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1.
Int. j. morphol ; 42(1): 216-224, feb. 2024. ilus
Article in English | LILACS | ID: biblio-1528818

ABSTRACT

SUMMARY: Senile osteoporosis is mainly caused by reduced osteoblast differentiation and has become the leading cause of fractures in the elderly worldwide. Natural organics are emerging as a potential option for the prevention and treatment of osteoporosis. This study was designed to study the effect of resveratrol on osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) in osteoporosis mice. A mouse model of osteoporosis was established by subcutaneous injection of dexamethasone and treated with resveratrol administered by gavage. In vivo and in vitro, we used western blot to detect protein expression, and evaluated osteogenic differentiation of BMSCs by detecting the expression of osteogenic differentiation related proteins, calcium deposition, ALP activity and osteocalcin content. Resveratrol treatment significantly increased the body weight of mice, the level of serum Ca2+, 25(OH)D and osteocalcin, ration of bone weight, bone volume/total volume, trabecular thickness, trabecular number, trabecular spacing and cortical thickness in osteoporosis mice. In BMSCs of osteoporosis mice, resveratrol treatment significantly increased the expression of Runx2, osterix (OSX) and osteocalcin (OCN) protein, the level of calcium deposition, ALP activity and osteocalcin content. In addition, resveratrol treatment also significantly increased the expression of SIRT1, p-PI3K / PI3K and p-AKT / AKT in BMSCs of osteoporosis mice. In vitro, resveratrol increased the expression of SIRT1, p-PI3K / PI3K and p-AKT / AKT, Runx2, OSX and OCN protein, the level of calcium deposition, ALP activity and osteocalcin content in BMSCs in a concentration-dependent manner, while SIRT1 knockdown significantly reversed the effect of resveratrol. Resveratrol can attenuate osteoporosis by promoting osteogenic differentiation of bone marrow mesenchymal stem cells, and the mechanism may be related to the regulation of SIRT1/PI3K/AKT pathway.


La osteoporosis senil es causada principalmente por una diferenciación reducida de osteoblastos y se ha convertido en la principal causa de fracturas en las personas mayores en todo el mundo. Los productos orgánicos naturales están surgiendo como una opción potencial para la prevención y el tratamiento de la osteoporosis. Este estudio fue diseñado para estudiar el efecto del resveratrol en la diferenciación osteogénica de las células madre mesenquimales de la médula ósea (BMSC) en ratones con osteoporosis. Se estableció un modelo de osteoporosis en ratones mediante inyección subcutánea de dexametasona y se trató con resveratrol administrado por sonda. In vivo e in vitro, utilizamos Western blot para detectar la expresión de proteínas y evaluamos la diferenciación osteogénica de BMSC detectando la expresión de proteínas relacionadas con la diferenciación osteogénica, la deposición de calcio, la actividad de ALP y el contenido de osteocalcina. El tratamiento con resveratrol aumentó significativamente el peso corporal de los ratones, el nivel sérico de Ca2+, 25(OH)D y osteocalcina, la proporción de peso óseo, el volumen óseo/ volumen total, el espesor trabecular, el número trabecular, el espaciado trabecular y el espesor cortical en ratones con osteoporosis. En BMSC de ratones con osteoporosis, el tratamiento con resveratrol aumentó significativamente la expresión de las proteínas Runx2, osterix (OSX) y osteocalcina (OCN), el nivel de deposición de calcio, la actividad de ALP y el contenido de osteocalcina. Además, el tratamiento con resveratrol también aumentó significativamente la expresión de SIRT1, p-PI3K/PI3K y p-AKT/AKT en BMSC de ratones con osteoporosis. In vitro, el resveratrol aumentó la expresión de las proteínas SIRT1, p-PI3K/PI3K y p- AKT/AKT, Runx2, OSX y OCN, el nivel de deposición de calcio, la actividad de ALP y el contenido de osteocalcina en BMSC de manera dependiente de la concentración, mientras que La caída de SIRT1 revirtió significativamente el efecto del resveratrol. El resveratrol puede atenuar la osteoporosis al promover la diferenciación osteogénica de las células madre mesenquimales de la médula ósea, y el mecanismo puede estar relacionado con la regulación de la vía SIRT1/PI3K/AKT.


Subject(s)
Animals , Male , Mice , Osteoporosis/drug therapy , Resveratrol/administration & dosage , Osteogenesis/drug effects , Cell Differentiation/drug effects , Blotting, Western , Disease Models, Animal , Sirtuin 1 , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/drug effects , Resveratrol/pharmacology , Mice, Inbred C57BL
2.
Int. j. morphol ; 42(1): 154-161, feb. 2024. ilus, tab
Article in English | LILACS | ID: biblio-1528830

ABSTRACT

SUMMARY: Esophageal cancer is one of the most aggressive gastrointestinal cancers. Invasion and metastasis are the main causes of poor prognosis of esophageal cancer. SPRY2 has been reported to exert promoting effects in human cancers, which controls signal pathways including PI3K/AKT and MAPKs. However, the expression of SPRY2 in esophageal squamous cell carcinoma (ESCC) and its underlying mechanism remain unclear. In the present study, we aimed to investigate the detailed role of SPRY2 in the regulation of cell proliferation, invasion and ERK/AKT signaling pathway in ESCC. It was identified that the expression level of SPRY2 in ESCC was remarkably decreased compared with normal tissues, and it was related to clinicopathologic features and prognosis ESCC patients. The upregulation of SPRY2 expression notably inhibited the proliferation, migration and invasion of Eca-109 cells. In addition, the activity of ERK /AKT signaling was also suppressed by the SPRY2 upregulation in Eca-109 cells. Our study suggests that overexpression of SPRY2 suppress cancer cell proliferation and invasion of by through suppression of the ERK/AKT signaling pathways in ESCC. Therefore, SPRY2 may be a promising prognostic marker and therapeutic target for ESCC.


El cáncer de esófago es uno de los cánceres gastrointestinales más agresivos. La invasión y la metástasis son las principales causas de mal pronóstico del cáncer de esófago. Se ha informado que SPRY2 ejerce efectos promotores en los cánceres humanos, que controla las vías de señales, incluidas PI3K/AKT y MAPK. Sin embargo, la expresión de SPRY2 en el carcinoma de células escamosas de esófago (ESCC) y su mecanismo subyacente aún no están claros. En el presente estudio, nuestro objetivo fue investigar el papel detallado de SPRY2 en la regulación de la proliferación celular, la invasión y la vía de señalización ERK/AKT en ESCC. Se identificó que el nivel de expresión de SPRY2 en ESCC estaba notablemente disminuido en comparación con los tejidos normales, y estaba relacionado con las características clínico-patológicas y el pronóstico de los pacientes con ESCC. La regulación positiva de la expresión de SPRY2 inhibió notablemente la proliferación, migración e invasión de células Eca-109. Además, la actividad de la señalización de ERK/AKT también fue suprimida por la regulación positiva de SPRY2 en las células Eca-109. Nuestro estudio sugiere que la sobreexpresión de SPRY2 suprime la proliferación y la invasión de células cancerosas mediante la supresión de las vías de señalización ERK/AKT en ESCC. Por lo tanto, SPRY2 puede ser un marcador de pronóstico prometedor y un objetivo terapéutico para la ESCC.


