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OBJECTIVE: To campare biomechanical effects of different postural compression techniques on three-dimensional model of lumbar disc herniation (LDH) by finite element analysis. METHODS: Lumbar CT image of a 48-year-old female patient with LDH (heighted 163 cm, weighted 53 kg) was collected. Mimics 20.0, Geomagic Studio, Solidwords and other software were used to establish three-dimensional finite element model of LDH on L4,5 segments. Compression techniques under horizontal position, 30° forward bending and 10° backward extension were simulated respectively. After applying the pressure, the effects of compression techniques under different positions on stress, strain and displacement of various tissues of intervertebral disc and nerve root were observed. RESULTS: L4, 5 segment finite element model was successfully established, and the model was validated. When compression manipulation was performed on the horizontal position, 30° flexion and 10° extension, the annular stress were 0.732, 5.929, 1.286 MPa, the nucleus pulposus stress were 0.190, 1.527, 0.295 MPa, and the annular strain were 0.097, 0.922 and 0.424, the strain sizes of nucleus pulposus were 0.153, 1.222 and 0.282, respectively. The overall displacement distance of intervertebral disc on Y direction were -3.707, -18.990, -4.171 mm, and displacement distance of nerve root on Y direction were +7.836, +5.341, +3.859 mm, respectively. The relative displacement distances of nerve root and intervertebral disc on Y direction were 11.543, 24.331 and 8.030 mm, respectively. CONCLUSION: Compression manipulation could make herniated intervertebral disc produce contraction and retraction trend, by increasing the distance between herniated intervertebral disc and nerve root, to reduce symptoms of nerve compression, to achieve purpose of treatment for patients with LDH, in which the compression manipulation is more effective when the forward flexion is 30°.
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Análisis de Elementos Finitos , Desplazamiento del Disco Intervertebral , Vértebras Lumbares , Humanos , Desplazamiento del Disco Intervertebral/fisiopatología , Femenino , Persona de Mediana Edad , Vértebras Lumbares/fisiopatología , Postura , Fenómenos Biomecánicos , Imagenología TridimensionalRESUMEN
CBP/p300 interacting transactivator with Glu/Asp-rich carboxy-terminal domain 1 (CITED1) is a transcriptional activator belonging to the non-DNA-binding transcription co-regulator family. It regulates diverse pathways, including the transforming growth factor/bone morphogenetic protein/SMAD, estrogen, Wnt-ß-catenin, and androgen-AR signaling pathways, by binding to CBP/p300 co-activators through its conserved transactivation domain CR2. CITED1 plays an important role in embryonic development and a certain regulatory role in the occurrence and development of various tumors. In this article, the biological characteristics, expression regulation, participating signaling pathways, and potential roles of CITED1 in the clinical diagnosis and treatment of tumors are reviewed.
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Proteínas Reguladoras de la Apoptosis , Neoplasias , Transactivadores , Humanos , Estrógenos , Neoplasias/genética , Factores de Transcripción , Proteínas Reguladoras de la Apoptosis/genética , Transactivadores/genéticaRESUMEN
BACKGROUND: Chemoattractant is critical to recruitment of osteoclast precursors and stimulates tumor bone metastasis. However, the role of chemoattractant in bone metastasis of colorectal cancer (CRC) is still unclear. METHODS: Histochemistry analysis and TRAP staining were utilized to detect the bone resorption and activation of osteoclasts (OCs) after administration of CCL7 neutralizing antibody or CCR1 siRNA. qRT-PCR analysis and ELISA assay were performed to detect the mRNA level and protein level of chemoattractant. BrdU assay and Tunel assay were used to detect the proliferation and apoptosis of osteoclast precursors (OCPs). The migration of OCPs was detected by Transwell assay. Western blots assay was performed to examine the protein levels of pathways regulating the expression of CCL7 or CCR1. RESULTS: OCPs-derived CCL7 was significantly upregulated in bone marrow after bone metastasis of CRC. Blockage of CCL7 efficiently prevented bone resorption. Administration of CCL7 promoted the migration of OCPs. Lactate promoted the expression of CCL7 through JNK pathway. In addition, CCR1 was the most important receptor of CCL7. CONCLUSION: Our study indicates the essential role of CCL7-CCR1 signaling for recruitment of OCPs in early bone metastasis of CRC. Targeting CCL7 or CCR1 could restore the bone volume, which could be a potential therapeutical target. Video Abstract.
