RESUMEN
CONTEXT: Sinisan (SNS) has been used to treat psychosomatic diseases of the digestive system. But little is known about how SNS affects water immersion restraint stress (WIRS). OBJECTIVE: To study the effects of SNS on colonic tissue injury in the WIRS model. MATERIALS AND METHODS: Forty-eight Kunming (KM) mice were randomized into 6 groups (n = 8): The control and WIRS groups receiving deionized water; the SNS low-dose (SL, 3.12 g/kg/d), SNS middle-dose (SM, 6.24 g/kg/d), SNS high-dose (SH, 12.48 g/kg/d), and diazepam (DZ, 5 mg/kg/d) groups; each with two daily administrations for 5 consecutive days. The 5 treatment groups were subjected to WIRS for 24 h on day 6. The effects of SNS on colon tissue injury caused by WIRS were assessed by changes in colon histology, inflammatory cytokines, brain-gut peptides, and tight junction (TJ) proteins levels. 16S rRNA gene sequencing was used to detect the regulation of the gut microbiota. RESULTS: SNS pretreatment significantly reduced TNF-α (0.75- to 0.81-fold), IL-6 (0.77-fold), and IFN-γ (0.69-fold) levels; and increased TJ proteins levels, such as ZO-1 (4.06- to 5.27-fold), claudin-1 (3.33- to 5.14-fold), and occludin (6.46- to 11.82-fold). However, there was no significant difference between the levels of substance P (SP) and vasoactive intestinal peptide (VIP) in the control and WIRS groups. SNS regulated the composition of gut microbiota in WIRS mice. CONCLUSION: The positive effects of SNS on WIRS could provide a theoretical basis to treat stress-related gastrointestinal disorders.
Asunto(s)
Microbioma Gastrointestinal , Ratones , Animales , Mucosa Intestinal , Inmersión , ARN Ribosómico 16S , Colon/patología , Proteínas de Uniones Estrechas/metabolismo , Agua/farmacologíaRESUMEN
ABSTRACT: Prolonged and intense stress can exceed the body's normal self-regulation and limited compensatory and repair capacity, resulting in pathological damage to the body. In this study, we established a rat stress myocardial injury (SMI) model to explore the protective effect of melatonin (MLT) on SMI and its possible mechanisms of action. Adult female Sprague Dawley (SD) rats were randomly divided into 5 groups: blank control group (NC), SMI group, MLT low-dose group, MLT medium-dose group, and MLT high-dose group, and 10 rats in each group were used to establish a SMI model by the water immersion restraint method. We observed the changes in body weight and tail vein glucose of each group. Serum levels of corticosterone (Cort), creatine kinase isoenzyme (CK-MB), and Troponin â (Tn-â ) and activity of lactic acid dehydrogenase were measured by ELISA. Transcriptome sequencing was used to find differentially expressed genes in the control and model groups, and the results were verified by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR). HE staining was used to visualize the pathological changes in the heart tissue of each group, and Western blot was used to study the differences in protein expression in the cardiomyocytes of each group to further corroborate the results. The body weight growth rate of rats in the SMI group was significantly lower than that of the NC group ( P < 0.01), and the body weight growth rate of rats in the MLT high-dose group was significantly higher than that of the SMI group ( P < 0.05) with no significant difference compared with the NC group rats. The mean blood glucose of rats in the SMI group was significantly higher compared with the NC group ( P < 0.001), while the mean blood glucose of rats in the MLT administration groups was dose-dependently reduced compared with the SMI group. By RNA-seq and bioinformatics tools such as KEGG and Gene ontology, we found that the circadian clock-related genes Ciart , Arnt1 , Per1 , and Dbp were significantly downregulated in the SMI group during water immersion stress, and differentially expressed genes were enriched in the p38MAPK signaling pathway and p53 signaling pathway. Moreover, genes related to inflammation and apoptosis were differentially expressed. ELISA results showed that Cort, CK-MB, and Tn-â levels were significantly higher in the SMI group compared with the NC group ( P < 0.01) and melatonin reduced the levels of Cort, CK-MB, and Tn-â and decreased lactic acid dehydrogenase activity in rat serum. HE staining results showed that melatonin could attenuate stress-generated myocardial injury. Western blot showed that melatonin reduced the expression of p38MAPK, p53, Bax, and caspase-3 and increased the expression of Bcl-2 protein in rat heart. Melatonin can inhibit myocardial injury caused by water immersion, and its mechanism of action may be related to the regulation of the expression of circadian clock genes such as Ciart , Arnt1 , Per1 , and Dbp ; the inhibition of the expression of proapoptotic proteins such as p38MAPK, p53, Bax, and caspase-3; and the increase of the expression of Bcl-2 antiapoptotic protein.
