RESUMEN

Background: Acid-sensing ion channel 1a (ASIC1a) plays a critical role in physiological and pathological processes. To further elucidate the biological functions of ASICs and their relationships with disease occurrence and development, it is advantageous to investigate and develop additional regulatory factors for ASICs. Methods: In this study, cation exchange chromatography was used to separate seven chromatographic components from Naja naja atra venom. Capillary electrophoresis was employed to detect that Ⅶ peak component containing a main protein Ⅶ-2, which could bind to ASIC1a. The analgesic effects of Ⅶ-2 protein were determined using hot plate methods, and ASIC1a expression in spinal cord tissue from rats with inflammatory pain was detected using western blot. Results: The purified Ⅶ-2 protein named Naja naja atra venom-Ⅶ-2 (NNAV-Ⅶ-2) was obtained by Sephadex G-50 gel filtration, which exhibited a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular weight of 6.7 kD. Remarkably, the NNAV-Ⅶ-2 protein demonstrated a significant analgesic effect and downregulated ASIC1a expression in the spinal cord tissue of rats with inflammatory pain. Conclusions: The analgesic mechanism of the NNAV-Ⅶ-2 protein may be associated with its binding to ASIC1a, consequently downregulating ASIC1a expression in neural tissues.

RESUMEN

Ibuprofen (IBP) is a typical nonsteroidal anti-inflammatory drug with a wide range of applications, large dosages, and environmental durability. Therefore, ultraviolet-activated sodium percarbonate (UV/SPC) technology was developed for IBP degradation. The results showed that IBP could be efficiently removed using UV/SPC. The IBP degradation was enhanced with prolonged UV irradiation time, with the decreasing IBP concentration and the increasing SPC dosage. The UV/SPC degradation of IBP was highly adaptable to pH ranging from 4.05 to 8.03. The degradation rate of IBP reached 100% within 30 min. The optimal experimental conditions for IBP degradation were further optimized using response surface methodology. IBP degradation rate reached 97.3% under the optimal experimental conditions: 5 µM of IBP, 40 µM of SPC, 7.60 pH, and UV irradiation for 20 min. Humic acid, fulvic acid, inorganic anions, and natural water matrix inhibited the IBP degradation to varying degrees. Scavenging experiments of reactive oxygen species indicated that hydroxyl radical played a major role in the UV/SPC degradation of IBP, while carbonate radical played a minor role. Six IBP degradation intermediates were detected, and hydroxylation and decarboxylation were proposed as the primary degradation pathways. An acute toxicity test, based on the inhibition of luminescence in Vibrio fischeri, indicated that the toxicity of IBP during UV/SPC degradation decreased by 11%. An electrical energy per order value of 3.57 kWh m-3 indicated that the UV/SPC process was cost-effective in IBP decomposition. These results provide new insights into the degradation performance and mechanisms of the UV/SPC process, which can potentially be used for practical water treatment in the future.

Asunto(s)

Contaminantes Químicos del Agua , Purificación del Agua , Radical Hidroxilo , Ibuprofeno/toxicidad , Carbonatos , Especies Reactivas de Oxígeno , Contaminantes Químicos del Agua/toxicidad , Oxidación-Reducción , Rayos Ultravioleta

RESUMEN

Cement stabilized soil (CSS) yields wide application as a routine cementitious material due to cost-effectiveness. However, the mechanical strength of CSS impedes development. This research assesses the feasible combined enhancement of unconfined compressive strength (UCS) and flexural strength (FS) of construction and demolition (C&D) waste, polypropylene fiber, and sodium sulfate. Moreover, machine learning (ML) techniques including Back Propagation Neural Network (BPNN) and Random Forest (FR) were applied to estimate UCS and FS based on the comprehensive dataset. The laboratory tests were conducted at 7-, 14-, and 28-day curing age, indicating the positive effect of cement, C&D waste, and sodium sulfate. The improvement caused by polypropylene fiber on FS was also evaluated from the 81 experimental results. In addition, the beetle antennae search (BAS) approach and 10-fold cross-validation were employed to automatically tune the hyperparameters, avoiding tedious effort. The consequent correlation coefficients (R) ranged from 0.9295 to 0.9717 for BPNN, and 0.9262 to 0.9877 for RF, respectively, indicating the accuracy and reliability of the prediction. K-Nearest Neighbor (KNN), logistic regression (LR), and multiple linear regression (MLR) were conducted to validate the BPNN and RF algorithms. Furthermore, box and Taylor diagrams proved the BAS-BPNN and BAS-RF as the best-performed model for UCS and FS prediction, respectively. The optimal mixture design was proposed as 30% cement, 20% C&D waste, 4% fiber, and 0.8% sodium sulfate based on the importance score for each variable.