Subject(s)
Humans , Esophageal Neoplasms/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Esophageal Squamous Cell Carcinoma/metabolism , Membrane Proteins/metabolism , Immunohistochemistry , Biomarkers, Tumor , Blotting, Western , Extracellular Signal-Regulated MAP Kinases , Cell Proliferation , Proto-Oncogene Proteins c-akt
3.
Int. j. morphol ; 41(5): 1348-1356, oct. 2023.
Article in English | LILACS | ID: biblio-1521029

ABSTRACT

SUMMARY: Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that is highly expressed in various types of cancers including breast cancer. However, the role of AhR with its endogenous ligand 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE) on the progression of breast cancer remains poorly understood. We aimed to investigate cell proliferation and migration states in breast cancer after activating AhR with the endogenous ligand ITE. Breast cancer tissue was evaluated by cell lines, immunohistochemistry, reverse transcription-polymerase chain reaction, cell proliferation, flow cytometry, migration assays and western blot techniques. We found that AhR was widely expressed in breast cancer tissues and metastasis lymph node tissues, but not in normal tissues. The expression AhR was independent between the age, grades and TNM classifications for breast cancer tissues. ITE treatment significantly induced the activation of AhR in a time-dependent manner in both MCF-7 and T47D breast cancer cell lines. Meanwhile, ITE did not affect the cell migration but significantly suppressed the cell proliferation in estrogen receptor positive (ER+) MCF-7 andT47D cells, which probably attribute to the induction of cell cycle arrest in G1 phase and shortened S phase. Further mechanism study showed that ERK1/2 and AKT signaling were required for the activation of AhR in MCF-7 cells. These data suggest that AhR is a potential new target for treating patients with breast cancer. ITE may be more potentially used for therapeutic intervention for breast cancer with the kind of ER(+).


El receptor de hidrocarburo de arilo (AhR) es un factor de transcripción activado por ligando que se expresa en gran medida en varios tipos de cáncer, incluido el cáncer de mama. Sin embargo, el papel de AhR con su ligando endógeno 2- (1'H-indol-3'-carbonil)-tiazol-4-ácido carboxílico metil éster (ITE) en la progresión del cáncer de mama sigue siendo poco conocido. Nuestro objetivo fue investigar la proliferación celular y los estados de migración en el cáncer de mama después de activar AhR con el ligando endógeno ITE. El tejido de cáncer de mama se evaluó mediante líneas celulares, inmunohistoquímica, reacción en cadena de la polimerasa con transcriptasa inversa, proliferación celular, citometría de flujo, ensayos de migración y técnicas de transferencia Western. Descubrimos que AhR se expresó ampliamente en tejidos de cáncer de mama y en linfonodos con metástasis, pero no en tejidos normales. La expresión AhR fue independiente entre la edad, grados y clasificaciones TNM para tejidos de cáncer de mama. El tratamiento con ITE indujo significativamente la activación de AhR de manera dependiente del tiempo en las líneas celulares de cancer de mama MCF-7 y T47D. Mientras tanto, ITE no afectó la migración celular, pero suprimió significativamente la proliferación celular en células MCF-7 y T47D con receptor de estrógeno positivo (ER+), lo que probablemente se atribuye a la inducción de la detención del ciclo celular en la fase G1 y la fase S acortada. Un estudio adicional del mecanismo mostró que las señales de ERK1/2 y AKT eran necesarias para la activación de AhR en las células MCF-7. Estos datos sugieren que AhR es un nuevo objetivo potencial para el tratamiento de pacientes con cáncer de mama. ITE puede ser utilizado más potencialmente en la intervención terapéutica para el cáncer de mama con el tipo de ER (+).


Subject(s)
Humans , Female , Thiazoles/administration & dosage , Breast Neoplasms/pathology , Receptors, Aryl Hydrocarbon/drug effects , Indoles/administration & dosage , Thiazoles/pharmacology , Immunohistochemistry , Receptors, Estrogen , Blotting, Western , Cytochrome P-450 CYP1A1/genetics , Cell Line, Tumor/drug effects , Cell Proliferation/drug effects , Cell Migration Assays , Cytochrome P-450 CYP1B1/genetics , Flow Cytometry , Indoles/pharmacology
4.
Int. j. morphol ; 41(4): 1152-1157, ago. 2023.
Article in English | LILACS | ID: biblio-1514346

ABSTRACT

SUMMARY: To investigate changes of MMP-9 in the rat spleen and hypoxia-induced microvascular basement membrane under high altitude hypoxia. Thirty male specific pathogen-free Sprague Dawley rats were randomly divided into control and hypoxia groups, with 15 rats in each group. The rats in the control group were placed in Dingxi City, Gansu Province (2080 m above sea level) for 30 days. Rats in the hypoxia group were raised in a hypoxic environment in Maduo County, Qinghai Province (4300 m above sea level), for 30 days to establish a hypoxic rat model. Routine blood tests, MMP-9 mRNA, MMP-9 protein, and the spleen microvascular basement membrane were detected. (1) Compared with the control group, the red blood cell count, hemoglobin, and hematocrit levels of the rats in the hypoxia group were all increased; thus, a hypoxia model was successfully established. (2) Compared with the control group, the expression of MMP-9 mRNA and protein was significantly higher in the spleen of rats in the hypoxic group, and the difference was statistically significant (P <0.05). (3) Compared with the control group, the blood vessel basement membrane in the spleen of the hypoxia group was degraded. Under natural low air pressure and high altitude conditions, the expression of MMP-9 in rat spleen tissue increases and participates in the degradation of the microvascular basement membrane.


El objetivo de este trabajo fue investigar los cambios de la MMP-9 en el bazo de la rata y la membrana basal microvascular inducida bajo hipoxia a gran altura. Treinta ratas macho Sprague Dawley, libres de patógenos específicos, se dividieron aleatoriamente en dos grupos de 15 ratas cada uno, un grupo control y un grupo hipoxia. Durante 30 días las ratas del grupo control estuvieron en la ciudad de Dingxi, provincia de Gansu (2080 m sobre el nivel del mar). Las ratas del grupo de hipoxia se criaron en un entorno hipóxico en el condado de Maduo, provincia de Qinghai (4300 m sobre el nivel del mar), durante 30 días para establecer un modelo de rata hipóxica. Se realizaron análisis de sangre de rutina, ARNm de MMP-9, proteína MMP-9 y de la membrana basal microvascular del bazo. En comparación con el grupo control, el recuento de glóbulos rojos, la hemoglobina y los niveles de hematocrito de las ratas del grupo de hipoxia aumentaron; por lo tanto, se estableció con éxito un modelo de hipoxia. En comparación con el grupo control, la expresión de ARNm y proteína de MMP-9 fue significativamente mayor en el bazo de las ratas del grupo hipóxico, siendo la diferencia estadísticamente significativa (P <0,05). En comparación con el grupo control, la membrana basal de los vasos sanguíneos estaba degradada en el bazo del grupo hipoxia. En condiciones naturales de baja presión atmosférica y gran altitud, la expresión de MMP-9 en el tejido del bazo de la rata aumenta y participa en la degradación de la membrana basal microvascular.