Asunto(s)
Melatonina , Daño por Reperfusión Miocárdica , Animales , Apoptosis , Glucemia/metabolismo , Peso Corporal , Caspasa 3/metabolismo , Forma MB de la Creatina-Quinasa/metabolismo , Femenino , Ácido Láctico/metabolismo , Ácido Láctico/farmacología , Melatonina/farmacología , Daño por Reperfusión Miocárdica/patología , Miocardio/metabolismo , Miocitos Cardíacos , Oxidorreductasas/metabolismo , Oxidorreductasas/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Ratas , Ratas Sprague-Dawley , Proteína p53 Supresora de Tumor/metabolismo , Agua/metabolismo , Agua/farmacología , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Chronic stress-induced brain injury (CSBI) is the organic damage of brain tissue caused by long-term psychological and environmental stress. However, there is no effective drug for the treatment of CSBI. The present study aimed to investigate possible mechanisms of CSBI and to explore related therapeutic targets. A rat model of CSBI was established by combining chronic restraint and cold water immersion. Our CSBI model was validated via Nissl staining, Western blotting, and behavioral tests. RNA sequencing (RNA-seq) was used to identify differentially expressed genes (DEGs) within brain tissue during CSBI. Both Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) analyses were performed to determine signaling pathways associated with CSBI-induced DEGs. Agonists/antagonists were used to validate the pharmacodynamics of potential therapeutic targets. A combination of chronic restraint and cold water immersion successfully induced a rat model of CSBI, as indicated by various markers of brain injury and cell apoptosis that were verified via Nissl staining, Western blotting, and behavioral tests. RNA-seq analysis identified 1131 DEGs in CSBI rats. Of these DEGs, 553 genes were up-regulated and 778 genes were down-regulated. GO and KEGG pathway analyses revealed that significant DEGs were predominantly related to membrane-bound ion channels, among which the potassium channel function was found to be significantly affected. Pharmacological experiments revealed that retigabine, a voltage-gated potassium channel opener, demonstrated a protective effect in CSBI rats. Taken together, our findings suggest that potassium channel function is disrupted in CSBI, and that potassium channel regulators may function as anti-CSBI drugs.
Asunto(s)
Lesiones Encefálicas/etiología , Fármacos Neuroprotectores/farmacología , Canales de Potasio/metabolismo , Estrés Psicológico/complicaciones , Animales , Encéfalo/efectos de los fármacos , Encéfalo/patología , Lesiones Encefálicas/patología , Lesiones Encefálicas/prevención & control , Carbamatos/farmacología , Carbamatos/uso terapéutico , Modelos Animales de Enfermedad , Femenino , Humanos , Fármacos Neuroprotectores/uso terapéutico , Fenilendiaminas/farmacología , Fenilendiaminas/uso terapéutico , Bloqueadores de los Canales de Potasio/farmacología , Canales de Potasio/agonistas , Canales de Potasio/genética , RNA-Seq , Ratas , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genéticaRESUMEN
Curzerene is a sesquiterpene and component used in oriental medicine. It was originally isolated from the traditional Chinese herbal medicine Curcuma rhizomes. In this study, anticancer activity of curzerene was examined in both in vitro and in vivo models. The result of the MTT assay showed that curzerene exhibited antiproliferative effects in SPC-A1 human lung adenocarcinoma cells in a time-dependent and dose-dependent manner. The anticancer IC50s were 403.8, 154.8, and 47.0 µM for 24, 48, and 72 hours, respectively. The flow cytometry analysis indicated curzerene arrested the cells in the G2/M cell cycle and promoted or induced apoptosis of SPC-A1 cells. The percentage of cells arrested in the G2/M phase increased from 9.26â% in the control group cells to 17.57â% in the cells treated with the highest dose (100 µM) of curzerene. Western blot and RT-PCR analysis demonstrated that curzerene induced the downregulation of GSTA1 protein and mRNA expressions in SPC-A1 cells. Tumor growth was significantly inhibited in SPC-A1 cell-bearing nude mice by using curzerene (135 mg/kg daily), meanwhile, curzerene did not significantly affect body mass and the organs of the mice, which may indicate that curzerene has limited toxicity and side effects in vivo. In conclusion, curzerene could inhibit the proliferation of SPC-A1 human lung adenocarcinoma cells line in both in vitro and in vivo models. Focusing on its relationship with GSTA1, curzerene could induce the downregulation of GSTA1 protein and mRNA expressions in SPC-A1 cells. Curzerene might be used as an anti-lung adenocarcinoma drug candidate compound for further development.
Asunto(s)
Adenocarcinoma/tratamiento farmacológico , Antineoplásicos/farmacología , Curcuma/química , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Neoplasias Pulmonares/tratamiento farmacológico , Extractos Vegetales/química , Sesquiterpenos/farmacología , Adenocarcinoma del Pulmón , Animales , Antineoplásicos/química , Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Femenino , Glutatión Transferasa/genética , Glutatión Transferasa/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Sesquiterpenos/química , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Short-chain acyl-CoA dehydrogenase (SCAD), a key enzyme of fatty acid ß-oxidation, plays an important role in cardiac hypertrophy. However, its effect on the cardiomyocyte apoptosis remains unknown. We aimed to determine the role of SCAD in tert-butyl hydroperoxide (tBHP)-induced cardiomyocyte apoptosis. The mRNA and protein expression of SCAD were significantly down-regulated in the cardiomyocyte apoptosis model. Inhibition of SCAD with siRNA-1186 significantly decreased SCAD expression, enzyme activity and ATP content, but obviously increased the content of free fatty acids. Meanwhile, SCAD siRNA treatment triggered the same apoptosis as cardiomyocytes treated with tBHP, such as the increase in cell apoptotic rate, the activation of caspase3 and the decrease in the Bcl-2/Bax ratio, which showed that SCAD may play an important role in primary cardiomyocyte apoptosis. The changes of phosphonate AMP-activated protein kinase α (p-AMPKα) and Peroxisome proliferator-activated receptor α (PPARα) in cardiomyocyte apoptosis were consistent with that of SCAD. Furthermore, PPARα activator fenofibrate and AMPKα activator AICAR treatment significantly increased the expression of SCAD and inhibited cardiomyocyte apoptosis. In conclusion, for the first time our findings directly demonstrated that SCAD may be as a new target to prevent cardiomyocyte apoptosis through the AMPK/PPARα/SCAD signal pathways.
