RESUMO
BACKGROUND: After myocardial infarction, the lost myocardium is replaced by fibrotic tissue, eventually progressively leading to myocardial dysfunction. Direct reprogramming of fibroblasts into cardiomyocytes via the forced overexpression of cardiac transcription factors Gata4, Mef2c, and Tbx5 (GMT) offers a promising strategy for cardiac repair. The limited reprogramming efficiency of this approach, however, remains a significant challenge. METHODS: We screened seven factors capable of improving direct cardiac reprogramming of both mice and human fibroblasts by evaluating small molecules known to be involved in cardiomyocyte differentiation or promoting human-induced pluripotent stem cell reprogramming. RESULTS: We found that vitamin C (VitC) significantly increased cardiac reprogramming efficiency when added to GMT-overexpressing fibroblasts from human and mice in 2D and 3D model. We observed a significant increase in reactive oxygen species (ROS) generation in human and mice fibroblasts upon Doxy induction, and ROS generation was subsequently reduced upon VitC treatment, associated with increased reprogramming efficiency. However, upon treatment with dehydroascorbic acid, a structural analog of VitC but lacking antioxidant properties, no difference in reprogramming efficiency was observed, suggesting that the effect of VitC in enhancing cardiac reprogramming is partly dependent of its antioxidant properties. CONCLUSIONS: Our findings demonstrate that VitC supplementation significantly enhances the efficiency of cardiac reprogramming, partially by suppressing ROS production in the presence of GMT.
Assuntos
Antioxidantes , Ácido Ascórbico , Humanos , Camundongos , Animais , Espécies Reativas de Oxigênio , Ácido Ascórbico/farmacologia , Antioxidantes/farmacologia , Reprogramação Celular , Proteínas com Domínio T/genética , Fatores de Transcrição MEF2/genética , Miócitos Cardíacos , Vitaminas , FibroblastosRESUMO
BACKGROUND: Direct cardiac reprogramming is currently being investigated for the generation of cells with a true cardiomyocyte (CM) phenotype. Based on the original approach of cardiac transcription factor-induced reprogramming of fibroblasts into CM-like cells, various modifications of that strategy have been developed. However, they uniformly suffer from poor reprogramming efficacy and a lack of translational tools for target cell expansion and purification. Therefore, our group has developed a unique approach to generate proliferative cells with a pre-CM phenotype that can be expanded in vitro to yield substantial cell doses. METHODS: Cardiac fibroblasts were reprogrammed toward CM fate using lentiviral transduction of cardiac transcriptions factors (GATA4, MEF2C, TBX5, and MYOCD). The resulting cellular phenotype was analyzed by RNA sequencing and immunocytology. Live target cells were purified based on intracellular CM marker expression using molecular beacon technology and fluorescence-activated cell sorting. CM commitment was assessed using 5-azacytidine-based differentiation assays and the therapeutic effect was evaluated in a mouse model of acute myocardial infarction using echocardiography and histology. The cellular secretome was analyzed using mass spectrometry. RESULTS: We found that proliferative CM precursor-like cells were part of the phenotype spectrum arising during direct reprogramming of fibroblasts toward CMs. These induced CM precursors (iCMPs) expressed CPC- and CM-specific proteins and were selectable via hairpin-shaped oligonucleotide hybridization probes targeting Myh6/7-mRNA-expressing cells. After purification, iCMPs were capable of extensive expansion, with preserved phenotype when under ascorbic acid supplementation, and gave rise to CM-like cells with organized sarcomeres in differentiation assays. When transplanted into infarcted mouse hearts, iCMPs prevented CM loss, attenuated fibrotic scarring, and preserved ventricular function, which can in part be attributed to their substantial secretion of factors with documented beneficial effect on cardiac repair. CONCLUSIONS: Fibroblast reprogramming combined with molecular beacon-based cell selection yields an iCMP-like cell population with cardioprotective potential. Further studies are needed to elucidate mechanism-of-action and translational potential.
Assuntos
Infarto do Miocárdio , Miócitos Cardíacos , Camundongos , Animais , Miócitos Cardíacos/metabolismo , Remodelação Ventricular , Proteínas com Domínio T/genética , Fatores de Transcrição MEF2/genética , Infarto do Miocárdio/terapia , Infarto do Miocárdio/tratamento farmacológico , Fibroblastos , Reprogramação Celular/genéticaRESUMO
Context: Exosomes are biologically active, extracellular vesicles that are involved in tumor-related processes, including activating tumors, facilitating tumor growth, and promoting inflammation. Objective: The study intended to investigate microRNAs (miRNAs) in exosomes that are associated with colorectal cancer (CRC). Design: The research team performed bioinformatics analysis, extracting RNA-sequencing (RNA-seq) datasets from the Cancer Genome Atlas (TCGA); ExoRBase, a database of different types of RNA information that scientists have extracted from human exosomes; and the Gene Expression Omnibus (GEO) databases, and analyzed the data. Setting: The study took place at the Department of Colorectal Surgery at the Chinese Academy of Medical Sciences and Peking Union Medical College in Beijing, China. Participants: From October 2020 to March 2021, a total of 28 CRC patients who underwent curative resection at the National Cancer Center were enrolled. Tumor samples and tumor-adjacent normal sample were obtained from these CRC patients. Postoperative pathological characteristics all shown adenocarcinoma. The research team recruited participants from the hospitals connected with CAMS and PUMC and obtained written informed consent from them for publication of a case report and any accompanying images. The Ethics Committee of the Cancer Institute (Hospital), CAMS & PUMC has officially recognized the study (NCC 2017-YZ-026). Outcome Measures: The research team: (1) extracted RNA-seq datasets from the TCGA, exoRBase and GEO and analyzed the differentially expressed genes (DEGs); (2) performed a cluster analysis of variant genes using weighted gene co-expression network analysis (WGCNA);(3) verified expression of myocyte enhancer factor 2C-genecards (MEF2C) and cluster of differentiation 36 (CD36) in CRC tissues; (4) explored the biological function of the MEF2C by performing proliferation, migration, and