RESUMO
Myocardial 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) metabolizes a nucleoside 2',3'-cyclic phosphate to a nucleoside 2'-phosphate. Recently, the roles of CNPase in the pathophysiological processes of heart failure have emerged. The mitochondrial acylome subjected to SIRT3 regulation give us comprehensive understanding of acylation modifications to a vast array of protein targets, and the list of acetylated mitochondrial proteins is still growing. However, it remains elusive whether CNPase is subjected to the regulation of acetylation and deacetylation, and the effects of which on CNPase enzymatic activity are still unknown. In this study, the mitochondrial distribution of CNPase was identified by immunofluorescence and cytosol/mitochondria fractioning. The immunofluorescence staining pattern of CNPase and Sirt3 overlapped on the same focal plane. Moreover, Sirt3 associates directly with CNPase, and the CNPase enzymatic activity was subjected to Sirt3 activity. Then biochemical methods using acetic anhydride was employed to acetylate the CNPase proteins, the enzymatic activity of CNPase decreased. Furthermore, co-immunoprecipitation coupled mass spectrometry identifies K196, K379, K128 as the main acetylation sites. Molecular dynamic simulation shows that acetylation modification suppressed the CNPase enzymatic activity through decreasing the opening probability of the binding pocket and restricting substrate accessibility. Together with these findings, this study reveals a molecular mechanism underlying Sirt3 regulating CNPase enzymatic activity, and suggests that targeting CNPase's post-translational modifications represents a promising therapeutic strategy.
Assuntos
2',3'-Nucleotídeo Cíclico Fosfodiesterases/metabolismo , Sirtuínas/metabolismo , Acilação , Animais , Células Cultivadas , Ratos , Especificidade por SubstratoRESUMO
Sepsis is a severe inflammatory disorder that can lead to multiple organ injury. Isosteviol sodium (STV-Na) is a terpenoid derived from stevioside that exerts anti-inflammatory, antioxidant and antiapoptotic activities. However, the influence of STV-Na on sepsis remains unknown. Here, we assessed the potential effects of STV-Na on sepsis and multiple organ injury induced by lipopolysaccharide (LPS). We found that STV-Na increased the survival rate of mice treat with LPS, significantly improved the functions of the heart, lung, liver, and kidney, reduced the production of inflammatory cytokines and decreased macrophage infiltration. Moreover, Multiorgan metabolomics analysis demonstrated that glutathione metabolism, purine metabolism, glycerophospholipid metabolism and pantothenate and CoA biosynthesis, were significantly altered by STV-Na. This study provides novel insights into the metabolite changes of multiple organ injury in septic mice, which may help characterize the underlying mechanism and provide an improved understanding of the therapeutic effects of STV-Na on sepsis.
Assuntos
Anti-Inflamatórios/uso terapêutico , Diterpenos do Tipo Caurano/uso terapêutico , Insuficiência de Múltiplos Órgãos/tratamento farmacológico , Sepse/tratamento farmacológico , Animais , Anti-Inflamatórios/farmacologia , Diterpenos do Tipo Caurano/farmacologia , Glutationa/metabolismo , Glicerofosfolipídeos/metabolismo , Rim/efeitos dos fármacos , Rim/metabolismo , Rim/patologia , Lipopolissacarídeos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Pulmão/patologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Masculino , Metabolômica , Camundongos Endogâmicos BALB C , Insuficiência de Múltiplos Órgãos/etiologia , Insuficiência de Múltiplos Órgãos/imunologia , Insuficiência de Múltiplos Órgãos/metabolismo , Miocárdio/metabolismo , Miocárdio/patologia , Ácido Pantotênico/metabolismo , Purinas/metabolismo , Sepse/complicações , Sepse/imunologia , Sepse/metabolismo , Baço/efeitos dos fármacos , Baço/metabolismo , Baço/patologiaRESUMO