RESUMEN

In this article, probabilistic hesitant fuzzy linguistic preference relations (PHFLPRs) are proposed to present the qualitative pairwise preference information of decision makers (DMs) with hesitation and probability uncertainty assessments. The measurements and improvements of additive consistency and consensus of PHFLPRs are investigated in group decision making (GDM). First, a new concept of probabilistic hesitant fuzzy linguistic term sets is defined. Second, the consistency and consensus measurements are established to survey the additive consistency and consensus levels of PHFLPRs. Subsequently, an optimization model is developed to improve the unacceptably additive consistent PHFLPR. By optimizing the unacceptable consensual PHFLPRs with repeating additive consistency improvement, the acceptably additive consistent and consensual PHFLPRs are obtained, based on which DMs' weights are determined objectively and then, the collective PHFLPR is aggregated from individual PHFLPRs. Alternatives' priority weights are derived from the collective PHFLPR as GDM. Finally, an example about failure criticality analysis is given, and a comparison analysis is presented.

Asunto(s)

Algoritmos , Lógica Difusa , Consenso , Toma de Decisiones , Lingüística

RESUMEN

OBJECTIVE: To establish a micellar electrokinetic capillary chromatography-based method for identification and quantitative detection of interleukin-12 (IL-12) and analysis of its unfolding process. METHODS: An uncoated fused-silica capillary (inner diameter 50 µm) with a total length of 48.5 cm (40 cm to the detector) was used for the experiment. The factors influencing the separation efficiency of IL-12 were analyzed, and a standard curve of IL-12 concentration was established. The mixture of IL-12 and anti-IL-12 antibody was incubated in a water bath at 38 ℃ for 40 min, and capillary electrophoresis was then performed under the same conditions. The results were compared with those of IL-12 and anti-IL-12 antibody to identify IL-12. IL-12 and dithiothreitol (DTT) were incubated at 60 ℃ in water bath for different lengths of times, and the unfolding process of IL-12 was analyzed based on electrophoresis results of IL-12 in different states. RESULTS: A micellar capillary electrophoresis on-line sweep method was established with 80 mmol/L borate (pH=9.3) containing 30 mmol/L sodium dodecyl sulfate (SDS) as the buffer solution. This system showed a good linear relationship between the peak area and the mass concentration of IL-12 with a linear correlation coefficient of 0.9991 within the linear range of 2 to 120 ng/L. As the incubation time of IL-12 and DTT prolonged, the disulfide bond of IL-12 gradually opened and resulted in distinct changes in the protein peak. CONCLUSIONS: This capillary electrophoresis-based method is simple and sensitive for IL-2 analysis and allows rapid detection of changes in IL-12 content in the setting of tumors and analysis of the possible causes.

Asunto(s)

Cromatografía Capilar Electrocinética Micelar , Electroforesis Capilar , Interleucina-12 , Micelas , Desplegamiento Proteico

RESUMEN

Protein C (PC) plays an important role in the balance of coagulation and anticoagulation. Thus, the detection of PC activity is diagnostically significant for patients with cardiovascular diseases. Presently, the key methods to detect PC activity are the chromogenic assay and activated partial thromboplastin time (APTT) test. PROTAC used in the chromogenic assay is isolated from Agkistrodon contortrix venom as protein C activator (PCA). However, the use of the chromogenic assay is limited because of the high price of PROTAC. In this study, PCA was successfully purified from Agkistrodon acutus venom (AAV) by ion-exchange and gel chromatography. PCA from AAV has a relative molecular mass of 24 kD, calculated from the measurement of 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The components of PCA were identified by MALDI-TOF/TOF-MS and mascot searches revealed that the coverage rate between PCA and zinc metalloproteinase AaPA from AAV was 21%. The chromogenic assay and APTT test were used to measure the enzymatic activity of PCA, and the results showed that PCA from AAV could specifically activate PC. In summary, the chromogenic assay described herein is highly sensitive and easy to perform.