invasion assays; and (5) used a chromatin immunoprecipitation (ChIP) experiment to analyze mechanisms to reveal CD36 transcription regulated by exosomal MEF2C. Results: A significant mean difference in exosomal MEF2C existed between normal and tumor tissues. By performing a correlation analysis, the research team found 609 potential target points of exosomal MEF2C (r > 0.5, P < .05). Weighted correlation network analysis (WGCNA) and protein-protein interaction (PPI) network analysis indicated that CD36 may be the target of exosomal MEF2C. Univariate, multivariate, and Kaplan-Meier analyses showed that CD36 was closely related to the overall survival (OS) of CRC patients. Obvious differences existed in the expression levels of MEF2C and CD36 in CRC and normal tissues according to qPCR and immunohistochemical assays. Functional-experiments analysis in vitro showed that exosomal-MEF2C could be considered as an antioncogene. Mechanistically, ChIP assays showed that MEF2C regulated the transcriptional level of CD 36; thus, the expression of CD36 increased significantly. Conclusion: MEF2C is a potential biomarker of a favorable prognosis in CRC and is related to the progression of CRC. Moreover, the MEF2C-CD36 pathway may reveal the tumor regulation mechanism in CRC. The exosomal MEF2C was the hub gene in exosomes, with CD36 was identified as the potential target. Exosomal MEF2C may be a promising molecular biomarker for predicting a good prognosis and may have potential as a medical target for CRC.
Assuntos
Neoplasias Colorretais , MicroRNAs , Humanos , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , MicroRNAs/genética , Perfilação da Expressão Gênica , Prognóstico , China , Fatores de Transcrição MEF2/genéticaRESUMO
The study aimed to investigate the effects of galangin on learning and memory impairments and Akt/MEF2 D/Beclin-1 signaling pathway in APP/PS1 double-transgenic mice. The mice in this experiment were divided into the normal group, model group, low-(25 mg·kg~(-1)), medium-(50 mg·kg~(-1)), and high-dose(100 mg·kg~(-1)) galangin groups, donepezil(3 mg·kg~(-1)) group, Akt inhibitor(25 mg·kg~(-1)) group, and autophagy inhibitor(30 mg·kg~(-1)) group, with ten in each group, and administered with the corresponding drugs for 30 successive days. On the 24 th day of medication, the water maze and dark avoidance tests were performed. The levels of p-tau, ß-amyloid peptide 1-42(Aß_(42)), acetylcholinesterase(AChE), ß-site amyloid precursor protein cleaving enzyme 1(BACE1), and amyloid precursor protein(APP) in hippocampus were detected by ELISA, the Beclin-1 mRNA expression by RT-PCR, the expression of Aß_(42) and glial fibrillary acidic protein(GFAP) by immunohistochemistry, and the expression of myocyte enhancer factor 2 D(MEF2 D) by immunofluorescence assay. The pathological changes in hippocampus were observed after HE staining, and the expression of Akt, MEF2 D, and Beclin-1 in hippocampus were assayed by Western blot. These results showed that compared with the normal group, the model group exhibited prolonged swimming time, increased number of errors and electric shocks, up-regulated p-tau, Aß_(42), APP, AChE, BACE1, GFAP, and Beclin-1, shortened incubation period, decreased p-Akt and MEF2 D, and obvious hippocampal injury. Compared with the model group, donepezil and galangin shortened the swimming time, reduced the number of errors and electric shocks, down-regulated the expression of p-tau, Aß_(42), APP, AChE, BACE1, GFAP, and Beclin-1, prolonged the incubation period, up-regulated p-Akt and MEF2 D, and improved the pathological changes in hippocampus. Compared with the autophagy inhibitor group, galangin prolonged the swimming time, elevated the number of errors and electric shocks, enhanced the expression of p-tau, Aß_(42), APP, AChE, BACE1, GFAP, and Beclin-1, shortened the incubation period, and diminished the expression of p-Akt and MEF2 D. In conclusion, galangin improves the learning and memory impairments and hippocampal neuron injury of APP/PS1 mice, which may be related to its regulation of Akt/MEF2 D/Beclin-1 signaling pathway.
Assuntos
Doença de Alzheimer , Precursor de Proteína beta-Amiloide , Acetilcolinesterase , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/genética , Secretases da Proteína Precursora do Amiloide/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Ácido Aspártico Endopeptidases/genética , Ácido Aspártico Endopeptidases/metabolismo , Proteína Beclina-1/genética , Proteína Beclina-1/metabolismo , Proteína Beclina-1/farmacologia , Modelos Animais de Doenças , Donepezila/metabolismo , Donepezila/farmacologia , Donepezila/uso terapêutico , Flavonoides , Hipocampo , Fatores de Transcrição MEF2 , Aprendizagem em Labirinto , Transtornos da Memória , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de SinaisRESUMO
Taurine (2-aminoethanesulfonic acid) is a free amino acid found abundantly in mammalian tissues. Increasing evidence suggests that taurine plays a role in the maintenance of skeletal muscle function and increase of exercise capacity. Most energy drinks contain this amino acid; however, there is insufficient research on the effects of long-term, low-dose supplementation of taurine. In this study, we investigated the effects of long-term administration of taurine at low doses on aging in rodents. In Experiment 1, we examined age-related changes in aging Sprague-Dawley (SD) rats (32-92 weeks old) that O2 consumption and spontaneous activity decreased significantly with aging. In Experiment 2, we examined the effects of long-term (21-week) administration of taurine on healthy aging SD rats. SD rats were stabilized for 32-34 weeks and divided into three groups, administrated water (control), 0.5% taurine (25 mg/kg body weight (BW)/day), or 1% taurine (50 mg/kg BW/day) from age 34 to 56 weeks (5 days/week, 5 mL/kg BW). Our findings suggest that long-term administration of taurine at relatively low dose could attenuate the age-related decline in O2 consumption and spontaneous locomotor activity. Upon intestinal absorption, taurine might modulate age-related changes in respiratory metabolism and skeletal muscle function via peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), succinate dehydrogenase (SDH), cytochrome c (Cycs), myocyte enhancer factor 2A (MEF2A), glucose transporter 4 (GLUT4), and myoglobin, which are regulated by the activation of AMP-activated protein kinase (AMPK). This article examines the mechanism underlying the effects of taurine on age-related changes, which may have potential clinical implications.
