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1.
Nat Commun ; 12(1): 3253, 2021 05 31.
Artigo em Inglês | MEDLINE | ID: mdl-34059674

RESUMO

Muscle stem cell function has been suggested to be regulated by Acetyl-CoA and NAD+ availability, but the mechanisms remain unclear. Here we report the identification of two acetylation sites on PAX7 that positively regulate its transcriptional activity. Lack of PAX7 acetylation reduces DNA binding, specifically to the homeobox motif. The acetyltransferase MYST1 stimulated by Acetyl-CoA, and the deacetylase SIRT2 stimulated by NAD +, are identified as direct regulators of PAX7 acetylation and asymmetric division in muscle stem cells. Abolishing PAX7 acetylation in mice using CRISPR/Cas9 mutagenesis leads to an expansion of the satellite stem cell pool, reduced numbers of asymmetric stem cell divisions, and increased numbers of oxidative IIA myofibers. Gene expression analysis confirms that lack of PAX7 acetylation preferentially affects the expression of target genes regulated by homeodomain binding motifs. Therefore, PAX7 acetylation status regulates muscle stem cell function and differentiation potential to facilitate metabolic adaptation of muscle tissue.


Assuntos
Autorrenovação Celular/genética , Músculo Esquelético/lesões , Fator de Transcrição PAX7/metabolismo , Regeneração/genética , Células Satélites de Músculo Esquelético/fisiologia , Acetilação , Animais , Células COS , Sistemas CRISPR-Cas , Cardiotoxinas/administração & dosagem , Cardiotoxinas/toxicidade , Diferenciação Celular/genética , Chlorocebus aethiops , Modelos Animais de Doenças , Técnicas de Silenciamento de Genes , Histona Acetiltransferases/genética , Histona Acetiltransferases/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Músculo Esquelético/citologia , Músculo Esquelético/efeitos dos fármacos , Mutagênese , Cultura Primária de Células , Regiões Promotoras Genéticas , Células Sf9 , Sirtuína 2/genética , Sirtuína 2/metabolismo , Spodoptera , Ativação Transcricional
2.
Int J Mol Sci ; 22(11)2021 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-34071003

RESUMO

Superoxide dismutase (SOD) is a major antioxidant enzyme for superoxide removal, and cytoplasmic SOD (SOD1) is expressed as a predominant isoform in all cells. We previously reported that renal SOD1 deficiency accelerates the progression of diabetic nephropathy (DN) via increasing renal oxidative stress. To evaluate whether the degree of SOD1 expression determines regeneration capacity and sarcopenic phenotypes of skeletal muscles under incipient and advanced DN conditions, we investigated the alterations of SOD1 expression, oxidative stress marker, inflammation, fibrosis, and regeneration capacity in cardiotoxin (CTX)-injured tibialis anterior (TA) muscles of two Akita diabetic mouse models with different susceptibility to DN, DN-resistant C57BL/6-Ins2Akita and DN-prone KK/Ta-Ins2Akita mice. Here, we report that KK/Ta-Ins2Akita mice, but not C57BL/6-Ins2Akita mice, exhibit delayed muscle regeneration after CTX injection, as demonstrated by the finding indicating significantly smaller average cross-sectional areas of regenerating TA muscle myofibers relative to KK/Ta-wild-type mice. Furthermore, we observed markedly reduced SOD1 expression in CTX-injected TA muscles of KK/Ta-Ins2Akita mice, but not C57BL/6-Ins2Akita mice, along with increased inflammatory cell infiltration, prominent fibrosis and superoxide overproduction. Our study provides the first evidence that SOD1 reduction and the following superoxide overproduction delay skeletal muscle regeneration through induction of overt inflammation and fibrosis in a mouse model of progressive DN.


Assuntos
Nefropatias Diabéticas/complicações , Músculo Esquelético/efeitos dos fármacos , Regeneração Nervosa/efeitos dos fármacos , Sarcopenia/etiologia , Superóxido Dismutase-1/efeitos dos fármacos , Animais , Cardiotoxinas/toxicidade , Colágeno Tipo I/biossíntese , Colágeno Tipo I/genética , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/genética , Nefropatias Diabéticas/enzimologia , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/patologia , Progressão da Doença , Indução Enzimática/efeitos dos fármacos , Fibrose , Regulação Enzimológica da Expressão Gênica , Predisposição Genética para Doença , Mesângio Glomerular/patologia , Inflamação , Insulina/deficiência , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Músculo Esquelético/enzimologia , Músculo Esquelético/patologia , Músculo Esquelético/fisiologia , Estresse Oxidativo/efeitos dos fármacos , Superóxido Dismutase-1/biossíntese , Superóxido Dismutase-1/genética , Superóxido Dismutase-1/fisiologia , Superóxidos/metabolismo
3.
Arterioscler Thromb Vasc Biol ; 41(3): 1019-1031, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33472401

