RESUMEN
Parkinson's disease (PD) is a common neurodegenerative disorder characterized by the gradual loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc), resulting in reduced dopamine levels in the striatum and eventual onset of motor symptoms. Linalool (3,7-dimethyl-1,6-octadien-3-ol) is a monoterpene in aromatic plants exhibiting antioxidant, antidepressant, and anti-anxiety properties. The objective of this study is to evaluate the neuroprotective impacts of linalool on dopaminergic SH-SY5Y cells, primary mesencephalic and cortical neurons treated with 1-methyl-4-phenylpyridinium ion (MPP+), as well as in PD-like mice induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Cell viability, α-tubulin staining, western blotting, immunohistochemistry and behavioral experiments were performed. In MPP+-treated SH-SY5Y cells, linalool increased cell viability, reduced neurite retraction, enhanced antioxidant defense by downregulation of apoptosis signaling (B-cell lymphoma 2 (Bcl-2), cleaved caspase-3 and poly ADP-ribose polymerase (PARP)) and phagocyte NADPH oxidase (gp91phox), as well as upregulation of neurotrophic signaling (brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF)) and nuclear factor-erythroid 2 related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) pathway. In MPP+-treated primary mesencephalic neurons, linalool enhanced the expressions of tyrosine hydroxylase (TH), Sirtuin 1 (SirT1), and parkin. In MPP+-treated primary cortical neurons, linalool upregulated protein expression of SirT1, γ-Aminobutyric acid type A-α1 (GABAA-α1), and γ-Aminobutyric acid type B (GABAB). In PD-like mice, linalool attenuated the loss of dopamine neurons in SNpc. Linalool improved the motor and nonmotor behavioral deficits and muscle strength of PD-like mice. These findings suggest that linalool potentially protects dopaminergic neurons and improves the impairment symptoms of PD.
Asunto(s)
Monoterpenos Acíclicos , Neuroblastoma , Fármacos Neuroprotectores , Enfermedad de Parkinson , Humanos , Ratones , Animales , Enfermedad de Parkinson/metabolismo , Neuronas Dopaminérgicas/metabolismo , Antioxidantes/metabolismo , Odorantes , Sirtuina 1/metabolismo , Fármacos Neuroprotectores/farmacología , Neuroblastoma/metabolismo , 1-Metil-4-fenilpiridinio , Fuerza Muscular , Modelos Teóricos , Ácido gamma-Aminobutírico/metabolismoRESUMEN
Autophagy facilitates the degradation of organelles and cytoplasmic proteins in a lysosome-dependent manner. It also plays a crucial role in cell damage. Whether loganin affects autophagy in chronic constriction injury (CCI)-induced neuropathic pain remains unclear. We investigated the neuroprotective effect of loganin on the autophagic-lysosomal pathway in the rat CCI model. Sprague-Dawley rats were divided into sham, CCI, sham + loganin, and CCI + loganin. Loganin (5 mg/kg/day) was intraperitoneally injected once daily, and rats were sacrificed on day 7 after CCI. This study focused on the mechanism by which loganin modulates autophagic flux after CCI. CCI enhanced the autophagic marker LC3B-II in the ipsilateral spinal cord. The ubiquitin-binding protein p62 binds to LC3B-II and integrates into autophagosomes, which are degraded by autophagy. CCI caused the accumulation of p62, indicating the interruption of autophagosome turnover. Loganin significantly attenuated the expression of Beclin-1, LC3B-II, and p62. Double immunofluorescence staining was used to confirm that LC3B-II and p62 were reduced by loganin in the spinal microglia and astrocytes. Loganin also lessened the CCI-increased colocalization of both proteins. Enhanced lysosome-associated membrane protein 2 (LAMP2) and pro-cathepsin D (pro-CTSD) in CCI rats were also attenuated by loganin, suggesting that loganin improves impaired lysosomal function and autophagic flux. Loganin also attenuated the CCI-increased apoptosis protein Bax and cleaved caspase-3. Loganin prevents CCI-induced neuropathic pain, which could be attributed to the regulation of neuroinflammation, neuronal autophagy, and associated cell death. These data suggest autophagy could be a potential target for preventing neuropathic pain.
Asunto(s)
Glicósidos Cardíacos , Neuralgia , Animales , Ratas , Autofagia , Constricción , Hiperalgesia/etiología , Hiperalgesia/complicaciones , Glicósidos Iridoides , Neuralgia/tratamiento farmacológico , Neuralgia/etiología , Neuralgia/metabolismo , Ratas Sprague-DawleyRESUMEN
Xanthine-based KMUP-1 was shown to inhibit phosphodiesterases (PDEs) and modulate G-protein coupled receptors (GPCRs) to lower hyperlipidemia and body weight. This study further investigated whether KMUP-1 affects adipogenesis and lipolysis in 3T3-L1 preadipocytes. KMUP-1 (1â»40 µM) concentration-dependently attenuated Oil Red O (ORO) staining and decreased triglyceride (TG) accumulation, indicating adipogenesis inhibition in 3T3-L1 cells. In contrast, the ß-agonist ractopamine increased ORO staining and TG accumulation and adipogenesis. KMUP-1 (1â»40 µM) also reduced MAPKs/Akt/PPARγ expression, PPARγ1/PPARγ2 mRNA, and p-ERK immunoreactivity at the adipogenesis stage, but enhanced hormone sensitive lipase (HSL) immunoreactivity at the lipolysis stage. Addition of protein kinase A (PKA) or protein kinase G (PKG) antagonist (KT5720 or KT5728) to adipocytes did not affect HSL immunoreactivity. However, KMUP-1 did increase HSL immunoreactivity and the effect was reduced by PKA or PKG antagonist. Simvastatin, theophylline, caffeine, and sildenafil, like KMUP-1, also enhanced HSL immunoreactivity. Phosphorylated HSL (p-HSL) was enhanced by KMUP-1, indicating increased lipolysis in mature 3T3-L1 adipocytes. Decreases of MAPKs/Akt/PPARγ during adipogenesis contributed to inhibition of adipocyte differentiation, and increases of PKA/PKG at lipolysis contributed to HSL activation and TG hydrolysis. Taken together, the data suggest that KMUP-1 can inhibit hyperadiposity in 3T3-L1 adipocytes.
