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We investigated Alongshan virus infection in reindeer in northeastern China. We found that 4.8% of the animals were viral RNA-positive, 33.3% tested positive for IgG, and 19.1% displayed neutralizing antibodies. These findings suggest reindeer could serve as sentinel animal species for the epidemiologic surveillance of Alongshan virus infection.
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Anticuerpos Antivirales , Reno , Animales , Reno/virología , China/epidemiología , Anticuerpos Antivirales/sangre , Anticuerpos Neutralizantes/sangre , Infecciones por Bunyaviridae/veterinaria , Infecciones por Bunyaviridae/epidemiología , Infecciones por Bunyaviridae/virología , ARN Viral , Inmunoglobulina G/sangreRESUMEN
Hantaan virus (HTNV) is responsible for hemorrhagic fever with renal syndrome (HFRS), primarily due to its ability to inhibit host innate immune responses, such as type I interferon (IFN-I). In this study, we conducted a transcriptome analysis to identify host factors regulated by HTNV nucleocapsid protein (NP) and glycoprotein. Our findings demonstrate that NP and Gc proteins inhibit host IFN-I production by manipulating the retinoic acid-induced gene I (RIG-I)-like receptor (RLR) pathways. Further analysis reveals that HTNV NP and Gc proteins target upstream molecules of MAVS, such as RIG-I and MDA-5, with Gc exhibiting stronger inhibition of IFN-I responses than NP. Mechanistically, NP and Gc proteins interact with tripartite motif protein 25 (TRIM25) to competitively inhibit its interaction with RIG-I/MDA5, suppressing RLR signaling pathways. Our study unveils a cross-talk between HTNV NP/Gc proteins and host immune response, providing valuable insights into the pathogenic mechanism of HTNV.
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Virus Hantaan , Interferón Tipo I , Interferón Tipo I/metabolismo , Virus Hantaan/genética , Virus Hantaan/metabolismo , Proteínas de Motivos Tripartitos/genética , Proteínas de Motivos Tripartitos/metabolismo , Transducción de Señal , Inmunidad Innata , Proteína 58 DEAD Box/genética , Proteína 58 DEAD Box/metabolismoRESUMEN
This study aimed to analyze the clinical significance of serum ferritin, procalcitonin (PCT), and C-reactive protein (CRP) in patients with hemorrhagic fever with renal syndrome (HFRS). The demographical, clinical, and laboratory data of 373 patients with HFRS in northeastern China were retrospectively analyzed. The levels of serum ferritin and PCT in severe patients (n = 108) were significantly higher than those in mild patients (n = 265, p < 0.001) and associated with HFRS severity. The area under the receiver operating characteristic curve (AUC) values of serum ferritin and PCT for predicting the severity of HFRS were 0.732 (95% CI 0.678-0.786, p < 0.001) and 0.824 (95% CI 0.773-0.875, p < 0.001), respectively, showing sensitivity and specificity of 0.75 and 0.88 for serum ferritin, and 0.76 and 0.60 for PCT. The CRP level in HFRS with bacterial co-infection (n = 115) was higher than that without bacterial co-infection (n = 258, p < 0.001). The AUC value of CRP for predicting bacterial co-infection was 0.588 (95% CI 0.525-0.652, p < 0.001), showing sensitivity and specificity of 0.43 and 0.76, respectively. The serum ferritin level in non-survivors (n = 14) was significantly higher than in survivors (n = 359, p < 0.001). The AUC value of serum ferritin for predicting mortality was 0.853 (95% CI 0.774-0.933, p < 0.001), showing sensitivity and specificity of 0.933 and 0.739. Serum ferritin and PCT have a robust association with HFRS severity and mortality, which may be promising predictors, and CRP is an effective biomarker to assess bacterial co-infection in HFRS.