RESUMO

Drug-induced cardiotoxicity is a significant clinical issue, with many drugs in the market being labeled with warnings on cardiovascular adverse effects. Treatments are often prematurely halted when cardiotoxicity is observed, which limits their therapeutic potential. Moreover, cardiotoxicity is a major reason for abandonment during drug development, reducing available treatment options for diseases and creating a significant financial burden and disincentive for drug developers. Thus, it is important to minimize the cardiotoxic effects of medications that are in use or in development. To this end, identifying patients at a higher risk of developing cardiovascular adverse effects for the drug of interest may be an effective strategy. The discovery of human induced pluripotent stem cells has enabled researchers to generate relevant cell types that retain a patient's own genome and examine patient-specific disease mechanisms, paving the way for precision medicine. Combined with the rapid development of pharmacogenomic analysis, the ability of induced pluripotent stem cell-derivatives to recapitulate patient-specific drug responses provides a powerful platform to identify subsets of patients who are particularly vulnerable to drug-induced cardiotoxicity. In this review, we will discuss the current use of patient-specific induced pluripotent stem cells in identifying populations who are at risk to drug-induced cardiotoxicity and their potential applications in future precision medicine practice. Graphic Abstract: A graphic abstract is available for this article.


Assuntos
Cardiotoxicidade/etiologia , Cardiotoxinas/toxicidade , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Arritmias Cardíacas/induzido quimicamente , Avaliação Pré-Clínica de Medicamentos/métodos , Marcadores Genéticos , Humanos , Células-Tronco Pluripotentes Induzidas/patologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Contração Miocárdica/efeitos dos fármacos , Miocardite/induzido quimicamente , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Miócitos Cardíacos/fisiologia , Testes Farmacogenômicos/métodos , Polimorfismo de Nucleotídeo Único , Medicina de Precisão/métodos , Fatores de Risco
4.
Anesthesiology ; 133(5): 1077-1092, 2020 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-32915958

RESUMO

BACKGROUND: Local anesthetics, particularly potent long acting ones such as bupivacaine, can cause cardiotoxicity by inhibiting sodium ion channels; however, the impact of left ventricular hypertrophy on the cardiotoxicity and the underlying mechanisms remain undetermined. Transient receptor potential canonical (TRPC) channels are upregulated in left ventricular hypertrophy. Some transient receptor potential channel subtypes have been reported to pass relatively large cations, including protonated local anesthetics; this is known as the "pore phenomenon." The authors hypothesized that bupivacaine-induced cardiotoxicity is more severe in left ventricular hypertrophy due to upregulated TRPC channels. METHODS: The authors used a modified transverse aortic constriction model as a left ventricular hypertrophy. Cardiotoxicity caused by bupivacaine was compared between sham and aortic constriction male rats, and the underlying mechanisms were investigated by recording sodium ion channel currents and immunocytochemistry of TRPC protein in cardiomyocytes. RESULTS: The time to cardiac arrest by bupivacaine was shorter in aortic constriction rats (n =11) than in sham rats (n = 12) (mean ± SD, 1,302 ± 324 s vs. 1,034 ± 211 s; P = 0.030), regardless of its lower plasma concentration. The half-maximal inhibitory concentrations of bupivacaine toward sodium ion currents were 4.5 and 4.3 µM, which decreased to 3.9 and 2.6 µM in sham and aortic constriction rats, respectively, upon coapplication of 1-oleoyl-2-acetyl-sn-glycerol, a TRPC3 channel activator. In both groups, sodium ion currents were unaffected by QX-314, a positively charged lidocaine derivative, that hardly permeates the cell membrane, but was significantly decreased with QX-314 and 1-oleoyl-2-acetyl-sn-glycerol coapplication (sham: 79 ± 10% of control; P = 0.004; aortic constriction: 47± 27% of control; P = 0.020; n = 5 cells per group). Effects of 1-oleoyl-2-acetyl-sn-glycerol were antagonized by a specific TRPC3 channel inhibitor. CONCLUSIONS: Left ventricular hypertrophy exacerbated bupivacaine-induced cardiotoxicity, which could be a consequence of the "pore phenomenon" of TRPC3 channels upregulated in left ventricular hypertrophy.


Assuntos
Anestésicos Locais/toxicidade , Bupivacaína/toxicidade , Cardiotoxinas/toxicidade , Hipertrofia Ventricular Esquerda/induzido quimicamente , Hipertrofia Ventricular Esquerda/metabolismo , Canais de Potencial de Receptor Transitório/biossíntese , Animais , Expressão Gênica , Células HEK293 , Humanos , Hipertrofia Ventricular Esquerda/genética , Masculino , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Ratos , Ratos Sprague-Dawley , Canais de Potencial de Receptor Transitório/genética
5.
Toxicology ; 441: 152508, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32525084