Asunto(s)
Adipocitos/citología , Adipogénesis/efectos de los fármacos , Piperidinas/farmacología , Transducción de Señal/efectos de los fármacos , Triglicéridos/metabolismo , Xantinas/farmacología , Células 3T3-L1 , Adipocitos/efectos de los fármacos , Adipocitos/metabolismo , Animales , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Lipólisis/efectos de los fármacos , Ratones , Proteínas Quinasas Activadas por Mitógenos/metabolismo , PPAR gamma/metabolismo , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Esterol Esterasa/metabolismoRESUMEN
This study examined the effects of eugenosedin-A (Eu-A) in a streptozotocin (STZ)/nicotinamide-induced rat model of type II diabetes mellitus (T2DM). Six-week-old Sprague-Dawley rats were randomly divided into three groups: (1) RD group, normal rats fed a regular diet (RD), (2) DM group, T2DM rats fed a high-fat diet, and (3) Eu-A group, T2DM rats fed a high fat diet plus oral Eu-A (5 mg/kg/day). After 30 days, the DM group had higher body weight, higher blood glucose and lower insulin levels than the RD group. The DM group also had increased protein expression of glycogen synthase kinase (GSK) in liver and skeletal muscle and decreased protein expression of insulin receptor (IR), insulin receptor substrate-1 (IRS-1), IRS-2, AMP-activated protein kinase (AMPK), glucose transporter-4 (GLUT-4), glucokinase (GCK), and peroxisome proliferator-activated receptor γ (PPAR-γ). STZ/nicotinamide-induced T2DM increased the expression of mitogen-activated protein kinases (MAPKs: p38, ERK, JNK) and inflammatory p65 protein. In the Eu-A treated T2DM rats, however, blood glucose was attenuated and the insulin concentration stimulated. Changes in IR, IRS-1 and IRS-2 proteins as well as AMPK, GLUT-4, GCK, GSK, PPAR-γ, MAPKs, and inflammatory p65 proteins were ameliorated. These results suggested that Eu-A alleviates STZ/nicotinamide-induced hyperglycemia by improving insulin levels and glucose metabolism, and inhibiting the MAPKs- and p65-mediated inflammatory pathway.
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Diabetes Mellitus Experimental/tratamiento farmacológico , Hiperglucemia/tratamiento farmacológico , Hipoglucemiantes/farmacología , Proteínas Quinasas Activadas por Mitógenos/genética , Piperazinas/farmacología , Animales , Glucemia/metabolismo , Diabetes Mellitus Experimental/inducido químicamente , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patología , Dieta Alta en Grasa/efectos adversos , Regulación de la Expresión Génica , Glucoquinasa/genética , Glucoquinasa/metabolismo , Transportador de Glucosa de Tipo 4/genética , Transportador de Glucosa de Tipo 4/metabolismo , Glucógeno Sintasa Quinasas/genética , Glucógeno Sintasa Quinasas/metabolismo , Hiperglucemia/inducido químicamente , Hiperglucemia/genética , Hiperglucemia/patología , Insulina/sangre , Proteínas Sustrato del Receptor de Insulina/genética , Proteínas Sustrato del Receptor de Insulina/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Masculino , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Niacinamida , PPAR gamma/genética , PPAR gamma/metabolismo , Ratas , Ratas Sprague-Dawley , Receptor de Insulina/genética , Receptor de Insulina/metabolismo , Transducción de Señal , Estreptozocina , Factor de Transcripción ReIA/genética , Factor de Transcripción ReIA/metabolismoRESUMEN
BACKGROUND: To identify the genetic defects and investigate the possible mechanism of cataract genesis in a five-generation family with autosomal dominant congenital posterior polar cataracts. METHODS: Clinical data were collected, and the lens phenotypes of the affected members in this family were recorded by slit lamp photography. Genomic DNA was isolated from peripheral blood using QIAamp DNA Blood Mini Kits. Twenty-three mutational hot spots associated with autosomal dominant congenital posterior polar cataracts were screened by PCR-based DNA sequencing. Properties and structural models of wild-type and mutant alpha-B (αB)-crystallin (CRYAB) were generated and analyzed using SWISS-MODEL. RESULTS: All affected individuals in this family started to exhibit poor vision at the age of 8-10 years. The lens opacity consisted of a single, well-defined plaque, 0.5-3 mm in diameter, which was confined to the posterior pole of the lens. DNA sequencing analysis of the affected members showed a novel, heterozygous missense mutation c.59C > G (P20R) in exon 1 of the CRYAB gene. This mutation was not found in 10 unaffected family members, or in 200 unaffected and unrelated individuals, thereby excluding the possibility that it is a rare polymorphism. Data generated using the ProtScale and PyMOL programs revealed that the mutation altered the stability and solubility of the αB-crystallin protein. CONCLUSIONS: This study reported a novel c.59C > G (P20R) missense mutation in CRYAB in a five-generation Chinese family with posterior polar cataract.