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Neoplasias Óseas , Quimiocina CCL7 , Neoplasias Colorrectales , Osteoclastos , Osteólisis , Neoplasias Óseas/metabolismo , Neoplasias Óseas/secundario , Huesos/metabolismo , Huesos/patología , Quimiocina CCL7/metabolismo , Factores Quimiotácticos/metabolismo , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Humanos , Osteoclastos/patología , Osteólisis/metabolismo , Regulación hacia ArribaRESUMEN
BACKGROUND: Bone metastasis of colorectal cancer (CRC) often indicates a poor prognosis. Osteolysis can be observed in metastatic sites, implying an aberrant activation of osteoclasts. However, how osteoclastogenesis is regulated in metastatic microenvironment caused by colorectal cancer is still unclear. METHODS: In this study, mice bone metastatic model of CRC was established through injection of MC-38 or CT-26 cells. BrdU assays showed primary CD115 ( +) osteoclast precursors (OCPs) proliferated at the first 2 weeks. Transcriptomic profiling was performed to identify differentially expressing genes and pathways in OCPs indirectly co-cultured with CRC cells RESULTS: The expression of IL4Rα was found to be significantly upregulated in OCPs stimulated by tumor conditioned medium (CM). Further investigation indicated that IL-4 signaling regulated proliferation of OPCs through interacting with type I IL4 receptor, and neutrophils were the main source of IL-4 in bone marrow. The proliferation of OCPs can be inhibited in IL4 deficiency mice. In addition, ERK pathway was activated by IL4/IL4R signaling. Ravoxertinib, an ERK antagonists, could significantly prevent bone destruction through inhibiting the proliferation of OCPs. CONCLUSION: Our study indicates the essential role of IL4/IL4R signaling for the proliferation of OCPs in early metastasis of CRC predominantly through activating ERK pathway, which remarkedly impacts the number of osteoclasts in later stage and leads to osteolytic lesions. Moreover, Ravoxertinib could be a new therapeutical target for bone metastasis of CRC.
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Neoplasias Óseas , Neoplasias Colorrectales , Interleucina-4/metabolismo , Receptores de Interleucina-4/metabolismo , Animales , Apoptosis , Neoplasias Óseas/genética , Neoplasias Óseas/metabolismo , Neoplasias Óseas/secundario , Proliferación Celular , Células Cultivadas , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Interleucina-4/genética , Masculino , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Osteoclastos/citología , Osteólisis , Receptores de Interleucina-4/genética , Transducción de Señal , Tibia/diagnóstico por imagen , Tibia/metabolismoRESUMEN
BRAF mutations, primarily sensitizing mutations, such as BRAFV600E , have been proven to response to the BRAF inhibitor, Dabrafenib combined with trametinib therapy, but there have been no data demonstrating that it has activity against NSCLC-related brain metastases (BM). How patients harboring BRAFS365L mutation (a rare mutation following BRAFV600E -inhibitor treatment) in NSCLC is unknown. Vemurafenib, another BRAF inhibitor, can reverse the resistance that develops with the BRAFS365L mutation following dabrafenib combined with trametentinib treatment in melanoma, but none has been reported in NSCLC. Lung papillary cancer, as a rare typing, occupies about 4% of NSCLC. Hence, we reported the first case of a patient with BM of lung papillary carcinoma harboring a BRAFV600E mutation who benefited from dabrafenib combined with trametinib, and following the development of the BRAFS365L mutation, vemurafenib remained an effective therapeutic option. Moreover, we found that the next-generation sequencing (NGS) of cerebrospinal fluid (CSF) circulating tumor DNA (ctDNA) may potentially provide more accurate information about intracranial lesions than ctDNA in the blood serum, which will be a better detection method.