Assuntos
Envelhecimento/efeitos dos fármacos , Envelhecimento/metabolismo , Músculo Esquelético/fisiopatologia , Taurina/administração & dosagem , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Envelhecimento/genética , Animais , Citocromos c/metabolismo , Suplementos Nutricionais/análise , Humanos , Fatores de Transcrição MEF2/genética , Fatores de Transcrição MEF2/metabolismo , Masculino , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Oxigênio/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Condicionamento Físico Animal , Ratos , Ratos Sprague-Dawley , Succinato Desidrogenase/genética , Succinato Desidrogenase/metabolismoRESUMO
BACKGROUND: Delivery of therapeutic small interfering RNA (siRNA) via functionalized nanoparticles holds great promise for cancer therapy. However, developing a safe and efficient delivery carrier of siRNA is a challenging issue. METHODS: RGDfC peptide was used to modify the surface of selenium nanoparticles (SeNPs) to synthesize a biocompatible siRNA delivery vehicle (R-SeNPs), and MEF2D-siRNA was loaded onto R-SeNPs to prepare a functionalized selenium nanoparticle R-Se@MEF2D-siRNA. The chemical properties of R-SeNPs were characterized, and the anticancer efficacy as well as related mechanisms of R-Se@MEF2D-siRNA were further explored. RESULTS: R-Se@MEF2D-siRNA was significantly taken up by SKOV3 cells and could enter SKOV3 cells mainly in the clathrin-associated endocytosis way. The result of in vitro siRNA release demonstrated that R-Se@MEF2D-siRNA could release MEF2D-siRNA quicker in a microenvironment simulating a lysosomal environment in tumor cells compared to a normal physiological environment. The results of qRT-PCR assay proved that R-Se@MEF2D-siRNA could effectively silence the expression of the MEF2D gene in SKOV3 cells. R-Se@MEF2D-siRNA remarkably suppressed the proliferation of SKOV3 cells and further triggered its apoptosis. In addition, R-Se@MEF2D-siRNA had the capability to disrupt mitochondrial membrane potential (MMP) in SKOV3 cells and resulted in the overproduction of reactive oxygen species (ROS), indicating that mitochondrial dysfunction and ROS generation played an important role in the apoptosis of SKOV3 cells induced by R-Se@MEF2D-siRNA. In vivo, R-Se@MEF2D-siRNA also exhibited excellent antitumor activity mainly through decreasing tumor cells proliferation and triggering their apoptosis in tumor-bearing nude mice. CONCLUSION: R-Se@MEF2D-siRNA provides an alternative strategy for ovarian cancer treatment in the clinic.
Assuntos
Inativação Gênica , Nanopartículas/química , Neoplasias Ovarianas/terapia , RNA Interferente Pequeno/química , RNA Interferente Pequeno/genética , Selênio/química , Animais , Apoptose/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Portadores de Fármacos/química , Feminino , Humanos , Fatores de Transcrição MEF2/deficiência , Fatores de Transcrição MEF2/genética , Camundongos , Camundongos Nus , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Espécies Reativas de Oxigênio/metabolismo , Microambiente Tumoral/genéticaRESUMO
BACKGROUND: The incidence of CHD is the highest among birth defects and is increasing year to year. CHD seriously harms the health of infants and young children and presents a large economic burden to families and society. The pathogenesis of CHD and preventive measures are the focus of current research. Our research aimed to explore the intervention effect of folic acid on heart abnormalities resulting from sodium arsenic (NaAsO2) exposure during the periconception period. METHODS: Sixty 35-day-old female SD rats were randomly divided into 5 groups with 12 rats in each group. Group A was the control group. The rats were given distilled water and ordinary chow. The rats in group B were given distilled water containing 75 mg/L NaAsO2 and ordinary chow. The rats in groups C, D, and E were given distilled water containing 75 mg/L NaAsO2 and chow containing 0.53 mg/kg, 5.3 mg/kg, and 10.6 mg/kg folic acid, respectively. The general condition of the embryos and the histopathology of the embryonic hearts were examined. The acetylation levels of histone H3K9 in heart tissues and the expression levels of Mef2C (which is related to heart development) were observed. RESULTS: The embryo weight and placental weight of groups B-E were significantly lower than those of group A (P < 0.05). The heart malformation rate of the fetal rats in groups B-E was significantly higher than that of the fetal rats in group A (P < 0.05). We found that the level of H3K9 acetylation in fetal rat cardiomyocytes in groups B-E was significantly higher than that in group A (P < 0.05) and that the level of H3K9 acetylation in groups C-E was lower than that in group B (P < 0.05). The mRNA level of Mef2C in fetal rat cardiomyocytes in group B-E was significantly higher than that in group A (P < 0.05), and the mRNA level of Mef2C in groups C-E was significantly lower than that in group B (P < 0.05). CONCLUSION: Supplementation with folic acid during the periconception period can interfere with the toxic effects of arsenic on the heart. The mechanism may be that lowering the acetylation levels of histone H3K9 in heart tissues leads to decreased expression levels of Mef2C, which may play a protective role in heart development in fetal rats.