RESUMO

Doxorubicin (DOX) is one of the most effective and irreplaceable chemotherapeutic agents but its clinical use is limited due to its cardiotoxicity. Glycyrrhizin(GL) has been applied to liver disorders for long. However, little is known that if GL could be meaningful in attenuating cardiotoxicity. The aim of this study is to investigate the cardioprotective effects of GL in DOX-induced cardiotoxicity (DIC) and the underlying mechanism. Here, H9c2 cardiomyoblasts, Neonatal rat cardiomyocytes (NRCMs), and Rats were introduced as test models. A single dose of 20 mg/kg DOX (i.p.) was applied to induce acute cardiotoxicity in vivo, as reflected by growth inhibition, increased levels of AST and CK-MB, and reduction of SOD activity, while GL (25 or 50 mg/kg/d, 14 d, i.p.) could counteract these effects. Moreover, pre-incubation with GL (0.8 mM for 12 h) in H9c2 cells protected against DOX-induced cytotoxicity, oxidative stress and depolarization of mitochondrial membrane potential (MMP). Besides, Western blot analysis showed that DOX upregulated the expression of LC3 II and p62 whereas GL reversed that both in vitro and in vivo and improved the obstructed autophagy flux in DOX-treated H9c2 cells with an autophagy inhibitor Bafilomycin A1 (Baf A1, 50 nM, 2 h). It has been previously documented that High-mobility group box 1 (HMGB1) was involved in DIC. In our work, knockdown of HMGB1 significantly increased cell viability and LC3 II level in H9c2, suggesting HMGB1 was crucial in DOX-induced autophagy-triggering cell death. Intriguingly, GL is a direct inhibitor of HMGB1. We found that GL downregulated Akt/mTOR autophagy signaling pathway in DOX-treated H9c2 cells. More importantly, in non-silencing H9c2 cells (transfected with negative control siRNA) cells, the expression of phospho-Akt, phospho-mTOR, p62, and LC3 II was significantly decreased with GL pretreament compared to DOX alone. However, in H9c2/HMGB1-(transfected with HMGB1 siRNA) cells exposed to DOX, the expression of p-Akt, p-mTOR, p62, LC3 II had no statistical difference with or without GL, revealing that HMGB1 mediated the cardioprotective action of GL in DIC. Taken together, our findings demonstrate that improved autophagy flux via HMGB1-dependent Akt/mTOR signaling pathway might contribute to attenuate DIC and go a novel insight into the underlying mechanisms of GL's cardioprotective action. GL could be a potential candidate for the prevention of DIC.


Assuntos
Antibióticos Antineoplásicos/toxicidade , Autofagia/efeitos dos fármacos , Cardiotoxinas/toxicidade , Doxorrubicina/toxicidade , Ácido Glicirrízico/farmacologia , Proteína HMGB1/metabolismo , Coração/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Animais , Western Blotting , Cardiotoxinas/antagonistas & inibidores , Linhagem Celular , Doxorrubicina/antagonistas & inibidores , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley
6.
PLoS One ; 15(5): e0232413, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32384080

RESUMO

BACKGROUND: Acute myocardial infarction (AMI) remains the most common cause of morbidity and mortality worldwide. The present study was directed to investigate the beneficial effects of benfotiamine pre- and post-treatments in isoproterenol (ISO)-induced MI in rats. METHODS: Myocardial heart damage was induced by subcutaneous injection of ISO (150 mg/kg) once daily for two consecutive days. Benfotiamine (100 mg/kg/day) was given orally for two weeks before or after ISO treatment. RESULTS: ISO administration revealed significant changes in electrocardiographic recordings, elevation of levels of cardiac enzymes; creatinine kinase (CK-MB) and troponin-I (cTn-I), and perturbation of markers of oxidative stress; nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, malondialdehyde (MDA), reduced glutathione (GSH), superoxide dismutase (SOD) and glutathione peroxidase (GPx) and markers of inflammation; protein kinase C (PKC), nuclear factor-kappa B (NF-κB) and metalloproteinase-9 (MMP-9). The apoptotic markers (caspase-8 and p53) were also significantly elevated in ISO groups in addition to histological alterations. Groups treated with benfotiamine pre- and post-ISO administration showed significantly decreased cardiac enzymes levels and improved oxidative stress, inflammatory and apoptotic markers compared to the ISO groups. CONCLUSION: The current study highlights the potential role of benfotiamine as a promising agent for prophylactic and therapeutic interventions in myocardial damage in several cardiovascular disorders via NADPH oxidase inhibition.


Assuntos
Inibidores Enzimáticos/uso terapêutico , Infarto do Miocárdio/tratamento farmacológico , NADPH Oxidases/antagonistas & inibidores , Tiamina/análogos & derivados , Animais , Biomarcadores/metabolismo , Cardiotoxinas/toxicidade , Modelos Animais de Doenças , Eletrocardiografia , Humanos , Mediadores da Inflamação/metabolismo , Isoproterenol/toxicidade , Masculino , Infarto do Miocárdio/induzido quimicamente , Infarto do Miocárdio/metabolismo , Miocárdio/metabolismo , Miocárdio/patologia , Estresse Oxidativo/efeitos dos fármacos , Ratos , Ratos Wistar , Tiamina/uso terapêutico
7.
Toxicol Lett ; 319: 40-48, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31706004

RESUMO

Two synthetic tryptamines, namely [3-[2-(diethylamino)ethyl]-1H-indol-4-yl] acetate (4-AcO-DET) and 3-[2-[ethyl(methyl)amino]ethyl]-1H-indol-4-ol (4-HO-MET), are abused by individuals seeking recreational hallucinogens. These new psychoactive substances (NPSs) can cause serious health problems because their adverse effects are mostly unknown. In the present study, we evaluated the cardiotoxicity of 4-AcO-DET and 4-HO-MET using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, electrocardiography (ECG), and the human ether-a-go-go-related gene (hERG) assay. In addition, we analyzed the expression level of p21 (CDC42/RAC)-activated kinase 1 (PAK1), which is known to play various roles in the cardiovascular system. In the MTT assay, 4-AcO-DET- and 4-HO-MET-treated H9c2 cells proliferated in a concentration-dependent manner. Moreover, both substances increased QT intervals (as determined using ECG) in Sprague-Dawley rats and inhibited potassium channels (as verified by the hERG assay) in Chinese hamster ovary cells. However, there was no change in PAK1 expression. Collectively, the results indicated that 4-AcO-DET and 4-HO-MET might cause adverse effects on the cardiovascular system. Further studies are required to confirm the relationship between PAK1 expression and cardiotoxicity. The findings of the present study would provide science-based evidence for scheduling the two NPSs.