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Lung adenocarcinoma remains a threat to human health due to its high rate of recurrence and distant metastasis. However, the molecular mechanism underlying lung adenocarcinoma metastasis remains yet incompletely understood. Here, we show that upregulated expression of polypeptide N-acetylgalactosaminyltransferase6 (GALNT6) in lung adenocarcinoma is associated with lymph node metastasis and poor prognosis. In lung adenocarcinoma cells, GALNT6 over-expression promoted epithelial-mesenchymal transition (EMT), wound healing, and invasion which could be significantly reversed by GALNT6 silencing. GALNT6 silencing also mitigated the metastasis of lung adenocarcinoma and prolonged the survival of xenograft tumor-bearing mice. Furthermore, GALNT6 directly interacted with, and O-glycosylated chaperone protein GRP78, which promoted EMT by enhancing the MEK1/2/ERK1/2 signaling in lung cancer cells. Therefore, GALNT6 is emerging as novel positive regulator for the malignancy of human lung adenocarcinoma. Targeting GALNT6-GRP78-MEK1/2/ERK1/2 may thus represent a new avenue to develop therapeutics against lung cancer metastasis.
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Adenocarcinoma del Pulmón/enzimología , Movimiento Celular , Proteínas de Choque Térmico/metabolismo , Neoplasias Pulmonares/enzimología , N-Acetilgalactosaminiltransferasas/metabolismo , Células A549 , Adenocarcinoma del Pulmón/genética , Adenocarcinoma del Pulmón/secundario , Adulto , Anciano , Anciano de 80 o más Años , Animales , Chaperón BiP del Retículo Endoplásmico , Transición Epitelial-Mesenquimal , Femenino , Regulación Neoplásica de la Expresión Génica , Glicosilación , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Masculino , Ratones Endogámicos BALB C , Ratones Desnudos , Persona de Mediana Edad , N-Acetilgalactosaminiltransferasas/genética , Invasividad Neoplásica , Procesamiento Proteico-Postraduccional , Transducción de Señal , Carga Tumoral , Polipéptido N-AcetilgalactosaminiltransferasaRESUMEN
Brain metastasis (BM) is a leading cause of mortality in patients with non-small cell lung cancer (NSCLC). However, the molecular mechanisms underlying BM of NSCLC remain largely unknown because of the lack of models to accurately investigate such a dynamic and complex process. Here we developed a multi-organ microfluidic chip as a new methodological platform to study BM. The chip consisted of two bionic organ units - an upstream "lung" and a downstream "brain" characterized by a functional "blood-brain barrier (BBB)" structure, allowing real-time visual monitoring of the entire BM process, from the growth of primary tumor to its breaking through the BBB, and finally reaching the brain parenchyma. The chip was verified by lung cancer cell lines with differing metastatic abilities and then applied for the BM research where we first demonstrated that the protein expression of Aldo-keto reductase family 1 B10 (AKR1B10) was significantly elevated in lung cancer BM. Silencing AKR1B10 in brain metastatic tumor cells suppressed their extravasation through the BBB in the in vitro Transwell model, in our ex vivo microfluidic chip, as well as the in vivo model of brain metastasis in nude mice. Moreover, AKR1B10 downregulated the expression of matrix metalloproteinase (MMP)-2 and MMP-9 via MEK/ERK signaling in metastatic lung cancers. These data suggest that our multi-organ microfluidic chip is a practical alternative to study BM pathogenesis, and AKR1B10 is a diagnostic biomarker and a prospective therapeutic target for NSCLC BM. STATEMENT OF SIGNIFICANCE: Brain metastasis (BM) of non-small cell lung cancer (NSCLC) is a complex cascade, and in particular, the process of lung cancer cells penetrating the blood-brain barrier (BBB) is very unique. However, due to the lack of reliable models that can faithfully mimic the dynamic process of BBB breaking, its molecular mechanisms have not well elucidated so far. In addition, although Aldo-keto reductase family 1 B10 (AKR1B10) has been implicated to the tumor development of liver cancer and many other cancers, little is known on its roles in the BM. Here, we established a multi-organ microfluidic bionic chip platform to recapitulate the entire BM process, and applied it to the BM pathology research, especially BBB extravasation. By using the chip and traditional models synergistically, we first demonstrated that AKR1B10 was significantly elevated in lung cancer BM, and defined the value of AKR1B10 as a diagnostic serum biomarker for lung cancer patients suffering from BM. Further, we investigated the role and mechanisms of AKR1B10 in BM that it promotes the extravasation of cancer cells through the BBB.