Subject(s)
Animals , Male , Rats , Spleen/pathology , Basement Membrane/pathology , Matrix Metalloproteinase 9 , Altitude Sickness , Blotting, Western , Rats, Sprague-Dawley , Microscopy, Electron, Transmission , Disease Models, Animal
5.
Int. j. morphol ; 41(3): 811-818, jun. 2023. ilus
Article in English | LILACS | ID: biblio-1514286

ABSTRACT

SUMMARY: The objective of this study was to investigate the mechanism of prenatal stress on the cognitive function of offspring, and clarify the change of histone deacetylase 2 (HDAC2) expression in hippocampal neurons of offspring. 16 pregnant SD rats were randomly divided into control group and stress group, with eight rats in each group. The stress group received restrained stress from 15 to 21 days of pregnancy, while the control group did not receive any treatment. Anxiety-like behavior and spatial memory, learning and memory ability were detected in open field, elevated plus maze, novel object recognition test, and Barnes maze. Nissl staining was used to detect the function of hippocampal neurons. Western blot was used to detect the expression of HDAC2 protein in hippocampal neurons of adult offspring. Immunofluorescence staining was used to detect the expression of HDAC2 protein and hippocampal neurogenesis. The learning and memory ability of adult offspring was decreased. The prenatal stress damaged the function of hippocampal neurons , the expression of HDAC2 was down-regulated, and the number of neurons was reduced. Maternal prenatal stress can down- regulate the expression of HDAC2 in the hippocampus of offspring, inhibits hippocampal neurogenesis and impairs the cognitive function.


El objetivo de este estudio fue investigar el mecanismo del estrés prenatal en la función cognitiva de la descendencia y aclarar el cambio de la expresión de la histona desacetilasa 2 (HDAC2) en las neuronas del hipocampo de la descendencia. 16 ratas SD preñadas se dividieron aleatoriamente en un grupo de control y un grupo de estrés, con ocho ratas en cada grupo. El grupo de estrés recibió estrés durante 15 a 21 días de pre, preñez, mientras que el grupo de control no recibió ningún tratamiento. El comportamiento similar a la ansiedad y la memoria espacial, el aprendizaje y la capacidad de memoria se detectaron en campo abierto, laberinto en cruz elevado, prueba de reconocimiento de objetos novedosos y laberinto de Barnes. La tinción de Nissl se utilizó para detectar la función de las neuronas del hipocampo. Se utilizó Western blot para detectar la expresión de la proteína HDAC2 en las neuronas del hipocampo de la descendencia adulta. La tinción de inmunofluorescencia se utilizó para detectar la expresión de la proteína HDAC2 y la neurogénesis del hipocampo. La capacidad de aprendizaje y memoria de la descendencia adulta se redujo. El estrés prenatal dañó la función de las neuronas del hipocampo, se reguló negativamente la expresión de HDAC2 y se redujo el número de neuronas. El estrés prenatal materno puede regular a la baja la expresión de HDAC2 en el hipocampo de la descendencia, inhibe la neurogénesis del hipocampo y deteriora la función cognitiva.


Subject(s)
Animals , Female , Pregnancy , Rats , Prenatal Exposure Delayed Effects , Stress, Psychological , Histone Deacetylase 2/metabolism , Cognitive Dysfunction , Immunohistochemistry , Blotting, Western , Rats, Sprague-Dawley , Neurogenesis , Epigenomics , Open Field Test , Elevated Plus Maze Test , Hippocampus , Learning , Memory
6.
Int. j. morphol ; 41(2): 431-436, abr. 2023. ilus, tab
Article in Spanish | LILACS | ID: biblio-1440308

ABSTRACT

La enfermedad periodontal es una de las principales causas de pérdida dentaria. Clínicamente, esta patología, mediada por la desregulación del sistema inmune producto de una disbiosis ocurrida en el surco gingival, inicia con la inflamación de la encía y evoluciona con el daño irreversible de los tejidos que rodean el diente. El hueso alveolar es uno de los tejidos afectados esta patología, esto debido a la activación de osteoclastos por la sobreexpresión de la proteína RANKL en el huésped. El propósito de este trabajo es determinar el nivel de sobreexpresión de RANKL, en un modelo de células tumorales U2OS, frente a la infección con Porphyromonas gingivalis y Prevotella intermedia. Para identificar el nivel de RANKL, se definieron cuatro grupos: Un grupo control, no tratado; Grupo PG, tratado con P. gingivalis; Grupo PI, tratado con P. Intermedia; y un grupo PG+PI, tratado con ambas bacterias. El nivel relativo de la proteína RANKL fue determinado en el sobrenadante y en los extractos celulares de manera independiente, mediante la técnica Western blot. En sobrenadantes, el grupo PG mostró mayores niveles de RANKL comparados con PI (p < 0,05). En extractos celulares los niveles fueron mayores en el grupo PG+PI (p < 0,05). El grupo PI mostró los niveles más bajos de RANKL. La infección polimicrobiana resulta en una mayor expresión de RANKL en células tumorales U2OS, mientras que frente a la infección P. gingivalis, se observó mayor cantidad de RANKL soluble.


SUMMARY: Periodontal disease is one of the main causes of tooth loss. Clinically, this pathology, mediated by the deregulation of the immune system due to a dysbiosis occurred in the gingival sulcus, begins with the inflammation of the gum and evolves with the irreversible damage of the tissues that surround the tooth. Alveolar bone is one of the most affected tissues by this disease, due to the activation of osteoclasts by the upregulation of RANKL in the host. The aim of this study is to determine the increase of RANKL, in a U2OS tumor cells model, inoculated with Porphyromonas gingivalis and Prevotella intermedia. To identify the level of RANKL, four groups were defined: A control group, not treated; PG group, treated with P.gingivalis; PI group, treated with P. intermedia; and a PG+PI group, treated with both bacteria. The relative level of RANKL was determined in the supernatant and cell extracts independently, using the Western blot technique. In supernatants, the PG group showed higher RANKL levels compared to PI (p < 0.05). In cell extracts the levels were higher in the PG+PI group (p < 0.05.). The PI group showed the lowest levels of RANKL.Polymicrobial infection results in a greater expression of of soluble RANKL was observed.