Assuntos
Arsenitos , Coração Fetal/efeitos dos fármacos , Ácido Fólico/farmacologia , Cardiopatias Congênitas/prevenção & controle , Compostos de Sódio , Acetilação , Animais , Cardiotoxicidade , Feminino , Coração Fetal/anormalidades , Coração Fetal/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Idade Gestacional , Cardiopatias Congênitas/induzido quimicamente , Cardiopatias Congênitas/embriologia , Cardiopatias Congênitas/metabolismo , Histonas/metabolismo , Fatores de Transcrição MEF2/genética , Fatores de Transcrição MEF2/metabolismo , Exposição Materna , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Gravidez , Ratos Sprague-DawleyRESUMO
OBJECTIVE: To investigate the effect of Xiaozhong Zhitong Ointment(XZZTO) on remodeling and repair of skeletal muscle injury in rats based on the expression mechanism of microRNA. METHODS: The rat gastrocnemius injury model was established by blunt contusion model. The expression of MEF2 gene and protein in gastrocnemius muscle was detected by quantitative PCR at 4, 7, 14 and 21 days after injury with XZZTO. The mechanism of the effect of XZZTO on the muscle remodeling and repair of rat gastrocnemius contusion model was discussed. RESULTS: The expression level of MEF2 in the treatment group was significantly higher than that of the control group and model group, which further confirmed the important role of MEF2 in inducing skeletal muscle remodeling and repair process in the topical drugs. The expression of MEF2 increased at 7 days after injury and remained at a high level until 21 days after injury. Compared with the model group, the peak expression period was about 14 days, and then returned to the general state. CONCLUSIONS: The expression level of MEF2 shows an upward trend. Even 21 days after injury, the expression of MEF2 dose not show a significant downward trend. It can be seen that XZZTO can promote the expression of MEF2. At the same time, XZZTO can regulate the regeneration and repair of skeletal muscle. Therefore, XZZTO can play a regeneration and repair role after skeletal muscle injury through gene regulation.
Assuntos
Fatores de Transcrição MEF2/genética , Músculo Esquelético , Animais , Contusões , Trato Gastrointestinal , Regulação da Expressão Gênica , Pomadas , Proteínas , RNA Mensageiro , RatosRESUMO
Cardiac hypertrophy has become a major cardiovascular problem wordwide and is considered the early stage of heart failure. Treatment and prevention strategies are needed due to the suboptimal efficacy of current treatment methods. Recently, many studies have demonstrated the important role of histone acetylation in myocardium remodelling along with cardiac hypertrophy. A Chinese herbal extract containing anacardic acid (AA) is known to possess strong histone acetylation inhibitory effects. In previous studies, we demonstrated that AA could reverse alcohol-induced cardiac hypertrophy in an animal model at the foetal stage. Here, we investigated whether AA could attenuate cardiac hypertrophy through the modulation of histone acetylation and explored its potential mechanisms in the hearts of transverse aortic constriction (TAC) mice. This study showed that AA attenuated hyperacetylation of acetylated lysine 9 on histone H3 (H3K9ac) by inhibiting the expression of p300 and p300/CBP-associated factor (PCAF) in TAC mice. Moreover, AA normalized the transcriptional activity of the heart nuclear transcription factor MEF2A. The high expression of cardiac hypertrophy-linked genes (ANP, ß-MHC) was reversed through AA treatment in the hearts of TAC mice. Additionally, we found that AA improved cardiac function and survival rate in TAC mice. The current results further highlight the mechanism by which histone acetylation is controlled by AA treatment, which may help prevent and treat hypertrophic cardiomyopathy.
Assuntos
Ácidos Anacárdicos/farmacologia , Cardiomegalia/prevenção & controle , Histona Acetiltransferases/antagonistas & inibidores , Pressão/efeitos adversos , Acetilação , Animais , Cardiomegalia/etiologia , Cardiomegalia/patologia , Modelos Animais de Doenças , Fatores de Transcrição MEF2/genética , Fatores de Transcrição MEF2/metabolismo , Masculino , Camundongos , Fatores de Transcrição de p300-CBP/genética , Fatores de Transcrição de p300-CBP/metabolismoRESUMO
OBJECTIVE@#To investigate the effect of Xiaozhong Zhitong Ointment(XZZTO) on remodeling and repair of skeletal muscle injury in rats based on the expression mechanism of microRNA.@*METHODS@#The rat gastrocnemius injury model was established by blunt contusion model. The expression of MEF2 gene and protein in gastrocnemius muscle was detected by quantitative PCR at 4, 7, 14 and 21 days after injury with XZZTO. The mechanism of the effect of XZZTO on the muscle remodeling and repair of rat gastrocnemius contusion model was discussed.@*RESULTS@#The expression level of MEF2 in the treatment group was significantly higher than that of the control group and model group, which further confirmed the important role of MEF2 in inducing skeletal muscle remodeling and repair process in the topical drugs. The expression of MEF2 increased at 7 days after injury and remained at a high level until 21 days after injury. Compared with the model group, the peak expression period was about 14 days, and then returned to the general state.@*CONCLUSIONS@#The expression level of MEF2 shows an upward trend. Even 21 days after injury, the expression of MEF2 dose not show a significant downward trend. It can be seen that XZZTO can promote the expression of MEF2. At the same time, XZZTO can regulate the regeneration and repair of skeletal muscle. Therefore, XZZTO can play a regeneration and repair role after skeletal muscle injury through gene regulation.