Assuntos
Cardiotoxinas/toxicidade , Alucinógenos/toxicidade , Triptaminas/toxicidade , Animais , Células CHO , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cricetulus , Canal de Potássio ERG1/metabolismo , Eletrocardiografia , Masculino , Miócitos Cardíacos/efeitos dos fármacos , Bloqueadores dos Canais de Potássio/toxicidade , Ratos , Ratos Sprague-Dawley , Quinases Ativadas por p21/biossíntese , Quinases Ativadas por p21/genética
8.
Oxid Med Cell Longev ; 2019: 3585390, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31827673

RESUMO

Myoblast fusion is an essential step in skeletal muscle development and regeneration. NADPH oxidase 4 (Nox4) regulates cellular processes such as proliferation, differentiation, and survival by producing reactive oxygen species (ROS). Insulin-like growth factor 1 induces muscle hypertrophy via Nox4, but its function in myoblast fusion remains elusive. Here, we report a ROS-dependent role of Nox4 in myoblast differentiation. Regenerating muscle fibers after injury by cardiotoxin had a lower cross-sectional area in Nox4-knockout (KO) mice than myofibers in wild-type (WT) mice. Diameters and fusion index values of myotubes differentiated from Nox4-KO primary myoblasts were significantly lower than those of myotubes derived from WT myoblasts. However, no difference was observed in the differentiation index and expression of MyoD, myogenin, and myosin heavy chain 3 (MHC) between KO and WT myotubes. The decreased fusion index was also observed during differentiation of primary myoblasts and C2C12 cells with suppressed Nox4 expression. In contrast, in C2C12 cells overexpressing Nox4, the fusion index was increased, whereas the differentiation index and MHC and myogenin protein expression were not affected compared to control. Interestingly, the expression of myomaker (Tmem8c), a fusogenic protein that controls myoblast fusion, was reduced in Nox4-knockdown C2C12 cells. The myomaker expression level was proportional to the cellular ROS level, which was regulated by of Nox4 expression level. These results suggests that Nox4 contributes to myoblast fusion, possibly through the regulation of myomaker expression via ROS production, and that Nox4-dependent ROS may promote skeletal muscle regeneration and growth.


Assuntos
Músculo Esquelético/fisiologia , NADPH Oxidase 4/metabolismo , Animais , Cardiotoxinas/toxicidade , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína MyoD/metabolismo , Mioblastos/citologia , Mioblastos/metabolismo , Miogenina/metabolismo , Cadeias Pesadas de Miosina/metabolismo , NADPH Oxidase 4/antagonistas & inibidores , NADPH Oxidase 4/genética , Pirazóis/farmacologia , Piridinas/farmacologia , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Regeneração/efeitos dos fármacos
9.
J Environ Pathol Toxicol Oncol ; 38(2): 143-152, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31679277

RESUMO

The current study evaluated the cardioprotective activity of genistein in cases of doxorubicin-(Dox) induced cardiac toxicity and a probable mechanism underlying this protection, such as an antioxidant pathway in cardiac tissues. Animals used in this study were categorized into four groups. The first group was treated with sodium carboxymethylcellulose (0.3%; CMC-Na) solution. The second group received Dox (3.0 mg/kg, i.p.) on days 6, 12, 18, and 24. The third and fourth groups received Dox (3 mg/kg, i.p.) on days 6, 12, 18, and 24 and received protective doses of genistein (100 [group 3] and 200 [group 4] mg/kg/day, p.o.) for 30 days. Treatment with genistein significantly improved the altered cardiac function markers and oxidative stress markers. This was coupled with significant improvement in cardiac histopathological features. Genistein enhanced the Nrf2 and HO-1 expression, which showed protection against oxidative insult induced by Dox. Terminal deoxynucleotidyl transferase dUTP nick end labeling assay showed substantial inhibition of apoptosis by genistein in myocardia. The study showed that genistein has a strong reactive oxygen species scavenging property and potentially (P ≤ .001) decreases the lipid peroxidation as well as inhibits DNA damage in cardiac toxicity induced by Dox. In conclusion, the potential antioxidant effect of genistein may be because of its modulatory effect on Nrf2/HO-1 signalling pathway and by this means exhibits cardioprotective effects from Dox-induced oxidative injury.