Asunto(s)

Agkistrodon , Activadores de Enzimas/aislamiento & purificación , Activadores de Enzimas/farmacología , Proteína C/agonistas , Venenos de Serpiente/química , Agkistrodon/metabolismo , Animales , Electroforesis en Gel de Poliacrilamida , Activación Enzimática/efectos de los fármacos , Activadores de Enzimas/química , Proteína C/metabolismo , Conejos , Venenos de Serpiente/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción

RESUMEN

(1) The alloy material 20CrMnTiH is widely used in gear manufacturing, but difficult to process, and its quantity (efficiency) and quality (surface quality) are generally negative correlation indicators. As a difficult but realistic problem, it is of important practical significance to explore how to efficiently grind high-precision low-carbon alloy gear workpieces. (2) Firstly, the pixel method was applied to analyze the grinding principles and explore the grinding parameters-the grinding wheel speed and grinding wheel frame moving speed-as well as the feed rate, which impacts the grinding indicators. Secondly, based on the ceramic microcrystalline corundum grinding wheel and the 20CrMnTiH gear workpiece, controlled experiments with 28 groups of grinding parameters were conducted. Moreover, the impact curves of the grinding parameters on the grinding indicators-the grinding efficiency, grinding wheel life, and surface roughness-were obtained by the multiple linear regression method. Finally, the multi-objective optimization method was used to comprehensively optimize the grinding process. (3) Compared with the traditional grinding process, under optimized grinding parameters, the 20CrMnTiH gear workpieces have a lower surface roughness and a longer grinding wheel life, and require a shorter time to achieve grinding accuracy. (4) The grinding experiments showed that the grinding parameters are linearly related to the grinding indicators. The optimization results show that the precision, efficiency, and economy of the 20CrMnTiH gear grinding process have been improved via the comprehensive optimization of the grinding parameters.

RESUMEN

Objective To investigate a protocol for the efficient differentiation of human induced pluripotent stem cells (hiPSCs) into insulin-producing cells (IPCs) in vitro. Methods Lysine-specific demethylase 1 (LSD1) gene was knocked down in hiPSCs by RNAi. A four-step method was performed to induce the differentiation of hiPSCs into IPCs. The differentiation efficiency of IPCs was analyzed by flow cytometry. Real-time quantitative PCR was used to detect the mRNA levels of LSD1, OCT4, SOX17, FOXA2, PDX1, PAX4, PAX6, HNF6, TCF1, NKX6.1, GLUT2, GK, insulin and MAFA. The expression and localization of PDX1 and insulin were determined by immunofluorescence technique. DTZ staining and transmission electron microscopy were used to observe the secretion and distribution of intracellular insulin-containing granules in IPCs. In addition, the yield of insulin and C-peptide of IPCs were tested by ELISA. Results Compared with the control, the LSD1 knockdown group showed a higher differentiation efficiency of IPCs and the mRNA expression of pancreatic islet ß-cell development-related genes SOX17, PDX1, PAX4 and insulin were significantly up-regulated. IPCs from the LSD1 knockdown group co-expressed mature ß-cell specific markers PDX1 and insulin. In the LSD1 knockdown group, IPCs released insulin as secretory vesicles in response to glucose stimuli, and the yield of insulin or C-peptide reached 1/6 of adult human islets (only 1/8 in the control group). Conclusion Knockdown of LSD1 can promote the efficient differentiation of hiPSCs into IPCs in vitro.

Asunto(s)

Células Madre Pluripotentes Inducidas , Células Secretoras de Insulina , Diferenciación Celular , Glucosa , Histona Demetilasas , Humanos , Insulina