Assuntos
Animais , Ratos , Contusões , Trato Gastrointestinal , Regulação da Expressão Gênica , Fatores de Transcrição MEF2 , Genética , Músculo Esquelético , Pomadas , Proteínas , RNA MensageiroRESUMO
Oxytocin (OT) has gained attention not only as anxiolytic drug and as potential treatment option for autistic children; it also acts as a growth and differentiation factor in neuronal cells. While behavioral effects of OT have been studied in detail, knowledge about the cellular effects of OT is relatively sparse. In this study, we present evidence for three hypotheses: 1) OT leads to neurite retraction in hypothalamic neurons via the OT receptor (OTR) 2) The transcription factor MEF-2A is a central regulator of OT-induced neurite retraction, and 3) The MAPK pathway is critical for OT-induced MEF-2A activation. Incubation of rat hypothalamic H32â¯cells with 10â¯nM to 1⯵M OT, vasopressin, and the specific OTR agonist TGOT, over the course of 12â¯h resulted in a time-dependent, significant retraction of neurites. In addition, the size of the nuclear compartment increased, whereas the overall cell size remained unchanged. OT treatment for 10â¯h increased the cellular viability significantly, and this effect could be blocked by a specific OTR antagonist, providing evidence for a specific and pro-active effect of OT on neurite retraction, and not as an unspecific side effect of apoptosis. The molecular mechanism that controls OT-induced neurite retraction includes a reduced phosphorylation of the transcription factor MEF-2A at Serine 408 (S408). This dephosphorylation is under the control of the OTR-coupled MAPK pathway, as blocking MEK1/2 by U0126 inhibited MEF-2A activation and subsequent neurite retraction. The siRNA-mediated knockdown of MEF-2A prevented the OT-induced neurite retraction, providing direct evidence for a role of MEF-2A in morphological alterations induced by OT treatment. In summary, the present study reveals a previously unknown OTR-coupled MAPK-MEF-2A pathway, which is responsible for OT-induced neurite retraction of hypothalamic neurons.
Assuntos
Hipotálamo/citologia , Fatores de Transcrição MEF2/metabolismo , Neurônios/metabolismo , Ocitocina/farmacologia , Animais , Linhagem Celular , Tamanho do Núcleo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Fatores de Transcrição MEF2/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Neuritos/efeitos dos fármacos , Neuritos/metabolismo , Neurônios/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Ratos , Receptores de Ocitocina/metabolismo , Transcrição Gênica/efeitos dos fármacos , Vasopressinas/farmacologiaRESUMO
Shenfu Injection (SFI) is a classical Chinese medicine used to treat heart failure. Our previous study demonstrated that miRNAs underwent changes in rats with myocardial hypertrophy induced by abdominal aortic constriction. Interestingly, there was a significant change in miR-19a-3p, whose target gene is known to be associated with MEF2 signaling. However, whether and how SFI regulates miR-19a-3p in the treatment of myocardial hypertrophy has not been investigated. The purpose of the present study was to investigate the regulatory effect of SFI on miR-19a-3p in MEF2 signaling in the rat hypertrophic myocardium. We found that the miR-19a-3p expression level was significantly decreased in the hypertrophic myocardium, and MEF2A was the target gene of miR-19a-3p. The protein expressions of MEF2A, ß-MHC, BNP and TRPC1 were significantly increased in hypertrophic cardiomyocytes. MiR-19a-3p was up-regulated after SFI treatment, and the protein expressions of these genes were significantly decreased. In addition, miR-19a-3p over-expression in hypertrophic cardiomyocytes could decrease MEF2A mRNA and protein expressions, and anti-miR-19a-3p showed the opposite result. Our study provided substantial evidence that miR-19a-3p played a functional role in MEF2 signaling in myocardial hypertrophy. SFI attenuated cardiomyocyte hypertrophy probably through up-regulating or maintaining the miR-19a-3p levels and regulating the MEF2 signaling pathway.
Assuntos
Cardiomegalia/tratamento farmacológico , Medicamentos de Ervas Chinesas/administração & dosagem , MicroRNAs/genética , Regulação para Cima , Regiões 3' não Traduzidas , Animais , Cardiomegalia/genética , Cardiomegalia/metabolismo , Células Cultivadas , Modelos Animais de Doenças , Medicamentos de Ervas Chinesas/farmacologia , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica/efeitos dos fármacos , Injeções , Fatores de Transcrição MEF2/genética , Fatores de Transcrição MEF2/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Ratos , Transdução de SinaisRESUMO
Oxidative stress plays a vital role in many pathological processes of the cardiovascular diseases. However, the underlying mechanism remains unclear, especially on a transcription factor (TF) level. In this study, a new method, concatenated tandem array of consensus transcription factor response elements (catTFREs), and an Illumina-based RNA-seq technology were integrated to systematically investigate the role of TFs in hydrogen peroxide (H2O2)-induced oxidative stress in cardiomyocytes; the damage was then rescued by Danhong injection (DHI), a Chinese standardized product approved for cardiovascular diseases treatment. The overall gene expression revealed cell apoptosis and DNA repair were vital for cardiomyocytes in resisting oxidative stress. By comprehensively integrating the transcription activity of TFs and their downstream target genes, an important TFs-target network were constructed and 13 TFs were identified as critical TFs in DHI-mediated protection in H2O2-induced oxidative stress. By using the integrated approach, seven TFs of these 13 TFs were also identified in melatonin-mediated protection in H2O2-induced damage. Furthermore, the transcription activity of DNA-(apurinic or apyrimidinic site) lyase (Apex1), Myocyte-specific enhancer factor 2D (Mef2d) and Pre B-cell leukemia transcription factor 3 (Pbx3) was further verified in pluripotent stem cell-derived cardiomyocytes. This research offers a new understanding of cardiomyocytes in response to H2O2-induced oxidative stress and reveals additional potential therapeutic targets. The combination of two parallel omics datasets (corresponding to the transcriptome and proteome) can reduce the noise in high-throughput data and reveal the fundamental changes of the biological process, making it suitable and reliable for investigation of critical targets in many other complicated pathological processes.