Assuntos
Cardiotônicos/farmacologia , Cardiotoxinas/toxicidade , Doxorrubicina/toxicidade , Genisteína/farmacologia , Heme Oxigenase-1/genética , Fator 2 Relacionado a NF-E2/genética , Estresse Oxidativo/efeitos dos fármacos , Animais , Cardiotoxicidade/etiologia , Heme Oxigenase-1/metabolismo , Masculino , Fator 2 Relacionado a NF-E2/metabolismo , Ratos , Ratos Wistar
10.
Circ Res ; 125(9): 855-867, 2019 10 11.
Artigo em Inglês | MEDLINE | ID: mdl-31600125

RESUMO

Given that cardiovascular safety concerns remain the leading cause of drug attrition at the preclinical drug development stage, the National Center for Toxicological Research of the US Food and Drug Administration hosted a workshop to discuss current gaps and challenges in translating preclinical cardiovascular safety data to humans. This white paper summarizes the topics presented by speakers from academia, industry, and government intended to address the theme of improving cardiotoxicity assessment in drug development. The main conclusion is that to reduce cardiovascular safety liabilities of new therapeutic agents, there is an urgent need to integrate human-relevant platforms/approaches into drug development. Potential regulatory applications of human-derived cardiomyocytes and future directions in employing human-relevant platforms to fill the gaps and overcome barriers and challenges in preclinical cardiovascular safety assessment were discussed. This paper is intended to serve as an initial step in a public-private collaborative development program for human-relevant cardiotoxicity tools, particularly for cardiotoxicities characterized by contractile dysfunction or structural injury.


Assuntos
Cardiotoxicidade/epidemiologia , Cardiotoxinas/toxicidade , Educação/normas , Relatório de Pesquisa/normas , United States Food and Drug Administration/normas , Animais , Cardiotoxicidade/prevenção & controle , Avaliação Pré-Clínica de Medicamentos/métodos , Avaliação Pré-Clínica de Medicamentos/normas , Avaliação Pré-Clínica de Medicamentos/tendências , Educação/tendências , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/patologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Relatório de Pesquisa/tendências , Estados Unidos/epidemiologia , United States Food and Drug Administration/tendências
11.
Toxicol Appl Pharmacol ; 383: 114785, 2019 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-31629732

RESUMO

Celastrol (CS), an active triterpene derived from traditional Chinese medicine Tripterygium wilfordii Hook. f, has been used to treat chronic inflammation, arthritis and other diseases. However, it has been reported that CS can trigger cardiotoxicity and the molecular mechanism of heart injury induced by CS is not clear. Considering the wide application of Tripterygium wilfordii Hook. f in clinics, it is necessary to develop an accurate and reliable method to assess the safety of CS, and to elucidate as much as possible the mechanism of cardiotoxicity induced by CS. In this study, Ultra-performance liquid chromatography coupled with quadrupole time of flight mass spectrometry (UPLC-Q-TOF/MS)-based metabolomics revealed clues to the mechanism of CS-induced heart injury. Palmitic acid significantly increased in plasma from CS-treated rats, and this increase resulted in oxidative stress response in vivo. Excessive ROS further activate TNF signaling pathway and caspase family, which were obtained from the KEGG enrichment analysis of network toxicology strategy. Protein expression level of caspase-3, caspase-8, bax were significantly increased by western blot. Q-PCR also showed the similar results as western blot. It means that apoptosis plays a key role in the process of celastrol induced cardiotoxicity. Blocking this signal axis may be a potential way to protect myocardial tissue.


Assuntos
Cardiotoxinas/toxicidade , Redes e Vias Metabólicas/efeitos dos fármacos , Metabolômica/métodos , Tripterygium/toxicidade , Triterpenos/toxicidade , Animais , Cardiotoxicidade/metabolismo , Cardiotoxinas/metabolismo , Masculino , Redes e Vias Metabólicas/fisiologia , Ratos , Ratos Wistar , Tripterygium/metabolismo , Triterpenos/metabolismo
12.
Arch Toxicol ; 93(12): 3387-3396, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31664499

RESUMO

High-content screening (HCS) technology combining automated microscopy and quantitative image analysis can address biological questions in academia and the pharmaceutical industry. Various HCS experimental applications have been utilized in the research field of in vitro toxicology. In this review, we describe several HCS application approaches used for studying the mechanism of compound toxicity, highlight some challenges faced in the toxicological community, and discuss the future directions of HCS in regards to new models, new reagents, data management, and informatics. Many specialized areas of toxicology including developmental toxicity, genotoxicity, developmental neurotoxicity/neurotoxicity, hepatotoxicity, cardiotoxicity, and nephrotoxicity will be examined. In addition, several newly developed cellular assay models including induced pluripotent stem cells (iPSCs), three-dimensional (3D) cell models, and tissues-on-a-chip will be discussed. New genome-editing technologies (e.g., CRISPR/Cas9), data analyzing tools for imaging, and coupling with high-content assays will be reviewed. Finally, the applications of machine learning to image processing will be explored. These new HCS approaches offer a huge step forward in dissecting biological processes, developing drugs, and making toxicology studies easier.