Subject(s)
Periodontal Diseases/microbiology , Bacteria, Anaerobic/physiology , Bone Resorption/microbiology , RANK Ligand/metabolism , Cells, Cultured , Blotting, Western , Porphyromonas gingivalis/physiology , Prevotella intermedia/physiology , Cell Line, Tumor , Electrophoresis , RANK Ligand/analysis
7.
Int. j. morphol ; 41(2): 362-367, abr. 2023. ilus
Article in English | LILACS | ID: biblio-1440327

ABSTRACT

SUMMARY: Liver transplantation is the only available method to treat liver failure induced by chronic liver injury. We sought to determine whether the angiotensin-converting enzyme inhibitor, captopril, can inhibit the development of chronic liver injury induced by the hepatotoxic agent thioacetamide (TAA) in association with the suppression of inflammation (hsCRP, TNF-α, and IL-6) / hypoxia- inducible factor 1-alpha (HIF-1α) / profibrosis (TIMP-1, MMP-9, and α-SMA) axis that mediates liver injury. Therefore, the model group of rats was injected for eight weeks with 200 mg/kg TAA starting at week two. The protective group was pretreated with 150 mg/ kg captopril daily for two weeks prior to TAA injections and continued receiving both capropril and TAA agents until being humanely scrificed at week 10. We observed a substantial damage to liver tissue in the model group as demonstrated by a significant (p<0.0001) increase in blood and hepatic tissue levels of high sensitivity C-reactive protein (hsCRP), tumor necrosis factor-a (TNF-α), interleukin- 6 (L-6), HIF-1α, tissue inhibitor of metalloproteinases-1 (TIMP-1), matrix metalloproteinase-9 (MMP-9), alpha-smooth muscle actin (α-SMA), alanine aminotransferase (ALT), and aspartate aminotransferase (AST). All these parameters were significantly (p<0.0244) protected by captopril. Also, a significant (p<0.0001) positive correlation was observed between a-SMA (profibrosis) and the serum and tissue levels of hsCRP, TNF-α, HIF-1α, TIMP-1, MMP-9, and ALT. Thus, these findings suggest that the induction of chronic liver injury by the hepatotoxic compound, TAA is associated with the upregulation of inflammation/HIF-1α/profibrosis, with captopril exhibiting beneficial hepatic pleotropic effects.


El trasplante de hígado es el único método disponible para tratar la insuficiencia hepática inducida por una lesión hepática crónica. Buscamos determinar si el inhibidor de la enzima convertidora de angiotensina, captopril, puede inhibir el desarrollo de lesión hepática crónica inducida por el agente hepatotóxico tioacetamida (TAA) en asociación con la supresión de la inflamación (hsCRP, TNF-α e IL-6) / factor inducible por hipoxia 1-alfa (HIF-1α) / profibrosis (TIMP-1, MMP-9 y α- SMA) eje que media la lesión hepática. Por lo tanto, al grupo modelo de ratas se le inyectó durante ocho semanas 200 mg/kg de TAA a partir de la semana dos. El grupo protector fue pretratado con 150 mg/kg de captopril al día durante dos semanas antes de las inyecciones de TAA y continuó recibiendo capropril y agentes TAA hasta que fue sacrificado en la semana 10. Observamos un daño sustancial en el tejido hepático en el grupo modelo, como lo demuestra un aumento significativo (p<0,0001) de los niveles en sangre y tejido hepático de proteína C reactiva de alta sensibilidad (hsCRP), factor de necrosis tumoral-α (TNF-a), interleucina-6 (L-6), HIF-1α, inhibidor tisular de metaloproteinasas-1 (TIMP-1), metaloproteinasa de matriz-9 (MMP-9), actina de músculo liso alfa (α-SMA), alanina aminotransferasa (ALT) y aspartato aminotransferasa (AST). Todos estos parámetros estaban significativamente (p<0,0244) protegidos por captopril. Además, se observó una correlación positiva significativa (p<0,0001) entre α-SMA (profibrosis) y los niveles séricos y tisulares de hsCRP, TNF-α, HIF-1α, TIMP- 1, MMP-9 y ALT. Por lo tanto, estos hallazgos sugieren que la inducción de daño hepático crónico por el compuesto hepatotóxico, TAA, está asociada con la regulación al alza de la inflamación/HIF-1α/profibrosis, con captopril exhibiendo efectos pleotrópicos hepáticos beneficiosos.


Subject(s)
Animals , Male , Rats , Thioacetamide/toxicity , Captopril/administration & dosage , Chemical and Drug Induced Liver Injury/drug therapy , Fibrosis , Immunohistochemistry , Blotting, Western , Actins , Tumor Necrosis Factor-alpha , Tissue Inhibitor of Metalloproteinase-1 , Matrix Metalloproteinase 9 , Disease Models, Animal , Hepatocyte Nuclear Factor 1-alpha , Real-Time Polymerase Chain Reaction , Matrix Metalloproteinase Inhibitors , Inflammation , Liver/drug effects
8.
Int. j. morphol ; 41(2): 583-590, abr. 2023. ilus
Article in English | LILACS | ID: biblio-1440339

ABSTRACT

SUMMARY: Rheumatoid arthritis (RA) that affects the synovial knee joint causes swelling of the synovial membrane and tissue damage. Interleukin-17A (IL-17A) and the enzyme glycogen synthase kinase-3β (GSK3β) are involved in the pathogenesis of RA. The link between IL-17A, GSK3β, the oxidative stress, and the profibrogenic marker alpha-smooth muscle actin (α-SMA) with and without TDZD-8, GSK3β inhibitor has not been studied before. Consequently, active immunization of rats was performed to induce RA after three weeks using collagen type II (COII) injections. The treated group received daily injection of 1 mg/kg TDZD-8 for 21 days following the immunization protocol (COII+TDZD-8). Blood and synovium tissue samples were harvested at the end of the experiment. RA development was confirmed as corroborated by a substantial increase in blood levels of the highly specific autoantibody for RA, anti-citrullinated protein antibody as well as augmentation of reactive oxidative species (ROS) levels measured as lipid peroxidation. RA induction also increased synovium tissue levels of IL-17A and the profibrogenic marker, α-SMA. All these parameters seemed to be significantly (p<0.0001) ameliorated by TDZD-8. Additionally, a significant correlation between IL-17A, ROS, and α-SMA and biomarkers of RA was observed. Thus, knee joint synovium RA induction augmented IL-17A/GSK3β/ROS/α-SMA axis mediated arthritis in a rat model of RA, which was inhibited by TDZD-8.