Assuntos
Cardiotônicos/farmacologia , Reparo do DNA/efeitos dos fármacos , Medicamentos de Ervas Chinesas/farmacologia , Peróxido de Hidrogênio/antagonistas & inibidores , Miócitos Cardíacos/efeitos dos fármacos , Fatores de Transcrição/genética , Transcriptoma , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Linhagem Celular , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Peróxido de Hidrogênio/farmacologia , Fatores de Transcrição MEF2/genética , Fatores de Transcrição MEF2/metabolismo , Melatonina/farmacologia , Análise em Microsséries , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Estresse Oxidativo , Ligação Proteica , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Ratos , Elementos de Resposta , Fatores de Transcrição/metabolismoRESUMO
Cardiac hypertrophy is a complex process involving highly coordinated but tight regulation of multiple elements, such as in epigenetics, which make an important contribution to myocardium remodeling and cardiac hypertrophy. Epigenetic regulations, particularly histone acetylation, have been implicated in cardiac hypertrophy, however, the exact mechanism is still largely unknown. In the present study, we explored the potential attenuating effects of Chinese herbal extract anacardic acid on phenylephrine-induced cardiac hypertrophy and the underlying mechanism. The mouse cardiac hypertrophy model was established and the hearts were collected from C57BL/6 mice for further analyses. The data showed that anacardic acid modulated the cardiac genes expression and attenuated the phenylephrine-induced cardiac hypertrophy via the suppression of histone acetylases activity and downstream cardiac genes. In addition, anacardic acid abrogated histone and MEF2A acetylation and DNA-binding activity by blocking p300-HAT and PCAF-HAT activities. In addition, anacardic acid normalized the cardiac hypertrophy-related genes expressions (ANP, BNP, cTnT, cTnI, ß-MHC, and Cx43) induced by phenylephrine at the level of transcription and translation. In addition, anacardic acid did not affect the blood routine index, hepatic function, renal function, and myocardial enzymes. Therefore, anacardic acid may prove to be a candidate drug to cure hypertrophic cardiomyopathy.
Assuntos
Ácidos Anacárdicos/farmacologia , Cardiomegalia/metabolismo , Histona Acetiltransferases/antagonistas & inibidores , Acetilação , Animais , Cardiomegalia/induzido quimicamente , Cardiomegalia/diagnóstico , Cardiomegalia/tratamento farmacológico , Modelos Animais de Doenças , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Testes de Função Cardíaca , Histona Acetiltransferases/metabolismo , Histonas/metabolismo , Fatores de Transcrição MEF2/genética , Masculino , Camundongos , Miocárdio/metabolismo , Miocárdio/patologia , Fenilefrina/efeitos adversos , Ligação Proteica , Transcrição GênicaRESUMO
Houttuynia cordata Thunb ( H cordata), a medicinal plant, has anticancer activity, as it inhibits cell growth and induces cell apoptosis in cancer. However, the potential anti-cancer activity and mechanism of H cordata for human liver cancer cells is not well understood. Recently, we identified hypoxia-inducible factor (HIF)-1A, Forkhead box (FOX)O3, and MEF2A as proapoptotic factors induced by H cordata, suggesting that HIF-1A, FOXO3, and MEF2A contribute to the apoptosis of HepG2 hepatocellular carcinoma cells. FOXO3 transcription factors regulate target genes involved in apoptosis. H cordata significantly increased the mRNA and protein expression of HIF-1A and FOXO3 and stimulated MEF2A expression in addition to increased apoptosis in HepG2 cells within 24 hours. Therefore, we determined the potential role of FOXO3 on apoptosis and on H cordata-induced MEF2A in HepG2 cells. HIF-1A silencing by siRNA attenuated MEF2A and H cordata-mediated FOXO3 upregulation in HepG2 cells. Furthermore, H cordata-mediated MEF2A expression enhanced caspase-3 and caspase-7, which were abolished on silencing FOXO3 with siRNA. In addition, H cordata inhibited growth of human hepatocellular carcinoma xenografts in nude mice. Taken together, our results demonstrate that H cordata enhances HIF-1A/FOXO3 signaling, leading to MEF2A upregulation in HepG2 cells, and in parallel, it disturbs the expression of Bcl-2 family proteins (Bax, Bcl-2, and Bcl-xL), which results in apoptosis. Taken together, these findings demonstrate that H cordata promotes the activation of HIF-1A-FOXO3 and MEF2A pathways to induce apoptosis in human HepG2 hepatocellular carcinoma cells and is, therefore, a promising candidate for antitumor drug development.