Assuntos
Ensaios de Triagem em Larga Escala/métodos , Toxicologia/métodos , Animais , Cardiotoxinas/toxicidade , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Humanos , Nefropatias/induzido quimicamente , Testes de Mutagenicidade/métodos , Síndromes Neurotóxicas/etiologia
13.
Electrophoresis ; 40(23-24): 3108-3116, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31650569

RESUMO

Accurate profiling of the lipophilicity of amphoteric compounds might be complex and laborious. In the present work the lipophilicity of 12 anthracycline antibiotics-four parent drugs: doxorubicin, daunorubicin, epidoxorubicin, and epidaunorubicin and eight novel formamidyne derivatives with attached morpholine, hexamethylenoimine or piperidine rings-was determined based on novel approach using MEEKC. In the second stage, lipophilicity was correlated with anthracycline toxicity towards two cell lines. In rat cardiomyoblast cell line (h9c2) a significant correlation between the logP and toxicity was found. The anthracycline lipophilicity was not correlated with toxicity towards the endothelial hybrid cell line (EAhy.926). In conclusion, the lipophilicity of anthracyclines seems to determine their toxicity towards cardiomyoblasts but not on endothelial cells, suggesting a different mechanism of anthracyclines intercellular transport or extrusion in cardiomyoblast and endothelial cells.


Assuntos
Antraciclinas , Antibacterianos , Cardiotoxinas , Cromatografia Capilar Eletrocinética Micelar/métodos , Animais , Antraciclinas/análise , Antraciclinas/química , Antraciclinas/toxicidade , Antibacterianos/análise , Antibacterianos/química , Antibacterianos/toxicidade , Cardiotoxinas/análise , Cardiotoxinas/química , Cardiotoxinas/toxicidade , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Endotélio Vascular/citologia , Interações Hidrofóbicas e Hidrofílicas , Ratos
14.
Toxicol Appl Pharmacol ; 383: 114761, 2019 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-31533062

RESUMO

Recent developments of novel targeted therapies are contributing to the increased long-term survival of cancer patients; however, drug-induced cardiotoxicity induced by cancer drugs remains a serious problem in clinical settings. Nevertheless, there are few in vitro cell-based assays available to predict this toxicity, especially from the aspect of morphology. Here, we developed a simple two-dimensional (2D) morphological assessment system, 2DMA, to predict drug-induced cardiotoxicity in cancer patients using human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) with image-based high-content analysis in a high-throughput manner. To assess the effects of drugs on cardiomyocytes, we treated iPSC-CMs with 28 marketed pharmaceuticals and measured two key parameters: number of cell nuclei and sarcomere morphology. Drugs that significantly perturbed these two parameters at concentrations ≤30 times the human Cmax value were regarded as positive in the test. Based on these criteria, the sensitivity and specificity of the 2DMA system were 81% and 100%, respectively. Moreover, the translational predictability of 2DMA was comparable with that of a three-dimensional cardiotoxicity assay. RNA sequencing further revealed that the expression levels of several genes related to sarcomere components decreased following treatment with sunitinib, suggesting that inhibition of the synthesis of proteins that comprise the sarcomere contributes to drug-induced sarcomere disruption. Based on these features, the 2DMA system provides mechanistic insight with high predictability of cancer drug-induced cardiotoxicity in humans, and could thus contribute to the reduction of drug attrition rates at early stages of drug development.


Assuntos
Antineoplásicos/toxicidade , Cardiotoxinas/toxicidade , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Microscopia Eletrônica/métodos , Miócitos Cardíacos/efeitos dos fármacos , Cardiotoxicidade/patologia , Técnicas de Cultura de Células/métodos , Células Cultivadas , Corantes Fluorescentes/análise , Previsões , Humanos , Células-Tronco Pluripotentes Induzidas/química , Células-Tronco Pluripotentes Induzidas/patologia , Miócitos Cardíacos/química , Miócitos Cardíacos/patologia
15.
Toxins (Basel) ; 11(9)2019 09 12.
Artigo em Inglês | MEDLINE | ID: mdl-31547294

RESUMO

Cardiotoxins (CTXs) are suggested to exert their cytotoxicity through cell membrane damage. Other studies show that penetration of CTXs into cells elicits mitochondrial fragmentation or lysosome disruption, leading to cell death. Considering the role of AMPK-activated protein kinase (AMPK) in mitochondrial biogenesis and lysosomal biogenesis, we aimed to investigate whether the AMPK-mediated pathway modulated Naja atra (Taiwan cobra) CTX3 cytotoxicity in U937 human leukemia cells. Our results showed that CTX3 induced autophagy and apoptosis in U937 cells, whereas autophagic inhibitors suppressed CTX3-induced apoptosis. CTX3 treatment elicited Ca2+-dependent degradation of the protein phosphatase 2A (PP2A) catalytic subunit (PP2Acα) and phosphorylation of AMPKα. Overexpression of PP2Acα mitigated the CTX3-induced AMPKα phosphorylation. CTX3-induced autophagy was via AMPK-mediated suppression of the Akt/mTOR pathway. Removal of Ca2+ or suppression of AMPKα phosphorylation inhibited the CTX3-induced cell death. CTX3 was unable to induce autophagy and apoptosis in U937 cells expressing constitutively active Akt. Met-modified CTX3 retained its membrane-perturbing activity, however, it did not induce AMPK activation and death of U937 cells. These results conclusively indicate that CTX3 induces autophagy and apoptosis in U937 cells via the Ca2+/PP2A/AMPK axis, and suggest that the membrane-perturbing activity of CTX3 is not crucial for the cell death signaling pathway induction.