La artritis reumatoide (AR) que afecta la articulación sinovial de la rodilla provoca inflamación de la membrana sinovial y daño tisular. La interleucina-17A (IL-17A) y la enzima glucógeno sintasa quinasa-3β (GSK3β) están involucradas en la patogenia de la AR. No se ha estudiadol vínculo entre IL-17A, GSK3β, el estrés oxidativo y el marcador profibrogénico actina de músculo liso alfa (α-SMA) con y sin inhibidor de TDZD-8, GSK3β. En consecuencia, se realizó una inmunización activa de ratas para inducir la AR después de tres semanas usando inyecciones de colágeno tipo II (COII). El grupo tratado recibió una inyección diaria de 1 µg/ kg de TDZD-8 durante 21 días siguiendo el protocolo de inmunización (COII+TDZD-8). Se recogieron muestras de sangre y tejido sinovial al final del experimento. El desarrollo de AR se confirmó como lo corroboró el aumento sustancial en los niveles sanguíneos del autoanticuerpo altamente específico para AR, el anticuerpo antiproteína citrulinada, así como el aumento de los niveles de especies oxidativas reactivas (ROS) medidos como peroxidación lipídica. La inducción de AR también aumentó los niveles de tejido sinovial de IL-17A y el marcador profibrogénico, α-SMA. Todos estos parámetros parecían mejorar significativamente (p<0,0001) con TDZD-8. Además, se observó una correlación significativa entre IL- 17A, ROS y α-SMA y biomarcadores de AR. Por lo tanto, la inducción de AR en la sinovial de la articulación de la rodilla aumentó la artritis mediada por el eje IL-17A/GSK3β/ROS/α-SMA en un modelo de rata de AR, que fue inhibida por TDZD-8.


Subject(s)
Animals , Rats , Arthritis, Rheumatoid , Thiadiazoles/administration & dosage , Fibrosis , Immunohistochemistry , Blotting, Western , Actins , Immunization , Reactive Oxygen Species , Rats, Wistar , Interleukin-17 , Collagen Type II/administration & dosage , Disease Models, Animal , Glycogen Synthase Kinase 3 beta
9.
Journal of Southern Medical University ; (12): 52-59, 2023.
Article in Chinese | WPRIM | ID: wpr-971494

ABSTRACT

OBJECTIVE@#To investigate the effect of ANP32A silencing on invasion and migration of colon cancer cells and the influence of the activity of AKT signaling pathway on this effect.@*METHODS@#Colorectal cancer HCT116 and SW480 were transfected with a small interfering RNA targeting ANP32A via a lentiviral vector. At 24, 48 and 72 h after the transfection, the changes in cell proliferation and AKT activity in the cells were detected using MTT assay and Western blotting, respectively. HCT116 and SW480 cells were treated with the AKT agonist SC79 or its inhibitor MK2206 for 24, 48, 72 and 96 h, and the changes in cell migration and invasion ability were analyzed using Transwell chamber assay and cell proliferation was assessed using MTT assay. The effects of SC79 and MK2206 on migration and invasion abilities of HCT116 and SW480 cells with or without ANP32A silencing were examined using wound healing and Transwell chamber assays, and the changes in the expression of metadherin (MTDH), a factor associated with cells invasion and migration, was detected with Western blotting.@*RESULTS@#Lentivirus-mediated ANP32A silencing significantly down-regulated the activity of AKT and inhibited the proliferation of both HCT116 and SW480 cells (P < 0.01). The application of AKT inhibitor MK2206 obviously inhibited the proliferation, invasion and migration of the colorectal cancer cells (P < 0.05), while the AKT agonist SC79 significantly promoted the invasion and migration of the cells (P < 0.01). In HCT116 and SW480 cells with ANP32A silencing, treatment with MK2206 strongly enhanced the inhibitory effects of ANP32A silencing on cell invasion and migration (P < 0.05) and the expression of MTDH, while SC79 partially reversed these inhibitory effects (P < 0.01).@*CONCLUSION@#ANP32A silencing inhibits invasion and migration of colorectal cancer cells possibly by inhibiting the activation of the AKT signaling pathway.


Subject(s)
Humans , Proto-Oncogene Proteins c-akt , Cell Proliferation , Blotting, Western , Cell Movement , Colonic Neoplasms , Membrane Proteins , RNA-Binding Proteins/genetics , Nuclear Proteins
10.
Biomedical and Environmental Sciences ; (12): 135-145, 2023.
Article in English | WPRIM | ID: wpr-970301

ABSTRACT

OBJECTIVE@#This study investigated how the natural phytophenol and potent SIRT1 activator resveratrol (RSV) regulate necroptosis during Vibrio vulnificus (V. vulnificus)-induced sepsis and the potential mechanism.@*METHODS@#The effect of RSV on V. vulnificus cytolysin (VVC)-induced necroptosis was analyzed in vitro using CCK-8 and Western blot assays. Enzyme-linked immunosorbent assays and quantitative real-time polymerase chain reaction, western blot, and immunohistochemistry and survival analyses were performed to elucidate the effect and mechanism of RSV on necroptosis in a V. vulnificus-induced sepsis mouse model.@*RESULTS@#RSV relieved necroptosis induced by VVC in RAW264.7 and MLE12 cells. RSV also inhibited the inflammatory response, had a protective effect on histopathological changes, and reduced the expression level of the necroptosis indicator pMLKL in peritoneal macrophages, lung, spleen, and liver tissues of V. vulnificus-induced septic mice in vivo. Pretreatment with RSV downregulated the mRNA of the necroptosis indicator and protein expression in peritoneal macrophages and tissues of V. vulnificus-induced septic mice. RSV also improved the survival of V. vulnificus-induced septic mice.@*CONCLUSION@#Our findings collectively demonstrate that RSV prevented V. vulnificus-induced sepsis by attenuating necroptosis, highlighting its potency in the clinical management of V. vulnificus-induced sepsis.


Subject(s)
Animals , Mice , Necroptosis , Resveratrol/therapeutic use , Vibrio vulnificus , Sepsis/drug therapy , Blotting, Western
11.
Journal of Southern Medical University ; (12): 1063-1070, 2023.
Article in Chinese | WPRIM | ID: wpr-987023

ABSTRACT

OBJECTIVE@#To investigate the prognostic value of death-associated protein 5 (DAP5) in gastric cancer (GC) and its regulatory effect on aerobic glycolysis in GC cells.@*METHODS@#We analyzed DAP5 expression levels in GC and adjacent tissues and its association with survival outcomes of GC patients using public databases. We collected paired samples of GC and adjacent tissues from 102 patients undergoing radical resection of GC in our hospital from June, 2012 to July, 2017, and analyzed the correlation of DAP5 expression level detected immunohistochemically with the clinicopathological parameters of the patients. Cox regression analysis, Kaplan-Meier analysis, and ROC curves were used to explore the independent risk factors and the predictive value of DAP5 expression for 5-year survival of the patients. In the cell experiments, we observed the changes in aerobic glycolysis in MGC-803 cells following lentivirus-mediated DAP5 knockdown or overexpression by measuring glucose uptake and cellular lactate level and using qRT-PCR and Western blotting.@*RESULTS@#Analysis using the public databases showed that DAP5 was highly expressed in GC and correlated with tumor progression and poor survival outcomes of the patients (P < 0.05). In the clinical samples, DAP5 expression was significantly higher in GC than in the adjacent tissues (3.19±0.60 vs 1.00±0.12; t=36.863, P < 0.01), and a high expression of DAP5 was associated with a reduced 5-year survival rate of the patients (17.6% vs 72.5%; χ2=29.921, P < 0.05). A high DAP5 expression, T3-4, N2-3, and CEA≥5 ng/mL were identified as independent risk factors affecting 5-year survival outcomes of GC (P < 0.05), for which DAP5 expression showed a prediction sensitivity, specificity and accuracy of 73.2%, 80.4% and 79.0%, respectively. In MGC-803 cells, DAP5 knockdown significantly reduced glucose uptake, lactate level and the expressions of GLUT1, HK2 and LDHA, and DAP5 overexpression produced the opposite effects (P < 0.05).@*CONCLUSION@#A high expression of DAP5 in GC, which enhances cellular aerobic glycolysis to promote cancer progression, is correlated with a poor survival outcome and may serve as a biomarker for evaluating long-term prognosis of GC patients.