Asunto(s)
Catarata/genética , ADN/genética , Cristalino/metabolismo , Mutación , Cadena B de alfa-Cristalina/genética , Catarata/congénito , Catarata/metabolismo , Niño , China , Análisis Mutacional de ADN , Femenino , Humanos , Masculino , Linaje , Cadena B de alfa-Cristalina/metabolismoRESUMEN
Eugenosedin-A (Eu-A) effects on vascular endothelial dysfunction and oxidative stress in a hyperlipidemic rat model were investigated. Rats were randomly divided into four groups: two control groups and two treatment groups. The control rats received a regular diet or high fat diet (HFD); the treatment rats fed received an HFD with 5 mg/kg Eu-A or atorvastatin for 10 weeks. No changes in serotonin levels were observed in the four groups; norepinephrine levels were enhanced in the HFD group which was attenuated by Eu-A and atorvastatin. In the HFD group, the vascular reactivity was increased by vasoconstrictors (5-nonyloxytryptamine, 5-HT, and phenylephrine) and decreased by an endothelium-dependent vasorelaxant, carbachol. Protein levels of α1-adrenergic receptors (not 5-HT1B/2A), reactive oxygen species (ROS) p47(phox), p67(phox), and gp91(phox), and oxidative damage markers 3-nitrotyrosine (3-NT) and 4-hydroxy-2-nonenal (4-HNE) were increased, but endothelial nitric oxide synthase (eNOS), P-eNOS and vasodilator-stimulated phosphoprotein phosphorylation (P-VASP) were decreased. Catalase and superoxide dismutase (SOD-1 and SOD-2) proteins were increased, but glutathione peroxidase (GPx) was decreased in the aorta. Eu-A and atorvastatin reduced vasoconstrictor-induced aortic contractions that might be related to 5-HT1B/2A and α1-adrenergic receptors inhibitory activities. Eu-A and atorvastatin improved eNOS/P-eNOS, P-VASP, GPx, and malondialdehyde (MDA) levels, and decreased ROS and oxidative damage markers. Taken together, we suggest that Eu-A can ameliorate hyperlipidemia-induced vascular endothelial dysfunction and oxidative dysregulation.
Asunto(s)
Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Hiperlipidemias/complicaciones , NADPH Oxidasas/metabolismo , Piperazinas/farmacología , Receptores Adrenérgicos/metabolismo , Serotonina/metabolismo , Animales , Aorta Torácica , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Moléculas de Adhesión Celular/metabolismo , Masculino , Malondialdehído/metabolismo , Proteínas de Microfilamentos/metabolismo , Norepinefrina/sangre , Estrés Oxidativo/efectos de los fármacos , Fosfoproteínas/metabolismo , Fosforilación/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismo , Serotonina/sangre , Superóxido Dismutasa/metabolismoRESUMEN
In this study the effects of low-dose aspirin (5 mg/kg) on adhesion molecule and chemokine expression in a hyperlipidemic rat model. Six-week-old Sprague-Dawley (SD) rats were assigned to two control groups receiving either a regular diet or high-fat diet (HFD) and a treatment group fed HFD with 5 mg/kg aspirin for a 10-week period. Compared with the regular diet control group, the HFD control group had higher body weight, lower levels of high-density lipoprotein, higher concentrations of insulin, triglyceride, total cholesterol, and low-density lipoprotein, but no differences in blood glucose and glycated hemoglobin. The prothrombin time (PT) and activated partial thromboplastin time (aPTT) were clearly shortened in the HFD group. That group also had increased expression of intercellular adhesion molecule-1 (ICAM-1), ICAM-2, ICAM-3, vascular cell adhesion molecule (VCAM), platelet endothelial cell adhesion molecule (PECAM) and P-selectin in platelets and vascular adhesion protein-1 in lymphocyte and in aorta increased expressions of ICAM-1, ICAM-2, ICAM-3, VCAM, PECAM, E-selectin, monocyte chemoattractant protein-1 (MCP-1) and CCR2. The HFD rats also had increased PKCα, IκB kinase α (IKKα), p65, mitogen-activated protein kinases (MAPKs) (p38, c-Jun N-terminal kinases 1, extracellular signal-regulated kinase 1/2), and their phosphorylated forms. Low-dose aspirin improved HFD-induced hyperinsulinemia and hyperlipidemia, recovered PT and aPTT, inhibited upregulation of adhesion molecules and chemokines and reduced expression of PKCα, IKKα, p65, and MAPKs. Low-dose aspirin ameliorates HFD-induced hyperlipidemia and hyperinsulinemia, and prevents HFD-induced expression of adhesion molecules and chemokine formation.