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Tick-borne encephalitis virus (TBEV) is an important causative agent that causes neurological infections in humans and animals. In recent years, only few epidemiological surveys on TBEV have been conducted in China. The purpose of this study was to determine the prevalence and subtype of TBEV in ticks in northeastern (NE) China. A total of 3799 questing ticks were collected in NE China between April 2015 and June 2016. Ticks were pooled and tested for TBEV RNA using semi-nested reverse transcriptase-polymerase chain reaction. Positive pools were used to isolate the virus and amplify complete sequences, followed by sequence identity and phylogenetic analysis. TBEV RNA was detected in Ixodes persulcatus ticks at a total prevalence of 2.9% (6/143; 95% confidence interval: 1.2%-5.9%). Three TBEV strains were isolated (JL-T75, HLB-T74, and DXAL-T83) and showed 93.9%-99.1% nucleotide identities and 97.1%-99.5% amino acid identities in Far Eastern (FE) TBEV subtypes, and 82.9%-87.6% nucleotide identities and 92.9%-96.4% amino acid identities in other subtypes. For polyprotein, the JL-T75, HLB-T74, and DXAL-T83 strains showed 29, 50, and 55 amino acid residues, respectively, different from those in the TBEV vaccine (Senzhang) strain in China. Phylogenetic analysis revealed that these viruses were clustered in the FE-TBEV branch but formed distinct clades depending on the natural foci. The results of this study suggest that the FE-TBEV subtype is still endemic in I. persulcatus ticks in NE China, and the viruses in different natural foci in NE China are more likely to have genetic differences.
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Virus de la Encefalitis Transmitidos por Garrapatas/genética , Virus de la Encefalitis Transmitidos por Garrapatas/aislamiento & purificación , Ixodes/virología , Animales , Secuencia de Bases , China , Conformación de Ácido Nucleico , Filogenia , ARN Viral/genéticaRESUMEN
BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is the most common monogenic kidney disorder. Half of the patients would slowly progress to end-stage renal disease. However, the potential target for ADPKD treatment is still lacking. METHODS: Four ADPKD patients and two healthy family members were included in this study. The peripheral blood samples were obtained and tested by the whole exome sequencing (WES). The autosomal mutations in ADPKD patients were retained as candidate sites. The Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment, and protein-protein interaction network (PPI) analyses were performed by clusterProfiler R package. A dataset containing 18 ADPKD patients and three normal samples were downloaded from the Gene Expression Omnibus (GEO) database and analyzed using the limma R package. RESULTS: A total of six mutant genes were identified based on the dominant genetic pattern and most of them had not been reported to be associated with ADPKD. Furthermore, 19 harmful genes were selected according to the harmfulness of mutation. GO and KEGG enrichment analyses showed that the processes of single-organism cellular process, response to stimulus, plasma membrane, cell periphery, and anion binding as well as cyclic adenosine monophosphate (cAMP) signaling pathway and pathways in cancer were significantly enriched. Through integrating PPI and gene expression analyses, acyl-CoA thioesterase 13 (ACOT13), which has not been reported to be related to ADPKD, and prostaglandin E receptor 2 (PTGER2) were identified as potential genes associated with ADPKD. CONCLUSIONS: Through combination of WES, gene expression, and PPI network analyses, we identified ACOT13 and PTGER2 as potential ADPKD-related genes.
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Secuenciación del Exoma/métodos , Regulación de la Expresión Génica , Riñón Poliquístico Autosómico Dominante/genética , ARN/genética , Subtipo EP2 de Receptores de Prostaglandina E/genética , Tioléster Hidrolasas/genética , Femenino , Humanos , Masculino , Linaje , Riñón Poliquístico Autosómico Dominante/diagnóstico , Riñón Poliquístico Autosómico Dominante/metabolismo , Subtipo EP2 de Receptores de Prostaglandina E/biosíntesis , Tioléster Hidrolasas/biosíntesis , Tomografía Computarizada por Rayos XRESUMEN
BACKGROUND: The clinical characteristics of patients with confirmed 2019 novel coronavirus disease (COVID-19) in Jilin Province, China were investigated. METHODS: Clinical, laboratory, radiology, and treatment data of 41 hospitalized patients with confirmed COVID-19 were retrospectively collected. The population was stratified by disease severity as mild, moderate, or severe, based on guidelines of the National Health and Medical Commission of China. RESULTS: The 41 hospitalized patients with COVID-19 were studied, and the median age was 45 years (interquartile range [IQR], 31-53; range, 10-87 years) and 18 patients (43.9%) were female. All of the patients had recently visited Wuhan or other places (ie, Beijing, Thailand) or had Wuhan-related exposure. Common symptoms included fever (32[78%]) and cough (29[70.7%]). All patients were without hepatitis B/C virus hepatitis. CRP (C-reactive protein, 11.3 mg/L [interquartile range {IQR}, 2.45-35.2]) was elevated in 22 patients (53.7%), and cardiac troponin I (1.5 ng/mL [IQR, 0.8-5.0]) was elevated in 41 patients (100%). Chest computed tomographic scans showed bilateral ground glass opacity (GGO) or GGO with consolidation in the lungs of 27(65.9%) patients. 31(75.6%) patients had an abnormal electrocardiograph (ECG). Comparing the three groups, the levels of CRP and cardiac troponin I, GGO distribution in bilateral lungs, and electrocardiogram changes were statistically significant (p < 0.05). Cardiac troponin I had a strong positive correlation with CRP (r = 0.704, p = 0.042) and LDH (r = 0.738, p = 0.037). CONCLUSION: Significant differences among the groups suggest that several clinical parameters may serve as biomarkers of COVID-19 severity at hospital admission. Elevated cTnI could be considered as a predictor of severe COVID-19, reflecting the prognosis of patients with severe COVID-19. The results warrant further inspection and confirmation.