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Aldo-Ceto Reductasas/metabolismo , Neoplasias Encefálicas , Dispositivos Laboratorio en un Chip , Neoplasias Pulmonares , Técnicas Analíticas Microfluídicas , Modelos Biológicos , Proteínas de Neoplasias/metabolismo , Neoplasias Encefálicas/enzimología , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/secundario , Humanos , Neoplasias Pulmonares/enzimología , Neoplasias Pulmonares/patología , Metástasis de la Neoplasia , Células THP-1RESUMEN
A 67-year-old man from Jingzhou was admitted to the First Hospital Affiliated to Yangtze University in July 2013 with sudden onset of abdominal pain with dizziness for 12 h. The patient had sign of peritoneal irritation. Ultrasonography of the abdomen and pelvis showed hepatic fibrosis due to schistosomiasis. Computed tomography showed free gas in the peritoneal cavity. Plain abdominal radiography showed bilateral subdiaphragmatic accumulation of gas, perforation of the viscus, and radio-opacity in the left renal area. The patient underwent emergency exploratory laparotomy. At laparotomy, a moderate amount of muddy yellow pus was found in the intra-abdominal cavity. At the junction of the jejunum and ileum, about 250 cm from Treitz's ligament, there was an about 10-cm length of inflamed small bowel with perforation (3 mm in diameter) along the mesenteric border at the middle of the lesion. The patient underwent resection of the affected intestinal segment, along with end-to-end intestinal anastomosis. Histopathological examination revealed mucosal necrosis and hemorrhage with a large number of infiltrating eosinophils and neutrophils, and acute submucosal inflammation with a large number of infiltrating eosinophils and neutrophils associated with Schistosoma japonicum (S. japonicum) eggs. No intravascular adult parasite was found. Postoperatively, the patient was treated with praziquantel (30 mg/kg daily) for 4 d. The patient progressed well. To the best of our knowledge, this is the first case of small bowel perforation associated with eggs of S. japonicum.
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Parasitosis Intestinales/parasitología , Perforación Intestinal/parasitología , Intestino Delgado/parasitología , Peritonitis/parasitología , Schistosoma japonicum/aislamiento & purificación , Esquistosomiasis Japónica/parasitología , Dolor Abdominal/parasitología , Anciano , Animales , Biopsia , Procedimientos Quirúrgicos del Sistema Digestivo , Humanos , Parasitosis Intestinales/diagnóstico , Parasitosis Intestinales/terapia , Perforación Intestinal/diagnóstico , Perforación Intestinal/terapia , Intestino Delgado/cirugía , Peritonitis/diagnóstico , Peritonitis/terapia , Esquistosomiasis Japónica/diagnóstico , Esquistosomiasis Japónica/terapia , Esquistosomicidas/uso terapéutico , Resultado del TratamientoRESUMEN
BACKGROUND: Non-invasive prenatal testing (NIPT) by massively parallel sequencing is a useful clinical test for the detection of common fetal aneuploidies. While the accuracy of aneuploidy detection can approach 100%, results discordant with the fetus are occasionally reported. In this study we investigated the basis of a discordant T21 positive and T18 negative NIPT result associated with a T18 fetus confirmed by karyotyping. METHODS: Massively parallel sequencing was used to detect fetal DNA in maternal circulating plasma. The parental origin and nature of the fetal and placental aneuploidies were investigated by quantitative fluorescent PCR of short tandem repeat (STR) sequences and by copy number variation (CNV) sequencing. RESULTS: There was no evidence of T21 maternal mosaicism, T21 microchimerism or a vanishing twin to explain the discordant NIPT result. However, examination of multiple placental biopsies showed both T21 and T18 mosaicism, including one confined region with a significantly higher proportion of T21 cells. Based on fetal DNA fractions and average mosaicism levels, the effective T21 and T18 fetal DNA fractions should have been sufficient for the detection of both trisomies. CONCLUSIONS: In this pregnancy, we speculate that confined placental region(s) with higher proportions of T21 cells were preferentially releasing fetal DNAs into the maternal circulation. This study highlights placental mosaicism as a significant risk factor for discordant NIPT results.