Asunto(s)
Antiinflamatorios no Esteroideos/uso terapéutico , Aspirina/uso terapéutico , Moléculas de Adhesión Celular/sangre , Quimiocinas/sangre , Dieta Alta en Grasa/efectos adversos , Hiperlipidemias/prevención & control , Animales , Antiinflamatorios no Esteroideos/administración & dosificación , Aorta Torácica/efectos de los fármacos , Aorta Torácica/metabolismo , Aspirina/administración & dosificación , Aterosclerosis/etiología , Aterosclerosis/inmunología , Aterosclerosis/metabolismo , Aterosclerosis/prevención & control , Plaquetas/inmunología , Plaquetas/metabolismo , Peso Corporal/efectos de los fármacos , Moléculas de Adhesión Celular/biosíntesis , Quimiocinas/inmunología , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Hiperlipidemias/sangre , Hiperlipidemias/complicaciones , Hiperlipidemias/inmunología , Lípidos/sangre , Linfocitos/inmunología , Linfocitos/metabolismo , Masculino , Ratas Sprague-Dawley , Aumento de Peso/efectos de los fármacosRESUMEN
OBJECTIVES: Previous studies have shown eugenosedin-A, a 5-HT(1B/2A) and α(1)/α(2)/ß(1)-adrenergic blocker, is able to decrease cholesterol levels, hyperglycaemia and inflammation in hyperlipidaemic mice induced by high-fat diet (HFD). The aim of this study is to examine the effects of eugenosedin-A on the inhibition of adhesion molecules of platelets, the aorta and acyl-coenzymeA:cholesterol acyltransferase-1 (ACAT-1) of macrophages in a hyperlipidaemic rat model. METHODS: Six-week-old Sprague-Dawley rats were randomly divided into two control and treatment groups. The control rats received either a regular diet or HFD and the treatment groups were fed HFD with either 5 mg/kg eugenosedin-A or atorvastatin for a 10-week period. KEY FINDINGS: Compared with the two control groups, the HFD group had lower levels of high-density lipoprotein, higher concentrations of triglycerides, total cholesterol, low-density lipoprotein and insulin. The expression of adhesion molecules in platelets, aorta and monocyte-macrophage were enhanced by HFD. HFD also increased upstream proteins and their phosphorylated form in the aorta. In treatment groups, eugenosedin-A and atorvastatin improved HFD-induced hyperlipidaemia and levels of insulin. Eugenosedin-A reduced the upregulation of P-selectin, ICAM-1, ICAM-2, ICAM-3, VCAM, PECAM in platelets and inhibited E-selectin, ICAM-1, ICAM-2, ICAM-3, VCAM and PECAM protein levels in the aorta. Eugenosedin-A reduced the ACAT-1 protein expression of monocyte-macrophages. The expression of PKCα, MAPKs, IKKα and p65 and their phosphorylated form were reduced in treatment groups. CONCLUSIONS: Taken together, hyperlipidaemia enhances the expression of adhesion molecules and ACAT-1 protein, and eugenosedin-A ameliorates those increases. Through inhibition of MAPK- and p-65-mediated NF-κB pathway, eugenosedin-A decreases the quantity of adhesion molecules.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Dieta Alta en Grasa/efectos adversos , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , FN-kappa B/antagonistas & inhibidores , Piperazinas/farmacología , Transducción de Señal/efectos de los fármacos , Factor de Transcripción ReIA/antagonistas & inhibidores , Acetil-CoA C-Acetiltransferasa/metabolismo , Animales , Aorta/efectos de los fármacos , Aorta/metabolismo , Plaquetas/efectos de los fármacos , Plaquetas/metabolismo , Peso Corporal/efectos de los fármacos , Modelos Animales de Enfermedad , Selectina E/metabolismo , Hiperlipidemias/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Masculino , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , FN-kappa B/metabolismo , Distribución Aleatoria , Ratas , Ratas Sprague-Dawley , Factor de Transcripción ReIA/metabolismoRESUMEN
BACKGROUND AND PURPOSE: Spinal muscular atrophy (SMA) is a progressive neuromuscular disease. Since disease severity is related to the amount of survival motor neuron (SMN) protein, up-regulated functional SMN protein levels from the SMN2 gene are considered a major SMA drug-discovery strategy. In this study, we investigated the possible effects of triptolide, a diterpene triepoxide purified from Tripterygium wilfordii Hook. F., as a new compound for increasing SMN protein. EXPERIMENTAL APPROACH: The effects and mechanisms of triptolide on the production of SMA protein were determined by cell-based assays using the motor neuronal cell line NSC34 and skin fibroblasts from SMA patients. Wild-type (Smn(+/+) SMN2(-/-) , C57BL/6) and SMA-like (Smn(-/-) SMN2) mice were injected with triptolide (0.01 or 0.1 mg·kg(-1) ·day(-1) , i.p.) and their survival rate and level of change in SMN protein in neurons and muscle tissue measured. KEY RESULTS: In NSC34 cells and human SMA fibroblasts, pM concentrations of triptolide significantly increased SMN protein expression and the levels of SMN complex component (Gemin2 and Gemin3). In human SMA fibroblasts, triptolide increased SMN-containing nuclear gems and the ratio of full-length transcripts (FL-SMN2) to SMN2 transcripts lacking exon 7 (SMN2Δ7). Furthermore, in SMA-like mice, triptolide significantly increased SMN protein levels in the brain, spinal cord and gastrocnemius muscle. Furthermore, triptolide treatment increased survival and reduced weight loss in SMA-like mice. CONCLUSION AND IMPLICATIONS: Triptolide enhanced SMN protein production by promoting SMN2 activation, exon 7 inclusion and increasing nuclear gems, and increased survival in SMA mice, which suggests triptolide might be a potential candidate for SMA therapy.