Asunto(s)
Apoptosis , Butiril-CoA Deshidrogenasa/metabolismo , Miocitos Cardíacos/citología , Miocitos Cardíacos/enzimología , Proteínas Quinasas Activadas por AMP/metabolismo , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacología , Animales , Animales Recién Nacidos , Apoptosis/efectos de los fármacos , Cardiotónicos/farmacología , Muerte Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Fenofibrato/farmacología , Miocitos Cardíacos/efectos de los fármacos , PPAR alfa/metabolismo , ARN Interferente Pequeño/metabolismo , Ratas Sprague-Dawley , Reacción en Cadena en Tiempo Real de la Polimerasa , Ribonucleótidos/farmacología , terc-Butilhidroperóxido/farmacologíaRESUMEN
Context Oxymatrine (OMT) is beneficial to human health by exerting various biological effects. Objective To investigate the absorption mechanism of OMT and discover absorption enhancers using Madin-Darby canine kidney (MDCK) cell monolayers. Materials and methods Concentration effects on the transport of OMT were measured in the range of 1.0 × 10(-5)-1.0 × 10(-3) M in 2 h. Then, the effect of time, direction, temperature and pH on the transport of OMT at 10(-4) M was studied. Moreover, Papp of OMT was determined in the absence/presence of cyclosporine and surfactants at 100 µM to further confirm the relative transport mechanism. Results The Papp APâBL ranged from (3.040 ± 0.23) × 10(-6) to (3.697 ± 0.19) × 10(-6 )cm/s as the concentration varied from 10(-5) to 10(-3) M. OMT showed similar Papp at 4 and 37 °C (p > 0.05). Increasing the apical pH 7.4 and 8.0 resulted in Papp versus pH 5.0 (p < 0.01). Furthermore, in the presence of cyclosporine and surfactants including sodium citrate, sodium dodecyl sulphate (SDS) and deoxysodium cholate, Papp was (0.318 ± 0.033) × 10(-5), (0.464 ± 0.048) × 10(-5), (0.897 ± 0.115) × 10(-5) and (1.341 ± 0.122) × 10(-5 )cm/s, respectively. In the presence of surfactants, Papp significantly increased up to 1.5-4.3-fold (p < 0.05). Discussion and conclusion OMT transport across MDCK cell monolayers was by passive diffusion. Sodium citrate, SDS and deoxysodium cholate serve as excellent absorption enhancers which are useful for the related research improving the oral bioavailability of OMT.
Asunto(s)
Alcaloides/metabolismo , Células Epiteliales/metabolismo , Riñón/metabolismo , Quinolizinas/metabolismo , Reabsorción Renal , Animales , Citratos/farmacología , Ciclosporina/farmacología , Ácido Desoxicólico/farmacología , Difusión , Perros , Relación Dosis-Respuesta a Droga , Células Epiteliales/efectos de los fármacos , Concentración de Iones de Hidrógeno , Riñón/efectos de los fármacos , Cinética , Modelos Lineales , Células de Riñón Canino Madin Darby , Permeabilidad , Reabsorción Renal/efectos de los fármacos , Citrato de Sodio , Dodecil Sulfato de Sodio/farmacología , Tensoactivos/farmacología , TemperaturaRESUMEN
The noninvasive imaging of cell death, including apoptosis and necrosis, is an important tool for the assessment of degenerative diseases and in the monitoring of tumor treatments. Duramycin is a peptide of 19-amino acids. It binds specifically to phosphatidylethanolamine a novel molecular target for cell death. N-(2-(18)F-Fluoropropionyl)duramycin ([(18)F]FPDuramycin) was prepared as a novel positron emission tomography (PET) tracer from the reaction of duramycin with 4-nitrophenyl 2-[(18)F]fluoropropionate ([(18)F]NFP). Compared with control cells (viable tumor cells), the in vitro binding of [(18)F]FPDuramycin with apoptotic cells induced by anti-Fas antibody resulted in a doubling increase, while the binding of [(18)F]FPDuramycin with necrotic cells induced by three freeze and thaw cycles resulted in a threefold increase. Biodistribution study in mice exhibited its rapid blood and renal clearance and predominant accumulation in liver and spleen over 120 min postinjection. Small-animal PET/CT imaging with [(18)F]FPDuramycin proved to be a successful way to visualize in vivo therapeutic-induced tumor cell death. In summary, [(18)F]FPDuramycin seems to be a potential PET probe candidate for noninvasive visualization of in vivo cell death sites induced by chemotherapy in tumors.