Subject(s)
Humans , Stomach Neoplasms , Blotting, Western , Databases, Factual , Glucose , Lactates
12.
Journal of Southern Medical University ; (12): 1041-1046, 2023.
Article in Chinese | WPRIM | ID: wpr-987020

ABSTRACT

OBJECTIVE@#To investigate the changes in gray matter volume in depressive-like mice and explore the possible mechanism.@*METHODS@#Twenty-four 6-week-old C57 mice were randomized equally into control group and model group, and the mice in the model group were subjected to chronic unpredictable mild stimulation (CUMS) for 35 days. Magnetic resonance imaging was performed to examine structural changes of the grey matter volume in depressive-like mice. The expression of brain-derived neurotrophic factor (BDNF) in the grey matter of the mice was detected using Western blotting and immunofluorescence staining.@*RESULTS@#Compared with the control mice, the mice with CUMS showed significantly decreased central walking distance in the open field test (P < 0.05) and increased immobile time in forced swimming test (P < 0.05). Magnetic resonance imaging showed that the volume of the frontal cortex was significantly decreased in CUMS mice (P < 0.001, when the mass level was greater than or equal to 10 756, the FDRc was corrected with P=0.05). Western blotting showed that the expression of mature BDNF in the frontal cortex was significantly decreased in CUMS mice (P < 0.05), and its expression began to decrease after the exposure to CUMS as shown by immunofluorescence staining. The volume of different clusters obtained by voxel-based morphometry (VBM) analysis was correlated with the expression level of mature BDNF detected by Western blotting (P < 0.05).@*CONCLUSION@#The decrease of frontal cortex volume after CUMS is related with the reduction of mature BDNF expression in the frontal cortex.


Subject(s)
Animals , Mice , Blotting, Western , Brain-Derived Neurotrophic Factor , Cerebral Cortex , Depression/physiopathology , Frontal Lobe/pathology
13.
Chinese Journal of Cellular and Molecular Immunology ; (12): 642-648, 2023.
Article in Chinese | WPRIM | ID: wpr-981911

ABSTRACT

Objective To express the monkeypox virus (MPXV) A23R protein in Escherichia coli and purify by Ni-NTA affinity column, and to prepare mouse antiserum against MPXV A23R. Methods The recombinant plasmid pET-28a-MPXV-A23R was constructed and transformed into Escherichia coli BL21 to induce the expression of A23R protein. After optimizing the conditions of expression, A23R protein was highly expressed. Recombinant A23R protein was purified by Ni-NTA affinity column and identified by Western blot analysis. The purified protein was used to immunize mice for preparing the A23R polyclonal antibody, and the antibody titer was detected by ELISA. Results The expression of A23R recombinant protein reached the peak under the induced conditions of 0.6 mmol/L isopropyl-β-D-thiogalactoside (IPTG), 37 DegreesCelsius and 20 hours. The purity of the protein was about 96.07% and was identified by Western blot analysis. The mice were immunized with recombinant protein, and the titer of antibody reached 1:102 400 at the 6th week after immunization. Conclusion MPXV A23R is expressed highly and purified with a high purity and its antiserum from mouse is obtained with a high titre.


Subject(s)
Animals , Mice , Monkeypox virus , Antibodies , Enzyme-Linked Immunosorbent Assay , Blotting, Western , Recombinant Proteins , Escherichia coli/genetics
14.
Chinese Journal of Cellular and Molecular Immunology ; (12): 544-551, 2023.
Article in Chinese | WPRIM | ID: wpr-981897

ABSTRACT

Objective To prepare specific mouse monoclonal antibody (mAb) against human adenovirus type 55 Hexon protein (HAdV55 Hexon). Methods The Hexon genes of HAdV55, 3, 4, 7, 16 and 21 were chemically synthesized as templates for PCR amplification. The prokaryotic expression plasmids pET28a-HAdV55 Hexon and eukaryotic expression plasmids pCAGGS-HAdV3, 4, 7, 16, 21 and 55 Hexon were constructed respectively. The pET28a-HAdV55 Hexon plasmid was transformed into E. coli competent cell BL21 (DE3) and was induced by IPTG. After the purified inclusion body was denatured and renatured, Hexon55 protein was purified by tangential flow filtration system. pCAGGS-HAdV55 Hexon was used to immunize BALB/c mice by cupping, and HAdV55 Hexon protein was used to booster immunization. The anti-HAdV55 Hexon mAb was prepared by hybridoma technique and the titer and subclass were determined. The specificity of antibody was identified by Western blot using HEK293T cells transfected with pCAGGS-HAdV55 Hexon and by immunofluorescence assay (IFA) using BHK cells transfected with pCAGGS-HAdV55 Hexon. Both clones with high titer were selected, and the cross-reactivity of pCAGGS-HAdV3, 4, 7, 16, 21 and 55 Hexon transfected cells were analyzed by Western blot analysis and IFA. Results PET28a-HAdV55 Hexon and pCAGGS-HAdV55 Hexon, 3, 4, 7, 16 and 21 expression plasmids were successfully constructed. BL21 transformed with pET28a-HAdV55 Hexon was induced by IPTG. The HAdV55 Hexon protein was mainly expressed in the form of inclusion body. After denaturation and renaturation, the purified HAdV55 Hexon protein was obtained by ultrafiltration. Six hybridoma cell lines secreting HAdV55 Hexon mAb were obtained. The antibody subclass analysis showed that 2 strains were IgG2a subtypes and 4 strains were IgG2b. Two specific HAdV55 Hexon antibodies with high titer were obtained, and there was no cross-reactivity with HAdV3, 4, 7, 16, 21 Hexon. Conclusion The specific mice mAb against HAdV55 Hexon provides an experimental basis for establishing its antigen detection method.