Asunto(s)
Diterpenos/uso terapéutico , Fibroblastos/efectos de los fármacos , Neuronas Motoras/efectos de los fármacos , Atrofia Muscular Espinal/tratamiento farmacológico , Fármacos Neuroprotectores/uso terapéutico , Fenantrenos/uso terapéutico , Proteína 2 para la Supervivencia de la Neurona Motora/biosíntesis , Transcripción Genética/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Western Blotting , Peso Corporal/efectos de los fármacos , Técnicas de Cultivo de Célula , Línea Celular , Supervivencia Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Diterpenos/administración & dosificación , Diterpenos/aislamiento & purificación , Diterpenos/farmacología , Relación Dosis-Respuesta a Droga , Compuestos Epoxi/administración & dosificación , Compuestos Epoxi/aislamiento & purificación , Compuestos Epoxi/farmacología , Compuestos Epoxi/uso terapéutico , Fibroblastos/metabolismo , Fibroblastos/patología , Gemini de los Cuerpos Enrollados/efectos de los fármacos , Gemini de los Cuerpos Enrollados/metabolismo , Humanos , Estimación de Kaplan-Meier , Ratones , Ratones Noqueados , Estructura Molecular , Neuronas Motoras/metabolismo , Atrofia Muscular Espinal/metabolismo , Atrofia Muscular Espinal/patología , Fármacos Neuroprotectores/administración & dosificación , Fármacos Neuroprotectores/aislamiento & purificación , Fármacos Neuroprotectores/farmacología , Fenantrenos/administración & dosificación , Fenantrenos/aislamiento & purificación , Fenantrenos/farmacología , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteína 2 para la Supervivencia de la Neurona Motora/genética , Tripterygium/química , Regulación hacia ArribaRESUMEN
OBJECTIVE: To detect the sperm plasma membrane integrity (PMI) of varicocele (VC) patients using SYBR-14/PI fluorescent staining and flow cytometry, and investigate its clinical significance. METHODS: We collected semen samples from 120 men, including 30 grade-1 varicocele patients (VC1), 30 grade-2 (VC2), 30 grade-3 (VC3), and 30 normal fertile volunteer controls. Conventional semen analyses were performed by computer-assisted semen analysis (CASA). All the semen samples were washed with PBS and then subjected to SYBR-14/PI staining for the detection of sperm PMI by flow cytometry. The proportion of normal sperm with PMI was indicated as the percentage of sperm emitting green fluorescence (SYBR-14+/PI- %), sperm PMI was determined and sperm fertilization capacity predicted. RESULTS: Significant differences were detected in SYBR-14+/PI- and SYBR-14-/PI+ between the normal men and varicocele male patients (P < 0.01). The percentages of the sperm with PMI (SYBR-14+/PI- %) were remarkably lower in the VC1, VC2 and VC3 groups ([54.85 +/- 3.78]%, [45.37 +/- 4.12]% and [35.14 +/- 4.91]%) than in the normal controls ([70.79 +/- 6.71]%). SYBR-14+/PI-% was correlated positively with sperm motility (r=0.965, P < 0.01) and the percentage of grade a + b sperm (r = 0.874, P < 0.01), negatively with the percentage of grade d sperm (r = -0.965, P <0.01), but not significantly with pH, semen volume and liquefaction time (P > 0.05). CONCLUSION: SYBR-14/PI fluorescent staining and flow cytometry can quickly and exactly detect sperm PMI. Varicocele decreases sperm PMI, which might be an important cause of male infertility.
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Membrana Celular/patología , Varicocele/patología , Varicocele/fisiopatología , Estudios de Casos y Controles , Citometría de Flujo , Humanos , Masculino , Compuestos Orgánicos , Análisis de Semen , Motilidad Espermática , Espermatozoides , Coloración y EtiquetadoRESUMEN
Globozoospermia, as a severe teratozoospermia caused by gene mutations, is a rare congenital disease with main clinical manifestations of the round head of sperm and abnormality or absence of acrosome, and its precise mechanism is not yet clear. Studies show that the pathogenic genes associated with globozoospermia include SPATA16, PICK1, GOPC, Hrb, Csnk2a2 and bs. This paper outlines the progress in the studies of molecular genetics of globozoospermia, aiming to contribute to the molecular diagnosis and mechanism investigation of the disease.