Asunto(s)
Bacteriocinas/química , Muerte Celular , Imagen Molecular , Péptidos/química , Tomografía de Emisión de Positrones , Radiofármacos/química , Animales , Apoptosis , Bacteriocinas/síntesis química , Bacteriocinas/farmacocinética , Femenino , Humanos , Células Jurkat , Ratones , Ratones Desnudos , Péptidos/síntesis química , Péptidos/farmacocinética , Radiofármacos/síntesis química , Radiofármacos/farmacocinética , Distribución TisularRESUMEN
Non-alcoholic fatty liver disease (NAFLD) has recently emerged as a global public health concern. Currently, the cornerstone of NAFLD treatment is lifestyle modification and, if necessary, weight loss. However, compliance is a challenge, and this approach alone may not be sufficient to halt and treat the more serious disease development, so medication is urgently needed. Nevertheless, no medicines are approved to treat NAFLD. Glucagon-like peptide-1 (GLP-1) is an enteropeptide hormone that inhibits glucagon synthesis, promotes insulin secretion, and delays gastric emptying. GLP-1 has been found in recent studies to be beneficial for the management of NAFLD, and the marketed GLP-1 agonist drugs have different degrees of effectiveness for NAFLD while lowering blood glucose. In this article, we review GLP-1 and its physiological roles, the pathogenesis of NAFLD, the correlation between NAFLD and GLP-1 signaling, and potential strategies for GLP-1 treatment of NAFLD.
Asunto(s)
Diabetes Mellitus Tipo 2 , Enfermedad del Hígado Graso no Alcohólico , Humanos , Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológico , Péptido 1 Similar al Glucagón/farmacología , Péptido 1 Similar al Glucagón/fisiología , Glucagón/uso terapéutico , Glucemia , Secreción de Insulina , Receptor del Péptido 1 Similar al Glucagón , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Hipoglucemiantes/uso terapéuticoRESUMEN
OBJECTIVE: To investigate the absolute bioavailability of caffeic acid in rats and its intestinal absorption properties. METHOD: The absolute bioavailability (Fabs) of caffeic acid was obtained after iv (2 mg x kg(-1)) or ig (10 mg x kg(-1)) administration to rats. The intestinal absorption of caffeic acid was explored by the recirculating vascularly perfused rat intestinal preparation. Caco-2 cell model was applied to measure the permeability of caffeic acid from apical to basolateral said (A-B) and from basolateral to apical said (B-A). RESULT: A two-compartment pharmacokinetic model was best to describe the pharmacokinetics of caffeic acid following iv or ig administration. The Fabs of caffeic acid was 14. 7% , and its intestinal absorption was 12.4%. The values of Papp A-->B and Papp B-->A of caffeic acid were retained stable while its concentration was changed. The efflux ratio values in this study surveyed were above 2.0, and suggesting caffeic acid was active transport. CONCLUSION: Caffeic acid was shown to have poor permeability across the Caco-2 cells, low intestinal absorption and low oral bioavailability in rats.
Asunto(s)
Ácidos Cafeicos/farmacocinética , Absorción Intestinal , Animales , Disponibilidad Biológica , Células CACO-2 , Ácidos Cafeicos/metabolismo , Humanos , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
Physiological reflexes and anatomical barriers render traditional eye drop delivery inefficient. We previously reported that drug-loaded nanoparticles and microspheres prepared from montmorillonite and Eudragit polymers exhibited good sustained-release and lowered intraocular pressure. Here, we compared the performance of optimized formulations to select the most suitable formulation for glaucoma therapy. We found that the microspheres had much higher encapsulation efficiency and drug loading than nanoparticles. Moreover, cytocompatibility experiments demonstrated that nanoparticles showed more severe cytotoxicity than microspheres, probably due to their smaller particles, enhanced cell uptake, and intracellular solubility. Interestingly, the pre-corneal retention time of nanoparticles reflected a clear advantage over microspheres, while the duration of the pharmacological effect of nanoparticles was not as good as that of microspheres: compared with the nanoparticle depressurization duration of only 8 h, the microspheres continuously depressurized for 12 h. The slower release of the microspheres and its micro-interaction mechanism with the discontinuous mucin layer of the tear film led to the inconsistency between duration of pharmacodynamics and fluorescence ocular retention time. In summary, the lower cytotoxicity and longer pharmacological effect of microspheres indicate their potential advantages for glaucoma applications.
Asunto(s)
Mucinas , Nanopartículas , Preparaciones de Acción Retardada , Intercambio Iónico , Microesferas , Tamaño de la PartículaRESUMEN
To examine the roles of intracellular calcium in RANKL-induced bone marrow macrophages (BMMs) differentiation, the effects of intracellular calcium chelator BAPTA-AM on RANKL-induced BMMs differentiation, and the activation of its relating signal proteins (MAPKs, and the PI3K/Akt) were studied. BMMs were cultured with various concentrations of BAPTA-AM in the presence of M-CSF (25 ng/ml) and RANKL (25 ng/ml) for 7 days, osteoclastogenic ability, cytosolic free Ca(2+) concentration, osteoclast survival and the expression of phosphorylated ERK1/2, SAPK/JNK, Akt and p38 MAPK were measured by TRAP staining, spectrofluorometer and Western blotting. BAPTA-AM inhibited osteoclastogenesis and osteoclast survival of BMMs by RANKL induction. In osteoclasts without the pretreatment of BAPTA-AM, the increased response of [Ca(2+)](i) was observed within 15 min and the maximum was about 1.2 times that of control. This response was sustained for 30 min and returned to the control level at 1h after RANKL-inducing, and the increased response of [Ca(2+)](i) was completely abolished and sustained to at least 8h by BAPTA-AM. Although immunoblotting data revealed that RANKL could activate the phosphorylation of ERK1/2, SAPK/JNK, Akt and p38 MAPK, the expression of ERK1/2, Akt and p38 MAPK phosphorylation was inhibited by BAPTA-AM dose-dependently. These results revealed that BAPTA-AM inhibit osteoclastogenic ability of BMMs via suppressing the increase of [Ca(2+)](i) which lead to inhibit RANKL-induced the phosphorylation of ERK, Akt and p38 MAPK, but not JNK. This finding may be useful in the development of an osteoclastic inhibitor that targets intracellular signaling factors.