Subject(s)
Animals , Mice , Humans , Adenoviruses, Human/genetics , Escherichia coli/genetics , HEK293 Cells , Isopropyl Thiogalactoside , Blotting, Western , Immunoglobulin G , Antibodies, Monoclonal , Antibody Specificity , Mice, Inbred BALB C
15.
Chinese Journal of Cellular and Molecular Immunology ; (12): 539-543, 2023.
Article in Chinese | WPRIM | ID: wpr-981896

ABSTRACT

Objective To propose the blood detection strategies for human immunodeficiency virus (HIV) among blood donors, and provide reference for the detection, early diagnosis and transmission blocking of HIV. Methods A total of 117 987 blood samples from blood donors were screened using the third- and fourth-generation ELISA HIV detection reagents. Western blot analysis was used to verify the reactive results of the third-generation reagent alone, or both the third-generation and fourth-generation reagents. HIV nucleic acid test was carried out for those with negative test results of the third- and fourth-generation reagents. For those with positive results of the fourth-generation reagent only, nucleic acid test followed by a confirmatory test by Western blot analysis was carried out. Results 117 987 blood samples from blood donors were tested by different reagents. Among them, 55 were tested positive by both the third- and fourth-generation HIV detection reagents at the same time, accounting for 0.047% and 54 cases were confirmed HIV-positive by Western blot analysis, and 1 case was indeterminate, then turned positive during follow-up testing. 26 cases were positive by the third-generation reagent test alone, among which 24 cases were negative and 2 were indeterminate by Western blot analysis. The band types were p24 and gp160 respectively detected by Western blot analysis, and were confirmed to be HIV negative in follow-up testing. 31 cases were positive by the fourth-generation HIV reagent alone, among which 29 were negative by nucleic acid test, and 2 were positive according to the nucleic acid test.Western blot analysis was used to verify that the two cases were negative. However, after 2~4 weeks, the results turned positive when the blood sample was retested by Western blot analysis during the follow-up of these two cases. All the specimens that were tested negative by both the third- and fourth-generation HIV reagents were validated negative by HIV nucleic acid test. Conclusion A combined strategy with both third- and fourth-generation HIV detection reagents can play a complementary role in blood screening among blood donors. The application of complementary tests, such as nucleic acid test and Western blot analysis, can further improve the safety of blood supply, thus contributing to the early diagnosis, prevention, transmission and treatment of blood donors potentially infected by HIV.


Subject(s)
Humans , HIV Infections/diagnosis , HIV Antibodies , Blood Donors , HIV-1 , Blotting, Western , Nucleic Acids
16.
Chinese Journal of Cellular and Molecular Immunology ; (12): 456-462, 2023.
Article in Chinese | WPRIM | ID: wpr-981886

ABSTRACT

Objective To prepare a rabbit anti-mouse coiled-coil domain containing 189 (Ccdc189) polyclonal antibody. Methods The pET-28a-Ccdc189 prokaryotic expression plasmid was constructed and transformed into E.coli BL21. IPTG was used to induce the expression of Ccdc189 prokaryotic protein. Adult male New Zealand rabbits were immunized with purified recombinant protein to obtain rabbit anti-mouse Ccdc189 polyclonal antibody. The specificity of the polyclonal antibody was identified by Western blot analysis, indirect ELISA and immunofluorescence histochemical staining. Results The pET-28a-Ccdc189 recombinant plasmid was successfully constructed and the expression of the Ccdc189 recombinant protein was induced. ELISA revealed that the titer of the polyclonal antibody was 1:1 000 000. Western blot and immunofluorescence staining demonstrated that the Ccdc189 polyclonal antibody could specifically identify the Ccdc189 prokaryotic protein and the Ccdc189 protein in adult wild-type mouse testis. Conclusion A polyclonal antibody with high specificity against mouse Ccdc189 was successfully created.


Subject(s)
Rabbits , Male , Animals , Mice , Antibody Specificity , Antibodies , Enzyme-Linked Immunosorbent Assay , Blotting, Western , Recombinant Proteins , Escherichia coli/genetics
17.
Chinese Journal of Cellular and Molecular Immunology ; (12): 363-370, 2023.
Article in Chinese | WPRIM | ID: wpr-981875

ABSTRACT

Objective To investigate antigen optimization of Shisa like protein 1 (SHISAL1) for preparing mouse anti-human SHISAL1 polyclonal antibody and to identify the specificity of the prepared antibody. Methods Bioinformatics was employed to predict the antigenic epitope region of SHISAL1 protein, and then a polypeptide composed of amino acid residues from the site of 28 to 97 of SHISAL1, termed SHISAL1-N, was selected as the antigen. The coding region of SHISAL1-N was cloned by molecular cloning technique, and then it was inserted into pET-28a to generate pET28a-SHISAL1-N recombinant plasmid. The two recombinant plasmids pET28a-SHISAL1-N and pET28a-SHISAL1 were transformed into BL21 (DE3) bacteria and induced to express by IPTG. The two proteins were purified and immunized to female Kunming mice, respectively. The specificities and sensitivities of the acquired antibodies were detected by Western blot analysis, immunoprecipitation and immunofluorescent cytochemical staining. Results pET28a-SHISAL1-N recombinant plasmid was successfully constructed, and the two fused proteins, SHISAL1 and SHISAL1-N, were induced to express. Moreover, two types of SHISAL1 mouse polyclonal antibodies, derived from SHISAL1-N and SHISAL1 antigens, were obtained. Western blot results showed that the antibody prepared from SHISAL1 antigen was less specific and sensitive compared with the antibody prepared from SHISAL1-N antigen which could specifically identify different endogenous SHISAL1 protein. Immunoprecipitation results showed that SHISAL1-N antibody could specifically pull down SHIISAL1 protein in hepatocellular carcinoma cells and immunofluorescence results demonstrated that SHISAL1-N antibody could specifically bind to SHISAL1 protein in the cytoplasm. Conclusion We have optimized the SHISAL1 antigen and prepared the mouse anti-human SHISAL1 polyclonal antibodies successfully, which can be used for Western blot analysis, immunoprecipitation and immunofluorescence cytochemical staining.