Assuntos
Apoptose/efeitos dos fármacos , Carcinoma Hepatocelular/tratamento farmacológico , Proteína Forkhead Box O3/metabolismo , Houttuynia/química , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias Hepáticas/tratamento farmacológico , Extratos Vegetais/farmacologia , Animais , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Células Hep G2 , Humanos , Neoplasias Hepáticas/metabolismo , Fatores de Transcrição MEF2/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacosRESUMO
Danshensu (DSS) and tetramethylpyrazine (TMP) are active ingredients of Salvia miltiorrhiza Bge. and Ligusticum chuanxiong Hort that are widely used in oriental medicine. Structural combination of compounds with known biological activity may lead to the formation of a molecule with multiple properties or new function profile. In the current study, the neuroprotective effects of DT-010, a novel analogue in which TMP was coupled to DSS through an ester bond and two allyl groups at the carboxyl group, were evaluated in a cellular model of Parkinson's disease (PD). As evidenced by the increase in cell survival, as well as the decrease in the number of Hoechst-stained apoptotic nuclei and the level of intracellular accumulation of reactive oxygen species, DT-010 at 3-30 µM substantially protected against MPP+-induced neurotoxicity in both PC12 cells and primary cerebellar granule neurons, a protection that was more potent and efficacious than its parent molecules DSS and TMP. Very encouragingly, we found that DT-010, but not DSS or TMP, could enhance myocyte enhancer factor 2D (MEF2D) transcriptional activity using luciferase reporter gene assay. The neuroprotective effects of DT-010 could be blocked by pharmacologic inhibition of PI3K pathways with LY294002, or MEF2D pathway with short hairpin RNA-mediated knockdown of MEF2D. Furthermore, western blot analysis revealed that DT-010 potentiates Akt protein expression against MPP+ to down-regulate MEF2D inhibitor GSK3ß. Taken together, the results suggest that DT-010 prevents MPP+-induced neurotoxicity via enhancing MEF2D through the activation of PI3K/Akt/GSK3ß pathway. DT-010 may be a potential candidate for further preclinical study for preventing and treating PD.
Assuntos
Fatores de Transcrição MEF2/metabolismo , Fármacos Neuroprotetores/farmacologia , Pirazinas/farmacologia , Animais , Linhagem Celular , Células Cultivadas , Medicamentos de Ervas Chinesas/farmacologia , Ativação Enzimática/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Doença de Parkinson/tratamento farmacológico , Fosfatidilinositol 3-Quinases/metabolismo , Ratos , Ativação Transcricional/efeitos dos fármacosRESUMO
BACKGROUND: Premature ventricular contractions (PVCs) are common in the general population, and frequent PVCs may result in the poor quality of life or even the damage of cardiac function. We examined the efficacy and safety of a traditional Chinese medicine Wenxin Keli for the treatment of frequent PVCs among a relatively large Chinese cohort. METHODS: We performed a randomized, double-blind, placebo-controlled, parallel-group, multicenter trial. A total of 1200 eligible participants were randomly assigned in a ratio of 1:1 to receive Wenxin Keli or the placebo for 4 weeks. The primary and secondary endpoint was the change of PVC numbers and PVC-related symptoms after a 4-week treatment compared with baseline, respectively. In addition, vital signs, laboratory values, and electrocardiographic parameters were assessed in a safety analysis. RESULTS: At the initial evaluation, no significant differences in the baseline characteristics were observed between the Wenxin Keli group and the placebo group. A smaller number of PVCs was observed after the 4-week treatment than at baseline, in both the Wenxin Keli group (5686 ± 5940 vs. 15,138 ± 7597 beats/d, P < 0.001) and the placebo group (10,592 ± 8009 vs. 14,529 ± 5929 beats/d, P < 0.001); moreover, the Wenxin Keli group demonstrated a significantly greater reduction in the frequency of PVCs than the placebo group (P < 0.001). In a full analysis set, patients in the Wenxin Keli group exhibited significantly higher total effective responses in the reduction of PVCs compared to those in the placebo group (83.8% vs. 43.5%,P < 0.001). The per-protocol analysis yielded similar results (83.0% vs. 39.3%,P < 0.001). Treatment with Wenxin Keli also demonstrated superior performance compared to the placebo with respect to PVC-related symptoms. No severe adverse effects attributable to Wenxin Keli were reported. CONCLUSIONS: Wenxin Keli treatment effectively reduced the overall number of PVCs and alleviated PVC-related symptoms in patients without structural heart diseases and had no severe side effects.
Assuntos
Doença da Artéria Coronariana/genética , Fatores de Transcrição MEF2/genética , Doença da Artéria Coronariana/prevenção & controle , Método Duplo-Cego , Medicamentos de Ervas Chinesas/efeitos adversos , Medicamentos de Ervas Chinesas/uso terapêutico , Humanos , Mutação/genética , Infarto do Miocárdio/tratamento farmacológico , Infarto do Miocárdio/genética , Ensaios Clínicos Controlados Aleatórios como Assunto , Complexos Ventriculares Prematuros/tratamento farmacológico , Complexos Ventriculares Prematuros/genéticaRESUMO
UNLABELLED: Adult cardiac stem cells (CSCs) express many endogenous cardiogenic transcription factors including members of the Gata, Hand, Mef2, and T-box family. Unlike its DNA-binding targets, Myocardin (Myocd)-a co-activator not only for serum response factor, but also for Gata4 and Tbx5-is not expressed in CSCs. We hypothesised that its absence was a limiting factor for reprogramming. Here, we sought to investigate the susceptibility of adult mouse Sca1+ side population CSCs to reprogramming by supplementing the triad of GATA4, MEF2C, and TBX5 (GMT), and more specifically by testing the effect of the missing co-activator, Myocd. Exogenous factors were expressed via doxycycline-inducible lentiviral vectors in various combinations. High throughput quantitative RT-PCR was used to test expression of 29 cardiac lineage markers two weeks post-induction. GMT induced more than half the analysed cardiac transcripts. However, no protein was detected for the induced sarcomeric genes Actc1, Myh6, and Myl2. Adding MYOCD to GMT affected only slightly the breadth and level of gene induction, but, importantly, triggered expression of all three proteins examined (α-cardiac actin, atrial natriuretic peptide, sarcomeric myosin heavy chains). MYOCD + TBX was the most effective pairwise combination in this system. In clonal derivatives homogenously expressing MYOCD + TBX at high levels, 93% of cardiac transcripts were up-regulated and all five proteins tested were visualized. IN SUMMARY: (1) GMT induced cardiac genes in CSCs, but not cardiac proteins under the conditions used. (2) Complementing GMT with MYOCD induced cardiac protein expression, indicating a more complete cardiac differentiation program. (3) Homogeneous transduction with MYOCD + TBX5 facilitated the identification of differentiating cells and the validation of this combinatorial reprogramming strategy. Together, these results highlight the pivotal importance of MYOCD in driving CSCs toward a cardiac muscle fate.