Asunto(s)
Células de la Médula Ósea/efectos de los fármacos , Calcio/química , Diferenciación Celular/efectos de los fármacos , Quelantes/farmacología , Ácido Egtácico/análogos & derivados , Macrófagos/efectos de los fármacos , Proteínas Quinasas/metabolismo , Ligando RANK/fisiología , Animales , Western Blotting , Células de la Médula Ósea/citología , Quelantes/química , Ácido Egtácico/química , Ácido Egtácico/farmacología , Macrófagos/citología , Masculino , Ratones , Ratones Endogámicos BALB C , Osteoblastos/citología , Osteoblastos/efectos de los fármacos , Fosforilación , Espectrometría de FluorescenciaRESUMEN
Developing drugs against metabolic-related disorders, including obesity and hyperlipidemia, is of importance for human health. Here, a bioactive phytochemical, protodioscin, isolated from the rhizomes of Dioscorea nipponica (Rhizoma Dioscoreae Nipponicae), was identified for its anti-hyperlipidemic effect. In hyperlipidemic rats, the post-administration of protodioscin significantly reduced the time of blood coagulation. In addition, the blood levels of triglyceride, cholesterol, low-density and high-density lipoproteins were also changed accordingly. Taken together, this was the first report of an antihyperlipidemic effect of protodioscin. Its great availability in various vegetables and medicinal plants will be useful in developing health food supplement(s) and/or drug(s) against hyperlipidemia.
Asunto(s)
Dioscorea/química , Diosgenina/análogos & derivados , Hipolipemiantes/farmacología , Saponinas/farmacología , Animales , Coagulación Sanguínea/efectos de los fármacos , HDL-Colesterol/sangre , LDL-Colesterol/sangre , Diosgenina/química , Diosgenina/aislamiento & purificación , Diosgenina/farmacología , Hipolipemiantes/química , Hipolipemiantes/aislamiento & purificación , Masculino , Ratas , Ratas Sprague-Dawley , Saponinas/química , Saponinas/aislamiento & purificación , Triglicéridos/sangreRESUMEN
AIMS: Flavin adenine dinucleotide (FAD), participates in fatty acid ß oxidation as a cofactor, which has been confirmed to enhance SCAD activity and expression. However, the role of FAD on hypertensive vascular remodeling is unclear. In this study, we investigated the underlying mechanisms of FAD on vascular remodeling and endothelial homeostasis. MAIN METHODS: Morphological examination of vascular remodeling were analyzed with hematoxylin and eosin (HE) staining, Verhoeff's Van Gieson (EVG) staing, Dihydroethidium (DHE) staining and Sirius red staining. HUVECs apoptotic rate was detected by flow cytometry and HUVECs reactive oxygen species (ROS) was detected by DHE-probe. Enzymatic reactions were used to detect SCAD enzyme activity. The protein level was detected by Western Blots, the mRNA level was detected by quantitative real-time PCR. KEY FINDINGS: In vivo experiments, FAD significantly decreased blood pressure and ameliorated vascular remodeling by increasing SCAD expression, Nitric Oxide (NO) production and reducing ROS production. In vitro experiments, FAD protected against the tBHP induced injury in HUVEC, by increasing the activity of SCAD, increasing the elimination of free fatty acid (FFA), scavenging ROS, reducing apoptotic rate, thereby improving endothelial cell function. SIGNIFICANCE: FAD has a new possibility for preventing and treating hypertensive vascular remodeling.