Assuntos
Cardiotoxinas/toxicidade , Proteínas Cardiotóxicas de Elapídeos/toxicidade , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Cálcio/metabolismo , Humanos , Leucemia , Proteína Fosfatase 2/genética , Células U937
16.
Skelet Muscle ; 9(1): 20, 2019 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-31358063

RESUMO

BACKGROUND: Estrogen signaling is indispensable for muscle regeneration, yet the role of estrogen in the development of muscle inflammation, especially in the intramuscular T cell response, and the influence on the intrinsic immuno-behaviors of myofibers remain largely unknown. We investigated this issue using the mice model of cardiotoxin (CTX)-induced myoinjury, with or without estrogen level adjustment. METHODS: CTX injection i.m. (tibialis anterior, TA) was performed for preparing mice myoinjury model. Injection s.c. of 17ß-estradiol (E2) or estrogen receptor antagonist 4-OHT, or ovariectomy (OVX), was used to change estrogen level of animal models in vivo. Serum E2 level was evaluated by ELISA. Gene levels of estrogen receptor (ERs) and cytokines/chemokines in inflamed muscle were monitored by qPCR. Inflammatory infiltration was observed by immunofluorescence. Macrophage and T cell phenotypes were analyzed by FACS. Immunoblotting was used to assess protein levels of ERs and immunomolecules in C2C12 myotubes treated with E2 or 4-OHT, in the presence of IFN-γ. RESULTS: We monitored the increased serum E2 level and the upregulated ERß in regenerated myofibres after myotrauma. The absence of estrogen in vivo resulted in the more severe muscle inflammatory infiltration, involving the recruitment of monocyte/macrophage and CD4+ T cells, and the heightened proinflammatory (M1) macrophage. Moreover, estrogen signaling loss led to Treg cells infiltration decrease, Th1 response elevation in inflamed muscle, and the markedly expression upregulation of immunomolecules in IFN-γ-stimulated C2C12 myotubes in vitro. CONCLUSION: Our data suggest that estrogen is a positive intervention factor for muscle inflammatory response, through its effects on controlling intramuscular infiltration and phenotypes of monocytes/macrophages, on affecting accumulation and function of Treg cells, and on suppressing Th1 response in inflamed muscle. Our findings also imply an inhibition effect of estrogen on the intrinsic immune behaviors of muscle cells.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Estrogênios/imunologia , Macrófagos/imunologia , Músculo Esquelético/imunologia , Animais , Cardiotoxinas/toxicidade , Estradiol/sangue , Receptor beta de Estrogênio/genética , Receptor beta de Estrogênio/metabolismo , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/lesões , Músculo Esquelético/metabolismo , Miosite/imunologia , Miosite/metabolismo , Miosite/patologia , Regeneração/genética , Regeneração/imunologia , Transdução de Sinais/imunologia
17.
Int J Cardiol ; 292: 171-179, 2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31160077

RESUMO

BACKGROUND: The immunotherapy has revolutionized the world of oncology in the last decades with considerable advantages in terms of overall survival in cancer patients. The association of Pembrolizumab and Trastuzumab was recently proposed in clinical trials for the treatment of Trastuzumab-resistant advanced HER2-positive breast cancer. Although immunotherapies are frequently associated with a wide spectrum of immune-related adverse events, the cardiac toxicity has not been properly studied. PURPOSE: We studied, for the first time, the putative cardiotoxic and pro-inflammatory effects of Pembrolizumab associated to Trastuzumab. METHODS: Cell viability, intracellular calcium quantification and pro-inflammatory studies (analyses of the production of Interleukin 1ß, 6 and 8, the expression of NF-kB and Leukotriene B4) were performed in human fetal cardiomyocytes. Preclinical studies were also performed in C57BL6 mice by analyzing fibrosis and inflammation in heart tissues. RESULTS: The combination of Pembrolizumab and Trastuzumab leads to an increase of the intracellular calcium overload (of 3 times compared to untreated cells) and to a reduction of the cardiomyocytes viability (of 65 and 20-25%, compared to untreated and Pembrolizumab or Trastuzumab treated cells, respectively) indicating cardiotoxic effects. Notably, combination therapy increases the inflammation of cardiomyocytes by enhancing the expression of NF-kB and Interleukins. Moreover, in preclinical models, the association of Pembrolizumab and Trastuzumab increases the Interleukins expression of 40-50% compared to the single treatments; the expression of NF-kB and Leukotriene B4 was also increased. CONCLUSION: Pembrolizumab associated to Trastuzumab leads to strong cardiac pro-inflammatory effects mediated by overexpression of NF-kB and Leukotriene B4 related pathways.