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Infertilidad Masculina/genética , Espermatozoides/anomalías , Acrosoma , Humanos , Infertilidad Masculina/patología , Masculino , MutaciónRESUMEN
OBJECTIVES: Eugenosedin-A has been found to ameliorate high-fat diet (HFD)-induced hyperglycaemia and hyperlipidaemia in C57BL/6J mice. This study aimed to investigate the mechanisms of action of eugenosedin-A on endothelial function and inflammation in hyperlipidaemic mice. METHODS: C57BL/6J mice were randomly divided into two control groups and two treatment groups. The control mice received either a regular diet or HFD, and the treatment groups were fed HFD with either 5 mg/kg eugenosedin-A or atorvastatin for eight weeks. KEY FINDINGS: Mice fed a HFD had higher concentrations of nitrate (NO) but not prostaglandin E2 (PGE2), increased tumour necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) mRNA and inducible nitric oxide synthase (iNOS) proteins, but decreased endothelial nitric oxide synthase (eNOS) proteins. HFD-induced upregulation of iNOS is associated with p38, extracellular signal-regulated kinase (ERK), c-Jun-N-terminal kinase (JNK), PI3K and Akt/IKKα/p65. Eugenosedin-A and atorvastatin reduced HFD-induced TNF-α and IFN-γ mRNA, NO generation, upregulation of iNOS protein, and down-regulation of eNOS protein. Both agents inhibited p38, ERK, JNK and Akt/IKKα/p65 protein levels in the aorta. However, eugenosedin-A did not significantly reduce p38 in the liver. CONCLUSIONS: Our results showed an association between obesity-induced inflammation and altered levels of TNF-α, IFN-γ, p38, ERK, JNK and Akt/IKKα/p65. Eugenosedin-A, like atorvastatin, could inhibit p38, ERK, JNK, Akt/IKKα/p65 proteins, as well as TNF-α and IFN-γ mRNA during the regulation of the obesity-induced inflammatory process.
Asunto(s)
Antiinflamatorios/uso terapéutico , Endotelio Vascular/efectos de los fármacos , Ácidos Heptanoicos/uso terapéutico , Hiperlipidemias/tratamiento farmacológico , Inflamación/tratamiento farmacológico , Óxido Nítrico Sintasa de Tipo III/metabolismo , Piperazinas/uso terapéutico , Pirroles/uso terapéutico , Animales , Antiinflamatorios/farmacología , Aorta/efectos de los fármacos , Aorta/metabolismo , Atorvastatina , Grasas de la Dieta/efectos adversos , Regulación hacia Abajo , Endotelio Vascular/metabolismo , Femenino , Ácidos Heptanoicos/farmacología , Hiperlipidemias/inducido químicamente , Hiperlipidemias/metabolismo , Inflamación/inducido químicamente , Inflamación/metabolismo , Interferón gamma/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Ratones , Ratones Endogámicos C57BL , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Óxido Nítrico/metabolismo , Obesidad/complicaciones , Piperazinas/farmacología , Pirroles/farmacología , ARN Mensajero/metabolismo , Distribución Aleatoria , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
OBJECTIVE: To determine the impact of Ureaplasma urealyticum (Uu) infection on the integrity of sperm plasma membrane in infertile males. METHODS: Sixty-three semen samples were divided into a Uu infection group (n = 32) and a normal control group (n = 31). Conventional semen analyses were performed by computer-assisted semen analysis (CASA) and Uu detected by the culture method. The semen samples were washed with PBS and dyed by SYBR-14/PI double fluorescent staining, followed by detection of the integrity of sperm plasma membrane by flow cytometry. The percentage of the sperm with intact plasma membrane was indicated as the percentage of sperm emitting green fluorescence (SYBR-14+/PI-%). RESULTS: The Uu infection group showed a significantly decreased integrity of sperm plasma membrane ([45.14 +/- 10.69]%) and reduced percentage of grade a + b sperm ([23.29 +/- 8.81]%) as compared with the normal control group ([72.68 +/- 9.91]% and [46.32 +/- 9.54]%) (P < 0.01). But there were no significant differences in the semen volume, pH value, and sperm concentration between the two groups (P > 0.05). CONCLUSION: Uu infection decreases the integrity of sperm plasma membrane, which might be an important factor of male infertility.
Asunto(s)
Membrana Celular/patología , Infertilidad Masculina/patología , Espermatozoides/patología , Infecciones por Ureaplasma/patología , Adulto , Estudios de Casos y Controles , Citometría de Flujo , Humanos , Infertilidad Masculina/microbiología , Infertilidad Masculina/fisiopatología , Masculino , Compuestos Orgánicos , Análisis de Semen/métodos , Espermatozoides/metabolismo , Infecciones por Ureaplasma/fisiopatología , Ureaplasma urealyticum , Adulto JovenRESUMEN
Eugenosedin-B is able to block serotonin (5-HT) and alpha/beta receptors and to inhibit platelet aggregation. In Wistar rats, intravenous injections of eugenosedin-B (2.4, 7.2, 12 micromoL/kg) caused a dose-dependent decrease in blood pressure and heart rate. In contrast, intracisternal injection of eugenosedin-B (0.3, 0.03 micromoL) and an alpha2-antagonist yohimbine (0.03 micromoL) increased blood pressure and heart rate. Eugenosedin-B and yohimbine prevented hypotension induced by intracisternal injection of an alpha2-agonist clonidine (38 pmol). In in vitro experiments, eugenosedin-B (10, 10, 10 M) competitively antagonized norepinephrine-, clonidine-, and 5-HT (10 to 10 M)-induced vasocontractions in isolated rat aorta. It also competitively antagonized the isoproterenol (10 to 10 M)-induced positive inotropic effects in isolated rat atrium. These findings clearly suggest that eugenosedin-B possesses alpha1, alpha2, beta1, and 5-HT2A receptor blocking activities. In isolated rabbit ear artery sensitized with 16 mM K, eugenosedin-B antagonized 5-nonyloxytryptamine- and 5-HT-induced vasocontractions, indicating it also blocked 5-HT1B and 5-HT2A receptors. In radioligand-binding experiments, eugenosedin-B had significant binding affinities on alpha1, alpha2, beta1, 5-HT1B, and 5-HT2A receptors. In human platelets, eugenosedin-B inhibited epinephrine and 5-HT-induced aggregations. It also had competitive binding effects in human platelet with [H]yohimbine (alpha2), [H]ketanserin (5-HT2A). We conclude that hypotensive and vasorelaxant effects of eugenosedin-B can be attributed to its multiple actions on the blockade of 5-HT1B, 5-HT2A, alpha1/2 and beta1 receptors, and its ability to reduce platelet aggregation attributed to its blockade of alpha2 and 5-HT2A receptors.