Asunto(s)
Acil-CoA Deshidrogenasas/metabolismo , Activadores de Enzimas/uso terapéutico , Flavina-Adenina Dinucleótido/uso terapéutico , Hipertensión/tratamiento farmacológico , Remodelación Vascular/efectos de los fármacos , Animales , Presión Sanguínea/efectos de los fármacos , Activadores de Enzimas/farmacología , Flavina-Adenina Dinucleótido/farmacología , Células Endoteliales de la Vena Umbilical Humana , Humanos , Hipertensión/metabolismo , Hipertensión/fisiopatología , Masculino , Ratas Endogámicas SHR , Ratas WistarRESUMEN
OBJECTIVE: To observe the changes of short-chain acyl-CoA dehydrogenase (SCAD) expression on human umbilical vein endothelial cell (HUVEC) apoptosis and investigate its relationship with apoptosis. METHODS: The HUVEC was cultured normally for 2-3 days. The apoptotic model of HUVEC was established by tert-butyl hydrogen peroxide (tBHP). The HUVEC was treated by different concentrations of tBHP (0, 10, 20, 30, 40, 50 µmol/L) for 12 hours and different time (0, 3, 6, 9, 12 hours) with 50 µmol/L tBHP to establish the apoptotic model of HUVEC. The cell viability was detected by methyl thiazolyl tetrazolium (MTT), the mRNA expression of SCAD was determined by real-time polymerase chain reaction (PCR), the protein expression of SCAD was achieved by Western Blot. The best concentrate and time were determined to interfere the HUVEC to achieve the apoptotic model of HUVEC. The SCAD gene of HUVEC was knocked down by RNA interference sequence (siRNA274, siRNA414, siRNA679). The mRNA expression of SCAD, the protein expression of SCAD and the activity of SCAD enzyme were detected to achieve the best RNA interference sequence. The HUVEC was intervened by the best RNA interference sequence and tBHP. The cell activity and apoptosis rate, the enzyme activity of SCAD, the mRNA and protein expression of SCAD, the contents of reactive oxygen species (ROS), aderosine triphosphate (ATP) and free fatty acid (FFA) were detected to observe the effect of SCAD on apoptosis of HUVEC. RESULTS: (1) The cell viability, the mRNA expression and the protein expression of SCAD were decreased gradually in a concentration and time dependent manner with the increase of tBHP concentration and the prolongation of intervention time. The decline was most significant in the group of the 50 µmol/L tBHP to interfere HUVEC for 12 hours. (2) The siRNA679 transfection was the most significant in reducing SCAD mRNA and protein expressions among the three interference sequences (siRNA274, siRNA414, siRNA679). (3) Compare with blank control group, the cell viability was significantly decreased in the siRNA679 group (A value: 0.48±0.09 vs. 1.00±0.09, P < 0.01), the apoptotic rate of HUVEC was significantly increased [(29.96±2.09)% vs. (2.90±1.90)%, P < 0.01], the expression of SCAD mRNA and SCAD protein, the activity of SCAD enzyme and the content of ATP were significantly decreased [SCAD mRNA (2-ΔΔCt): 0.50±0.16 vs. 1.34±0.12, SCAD/α-Tubulin: 0.67±0.11 vs. 1.00±0.06, the activity of SCAD enzyme (kU/g): 0.38±0.04 vs. 0.53±0.04, the content of ATP (µmol/g): 0.14±0.02 vs. 0.19±0.01, all P < 0.05], the contents of FFA and ROS were significantly increased [FFA (nmol/g): 0.84±0.07 vs. 0.47±0.04, ROS (average fluorescence intensity): 647.5±23.7 vs. 434.2±46.5, both P < 0.01]. Meanwhile, SCAD siRNA treatment triggered the same apoptosis as HUVEC treated with tBHP. CONCLUSIONS: Down-regulation of SCAD may play an important role in HUVEC apoptosis. Increase in the expression of SCAD may become an important part in intervening HUVEC apoptosis.
Asunto(s)
Apoptosis , Butiril-CoA Deshidrogenasa/metabolismo , Células Endoteliales de la Vena Umbilical Humana , Supervivencia Celular , Humanos , Especies Reactivas de OxígenoRESUMEN
OBJECTIVE: To Study the changes of short-chain acyl-CoA dehydrogenase (SCAD) in heart failure (HF) after myocardial infarction (MI), and the effect of aerobic exercise on SCAD. METHODS: Healthy male Sprague-Dawley (SD) rats were divided into sham operation group (Sham group), sham operation swimming group (Sham+swim group), HF model group (LAD group) and HF swimming group (LAD+swim group) by random number table method, with 9 rats in each group. The left anterior descending branch of coronary artery (LAD) was ligated to establish a rat model of HF after MI. In Sham group, only one loose knot was threaded under the left coronary artery, and the rest operations were the same as those in LAD group. Rats in Sham+swim group and LAD+swim group were given swimming test for 1 week after operation (from 15 minutes on the 1st day to 60 minutes on the 5th day). Then they were given swimming endurance training (from the 2nd week onwards, 60 minutes daily, 6 times weekly, 10 weeks in a row). Tail artery systolic pressure (SBP) was measured before swimming endurance training and every 2 weeks until the end of the 10th week. Ten weeks after swimming training, echocardiography was performed to measure cardiac output (CO), stroke volume (SV), left ventricular ejection fraction (LVEF), shortening fraction (FS), left ventricular end-systolic diameter (LVESD), left ventricular end-diastolic diameter (LVEDD), left ventricular end-systolic volume (LVESV), and left ventricular end-diastolic volume (LVEDV). Morphological changes of heart were observed by Masson staining. Apoptosis of myocardial cells was detected by transferase-mediated deoxyuridine triphosphate-biotin nick end labeling stain (TUNEL) and apoptosis index (AI) was calculated. Reverse transcription-polymerase chain reaction (RT-PCR) and Western Blot were used to detect the mRNA and protein expression of myocardial SCAD respectively. In addition, the enzyme activity of SCAD, the content of adenosine triphosphate (ATP) and free fatty acid (FFA) in serum and myocardium were detected according to the kit instruction steps. RESULTS: Compared with Sham group, Sham+swim group showed SBP did not change significantly, with obvious eccentric hypertrophy and increased myocardial contractility, and LAD group showed persistent hypotension, obvious MI, thinning of left ventricle, and decreased myocardial systolic/diastolic function. Compared with LAD group, SBP, systolic/diastolic function and MI in LAD+swim