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ADN/sangre , Feto/citología , Placenta/citología , Diagnóstico Prenatal , Trisomía/diagnóstico , Trisomía/genética , Cromosomas Humanos Par 18/genética , ADN/genética , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Embarazo , Análisis de Secuencia de ADN , Síndrome de la Trisomía 18 , Adulto JovenAsunto(s)
Ácido Fólico/metabolismo , Cardiopatías Congénitas/genética , Metilenotetrahidrofolato Deshidrogenasa (NADP)/genética , Metilenotetrahidrofolato Reductasa (NADPH2)/genética , Polimorfismo Genético , Preescolar , Cistationina betasintasa/genética , Ácido Fólico/sangre , Predisposición Genética a la Enfermedad , Cardiopatías Congénitas/enzimología , Humanos , Lactante , Recién Nacido , Metiltransferasas/genética , Antígenos de Histocompatibilidad Menor , Factores de RiesgoRESUMEN
OBJECTIVE: To observe the killing effect of sodium abietate on adult male and female worms of Schistosoma japonicum in vitro. METHODS: The mice infected with cercariae of S. japonicum were sacrificed and perfused five weeks later, the adult worms obtained by the portal perfusion method, were cultivated in DMEM medium containing different concentrations of sodium abietate for 3 days, except the controls, then the worms were observed for the death and motility reducing. The worms were stained by hydrochloric acid carmine for the detection of the changes, and the protein of the worms was detected by using the ultraviolet ray-absorption and Bradford method. RESULT: After the treatment of sodium abietate, the mortality and motility reducing rate of adult worms were higher significantly than the controls; the effect of sodium abietate on male worms was more obvious than on female worms. The male worms' intestinal canal enlarged and appeared black or brown bands or spots after the treatment. The contents of the intestine of female worms were distributed asymmetrically, and the shape of some worms' ovaries was anomalism and the coloring was asymmetrical. Compared with the control group, the protein of adult male and female worms were reduced (P < 0.05). CONCLUSION: Sodium abietate could kill adult worms of S. japonicum in vitro. It may affect the protein metabolism of the worms.
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Abietanos/toxicidad , Antihelmínticos/toxicidad , Schistosoma japonicum/efectos de los fármacos , Esquistosomiasis Japónica/parasitología , Animales , Femenino , Masculino , Ratones , Schistosoma japonicum/fisiología , Esquistosomiasis Japónica/tratamiento farmacológicoRESUMEN
SR-A (class A macrophage scavenger receptor) is a transmembrane receptor that can bind many different ligands, including modified lipoproteins that are relevant to the development of vascular diseases. However, the precise endocytic pathways of SR-A/mediated ligands internalization are not fully characterized. In this study, we show that the SR-A/ligand complex can be endocytosed by both clathrin- and caveolae-dependent pathways. Internalizations of SR-A-lipoprotein (such as acLDL) complexes primarily go through clathrin-dependent endocytosis. In contrast, macrophage apoptosis triggered by SR-A-fucoidan internalization requires caveolae-dependent endocytosis. The caveolae-dependent process activates p38 kinase and JNK signaling, whereas the clathrin-mediated endocytosis elicits ERK signaling. Our results suggest that different SR-A endocytic pathways have distinct functional consequences due to the activation of different signaling cascades in macrophages.