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COVID-19/diagnóstico , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores/sangre , COVID-19/epidemiología , COVID-19/patología , COVID-19/fisiopatología , Niño , China/epidemiología , Femenino , Corazón/fisiopatología , Hospitalización , Humanos , Pulmón/diagnóstico por imagen , Pulmón/patología , Masculino , Persona de Mediana Edad , Neumonía Viral/diagnóstico , Neumonía Viral/epidemiología , Neumonía Viral/patología , Neumonía Viral/fisiopatología , Pronóstico , Estudios Retrospectivos , SARS-CoV-2 , Adulto JovenRESUMEN
Eight members of a big family with laboratory-confirmed COVID-19 pneumonia were admitted to First Hospital of Jilin University, Changchun, China, from 28 January to 5 February 2020. The clinical records, laboratory results, and chest computed tomography (CT) scans were retrospectively reviewed. Throat swab samples were positive for severe acute respiratory syndrome coronavirus 2, confirmed by the Center for Disease Control and Prevention of Changchun. All eight patients had fever of different degrees; and 6, 3, and 2 had cough; diarrhea; and sore throat. With disease progression, the percentage of lymphocytes in older patients increased, CT images worsened, and the ratio of lymphocytes increased when images revealed inflammation absorption. Although the CT images showed ground-glass opacities in the youngest patient, his lymphocyte count did not decrease with mild clinical symptoms, and the images showed that inflammation was quickly absorbed. Only the oldest patient developed critical illness. The C reaction protein (CRP) levels of Patient 5 increased significantly, and the rate of decline was the slowest, while his condition was the most severe. The clinical manifestations of COVID-19 in this family cluster varied with contact, age, and underlying disease. Lymphocyte count and quality of chest CT images appeared inversely associated with disease severity. CRP changes may be an indicator of disease severity and prognosis.
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Betacoronavirus , Técnicas de Laboratorio Clínico , Infecciones por Coronavirus/diagnóstico , Infecciones por Coronavirus/transmisión , Neumonía Viral/diagnóstico , Neumonía Viral/transmisión , Adulto , Anciano , Anciano de 80 o más Años , Betacoronavirus/patogenicidad , COVID-19 , Prueba de COVID-19 , China/epidemiología , Infecciones por Coronavirus/epidemiología , Familia , Femenino , Humanos , Pulmón/diagnóstico por imagen , Masculino , Persona de Mediana Edad , Pandemias , Linaje , Neumonía Viral/epidemiología , Estudios Retrospectivos , SARS-CoV-2 , Factores de Tiempo , Tomografía Computarizada por Rayos X , VirulenciaRESUMEN
Since its outbreak in Wuhan, Hubei Province China, 2019-coronavirus infected disease (COVID-19) had been widely spread all over the world, the control of which calls for a better understanding of its epidemiology and clinical characteristics. We included 12 confirmed cases of COVID-19 in First Affiliated Hospital of Jilin University from 23 January 2020 to 11 February 2020, which were retrospectively analyzed for epidemiological, demographic, clinical, laboratory, and radiological features. All the patients were confirmed by nucleic acid detection, the average age of whom was 45.25 years (range, 23-79 years). Most patients had a history of Wuhan traveling or had contact with Wuhan travelers or infected cases. Obvious family cluster was observed. Clinical manifestations included fever (12/12), fatigue (10/12), cough (6/12), sore throat (4/12), headache (3/12), and diarrhea (2/12). Only three out of eight patients had pneumonia manifestation on radiography. Most patients had a normal white blood cell (WBC) count and normal or reduced lymphocyte (LY) count. Pneumonia changes were observed in all the four patients who underwent a chest CT scan. Only one elderly patient developed severe pneumonia, while all the rest were mild disease and had a self-limiting course.