group were significantly improved [SBP (mmHg, 1 mmHg = 0.133 kPa): 119.5±4.4 vs. 113.2±4.5 at 4 weeks, 120.3±4.0 vs. 106.5±3.7 at 6 weeks, 117.4±1.3 vs. 111.0±2.3 at 8 weeks, 126.1±1.6 vs. 119.4±1.9 at 10 weeks; CO (mL/min): 59.10±6.31 vs. 33.19±4.76, SV (µL): 139.42±17.32 vs. 84.02±14.26, LVEF: 0.523±0.039 vs. 0.309±0.011, FS: (28.17±2.57)% vs. (15.93±3.64)%, LVEDD (mm): 8.80±0.19 vs. 9.35±0.30, LVESD (mm): 5.90±0.77 vs. 7.97±0.60, LVEDV (µL): 426.57±20.84 vs. 476.24±25.18, LVESV (µL): 209.50±25.18 vs. 318.60±16.10; AI: (20.4±1.4)% vs. (31.2±4.6)%; all P < 0.05]. Compared with Sham group, the mRNA and protein expression of myocardium SCAD, the activity of SCAD in Sham+swim group were significantly increased, the content of ATP was slightly increased, the content of serum FFA was significantly decreased, and the content of myocardial FFA was slightly decreased; conversely, the mRNA and protein expression of myocardium SCAD, the activity of SCAD and the content of ATP in LAD group were significantly decreased, the content of serum and myocardial FFA were significantly increased. Compared with LAD group, the mRNA and protein expression of myocardium SCAD, the content of ATP were significantly increased in LAD+swim group [SCAD mRNA (2-ΔΔCt): 0.52±0.16 vs. 0.15±0.01, SCAD/GAPDH (fold increase from Sham group): 0.94±0.08 vs. 0.60±0.11, ATP content (µmol/g): 52.8±10.1 vs. 14.7±6.1, all P < 0.05], the content of serum and myocardial FFA were significantly decreased [serum FFA (nmol/L): 0.11±0.03 vs. 0.29±0.04, myocardial FFA (nmol/g): 32.7±8.2 vs. 59.7±10.7, both P < 0.05], and the activity of SCAD was slightly increased (kU/g: 12.3±4.3 vs. 8.9±5.8, P > 0.05). CONCLUSIONS: The expression of SCAD in HF was significantly down-regulated, and the expression was significantly up-regulated after aerobic exercise intervention, indicating that swimming may improve the severity of HF by up-regulating the expression of SCAD.
Asunto(s)
Butiril-CoA Deshidrogenasa/metabolismo , Insuficiencia Cardíaca/metabolismo , Animales , Insuficiencia Cardíaca/etiología , Masculino , Infarto del Miocardio/complicaciones , Condicionamiento Físico Animal , Distribución Aleatoria , Ratas , Ratas Sprague-DawleyRESUMEN
The prognosis of colorectal cancer (CRC) is seriously affected by high intestinal mucosal permeability accompanied by increasing tumor load. Berberine, a natural plant-derived product, can protect the intestinal mucosal barrier and suppress tumor growth, but its effects on the intestinal mucosal barrier dysfunction of CRC have not yet been evaluated. Herein, we assessed the effects of berberine on the intestinal mucosal permeability of HCT116 tumor-bearing mice and the underlying mechanism. Berberine (6.25, 12.5, 25 mg/kg) was administered to tumor-bearing mice for 3 weeks by intraperitoneal injection, and saline was given to controls and models. Compared with the control group, tumor-bearing mice had increased intestinal mucosal permeability in the third week. Meanwhile, the body weight decreased by 4%-7%, the concentration of D-lactic acid in plasma increased, and the expressions of ZO1 and Occludin were down-regulated. The intestinal mucosa was impaired. Compared with the model group, berberine inhibited tumor growth in a dose-dependent manner (6.25, 12.5, 25 mg/kg), reduced the permeability of intestinal mucosa, and alleviated intestinal mucosal damage. HPLC showed that berberine decreased the content of polyamines in tumor tissue, whereas increased that in intestinal mucosa tissue. Western blot showed that berberine inhibited the expressions of ODC, C-MYC and HIF-1α, but up-regulated those of OAZ1 and SSAT. In short, berberine may exert antitumor effects by suppressing tumor growth and elevating the intestinal mucosal permeability.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Berberina/farmacología , Neoplasias Colorrectales/tratamiento farmacológico , Poliaminas/metabolismo , Animales , Antineoplásicos Fitogénicos/administración & dosificación , Berberina/administración & dosificación , Neoplasias Colorrectales/patología , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo/efectos de los fármacos , Células HCT116 , Humanos , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/patología , Ácido Láctico/sangre , Ratones , Ratones Desnudos , Ocludina/genética , Permeabilidad/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto , Proteína de la Zonula Occludens-1/genéticaRESUMEN
Glutathione S-transferase A1 (GSTA1) is a phase II detoxification enzyme and serves a crucial role in anti-cancer drug resistance. In our previous study, GSTA1 was identified to be highly expressed in various subtypes of non-small-cell lung cancer cell lines compared with human embryonic lung fibroblast cell line MRC-5. The aim of the present study was to investigate the effect of GSTA1 expression on the proliferation and apoptosis of A549 cells. GSTA1 expression was knocked down or with overexpressed using lentivirus particles. Western blot analysis and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) were used to assess the protein, and mRNA levels of GSTA1 in A549 cells, respectively. The effect of GSTA1 manipulation on cell proliferation and apoptosis were investigated in vitro using MTT assays, Hoechst 33258 staining and flow cytometry, and in vivo using A549 cell line xenografts in nude mice. The results of the western blot analysis and RT-qPCR revealed that stable cell models of GSTA1 knockdown, and overexpression were established. The data of the MTT assay indicated that the downregulation of GSTA1 significantly inhibited cell proliferation compared with si-control-transfected cells. These si-GSTA1 A549 cells exhibited typical morphological changes of apoptosis, including chromatin condensation and shrunken nuclei compared with the si-control counterparts. An AnnexinV-fluorescein isothiocyanate assay verified that the downregulation of GSTA1 significantly induced cell apoptosis in vitro. In addition, overexpression of GSTA1 significantly promoted tumor growth in vivo. Accordingly, downregulation of GSTA1 suppressed tumor growth. In conclusion, GSTA1 plays an important role in regulation of cell proliferation and cell apoptosis in A549 cell line.