Subject(s)
Animals , Female , Humans , Mice , Antibodies , Antibody Specificity , Blotting, Western , Cloning, Molecular , Epitopes/genetics
18.
Chinese Journal of Biotechnology ; (12): 2730-2742, 2023.
Article in Chinese | WPRIM | ID: wpr-981229

ABSTRACT

c-Myc protein encoded by c-Myc (cellular-myelocytomatosis viral oncogene) gene regulates the related gene expression through the Wnt/β-catenin signaling pathway, and has received extensive attention in recent years. The purpose of this study was to express Helicoverpa armigera c-Myc gene (Ha-c-Myc) by using prokaryotic expression system, prepare the polyclonal antibody, examine the spatio-temporal expression profile of Ha-c-Myc, and investigate the possible function of Ha-c-Myc in regulating H. armigera sterol carrier protein-2 (SCP-2) gene expression. The Ha-c-Myc gene was amplified by PCR and cloned into a prokaryotic expression plasmid pET-32a(+). The recombinant plasmid pET-32a-Ha-c-Myc was transformed into Escherichia coli BL21. IPTG was used to induce the expression of the recombinant protein. Protein was purified by Ni2+-NTA column and used to immunize New Zealand rabbits for preparing the polyclonal antibody. The Ha-c-Myc expression levels in different developmental stages (egg, larva, prepupa, pupa, and adult) of H. armigera and different tissues (midgut, fat body, head, and epidermis) of the prepupa were determined by real-time quantitative reverse transcription PCR (qRT-PCR). Ha-c-Myc siRNA was synthesized and transfected into H. armigera Ha cells. The relative mRNA levels of Ha-c-Myc and HaSCP-2 in Ha cells were detected by qRT-PCR. Results showed that the pET-32a-Ha-c-Myc recombinant plasmid was constructed. The soluble Ha-c-Myc protein of about 65 kDa was expressed in E. coli. The polyclonal antibody was prepared. Western blotting analysis suggested that the antibody had high specificity. Enzyme linked immunosorbent assay (ELISA) showed that the titer of the antibody was high. Ha-c-Myc gene expressed at all developmental stages, with high levels in the early and late instars of larva, and the prepupal stage. Tissue expression profiles revealed that Ha-c-Myc expressed in various tissues of prepupa, with high expression level in the midgut, but low levels in the epidermis and fat body. RNAi results showed that the knockdown of Ha-c-Myc expression significantly affected transcription of HaSCP-2, leading to a 50% reduction in HaSCP-2 mRNA expression level. In conclusion, the Ha-c-Myc was expressed through a prokaryotic expression system, and the polyclonal anti-Ha-c-Myc antibody was obtained. Ha-c-Myc may promote the expression of HaSCP-2 and play an important role in the lipid metabolism of H. armigera. These results may facilitate further study on the potential role and function mechanism of Ha-c-Myc in H. armigera and provide experimental data for exploring new targets of green pesticides.


Subject(s)
Animals , Rabbits , Escherichia coli/metabolism , Enzyme-Linked Immunosorbent Assay , Moths/genetics , Blotting, Western , Larva/genetics , Isoantibodies/metabolism , Antibody Specificity
19.
Braz. j. biol ; 83: 1-11, 2023. tab, graf, ilus
Article in English | LILACS, VETINDEX | ID: biblio-1468926

ABSTRACT

The telencephalon refers to the most highly developed and anterior part of the forebrain, consisting mainly of the cerebral hemispheres. The study determined Neuroglobin (Ngb) and Hypoxia-inducible factor (HIF-1α) expression in the telencephalon of yak and cattle, and compare the expression and distribution pattern of Ngb and HIF-1α in the two animals. Immunohistochemistry (IHC), quantitative real-time Polymerase Chain Reaction (qRT-PCR), and Western blot (WB) were employed to investigate Ngb and Hif-1α expression in the telencephalon of yak and cattle. mRNA and protein expressions of Ngb and HIF-1α showed positive in different tissues of the yak and cattle telencephalon. Ngb expression in tissues of the yak recorded higher as compare to cattle while HIF-1α expression was found higher in cattle than yak. The HIF-1α expression in some tissues of yak telencephalon was consistent with the cattle. The results documented that HIF-1α may have a direct or indirect synergistic effect on Ngb expression in the yak telencephalon to improve hypoxia adaptation. It is suggested that yak may need more Ngb expression for adaptation, but the expression of HIF-1α seems to be down-regulated during long-term adaptation, and the specific causes of this phenomenon needs to be further verified.


O telencéfalo refere-se à parte anterior e mais desenvolvida do prosencéfalo, consistindo principalmente dos hemisférios cerebrais. O estudo determinou a expressão de neuroglobina (Ngb) e fator indutível por hipóxia (HIF-1α) no telencéfalo de iaques e bovinos e comparou a expressão e o padrão de distribuição de Ngb e HIF-1α nos dois animais. Imuno-histoquímica (IHC), reação em cadeia da polimerase quantitativa em tempo real (qRT-PCR) e Western blot (WB) foram empregados para investigar a expressão de Ngb e Hif-1α no telencéfalo de iaques e bovinos. As expressões de mRNA e proteínas de Ngb e HIF-1α mostraram-se positivas em diferentes tecidos do telencéfalo de iaque e bovino. A expressão de Ngb nos tecidos do iaque foi registrada mais alta em comparação com o gado, enquanto a expressão do HIF-1α foi encontrada mais alta no gado do que no iaque. A expressão de HIF-1α em alguns tecidos do telencéfalo de iaque foi consistente com o gado. Os resultados documentaram que o HIF-1α pode ter um efeito sinérgico direto ou indireto na expressão de Ngb no telencéfalo de iaque para melhorar a adaptação à hipóxia. É sugerido que o iaque pode precisar de mais expressão de Ngb para adaptação, mas a expressão de HIF-1α parece ser regulada para baixo durante a adaptação de longo prazo, e as causas específicas desse fenômeno precisam ser verificadas.


Subject(s)
Animals , Cattle , Hypoxia-Inducible Factor 1/analysis , Neuroglobin/analysis , Telencephalon , Immunohistochemistry , Real-Time Polymerase Chain Reaction , Blotting, Western
20.
Braz. J. Pharm. Sci. (Online) ; 59: e211035, 2023. graf
Article in English | LILACS | ID: biblio-1505835

ABSTRACT

Abstract Compound Danshen Dripping Pills (CDDPs) have been used in clinical treatment to protect the heart from ischemia/reperfusion (IR) injury for many years. However, the underlying mechanism implicated in the protective effects remains to be explored. Here, we determined the effects of CDDPs in Sprague-Dawley rats with the IR model. Cardiac function in vivo was assessed by echocardiography. Transmission electron microscopy, histological and immunohistochemical techniques, Western blotting and recombinant adeno-associated virus 9 transfection were used to illustrate the effects of CDDPs on IR and autophagy. Our results showed that pretreatment with CDDPs decreased the level of serum myocardial enzymes and infarct size in rats after IR. Apoptosis evaluation showed that CDDPs significantly ameliorated the cardiac apoptosis level after IR. Meanwhile, CDDPs pretreatment increased myocardial autophagic flux, with upregulation of LC3B, downregulation of p62, and increased autophagosomes and autolysosomes. Moreover, the autophagic flux inhibitor chloroquine could increase IR injury, while CDDPs could partially reverse the effects. Furthermore, our results showed that the activation of AMPK/mTOR was involved in the cardioprotective effect exerted by CDDPs. Herein, we suggest that CDDPs partially protect the heart from IR injury by enhancing autophagic flux through the activation of AMPK/mTOR.


Subject(s)
Animals , Male , Rats , Reperfusion/classification , Reperfusion Injury/classification , Blotting, Western/instrumentation , Heart/physiopathology , Ischemia/classification , Echocardiography/methods , Microscopy, Electron, Transmission/methods , Infarction/pathology
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