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Apoptosis/fisiología , Caveolas/metabolismo , Endocitosis/fisiología , Sistema de Señalización de MAP Quinasas/fisiología , Macrófagos/metabolismo , Receptores Depuradores de Clase A/metabolismo , Animales , Antiulcerosos/farmacología , Apoptosis/efectos de los fármacos , Línea Celular , Activación Enzimática/efectos de los fármacos , Activación Enzimática/fisiología , MAP Quinasa Quinasa 4/genética , MAP Quinasa Quinasa 4/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Polisacáridos/farmacología , Receptores Depuradores de Clase A/genética , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Shenfu injection (the major components of which are ginsenosides compound, extract of Panax ginseng shown to have antioxidant properties) is a well-known important Chinese traditional medicine used for the treatment of various diseases especial for cardiac diseases. The precise mechanism of the biological actions of this plant is not fully understood, in order to elucidate the protection of cardiomyocytes. The aim of the present study was to investigate the effect of Shenfu injection on hypoxia/reoxygenation (H/R)-induced apoptosis and the expression of bcl-2 and caspase-3 in cultured neonatal rat cardiomyocytes in vitro. Ventricular myocytes were isolated from neonatal rat hearts and were exposed to 4 h of hypoxia followed by 16 h of reoxygenation. The results indicated that treatment with different doses of Shenfu injection protected cardiacmyocyte cultures from hypoxia/reoxygenation-induced apoptosis. Caspase-3 activation was decreased in hypoxic/reoxygenationed cardiomyocytes co-treated with Shenfu injection when compared to hypoxia/reoxygenation alone treated cultures. Expression of the Bcl-2 proteins was increased in Shenfu injection-treated cardiomyocytes subjected to hypoxia/reoxygenation. In conclusion, ginsenosides compound has obviously protective effects on cardiacmyocytes against apoptosis induced by hypoxia/reoxygenation injury, whose mechanisms probably involve the inhibition of down-regulation of Bcl-2 protein levels and sequential activation of caspase-3.
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Apoptosis/efectos de los fármacos , Medicamentos Herbarios Chinos/farmacología , Hipoxia/metabolismo , Miocitos Cardíacos/efectos de los fármacos , Animales , Animales Recién Nacidos , Cardiotónicos , Caspasa 3/efectos de los fármacos , Caspasa 3/metabolismo , Células Cultivadas , Medicamentos Herbarios Chinos/administración & dosificación , Ventrículos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Oxígeno , Proteínas Proto-Oncogénicas c-bcl-2/efectos de los fármacos , RatasRESUMEN
OBJECTIVE: To investigate effects of Shenfu injection on the concentrations of plasma tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6), activity of Nuclear Factor kappa B (NF-kappaB) and heart tissue ultrastructure during myocardial ischemia/reperfusion (I/R) injury in rats and its potential mechanism. METHODS: Myocardial ischemia/reperfusion (I/R) was produced by ligation and release of the left anterior descending coronary artery. Ischemia lasted for 30 min and reperfusion for 60 min. Twenty-four healthy male SD rats weighing 230-280 g were randomly divided into three groups (n = 8, each): Group I (Sham-operation group); Group II (I/R group); Group III (Shenfu group), in which Shenfu injection (10 ml/kg) was intraperitoneally injected 30 min before ischemia in animals with I/R. The plasma concentrations of IL-6 and TNF-alpha were measured by ELISA, and the heart was harvested for determination of NF-kappaB levels by Ecl-western blot analysis. Electron microscopy was used to study its ultrastructure. RESULTS: After reperfusion, NF-kappaB binding activity in myocardial nuclei and the plasma concentrations of IL-6 and TNF-alpha were significantly increased in Group II, compared with Group I (P < 0.01), and they were markedly reduced in Group III, compared with Group II (P < 0.01). In addition, electron microscopic examination showed more serious injury of the myocardium ultrastructure in Group II, while in Group III the myocardial ultrastructure was similar to normal state. CONCLUSIONS: Shenfu injection inhibits NF-kappaB activity in I/R myocardium and leads to down-regulation of proinflammatory cytokine expression, which might be one of the molecular mechanisms of Shenfu injection in cardioprotection.