Assuntos
Reprogramação Celular , Miócitos Cardíacos/citologia , Proteínas Nucleares/metabolismo , Células-Tronco/metabolismo , Proteínas com Domínio T/metabolismo , Transativadores/metabolismo , Animais , Diferenciação Celular , Células Cultivadas , Doxiciclina/toxicidade , Embrião de Mamíferos/metabolismo , Fator de Transcrição GATA4/metabolismo , Vetores Genéticos/metabolismo , Células HEK293 , Humanos , Lentivirus/genética , Fatores de Transcrição MEF2/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Miocárdio/metabolismo , Proteínas Nucleares/genética , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Proteínas com Domínio T/genética , Transativadores/genética , Fatores de Transcrição/metabolismoRESUMO
BACKGROUND: Chronic heart failure (CHF) is a complex clinical syndrome, and a serious stage of various heart diseases. Dysfunction of histone acetylation is involved in pathogenesis of CHF. Lujiao is a clinical and traditional prescription that has been previously used in the treatment of heart failure. The objective of our study was to explore the effects of traditional Chinese Medicine intervention with Lujiao prescription on hypertrophic cardiomyocytes with histone acetylation abnormality. METHODS: Myocardial cells from neonatal rats were stimulated via phenylephrine (PE) and then randomly divided into seven groups: normal group (without any treatment), model group (treated with saline), TSA group (treated with trichostatin A), perindopril group (treated with perindopril), and the high, medium, and low dose of Lujiao groups (treated with 2.4 g/mL, 1.2 g/mL, and 0.6 g/mL of Lujiao, respectively). The test drug of perindopril group or Lujiao group was derived from serum after drug treatment in rats. Real-time polymerase chain reaction and Western blot were performed to analyze expression of myocyte enhancer factor 2 (MEF-2), α-major histocompatibility complex (MHC), and ß-MHC and acetylation level of histone H3. RESULTS: Expressions of MEF-2 and ß-MHC were significantly increased after PE treatment and decreased after drug treatment. Expression of α-MHC mRNA was significantly reduced after PE treatment and increased after being treated with Lujiao prescription, perindopril, and TSA. The acetylation level of histone H3 decreased in rat myocardial cells stimulated by PE 48 for hours and this decrease was reversed after treatment with high and medium doses of Lujiao prescription, perindopril and TSA. CONCLUSION: Histone acetylation-MEF-2-α-MHC/ß-MHC axis was discovered in myocardial hypertrophy, and intervention of Lujiao prescription exhibited good effects.
Assuntos
Cardiomegalia/tratamento farmacológico , Histonas/metabolismo , Medicina Tradicional Chinesa , Miócitos Cardíacos/patologia , Acetilação , Animais , Células Cultivadas , Ácidos Hidroxâmicos/uso terapêutico , Fatores de Transcrição MEF2/fisiologia , Masculino , Miócitos Cardíacos/metabolismo , Cadeias Pesadas de Miosina/genética , Perindopril/uso terapêutico , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND: Pulmonary arterial hypertension (PAH) is a progressive disease of the pulmonary arterioles, characterized by increased pulmonary arterial pressure and right ventricular failure. The cause of PAH is complex, but aberrant proliferation of the pulmonary artery endothelial cells (PAECs) and pulmonary artery smooth muscle cells is thought to play an important role in its pathogenesis. Understanding the mechanisms of transcriptional gene regulation involved in pulmonary vascular homeostasis can provide key insights into potential therapeutic strategies. METHODS AND RESULTS: We demonstrate that the activity of the transcription factor myocyte enhancer factor 2 (MEF2) is significantly impaired in the PAECs derived from subjects with PAH. We identified MEF2 as the key cis-acting factor that regulates expression of a number of transcriptional targets involved in pulmonary vascular homeostasis, including microRNAs 424 and 503, connexins 37, and 40, and Kruppel Like Factors 2 and 4, which were found to be significantly decreased in PAH PAECs. The impaired MEF2 activity in PAH PAECs was mediated by excess nuclear accumulation of 2 class IIa histone deacetylases (HDACs) that inhibit its function, namely HDAC4 and HDAC5. Selective, pharmacological inhibition of class IIa HDACs led to restoration of MEF2 activity in PAECs, as demonstrated by increased expression of its transcriptional targets, decreased cell migration and proliferation, and rescue of experimental pulmonary hypertension models. CONCLUSIONS: Our results demonstrate that strategies to augment MEF2 activity hold potential therapeutic value in PAH. Moreover, we identify selective HDAC IIa inhibition as a viable alternative approach to avoid the potential adverse effects of broad spectrum HDAC inhibition in PAH.