Assuntos
Anticorpos Monoclonais Humanizados/toxicidade , Cardiotoxinas/toxicidade , Mediadores da Inflamação/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Trastuzumab/toxicidade , Animais , Anticorpos Monoclonais Humanizados/administração & dosagem , Antineoplásicos Imunológicos/administração & dosagem , Antineoplásicos Imunológicos/toxicidade , Cardiotoxinas/administração & dosagem , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Técnicas de Cocultura , Combinação de Medicamentos , Feminino , Humanos , Imunoterapia/efeitos adversos , Imunoterapia/métodos , Camundongos , Camundongos Endogâmicos C57BL , Trastuzumab/administração & dosagem
18.
Nutrients ; 11(4)2019 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-30999708

RESUMO

The effects of lactate on muscle mass and regeneration were investigated using mouse skeletal muscle tissue and cultured C2C12 cells. Male C57BL/6J mice were randomly divided into (1) control, (2) lactate (1 mol/L in distilled water, 8.9 mL/g body weight)-administered, (3) cardio toxin (CTX)-injected (CX), and (4) lactate-administered after CTX-injection (LX) groups. CTX was injected into right tibialis anterior (TA) muscle before the oral administration of sodium lactate (five days/week for two weeks) to the mice. Oral lactate administration increased the muscle weight and fiber cross-sectional area, and the population of Pax7-positive nuclei in mouse TA skeletal muscle. Oral administration of lactate also facilitated the recovery process of CTX-associated injured mouse TA muscle mass accompanied with a transient increase in the population of Pax7-positive nuclei. Mouse myoblast-derived C2C12 cells were differentiated for five days to form myotubes with or without lactate administration. C2C12 myotube formation with an increase in protein content, fiber diameter, length, and myo-nuclei was stimulated by lactate. These observations suggest that lactate may be a potential molecule to stimulate muscle hypertrophy and regeneration of mouse skeletal muscle via the activation of muscle satellite cells.


Assuntos
Músculo Esquelético/efeitos dos fármacos , Mioblastos/efeitos dos fármacos , Regeneração/efeitos dos fármacos , Lactato de Sódio/farmacologia , Animais , Cardiotoxinas/toxicidade , Linhagem Celular , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/crescimento & desenvolvimento , Músculo Esquelético/fisiologia , Distribuição Aleatória , Lactato de Sódio/administração & dosagem
19.
Toxicol Lett ; 307: 41-48, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-30817977

RESUMO

Doxorubicin (DOX) is widely used as a broad-spectrum anti-tumor anthracycline to treat various cancers. The serious adverse effects of DOX on cardiotoxicity limit its clinical application. There are several different mechanisms involved in DOX-induced cardiotoxicity. Oxidative stress (OS) is caused by an imbalance between reactive oxygen species (ROS) and endogenous antioxidants in response to injury, which can lead to myocardial toxicity. The aim of this review was to investigate the mechanisms underlying the effects of oxidative stress injury on myocardial toxicity, from three different aspects: the increase in downstream oxidative stress products, the reduction in upstream antioxidative stress products, and subcellular organelles. Finally, there are some anti-oxidative drugs that show efficacy in limiting DOX-induced cardiotoxicity. It is necessary to fully understand the toxicity of DOX to the myocardium and achieve symptomatic treatment.


Assuntos
Cardiotoxicidade/etiologia , Cardiotoxinas/toxicidade , Doxorrubicina/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Animais , Coração/efeitos dos fármacos , Humanos , Miocárdio/metabolismo
20.
Ecotoxicol Environ Saf ; 168: 378-387, 2019 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-30396134

RESUMO

A growing number of epidemiological surveys show that PM2.5 is an important promoter for the cardiovascular dysfunction induced by atmospheric pollution. PM2.5 is a complex mixture of solid and liquid airborne particles and its components determine the health risk of PM2.5to a great extent. However, the individual cardiotoxicities of different PM2.5 fractions are still unclear, especially in the cellular level. Here we used the neonatal rat cardiomyocytes (NRCMs) to evaluate the cardiac toxicity of PM2.5 exposure. The cytotoxicities of Total-PM2.5, water soluble components of PM2.5 (WS-PM2.5) and water insoluble components of PM2.5 (WIS-PM2.5), which include the cell viability, cell membrane damage, reactive oxygen species (ROS) generation, were examined with NRCMs in vitro. The results indicated that Total-PM2.5 or WIS-PM2.5 exposure significantly decreased the cell viability, induced the cell membrane damage and increased the ROS level in NRCMs at concentrations above 50 µg/mL. However, WS-PM2.5 exposure could induce the cytotoxicity on NRCMs until the concentration of WS-PM2.5 was raised to a higher concentration (75 µg/mL). Furthermore, the DNA damage was detected in NRCMs after 48 h of exposure with Total-PM2.5, WS-PM2.5 or WIS-PM2.5 (75 µg/mL) and the adverse effects on mitochondrial function and action potentials of NRCMs were detected only both in the Total-PM2.5 and WIS-PM2.5 treatment group. In summary, our project not only estimates the risk of PM2.5 on cardiac cells but also reveal that Total-PM2.5 and WIS-PM2.5 exposure were predominantly associated with the functional cardiotoxicities in NRCMs.


Assuntos
Cardiotoxinas/toxicidade , Miócitos Cardíacos/efeitos dos fármacos , Material Particulado/toxicidade , Animais , Animais Recém-Nascidos , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Dano ao DNA/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Miócitos Cardíacos/metabolismo , Ratos , Espécies Reativas de Oxigênio/metabolismo
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