RESUMEN

OBJECTIVE: To investigate the effects of Agkistrodon acutus venom protein C activator(PCA) on ultrastructure of human umbilical vein endothelial cells(HUVEC), and the levels of tissue factor(TF), vascular von Willebrand factor (vWF) and endothelin-1 secreted by HUVEC and to clarify the anti-thrombotic mechanism of PCA. METHODS: The experiments were divided into control group(DMEM), LPS group (LPS 0.1 µg/ml), PCA group(PCA 1 µg/ml) and PCA+LPS group (1 µg/ml PCA+ 0.1 µg/ml LPS). The morphology of endoplasmic reticulum, mitochondria and the number of autophagosome in HUVEC were observed by transmission electron microscopy. The TF, vWF and ET-1 were measured in the medium of each group by ELISA; RT-PCR was used to detect mRNA expression level of vWF and ET-1 in cells; and the protein expression level of TF in cells was detected by Western blot. RESULTS: Compared with the control group, the ultrastructural changes of HUVEC in the LPS group included the cell membrane getting rough, swelling of mitochondria and endoplasmic reticulum, and autophagosome increase, however, the ultrastructure differences between PCA and control group were not significant. Compared with the ultrastructure of HUVECs in LPS group, the swelling of mitochondria and endoplasmic reticulum disappeared in the LPS+PCA group, and the number of autophagosome decreased obviously. Compared with the control group, the content of ET-1, vWF and TF in cell culture supernatant, and the protein expression level of vWF, ET-1 gene and TF protein were significantly increased in LPS group (P<0.05); the expression levels of the 3 factors in the cell culture supernatant and cells in PCA group were not significantly different from the control group (P>0.05). The expression levels of TF, vWF and ET-1 in LPS group were significantly lower than those in LPS+PCA group (P<0.05). CONCLUSION: PCA(1 µg/ml) can reduce the ultrastructural changes of HUVEC induced by LPS, and inhibit the increase of TF, vWF and ET-1 secretion from HUVEC induced by LPS.

Asunto(s)

Agkistrodon , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Venenos de Serpiente/farmacología , Animales , Células Cultivadas , Endotelio Vascular , Humanos , Tromboplastina/efectos de los fármacos , Tromboplastina/metabolismo , Ponzoñas , Factor de von Willebrand

RESUMEN

CONTEXT: Recent studies show that the Agkistrodon acutus (Viperidae) (syn. Deinagkistrodon acutus) protein C activator (PCA) treats acute myocardial infarction and ischaemia-reperfusion animal models effectively, while the underlying mechanism remains unknown. OBJECTIVE: To study the effect of PCA on the injury of human umbilical vein endothelial cells (HUVECs) induced by H2O2 and the underlying mechanism. MATERIALS AND METHODS: Primary cultured HUVECs were pretreated with PCA (20, 40 and 80 µg/mL) for 1 h, then HUVEC apoptosis was induced by 300 µmol/mL H2O2. Apoptosis was analyzed by AnnexinV-FITC/PI, and reactive oxygen species (ROS) level was tested by flow cytometry. Colorimetric methods were used to detect the levels of NO and IL-1. In addition, real-time PCR and western blot analyses were used to detect the expression of eNOS and phospho-p38/MAPK. RESULTS: Morphological changes were induced by H2O2 in HUVECs. The cell survival rate was increased by 43.9, 64.0 and 80.6% in each PCA pretreated group (20, 40 and 80 µg/mL) compared to the model group. In each PCA pretreated group, oxidative stress level was also decreased to 54.7, 42.7 and 25.1%. Moreover, the level of IL-1 was decreased to 83.3, 62.2 and 30.7%. The level of NO was increased by 155.9, 232.4 and 317.6%. Apoptosis rate was decreased to 59.0, 47.7 and 32.7%. Phospho-p38 expression was downregulated, but eNOS expression was upregulated. DISCUSSION AND CONCLUSION: The results suggest that PCA can effectively protect the endothelial cells from injury induced by H2O2, which may be associated with antioxidation, upregulation of eNOS and downregulation of p38-MAPK.

Asunto(s)

Agkistrodon , Apoptosis/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Peróxido de Hidrógeno/toxicidad , Proteína C , Venenos de Víboras/farmacología , Animales , Apoptosis/fisiología , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Células Cultivadas , Relación Dosis-Respuesta a Droga , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Proteína C/metabolismo , Venenos de Víboras/aislamiento & purificación