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Medicamentos Herbarios Chinos/farmacología , Daño por Reperfusión Miocárdica/tratamiento farmacológico , FN-kappa B/antagonistas & inhibidores , Animales , Interleucina-6/sangre , Masculino , Daño por Reperfusión Miocárdica/metabolismo , Daño por Reperfusión Miocárdica/patología , Miofibrillas/ultraestructura , Ratas , Ratas Sprague-Dawley , Factor de Necrosis Tumoral alfa/análisisRESUMEN
Nitric oxide (.NO) regulates vascular function, and myoglobin (Mb) is a heme protein present in skeletal, cardiac, and smooth muscle, where it facilitates O(2) transfer. Human ferric Mb binds .NO to yield nitrosylheme and S-nitroso (S-NO) Mb (Witting, P. K., Douglas, D. J., and Mauk, A. G. (2001) J. Biol. Chem. 276, 3991-3998). Here we show that human ferrous oxy-myoglobin (oxyMb) oxidizes .NO, with a second order rate constant k = 2.8 +/- 0.1 x 10(7) M(-1).s(-1) as determined by stopped-flow spectroscopy. Mixtures containing oxyMb and S-nitrosoglutathione or S-nitrosocysteine added at 1.5-2 moles of S-nitrosothiol/mol oxyMb yielded S-NO oxyMb through trans-nitrosation equilibria as confirmed with mass spectrometry. Rate constants for the equilibrium reactions were k(forward) = 110 +/- 3 and k(reverse) = 16 +/- 3 M(-1).s(-1) for S-nitrosoglutathione and k(forward) = 293 +/- 5 and k(reverse) = 20 +/- 2 M(-1).s(-1) for S-nitrosocysteine. Incubation of S-NO oxyMb with Cu(2+) ions stimulated .NO release as measured with a .NO electrode. Similarly, Cu(2+) released .NO from Mb immunoprecipitated from cultured human vascular smooth muscle cells (VSMCs) that were pre-treated with diethylaminenonoate. No .NO release was observed from VSMCs treated with vehicle alone or immunoprecipitates obtained from porcine aortic endothelial cells with and without diethylaminenonoate treatment. Importantly, pre-constricted aortic rings relaxed in the presence of S-NO oxyMb in a cyclic GMP-dependent process. These data indicate that human oxyMb rapidly oxidizes .NO and that biologically relevant S-nitrosothiols can trans-(S)nitrosate human oxyMb. Furthermore, S-NO oxyMb can be isolated from cultured human VSMCs exposed to an exogenous .NO donor at physiologic concentration. The potential biologic implications of S-NO oxyMb acting as a source of .NO are discussed.
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Cisteína/análogos & derivados , Mioglobina/química , Óxido Nítrico/metabolismo , Nitrógeno/química , Animales , Aorta/patología , Células Cultivadas , Cobre/química , Cisteína/química , Electrodos , Electrones , Endotelio Vascular/citología , Humanos , Inmunoprecipitación , Cinética , Modelos Químicos , Músculo Liso/citología , Mioglobina/metabolismo , Óxido Nítrico/química , Oxígeno/metabolismo , Conejos , Proteínas Recombinantes/química , S-Nitrosoglutatión/química , S-Nitrosotioles/química , Espectrofotometría , Factores de TiempoRESUMEN
<p><b>OBJECTIVE</b>To investigate effects of Shenfu injection on the concentrations of plasma tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6), activity of Nuclear Factor kappa B (NF-kappaB) and heart tissue ultrastructure during myocardial ischemia/reperfusion (I/R) injury in rats and its potential mechanism.</p><p><b>METHODS</b>Myocardial ischemia/reperfusion (I/R) was produced by ligation and release of the left anterior descending coronary artery. Ischemia lasted for 30 min and reperfusion for 60 min. Twenty-four healthy male SD rats weighing 230-280 g were randomly divided into three groups (n = 8, each): Group I (Sham-operation group); Group II (I/R group); Group III (Shenfu group), in which Shenfu injection (10 ml/kg) was intraperitoneally injected 30 min before ischemia in animals with I/R. The plasma concentrations of IL-6 and TNF-alpha were measured by ELISA, and the heart was harvested for determination of NF-kappaB levels by Ecl-western blot analysis. Electron microscopy was used to study its ultrastructure.</p><p><b>RESULTS</b>After reperfusion, NF-kappaB binding activity in myocardial nuclei and the plasma concentrations of IL-6 and TNF-alpha were significantly increased in Group II, compared with Group I (P < 0.01), and they were markedly reduced in Group III, compared with Group II (P < 0.01). In addition, electron microscopic examination showed more serious injury of the myocardium ultrastructure in Group II, while in Group III the myocardial ultrastructure was similar to normal state.</p><p><b>CONCLUSIONS</b>Shenfu injection inhibits NF-kappaB activity in I/R myocardium and leads to down-regulation of proinflammatory cytokine expression, which might be one of the molecular mechanisms of Shenfu injection in cardioprotection.</p>