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Infecciones por Coronavirus/epidemiología , Infecciones por Coronavirus/patología , Neumonía Viral/epidemiología , Neumonía Viral/patología , Adulto , Anciano , Betacoronavirus/aislamiento & purificación , COVID-19 , China/epidemiología , Infecciones por Coronavirus/diagnóstico por imagen , Infecciones por Coronavirus/virología , Tos/etiología , Diarrea/etiología , Fatiga/etiología , Femenino , Fiebre/etiología , Cefalea/etiología , Humanos , Recuento de Leucocitos , Masculino , Persona de Mediana Edad , Pandemias , Faringitis/etiología , Neumonía Viral/diagnóstico por imagen , Neumonía Viral/virología , Estudios Retrospectivos , SARS-CoV-2 , Tomografía Computarizada por Rayos X , Adulto JovenRESUMEN
OBJECTIVES: To determine blood Brucella DNA loads between brucellosis patients and those without brucellosis. METHODS: The patient group included 350 brucellosis patients. The control was composed of 200 subjects without brucellosis. The extracted DNA from blood was tested by quantitative polymerase chain reaction (qPCR). The cutoff value was determined by receiver operating characteristic curve analysis. A portion of the brucellosis patients were monitored by qPCR during therapy. RESULTS: The detection limit of qPCR was between 1E+01cfu/µL and 1E+08cfu/µL. The standard curve R2 reached 0.998. The cutoff value was 4E+01cfu/µL, which was determined by comparison of the patient group and the control. The qPCR assay had a specificity of 100% and a sensitivity of 93.14%. The monitoring results showed that the Brucella DNA load decreased in most patients during the first 4 weeks of treatment. One patient with bad treatment compliance showed a rebound. CONCLUSIONS: The qPCR results were in accordance with the course of brucellosis in the clinic. The DNA load often reflects the situation of the Brucella-infected patient. The cutoff value provides an important reference of infection. This qPCR-based method can be used to assist in the diagnosis of brucellosis and to adjust the therapy.
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Brucella/aislamiento & purificación , Brucelosis/diagnóstico , ADN Bacteriano/sangre , Adulto , Pruebas de Aglutinación , Médula Ósea/microbiología , Brucella/efectos de los fármacos , Brucella/genética , Brucelosis/tratamiento farmacológico , Brucelosis/microbiología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Reacción en Cadena en Tiempo Real de la Polimerasa , Sensibilidad y EspecificidadRESUMEN
Chronic hepatic injury caused by hepatitis B and C virus (HBV and HCV) infection, high fat diet and alcohol intake has increased to be the critical promoter of hepatocellular carcinoma (HCC). These high risk factors set into motion a vicious cycle of hepatocyte death, inflammation and fibrosis that finally results in cirrhosis and HCC after several decades. However, the treatment options for HCC are very limited. Therefore, early treatment of liver injury may reduce the incidence and probability of HCC or delay the progression of HCC. Substantial ongoing research has focused on nontoxic biological macromolecules, mainly polysaccharides, which possess prominent efficacies on hepatoprotective activity. Based on these encouraging observations, a great deal of effort has been devoted to discovering novel polysaccharides for the development of effective therapeutics for hepatic injury. This review focuses on the protective effects of polysaccharides on liver injury, including hepatitis virus infection, nonalcoholic steatohepatitis, alcoholic liver disease and other hepatic injuries, and describes the underlying mechanisms.