RESUMEN

AIM: To assess the effects of protein C activator (PCA) from Agkistrodon halys snake venom on cardiac fibrosis in streptozotocin (STZ) induced diabetic rat model, and investigate the mechanisms of its action. METHODS: PCA was identified by one-dimensional reversed phase liquid chromatography - mass spectrometry/mass spectrometry. Male Sprague-Dawley rats (120-140 g) were randomly assigned to negative control (NC) and diabetic group. Diabetes was induced by STZ in high-fat diet fed rats. Diabetic group was subdivided into three groups: diabetic group (DM), diabetic group treated with PCA (0.5, 2, and 8 mg/kg), and diabetic group treated with metformin (5 mg/kg, positive control). NC and DM groups received the same volume of distilled water. Left ventricular mass index (LVWI) and collagen volume fraction were measured by hematoxylin and eosin and Masson staining. Transforming growth factor beta-1 (TGF-ß1) and interleukin 1 beta (IL-1ß) levels were determined by enzyme-linked immunosorbent assay. RESULTS: The diabetic rat model was successfully established by STZ induction and high-fat diet. Glucose level, LVWI, TGF-ß1 and IL-1ß level, and collagen volume fraction were significantly reduced in diabetic rats treated by PCA in a dose-dependent manner (P<0.050), especially in the high dose (8 mg/kg) group (P<0.010), compared to diabetes group. The high dose PCA had the same effect as metformin positive control in reducing the level of fasting blood glucose. PCA decreased the expression of MMP-2 and reduced that of TIMP-2. CONCLUSION: Our results indicate that PCA has anti-fibrotic effects and that it may be used to treat myocardial fibrosis.

Asunto(s)

Agkistrodon , Venenos de Crotálidos/química , Diabetes Mellitus Experimental/tratamiento farmacológico , Fibrosis Endomiocárdica/prevención & control , Oligopéptidos/farmacología , Animales , Glucemia/metabolismo , Western Blotting , Cromatografía de Fase Inversa , Colágeno/metabolismo , Relación Dosis-Respuesta a Droga , Fibrosis Endomiocárdica/sangre , Fibrosis Endomiocárdica/patología , Activación Enzimática , Ensayo de Inmunoadsorción Enzimática , Ventrículos Cardíacos/patología , Hipoglucemiantes/farmacología , Interleucina-1beta/sangre , Masculino , Metaloproteinasa 2 de la Matriz/metabolismo , Metformina/farmacología , Oligopéptidos/aislamiento & purificación , Proteína C/metabolismo , Ratas , Ratas Sprague-Dawley , Espectrometría de Masas en Tándem , Inhibidor Tisular de Metaloproteinasa-2/metabolismo , Factor de Crecimiento Transformador beta1/sangre

RESUMEN

OBJECTIVE: To investigate the effect of component I from agkistrodon acutus venomon (AAVC-I) the migration of human umbilical vein endothelial cells (HUVECs), and to elucidate the possible anti-angiogenic mechanism of AAVC-I. METHODS: The effect of AAVC-I on the migration of HUVECs which was cultivated in vitro and treated with AAVC-1 at four concentrations: 0, 20, 40, 80 microg/ml, was observed by methods of scratch wound-healing and Transwell assay. The expression level of mRNA and protein of P-selectin and intercellular cell adhension molecule-I (ICAM-1) were examined by RT-PCR and Western blot assay. RESULTS: Compared with the blank group, the migration ability of HUVECs in each AAVE-I treated group was reduced in a dose-dependent manner, and the expression level of the mRNA and protein of P-selectin and ICAM-1 were decreased. CONCLUSION: AAVC-I inhibits the migration of endothelial cell, which is acted by down-regulation of the expression content of mRNA and protein of P-selectin and ICAM-1.

Asunto(s)

Movimiento Celular/efectos de los fármacos , Venenos de Crotálidos/farmacología , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Cultivadas , Regulación hacia Abajo , Humanos , Molécula 1 de Adhesión Intercelular/metabolismo , Selectina-P/metabolismo , ARN Mensajero

RESUMEN

OBJECTIVE: To explore the association of fractalkine (FKN) and CD11c expressions oncommon carotid artery atherosclerotic plaques from apoE(-/-) mice with the severity of atherosclerotic lesions. METHODS: Totally 24 apoE(-/-) mice were divided into two groups and fed on a high-fat diet or a normal diet for 12 weeks. Then the blood lipids as well as the plaque area and vascular stenosis rate of the common carotid artery were measured to evaluate the severity of atherosclerotic lesions of the animals. Moreover, immunohistochemical staining was performed to examine the levels of FKN and CD11c expression. RESULTS: The plaque areas and vascular stenosis rates of the common carotid artery in the experimental group were remarkably larger than those in control group (about 4-fold and 2-fold, respectively). The level of FKN expression in the experimental group was 2 times of that in the control group (P<0.05), and the number of CD11c (+) cells in the plaques in the experimental group was about 4 times of than in the control group (P<0.05). CONCLUSION: The expressions of chemokine and FKN remarkably increase in apoE (-/-) atherosclerotic plaques, suggesting that chemokine and FKN may paly important roles in the development of atherosclerosis.

