kg x d) for 6 weeks). CONCLUSION: Ipt can reverse cardiac remodeling induced by isoproterenol in rats. The endothelial protective effect regulating effects of Ipt on the balance between the ET-1 and PGI2 system may be involved in its mechanisms.
Assuntos
Canais KATP/efeitos dos fármacos , Propilaminas/farmacologia , Remodelação Ventricular/efeitos dos fármacos , Animais , Endotelina-1/sangue , Hemodinâmica , Hidroxiprolina/metabolismo , Isoproterenol/farmacologia , Masculino , Miocárdio/metabolismo , Prostaglandinas I/sangue , Ratos , Ratos WistarRESUMO
AIMS: Endothelial cells play a pivotal role in vascular intimal inflammation during cardiovascular diseases. The chemerin/ChemR23 system in endothelial cells is one of physiological mechanisms that regulate inflammatory responses. Our previous studies indicated that stimulation of non-neuronal muscarinic receptor (NNMR) improved endothelial dysfunction. However, the relationship between the chemerin/ChemR23 signaling axis and NNMR in endothelial cell is poorly understood. Here, we first investigated whether the modulation of chemerin/ChemR23 signaling axis is involved in NNMR-mediated endothelial protection. MAIN METHODS: Cultured rat aortic endothelial cells (RAECs) were used. The ChemR23 protein expression and chemerin secretion were measured using Western blot analysis. The gene expression level of ChemR23 was examined with reverse transcriptase PCR (RT-PCR). The production of nitric oxide (NO) was determined by a nitrate reductase assay kit. KEY FINDINGS: A sharp decline of chemerin secretion and ChemR23 protein/gene expression was observed in RAECs after exposed to homocysteine at concentration of 0.5 mmol/L. Arecoline (10 µmol/L) pretreatment increased ChemR23 protein expression as well as mRNA expression, and enhanced the secretion of chemerin. Arecoline could also reverse the decreased ChemR23 mRNA expression induced by uric acid, high glucose, or oxidized low-density lipoprotein. Furthermore, the modulation of arecoline on chemerin/ChemR23 signaling axis was absolutely abolished in the presence of the nonselective muscarinic receptors antagonist atropine 1 µmol/L. Additionally, arecoline improved endothelial dysfunction by increasing the reduced NO production induced by uric acid, which was blocked by anti-ChemR23 antibody. SIGNIFICANCE: The chemerin/ChemR23 signaling axis participates in NNMR-mediated protection against endothelial dysfunction in cardiovascular system.
Assuntos
Células Endoteliais/patologia , Regulação da Expressão Gênica , Inflamação/fisiopatologia , Receptores de Quimiocinas/metabolismo , Receptores Muscarínicos/metabolismo , Adipocinas/metabolismo , Animais , Aorta Torácica/citologia , Aorta Torácica/metabolismo , Arecolina/farmacologia , Atropina/farmacologia , Western Blotting , Quimiocinas , Células Endoteliais/metabolismo , Homocisteína/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular , Masculino , Óxido Nítrico/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de Quimiocinas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacosRESUMO
Vascular endothelium plays an important role in regulating vascular homeostasis. Over the past years, it has become clear that endothelial dysfunction is a key event of pathophysiological changes in the initiation and progression of injuries induced by extreme environmental factors. The present review summarizes current understanding of vascular endothelial dysfunction induced by hypoxia, cold and heat, and provides the information for prevention and treatment of environmental exposure injuries.
Assuntos
Endotélio Vascular/fisiopatologia , Hipóxia/fisiopatologia , Lesões do Sistema Vascular , Meio Ambiente , Humanos , TemperaturaRESUMO
OBJECTIVE: High altitude pulmonary edema (HAPE), a life-threatening disease, has no biological markers used for the routine prevention, diagnosis and treatment. The aim of this study was to identify serum proteins differentially expressed in patients with HAPE for discovering essential biomarkers. METHODS: A complete serum proteomic analysis was performed on 10 HAPE patients and on 10 high altitude and 11 sea level healthy people as control using two-dimensional gel electrophoresis, followed by matrix-assisted laser desorption/ionization mass spectrometry and peptide mass fingerprinting. Finally, two most significantly changed proteins were validated by enzyme-linked immunosorbent assay (ELISA). RESULTS: Eight protein spots stained with differential intensity, respresenting 5 distinct proteins were identified in patients compared with healthy controls through analysis of these composite gels. Among them, four proteins, namely alpha 1-antitrypsin(alpha1-AT), Haptoglobin(Hp), apolipoprotein A-1 (apoA-1) and Complement C3 increased remarkably, while one protein, apolipoprotein A-IV (apoA-IV) decreased significantly. The variation of alpha1-AT and Haptoglobin, as detected by ELISA, was consistent with the results from proteomic analysis. CONCLUSIONS: It is well known that Hp, alpha1-AT and complement C3 are associated with inflammation and apoA-1 and apoA-IV play important roles in lipid absorption, transport and metabolism. Therefore, the significant expression changes of Hp, alpha1-AT and complement C3 and apoA-1 and apoA-IV between HAPE patients and their corresponding healthy controls highlight the role of inflammatory response system and lipid metabolism system in the pathophysiology of HAPE.
Assuntos
Altitude , Biomarcadores/sangue , Proteoma , Edema Pulmonar/sangue , Proteínas Sanguíneas/metabolismo , Estudos de Casos e Controles , Eletroforese em Gel Bidimensional , Ensaio de Imunoadsorção Enzimática , Humanos , Mapeamento de Peptídeos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
The incidence of deacclimatization to high altitude syndrome (DAHAS) prevailed up to 80% in highland troops, and 100% in manual workers, and severe DAHAS could significantly affects patients' health, work and life. So it is imperative to develop effective prevention and treatment measures for DAHAS. The present review analyzes effective prophylactic and therapeutic measures against DAHAS, implemented at our hospital.