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Hepatopatías/tratamiento farmacológico , Polisacáridos/farmacología , Polisacáridos/uso terapéutico , Sustancias Protectoras/farmacología , Sustancias Protectoras/uso terapéutico , Animales , Antineoplásicos/química , Antineoplásicos/aislamiento & purificación , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Susceptibilidad a Enfermedades , Humanos , Cirrosis Hepática/tratamiento farmacológico , Cirrosis Hepática/etiología , Cirrosis Hepática/metabolismo , Cirrosis Hepática/patología , Hepatopatías/etiología , Hepatopatías/patología , Hepatopatías Alcohólicas/tratamiento farmacológico , Hepatopatías Alcohólicas/etiología , Hepatopatías Alcohólicas/metabolismo , Hepatopatías Alcohólicas/patología , Polisacáridos/química , Polisacáridos/aislamiento & purificación , Sustancias Protectoras/química , Sustancias Protectoras/aislamiento & purificaciónRESUMEN
The diabetes mellitus (DM)-induced reduction of neurogenesis in the hippocampus is consequently accompanied by cognitive decline. The present study set out to define the critical role played by long noncoding RNA H19 (lncRNA H19) in the apoptosis of hippocampal neurons, as well as oxidative stress (OS) in streptozotocin (STZ)-induced DM mice through regulation of insulin-like growth factor 2 (IGF2) methylation. The expression of lncRNA H19 in the hippocampal neurons and surviving neurons were detected. Hippocampal neurons were cultured and transfected with oe-H19, sh-H19, oe-IGF2, or sh-IGF2, followed by detection of the expressions of IGF2 and apoptosis-related genes. Determination of the lipid peroxide and glutathione levels was conducted, while antioxidant enzyme activity was identified. The IGF2 methylation, the binding of lncRNA H19 to DNA methyltransferase, and the binding of lncRNA H19 to IGF2 promoter region were detected. DM mice exhibited high expressions of H19, as well as a decreased hippocampal neurons survival rate. Higher lncRNA H19 expression was found in DM. Upregulated lncRNA H19 significantly increased the expression of Bax and caspase-3 but decreased that of Bcl-2, thus promoting the apoptosis of hippocampal neuron. Besides, upregulation of lncRNA H19 induced OS. LncRNA H19 was observed to bind specifically to the IGF2 gene promoter region and promote IGF2 methylation by enriching DNA methyltransferase, thereby silencing IGF2 expression. Taken together, downregulated lncRNA H19 reduces IGF2 methylation and enhances its expression, thereby suppressing hippocampal neuron apoptosis and OS in STZ-induced (DM) mice.
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Metilación de ADN/genética , Diabetes Mellitus Experimental/genética , Diabetes Mellitus/genética , Factor II del Crecimiento Similar a la Insulina/genética , ARN Largo no Codificante/genética , Animales , Apoptosis/genética , Diabetes Mellitus/patología , Diabetes Mellitus Experimental/patología , Regulación de la Expresión Génica/genética , Impresión Genómica/genética , Hipocampo/metabolismo , Hipocampo/patología , Humanos , Metiltransferasas/genética , Ratones , Neuronas/metabolismo , Neuronas/patología , Estrés Oxidativo/genética , Regiones Promotoras Genéticas/genética , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteína X Asociada a bcl-2/genéticaRESUMEN
BACKGROUND/AIMS: Cerebral ischemia is considered to be the most common cause of stroke with high mortality. It occurs as a result of the damage of the hippocampal neurons with lymphocyte function-associated antigen (LFA)-1 being emphasized to play a role in the biological functions of hippocampal neurons. This study was conducted in order to investigate the effects of specific knockdown of LFA-1 expression by lentivirus had on the apoptosis of the hippocampal neurons, simulated by rat models of acute cerebral ischemia after cerebral lymphatic blockage. METHODS: A total of 60 Wistar rats were selected as subjects, among which 50 were used to establish models of the acute cerebral ischemia after cerebral lymphatic blockage, while the remaining 10 rats were treated with the sham operation. The underlying regulatory mechanisms regarding LFA-1 were analyzed with the treatment of si-LFA-1 and LFA-1 vector in the hippocampal CA1 area of brain tissues isolated from the rats with acute cerebral ischemia. The brain water content, electrolyte content, and blood-brain barrier permeability located in ischemic area of rats were measured. TUNEL staining and immunochemistry methods were employed in order to determine the apoptosis rate and positive levels of LFA-1, MMP-9, and Caspase-3. The mRNA and protein levels of related genes were also detected by means of RT-qPCR and western blot assay. RESULTS: The brain water content, Na+ and Ca+ contents, blood-brain barrier permeability, apoptosis rate, positive levels of LFA-1, MMP-9, and Caspase-3 were decreased, and the K+ content was increased in ischemic tissues treated with si-LFA-1. The mRNA and protein levels of LFA-1, MMP-9, Caspase-3, and Bax had all decreased, while the mRNA and protein levels of Bcl-2 were elevated in the hippocampal CA1 area of rat brain tissues treated with si-LFA-1. These situations could be reversed through the up-regulation of LFA-1. CONCLUSION: In conclusion, LFA-1 gene silencing could improve the acute cerebral ischemia after cerebral lymphatic blockage by inhibiting apoptosis of the hippocampal neurons in rats.