Asunto(s)

Aterosclerosis/metabolismo , Antígenos CD11/metabolismo , Quimiocina CX3CL1/metabolismo , Placa Aterosclerótica/patología , Animales , Aterosclerosis/patología , Dieta Alta en Grasa , Modelos Animales de Enfermedad , Ratones , Ratones Noqueados

RESUMEN

This study was purpose to investigate apoptosis pathway of leukemia K562 cells induced by anticoagulant fraction from Agkistrodon acutus venom (AVVC-1). The mitochondrial transmembrane potential (ΔΨm) of leukemia K562 cells was detected by flow cytometry with JC-1 single staining. The expression of cytochrome C in the mitochondrial of leukemia K562 cells was analyzed by Western blot after AVVC-1 treatment. The distribution of cytochrome C in leukemia K562 cells was measured by immuno-fluorescence test. The results showed that the potential of mitochondrial membrane decreased after treatment with different concentrations of AVVC-1 (12.5, 25, 50, 100 µg/ml) for 6 h (P < 0.01). The expression level of cytochrome C protein in mitochondria obviously declined after treatment with 30 µg/ml AVVC-1 for 48 h, and the fluorescent intensity of cytochrome C in cytosol was enhanced at the same time. It is concluded that AVVC-1-induced K562 cell apoptosis is related with mitochondrial damage, and cytochrome C may be a useful agent for investigating human leukemia therapy by using AVVC-1.

Asunto(s)

Agkistrodon , Apoptosis/efectos de los fármacos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias/metabolismo , Venenos de Serpiente/farmacología , Animales , Citocromos c/metabolismo , Humanos , Células K562

RESUMEN

A new capillary zone electrophoresis method (CZE) has been established for the interaction analysis of protein C (PC) with a protein C activator (PCA) from Agkistrodon acutus venom. The analysis was performed on an uncoated fused-silica capillary with 75 microm i.d. and a total length of 60.2 cm (50 cm to the detector) with a buffer solution of 50 mmol/L Tris-HCl (pH 7.4) and 198 nm of wavelength. The factors which influence the separation of the PCA, such as buffer solution and ion concentration, and the interaction between the PCA and PC incubated for different times at 37.5 degrees C were studied. The linear range was from 10 to 300 mg/L. The limit of detection was 3 mg/L (S/N = 3). The relative standard deviation (RSD) for the migration time of the PCA was 0.56%. The RSD for the peak area was 3.8% (n = 6). The equal volumes of the PCA (200 mg/L) and PC (60 mg/L) were incubated for five minutes, at which their binding rate reached the maximum. And no hydrolyzed peptide chain from PC was found in the electropherogram. The PCA from Agkistrodon acutus venom could activate PC directly through changing the space conformation of PC. The method is simple, and highly sensitive with high resolution, and will provide important theoretical basis for the rapid detection of venom proteins and their activities in the future.

Asunto(s)

Venenos de Crotálidos/química , Electroforesis Capilar/métodos , Oligopéptidos/química , Proteína C/química , Dominios y Motivos de Interacción de Proteínas

RESUMEN

To investigate the effects of component I from Agkistrodon acutus venom (AAVC-I) on the biological features of chronic myeloid leukemia cells, K562/A02 leukemia cells were cultured in the presence of AAVC-I (6.25 - 100 µg/ml) and the proliferation status was assayed by CCK-8 method. Morphological changes were observed by inversed microscope after Giemsa and Hochest 33258 staining, and cell apoptosis was detected by flow cytometry. Caspase 3 activity was tested by using Chromogenic Activity Assay Kit. The results showed that AAVC-I inhibited the growth of K562/A02 cells in time- and concentration-dependant manners, and the IC(50) at 48 h was 30.988 µg/ml. Giemsa and Hochest 33258 staining showed the typical apoptotic features in K562/A02 cells after induction with AAVC-I for 48 h. Flow cytometric analysis revealed that the percentage of the apoptotic cells reached from 0.88 up to 53.66 as the treated concentration was elevated from 0 to 50 µg/ml. Compared with the control group, the expression of caspase 3 in the tested group was enhanced in a dose-dependent manner (P < 0.05). It is concluded that AAVC-I can effectively inhibit the growth and promote apoptosis of K562/A02 cells. Elevated expression of caspase-3 may be attributed to the apoptosis of K562/A02 cells.