Assuntos
Doença da Altitude/terapia , Altitude , Aclimatação , Doença da Altitude/prevenção & controle , HumanosRESUMO
OBJECTIVE: Endothelial apoptosis plays an important role in the initiation of atherosclerosis. It would be useful to clarify whether activation of non-neuronal muscarinic receptor (NNMR) could prevent endothelial apoptosis and atherosclerosis. We investigated the effects of NNMR activation on regulating rat aortic endothelial cells (RAECs) apoptosis induced by homocysteine, an independent risk factor of atherosclerosis, and further studied its molecular mechanism. METHODS: RAECs were incubated using homocysteine at the concentration of 2.7 mmol/L for 36 h. RAECs were also pre-treated with carbachol or arecoline to examine their effects. RT-PCR was used to assess changes in the gene expression related to cell apoptosis. RESULTS: Incubation of RAECs with homocysteine at the concentration of 2.7 mmol/L resulted in morphologic changes, such as cellular shrinkage, membrane blebbing, chromatin condensation and margination. These could be attenuated by pretreatment with carbachol and arecoline at the concentration of 10 micromol/L for 12 h. Homocysteine induced apoptosis in RAECs and the molecular mechanisms were associated with the regulation of fas, fas-L and caspase-8 in the death receptor pathway, bcl-2, bcl-xL and bax in the mitochondrial pathway, caspase-12 in the endoplasmic reticulum pathway and caspase-3, caspase-6 and p53 as downstream effectors. Carbachol and arecoline attenuated the effects of homocysteine on genes in the death receptor pathway, in the mitochondrial pathway and in the downstream pathway. Atropine could reverse all of the effects of arecoline. CONCLUSION: Activation of NNMR by carbacol and arecoline inhibits homocysteine-induced endothelial cell apoptosis mainly through regulation of death receptor pathway, mitochondrial pathway and downstream effectors.
Assuntos
Apoptose , Células Endoteliais/citologia , Homocisteína/efeitos adversos , Receptores Muscarínicos/metabolismo , Animais , Aorta/citologia , Proteínas Reguladoras de Apoptose/metabolismo , Arecolina , Carbacol , Ciclo Celular , Retículo Endoplasmático/metabolismo , Células Endoteliais/efeitos dos fármacos , Mitocôndrias/metabolismo , RatosRESUMO
Acclimatization is a process of biological adaptation when exposed to environmental factors such as hypoxia, cold and heat for prolonged periods of time, where non-genetical variations play a role in allowing subjects to tolerate hypoxic, cold or hot environments. This review focuses on the characteristics and mechanisms of acclimatization found through major research advances by our institute. First, the mechanisms underlying the acclimatization to extreme environments are complex. In our investigations, the physiological changes of multiple systems including the nervous, circulatory, respiratory, and hemopoietic system were demonstrated when the acclimatization to hypoxia was developed, and the underlying significance of hypoxia-inducible factor-1 (HIF-1) was investigated. Second, it is suggested that the development of acclimatization to extreme environments is complicated. Hypoxia and cold coexist at high altitude. Our investigations revealed the characteristics of negative cross-relationship in the acclimatization to hypoxia and cold. And third, it is interesting for us to understand that acclimatization to extreme environments is transferable among individuals, and the characteristics of heat acclimatization-inducing factor (HAlF) were presented. The above findings will provide a theoretical guidance for protective operations and help to establish a solid foundation for future research related to acclimatization.
Assuntos
Aclimatação/fisiologia , Altitude , Temperatura Baixa , Meio Ambiente , Temperatura Alta , Humanos , Hipóxia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismoRESUMO
OBJECTIVE: To study the effects of iptakalim (IPT) on pressure-overload induced cardiac remodeling in rats, and investigate correlation between this protection effects and plasma PGI2 content. METHOD: The pressure-overload induced cardiac remodeling model was induced by abdominal aorta constriction for 6 weeks, and the rats were divided into 5 groups repectively: (1) sham group, (2) control group, (3) IPT 3 mg/kg group (IPT 3), (4) indomethacin 2 mg/kg group (Indo 2), (5) indomethacin 2 mg/kg + IPT 3 mg/kg group (Indo 2 + IPT 3). RM6000 eight channel physiological recorder was used to record haemodynamics index, heart weight was weighed and the cardiac remodeling index was calculated, HE stain and Masson's stain were employed to perform histological analysis, colorimetric method was used to detect the hydroxyproline content in cardiac tissue, radioimmunological method was used to measure the plasma PGI2 content. RESULTS: After 42 days of aortic banding, the hyperdynamic circulation state, cardiac remodeling and decreased plasma PGI2 content were observed in the model group compared with those in the sham group, which were effectively reserved by treatment with IPT 3 mg/kg. Single-use indomethacin led to further deterioration of this pathophysiological changes, however, combination administration of IPT 3 mg/kg prevented these from worsening characteristic by ameliorating hyperdynamic circulation state and cardiac remodeling, augmnent plasma PGI2 content. CONCLUSION: IPT can significantly reverse abdominal aorta binding/pressure-overload induced cardiac remodeling, its mechanism may contribute to binding K(ATP) channel in endothelial cells, ameliorating endothelium cells function, augmenting PGI2 synthesis and secretion.