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Isquemia Encefálica/genética , Isquemia Encefálica/terapia , Silenciador del Gen , Hipocampo/patología , Antígeno-1 Asociado a Función de Linfocito/genética , Animales , Apoptosis , Isquemia Encefálica/patología , Femenino , Terapia Genética , Hipocampo/citología , Hipocampo/metabolismo , Lentivirus/genética , Sistema Linfático/metabolismo , Sistema Linfático/patología , Masculino , Neuronas/citología , Neuronas/metabolismo , Neuronas/patología , Ratas WistarRESUMEN
This study is designed to investigate the role of basic fibroblast growth factor (bFGF) antisense oligonucleotide (ASODN) on the proliferation and differentiation of neural stem cells (NSCs) in rat models with focal cerebral infarction (CI). Seventy-five Sprague-Dawlay (SD) rats were randomly divided into the control, sham, middle cerebral artery occlusion (MCAO), MCAO + nonsense oligonucleotide (NODN), and MCAO + ASODN groups. Proliferation and differentiation of NSCs were detected by bromodeoxyuridine (BrdU) and immunofluorescence staining, respectively. ELISA was performed to detect the expressions of endogenous factors that include insulin-like growth factor 1 (IGF-1), glial cell line derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF), transforming growth factor-α1 (TGF-α1), bFGF, and nerve growth factor (NGF). Results show significant neurological deficits and focal CI in the MCAO and MCAO + NODN groups. An obvious increase of NSC proliferation, reactive proliferation of astrocytes in CI areas, differentiation of newly proliferated NSCs into mature neuronal cells, and expressions of endogenous growth factors exhibited in the MCAO, MCAO + NODN and MCAO + ASODN groups. Compared to the MCAO and MACO + NODN groups, the MCAO + ASODN group showed a significant decrease NSC proliferation and differentiation in CI areas as well as decrease expressions of endogenous growth factors. These findings may offer insight to help us understand more as to how bFGF ASODN can effectively suppress the proliferation and differentiation of NSCs. These findings are expected to help contribute to research for new targets in the treatment of focal CI. J. Cell. Biochem. 118: 3875-3882, 2017. © 2017 Wiley Periodicals, Inc.