Asunto(s)
Antagonistas Adrenérgicos alfa/farmacología , Antagonistas Adrenérgicos beta/farmacología , Plaquetas/efectos de los fármacos , Piperazinas/farmacología , Inhibidores de Agregación Plaquetaria/farmacología , Antagonistas de la Serotonina/farmacología , Estirenos/farmacología , Vasodilatadores/farmacología , Antagonistas Adrenérgicos alfa/administración & dosificación , Antagonistas Adrenérgicos beta/administración & dosificación , Animales , Aorta Torácica/efectos de los fármacos , Aorta Torácica/fisiología , Unión Competitiva , Plaquetas/metabolismo , Presión Sanguínea/efectos de los fármacos , Oído/irrigación sanguínea , Humanos , Hipotensión/inducido químicamente , Hipotensión/prevención & control , Técnicas In Vitro , Inyecciones Intravenosas , Inyecciones Intraventriculares , Masculino , Contracción Miocárdica/efectos de los fármacos , Piperazinas/administración & dosificación , Inhibidores de Agregación Plaquetaria/administración & dosificación , Conejos , Ensayo de Unión Radioligante , Ratas , Ratas Wistar , Receptor de Serotonina 5-HT1B/metabolismo , Receptor de Serotonina 5-HT2A/metabolismo , Receptores Adrenérgicos alfa 1/fisiología , Receptores Adrenérgicos alfa 2/fisiología , Antagonistas del Receptor de Serotonina 5-HT1 , Antagonistas del Receptor de Serotonina 5-HT2 , Antagonistas de la Serotonina/administración & dosificación , Estirenos/administración & dosificación , Vasoconstricción/efectos de los fármacos , Vasodilatadores/administración & dosificaciónRESUMEN
BACKGROUND: Soluble guanylyl cyclase (sGC)/cyclic guanosine monophosphate (cGMP)/protein kinase G (PKG) and Rho kinase (ROCK2) pathways are important in the regulation of prostate smooth muscle tone. This study is aimed to examine the relaxation activities of a sGC activator and PDE5A/ROCK2 inhibitor KMUP-1 in rat prostate and associated anti-proliferation activity in human prostatic epithelial cells. METHODS: The action characteristics of KMUP-1 were identified by isometric tension measurement, receptor binding assay, Western blotting and radioimmunoassay in rat prostate. Anti-proliferation activity of KMUP-1 in human prostatic epithelial PZ-HPV-7 cells was identified using flow cytometry and real time QRT-PCR. RESULTS: KMUP-1 inhibited phenylephrine-induced contractility in a concentration-dependent manner. KMUP-1 possessed potent alpha(1A/)alpha(1D)-adrenoceptor binding inhibition activity, increased cAMP/cGMP levels and increased the expression of sGC, PKG, and PKA protein in rat prostate. Moreover, KMUP-1 inhibited phenylephrine-induced ROCK2 expression. KMUP-1 inhibited cell growth, arrested the cell cycle at G(0)/G(1) phase and increased the expression of p21 in PZ-HPV-7 cells. CONCLUSIONS: These results broaden our knowledge of sGC/cGMP/PKG and ROCK2 regulation on the relaxation and proliferation of prostate, which may help in the design of benign prostate hyperplasia (BPH) therapies that target these signaling pathways. KMUP-1 possesses the potential benefit in the treatment of BPH by its alpha(1A/)alpha(1D)-adrenoceptor blockade, sGC activation, inhibition of PDE5A and ROCK2 and p21 protein enhancement, leading to attenuation of the smooth muscle tone and the proliferation of epithelial PZ-HPV-7 cells. The synergistic contribution of these pathways by KMUP-1 may benefit BPH patients with lower urinary tract symptoms.