RESUMEN
OBJECTIVE: To investigate whether heroin can directly induce apoptosis in primary cultured cortical neurons of rat's brain. METHODS: Cultured primary neurons cultures were obtained from cerebral cortex of embryo rats. After 7 days, the cells were incubated with different concentrations of heroin (purity-80%) for 24 hours. The neuronal survival was assessed by cell viability counting with fluorescent diacetate (FDA) staining. The morphological and biochemical changes were observed with Hoechst 33258 fluorescent staining and then analyzed by agarose gel electrophoresis, respectively. RESULTS: After treatment with different concentrations of heroin, the neurons showed a decreased survival rate in a dose dependent manner, and there was a significant difference in the survival rate between the heroin group and the control group (P < 0.05). When exposed to different concentrations of heroin, neurons exhibited the morphological and biochemical features of apoptosis, including cell shrinkage, neurite degeneration, network disappearance, condensation and aggregation of nuclear chromatin, and the formation of DNA ladders. With the increase of heroin concentration of rat's brain more apoptotic bodies were seen. CONCLUSION: Heroin can directly induce apoptosis in primary cultured cortical neurons in rat's brain.
Asunto(s)
Apoptosis/efectos de los fármacos , Corteza Cerebral/efectos de los fármacos , Heroína/farmacología , Neuronas/efectos de los fármacos , Animales , Núcleo Celular/efectos de los fármacos , Núcleo Celular/patología , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Corteza Cerebral/patología , Fragmentación del ADN/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Agar/métodos , Femenino , Heroína/química , Masculino , Neuronas/patología , Ratas , Ratas Sprague-Dawley , Coloración y EtiquetadoRESUMEN
Apigenin is a nonmutagenic flavonoid that has antitumor properties. Polyamines are ubiquitous cellular polycations, which play an important role in the proliferation and differentiation of cancer cells. Highly regulated pathways control the biosynthesis and degradation of polyamines. Ornithine decarboxylase (ODC) is the rate-limiting enzyme in the metabolism, and spermidine/spermine-N1-Acetyl transferase (SSAT) is the rate-limiting enzyme in the catabolism of polyamines. In the current study, the effect of increasing concentrations of apigenin on polyamine levels, ODC and SSAT protein expression, mRNA expression, cell proliferation and apoptosis, and the production of reactive oxygen species (ROS) was investigated in SW620 colon cancer cells. The results showed that apigenin significantly reduced cell proliferation, decreased the levels of spermidine and spermine, and increased previously downregulated putrescine contents. Apigenin also enhanced SSAT protein and mRNA levels and the production of reactive oxygen species in SW620 cells, though it had no significant effect on the levels of ODC protein or mRNA. Apigenin appears to decrease the proliferation rate of human SW620 cells by facilitating SSAT expression to induce polyamine catabolism and increasing ROS levels to induce cell apoptosis.
RESUMEN
Curcumol, a polyphenol compound derived from the rhizome of Curcuma, has been established as an antitumor compound against multiple types of cancer, including gastric (GC), lung, liver and breast cancer. However, the molecular mechanisms undelying its anticancer activity in GC are still unclear. In this study, the antitumor efficacy of curcumol was ascertained in human gastric adenocarcinoma MGC-803 cells. An MTT assay was used to assess the viability of the MGC-803 cells treated by curcumol. The results of the Annexin V/propidium iodide (PI) staining followed by fluorescence activated cell sorting (FACS) analysis demonstrated that the cell cycle was arrested in the G2/M phase by curcumol. Annexin V-FITC/PI double staining followed by FACS analysis revealed that curcumol induced apoptosis of MGC-803 cells. FACS analysis after the cells were loaded with a DFCH-DA probe revealed that the level of reactive oxygen species (ROS) increased after the cells were treated with curcumol. In adittion, FACS analysis after the cells were loaded with JC-1 revealed that the level of mitochondrial membrane potential (MMP) decreased after the cells were treated with curcumol. Furthermore, the downregulation of isocitrate dehydrogenase 1 (IDH1) was observed in the MGC-803 cells after being treated with curcumol as determined by western blotting and RT-qPCR. In conclusion, we elucidated the antitumor effect of curcumol on MGC-803 cells and the involved mechanisms related to the induction of apoptosis, the increase of ROS, the decrease of MMP and the downregulation of IDH1.