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Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Infarto Cerebral/metabolismo , Factor 2 de Crecimiento de Fibroblastos/antagonistas & inhibidores , Regulación de la Expresión Génica/efectos de los fármacos , Células-Madre Neurales/metabolismo , Oligodesoxirribonucleótidos Antisentido/farmacología , Animales , Infarto Cerebral/patología , Factor 2 de Crecimiento de Fibroblastos/biosíntesis , Células-Madre Neurales/patología , Ratas , Ratas Sprague-DawleyRESUMEN
This study aims to explore the effects of the TLR4 signaling pathway on the apoptosis of neuronal cells in rats with diabetes mellitus complicated with cerebral infarction (DMCI). A DMCI model was established with 40 Sprague Dawley rats, which were assigned into blank, sham, DM + middle cerebral artery occlusion (MCAO) and DM + MCAO + TAK242 groups. Superoxide dismutase (SOD) activity and malondialdehyde (MDA) content were measured. A TUNEL assay was applied for detecting cell apoptosis, and Western blotting was used for detecting the expression of TLR4, TNF-α, IL-1ß and apoptosis-related proteins. Compared with the blank and sham groups, there was an increase in cell apoptosis, expression of Bcl-2, Bax, cleaved caspase-3, TNF-α, IL-1ß and TLR4 proteins and MDA content and a decrease in SOD activity in the DM + MCAO and DM + MCAO + TAK242 groups. Compared with those in the DM + MCAO group, rats in the DM + MCAO + TAK242 group exhibited an increase in SOD activity and a decrease in cell apoptosis, expression of Bcl-2, Bax, cleaved caspase-3, TNF-α, IL-1ß and TLR4 proteins and MDA content. Inhibition of the TLR4 signaling pathway reduces neuronal cell apoptosis and nerve injury to protect the brain.
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Apoptosis , Diabetes Mellitus Experimental/metabolismo , Infarto de la Arteria Cerebral Media/metabolismo , Neuronas/metabolismo , Transducción de Señal , Receptor Toll-Like 4/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis/metabolismo , Encéfalo/metabolismo , Encéfalo/patología , Diabetes Mellitus Experimental/complicaciones , Infarto de la Arteria Cerebral Media/complicaciones , Interleucina-1beta/metabolismo , Masculino , Malondialdehído/metabolismo , Ratas Sprague-Dawley , Superóxido Dismutasa/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Amyloid ß peptide binding alcohol dehydrogenase (ABAD) decoy peptide (DP) can competitively antagonize binding of amyloid ß peptide to ABAD and inhibit the cytotoxic effects of amyloid ß peptide. Based on peptide aptamers, the present study inserted ABAD-DP into the disulfide bond of human thioredoxin (TRX) using molecular cloning technique to construct a fusion gene that can express the TRX1-ABAD-DP-TRX2 aptamer. Moreover, adeno-associated virus was used to allow its stable expression. Immunofluorescent staining revealed the co-expression of the transduced fusion gene TRX1-ABAD-DP-TRX2 and amyloid ß peptide in NIH-3T3 cells, indicating that the TRX1-ABAD-DP-TRX2 aptamer can bind amyloid ß peptide within cells. In addition, cell morphology and MTT results suggested that TRX1-ABAD-DP-TRX2 attenuated amyloid ß peptide-induced SH-SY5Y cell injury and improved cell viability. These findings confirmed the possibility of constructing TRX-based peptide aptamer using ABAD-DP. Moreover, TRX1-ABAD-DP-TRX2 inhibited the cytotoxic effect of amyloid ß peptide.
RESUMEN
OBJECTIVE: To construct TRX-ABAD-DP-TRX (T-A-T) fusion gene of a novel ABAD-DP aptamer through the insertion of ABAD-DP into the modified human thioredoxin (hTRX) and exploit the possibility of further applications for the gene therapy of Alzheimer's disease. METHODS: According to the designed sequence, the target fragments of TRX1, TRX2 and ABAD-DP were created by PCR (polymerase chain reaction) and then inserted into the multiple clone site of adeno-associated virus shuttle plasmid pSSHG-CMV with gene cloning technique. The corresponding fusion gene TRX1-ABAD-DP-TRX2 was identified by restriction enzymes digestion with EcoRI and BamHI. The recombinant adeno-associated virus (AAV/T-A-T) was produced in HeLa cells with linear polyethylenimine. The expression of T-A-T fusion gene and co-localization between T-A-T and Aß peptide in NIH 3T3 cells were examined by fluorescent immunohistochemistry. RESULTS: The size of fusogenic fragment TRX1-ABAD-DP-TRX2 was approximately 435 bp. And it was consistent with our design. T-A-T fusion gene was expressed in NIH 3T3 cells. Through co-expression, T-A-T aptamer and intracellular Aß peptide were co-localized. It indicated that T-A-T aptamer could bind Aß within NIH 3T3 cells. CONCLUSION: The TRX1-ABAD-DP-TRX2 fusion gene is successfully cloned and expressed. And it may provide rationales for further applications in the gene therapy of Alzheimer's disease's.