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Antagonistas Adrenérgicos alfa/farmacología , GMP Cíclico/metabolismo , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Piperidinas/farmacología , Próstata/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Xantinas/farmacología , 3',5'-GMP Cíclico Fosfodiesterasas/antagonistas & inhibidores , Animales , Western Blotting , Proliferación Celular/efectos de los fármacos , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de GMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 5 , Sinergismo Farmacológico , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Humanos , Técnicas In Vitro , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Masculino , Contracción Muscular/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Piperazinas/farmacología , Próstata/metabolismo , Próstata/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Purinas/farmacología , Ratas , Ratas Wistar , Receptores Adrenérgicos alfa/metabolismo , Citrato de Sildenafil , Sulfonamidas/farmacología , Sulfonas/farmacología , Tamsulosina , Quinasas Asociadas a rhoRESUMEN
KMUP-3 (7-[2-[4-(4-nitrobenzene)piperazinyl]ethyl]-1,3-dimethylxanthine) was investigated in guinea pig tracheal smooth muscle. Intratracheal instillation of tumor necrosis factor (TNF)-alpha (0.01 mg/kg/300 microl) induced bronchoconstriction, increases of lung resistance, and decreases of dynamic lung compliance. Instillation of KMUP-3 (0.5-2.0 mg/kg) reversed this situation. In isolated trachea precontracted with carbachol, KMUP-3 (10-100 microM)-caused relaxations were attenuated by epithelium removal and by pretreatments with an inhibitor of K(+) channel, tetraethylammonium (10 mm); K(ATP) channel, glibenclamide (1 microM); voltage-dependent K(+) channel, 4-aminopyridine (100 microM); Ca(2+)-dependent K(+) channel, charybdotoxin (0.1 microM) or apamin (1 microM); soluble guanylate cyclase (sGC), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1one (ODQ, 1 microM); nitric-oxide (NO) synthase, N(omega)-nitro-L-arginine methyl ester (L-NAME, 100 microM); and adenylate cyclase, SQ 22536 [9-(terahydro-2-furanyl)-9H-purin-6-amine] (100 microM). KMUP-3 (0.01-100 microM) induced increases of cGMP and cAMP in primary culture of tracheal smooth muscle cells (TSMCs). The increase in cGMP by KMUP-3 was reduced by ODQ and L-NAME; the increase in cAMP was reduced by SQ 22536. Western blot analysis indicated that KMUP-3 (1 microM) induced expression of protein kinase A (PKA)(ri) and protein kinase G (PKG)(1alpha 1beta) in TSMCs.SQ 22536 inhibited KMUP-3-induced expression of (PKA)(ri). On the contrary, ODQ inhibited KMUP-3-induced expression of PKG(1alpha 1beta) In epithelium-intact trachea, KMUP-3 increased the NO release. Activation of sGC, NO release, and inhibition of phosphodiesterases in TSMCs by KMUP-3 may result in increases of intracellular cGMP and cAMP, which subsequently activate PKG and PKA, efflux of K(+) ion, and associated reduction in Ca(2+) influx in vitro, indicating the action mechanism to protect against TNF-alpha-induced airway dysfunction in vivo.
Asunto(s)
Proteínas Quinasas Dependientes de GMP Cíclico/biosíntesis , GMP Cíclico/biosíntesis , Contracción Muscular/efectos de los fármacos , Óxido Nítrico/metabolismo , Piperidinas/farmacología , Respiración/efectos de los fármacos , Tráquea , Xantinas/farmacología , Adenilil Ciclasas/metabolismo , Animales , Cobayas , Técnicas In Vitro , Masculino , Músculo Liso/efectos de los fármacos , Músculo Liso/enzimología , Músculo Liso/metabolismo , Hidrolasas Diéster Fosfóricas/metabolismo , Canales de Potasio/metabolismo , Tráquea/efectos de los fármacos , Tráquea/enzimología , Tráquea/metabolismo , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
The cellular mechanisms of vasorelaxant effects of newly synthesized KMUP-3 and KMUP-4 were investigated in rat aortic smooth muscle (RASM). KMUP-3 (7-[2-[4-(4-nitrobenzene)piperazinyl]ethyl]-1,3-dimethylxanthine) and KMUP-4 (7-[2-[4-(2-nitrobenzene)piperazinyl]ethyl]-1,3-dimethylxanthine) elicited concentration-dependent relaxation of endothelium-intact and denuded RASM precontracted with phenylephrine. Relaxant responses were also produced by the PDE inhibitors theophylline, milrinone, rolipram, and zaprinast (1 nM-100 microM). The relaxant responses of KMUP-3 and KMUP-4 were reduced by endothelium removal and by the presence of the NOS inhibitor L-NAME (100 microM), the sGC inhibitor ODQ (1 microM), the adenylyl cyclase (AC) inhibitor SQ 22536 (100 microM), and the prostaglandin inhibitor indomethacin (10 microM). Additionally, the vasorelaxations of both agents were also attenuated by pretreatment with the nonselective K+ channel blocker TEA (10 mM), the KATP channel blocker glibenclamide (1 microM), the voltage-dependent K+ (KV) channel blocker 4-AP (100 microM), and Ca(2+)-dependent K+ (KCa) channel blockers apamin (1 microM) and charybdotoxin (ChTX, 0.1 microM). In addition, elevated extracellular K+ (80 mM) interferes with KMUP-3- and KMUP-4-induced vasorelaxations. Preincubation with both agents (1 microM) significantly enhanced the dilator responses of isoproterenol and SNP. KMUP-3 and KMUP-4 inhibited PDE activities and increased cAMP and cGMP levels in primary culture of RASM that were inhibited by SQ 22536 and ODQ, respectively. In cultured HUVECs, KMUP-3 and KMUP-4 (0.1 microM), more potent than YC-1, significantly increased the expression of eNOS protein. In summary, KMUP-3 and KMUP-4 induce aortic relaxations through both endothelium-dependent and -independent mechanisms. Mechanisms of vasorelaxation induced by both compounds involve multiple processes, such as accumulation of cyclic nucleotides partly as a result of PDE inhibition, K-channel activation, and indomethacin-sensitive endothelium function.