Asunto(s)

Apoptosis/efectos de los fármacos , Caspasa 3/metabolismo , Venenos de Crotálidos/farmacología , Leucemia/metabolismo , Animales , Proliferación Celular/efectos de los fármacos , Regulación Leucémica de la Expresión Génica , Humanos , Células K562 , Leucemia/patología

RESUMEN

The study was purposed to investigate the effect of extract of Agkistrodon Halys venom on proliferation and apoptosis of K562 cells. The inhibition of K562 cell proliferation was measured by MTT assay; The morphologic changes of K562 cells was observed by microscopy; the apoptosis of K562 cells was measured by flow cytometry; the activity of extracellular signal-regulated kinase (ERK) in K562 cells was detected by Western blot. The results showed that when K562 cells were treated with 0, 1, 10, 20 microg/ml of the extraction for 48 hours, the apoptosis rates were 2.1%, 21.3%, 49.7%, 70.1%, respectively. The proliferation of K562 cells was obviously inhibited in dose-dependent manner. Typical morphologic changes significantly appeared in the extract-treated K562 cells. The extract obviously inhibited the activity of ERK in K562 cells. It is concluded that the extract of Agkistrodon Halys' venom can inhibit the proliferation of K562 cells and induce apoptosis of K562 cells.

Asunto(s)

Agkistrodon , Apoptosis/efectos de los fármacos , Mezclas Complejas/farmacología , Venenos de Crotálidos/química , Animales , Proliferación Celular/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Células K562

RESUMEN

Myocardial ischemia reperfusion results in an increase in intracellular sodium concentration, which secondarily increases intracellular calcium via Na(+)-Ca2+ exchange, resulting in cellular injury. Endoxin is an endogenous medium of digitalis receptor and can remarkably inhibit Na+/K(+)-ATPase activity. Although the level of plasma endoxin is significantly higher during myocardial ischemia, its practical significance is unclear. This research is to investigate whether endoxin is one of important factors involved in myocardial ischemia reperfusion injury. Ischemia reperfusion injury was induced by 30 min of global ischemia and 30 min of reperfusion in isolated rat hearts. Heart rate (HR), left ventricular developed pressure (LVDP), and its first derivative (+/-dp/dtmax) were recorded. The endoxin contents, intramitochondrial Ca2+ contents, and the Na+/K(+)-ATPase activity in myocardial tissues were measured. Myocardial damages were evaluated by electron microscopy. The endoxin and intramitochondrial Ca2+ contents in myocardial tissues were remarkably higher, myocardial membrane ATPase activity was remarkably lower, the cardiac function was significantly deteriorated, and myocardial morphological damages were severe in myocardial ischemia reperfusion group vs. control. Anti-digoxin antiserum (10, 30 mg/kg) caused a significant improvement in cardiac function (LVDP and +/-dp/dtmax), Na+/K(+)-ATPase activity, and myocardial morphology, and caused a reduction of endoxin and intramitochondrial Ca2+ contents in myocardial tissues. In the present study, the endoxin antagonist, anti-digoxin antiserum, protected the myocardium against the damages induced by ischemia reperfusion in isolated rat hearts. The results suggest that endoxin might be one of main factors mediating myocardial ischemia reperfusion injury.

Asunto(s)

Daño por Reperfusión Miocárdica/metabolismo , Daño por Reperfusión Miocárdica/patología , Saponinas/biosíntesis , Animales , Calcio/metabolismo , Cardenólidos , Digoxina , Femenino , Humanos , Sueros Inmunes/biosíntesis , Técnicas In Vitro , Masculino , Mitocondrias Cardíacas/enzimología , Mitocondrias Cardíacas/metabolismo , Mitocondrias Cardíacas/ultraestructura , Daño por Reperfusión Miocárdica/enzimología , Ratas , Ratas Sprague-Dawley , Intercambiador de Sodio-Calcio/fisiología , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , ATPasa Intercambiadora de Sodio-Potasio/metabolismo