Heart failure is the end-stage of all cardiovascular diseases with a ~25% 5-year survival rate, and insufficient mitochondrial energy production to meet myocardial demand is the hallmark of heart failure. Mitochondrial components involved in the regulation of ATP production remain to be fully elucidated. Recently, roles of 2',3'-cyclic nucleotide-3'-phosphodiesterase (CNPase) in the pathophysiological processes of heart diseases have emerged, implicated by evidence that mitochondrial CNPase proteins are associated with mitochondrial integrity under metabolic stress. In this study, a zebrafish heart failure model was established, by employing antisense morpholino oligonucleotides and the CRISPR-Cas9 gene-editing system, which recapitulates heart failure phenotypes including heart dysfunction, pericardial edema, ventricular enlargement, bradycardia, and premature death. The translational implications of CNPase in the pathophysiological process of heart failure were tested in a pressure overload-induced heart hypertrophy model, which was carried out in rats through transverse abdominal aorta constriction (TAAC). AAV9-mediated myocardial delivery of CNPase mitigated the hypertrophic response through the specific hydrolysis of 2'-3'-cyclic nucleotides, supported by the decrease of cardiac hypertrophy and fibrosis, the integrity of mitochondrial ultrastructure, and indicators of heart contractility in the AAV9-TAAC group. Finally, the biometrics of a mitochondrial respiration assay carried out on a Seahorse cellular energy analyzer demonstrated that CNPase protects mitochondrial respiration and ATP production from AngII-induced metabolic stress. In summary, this study provides mechanistic insights into CNPase-2',3'-cyclic nucleotide metabolism that protects the heart from energy starvation and suggests novel therapeutic approaches to treat heart failure by targeting CNPase activity.
Assuntos
2',3'-Nucleotídeo Cíclico Fosfodiesterases/antagonistas & inibidores , Sistemas CRISPR-Cas , Cardiomegalia/prevenção & controle , Modelos Animais de Doenças , Metabolismo Energético , Mitocôndrias/fisiologia , Nucleotídeos Cíclicos/metabolismo , 2',3'-Nucleotídeo Cíclico Fosfodiesterases/genética , Animais , Masculino , Ratos , Ratos Sprague-Dawley , Peixe-ZebraRESUMO
Evidence demonstrates that M1 macrophage polarization promotes inflammatory disease. Here, we discovered that (R)-salbutamol, a ß2 receptor agonist, inhibits and reprograms the cellular metabolism of RAW264.7 macrophages. (R)-salbutamol significantly inhibited LPS-induced M1 macrophage polarization and downregulated expressions of typical M1 macrophage cytokines, including monocyte chemotactic protein-1 (MCP-1), interleukin-1ß (IL-1ß) and tumour necrosis factor α (TNF-α). Also, (R)-salbutamol significantly decreased the production of inducible nitric oxide synthase (iNOS), nitric oxide (NO) and reactive oxygen species (ROS), while increasing the reduced glutathione (GSH)/oxidized glutathione (GSSG) ratio. In contrast, (S)-salbutamol increased the production of NO and ROS. Bioenergetic profiles showed that (R)-salbutamol significantly reduced aerobic glycolysis and enhanced mitochondrial respiration. Untargeted metabolomics analysis demonstrated that (R)-salbutamol modulated metabolic pathways, of which three metabolic pathways, namely, (a) phenylalanine metabolism, (b) the pentose phosphate pathway and (c) glycerophospholipid metabolism were the most noticeably impacted pathways. The effects of (R)-salbutamol on M1 polarization were inhibited by a specific ß2 receptor antagonist, ICI-118551. These findings demonstrated that (R)-salbutamol inhibits the M1 phenotype by downregulating aerobic glycolysis and glycerophospholipid metabolism, which may propose (R)-salbutamol as the major pharmacologically active component of racemic salbutamol for the treatment of inflammatory diseases and highlight the medicinal value of (R)-salbutamol.
Assuntos
Albuterol/farmacologia , Polaridade Celular , Regulação da Expressão Gênica/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Metaboloma/efeitos dos fármacos , Agonistas de Receptores Adrenérgicos beta 2/farmacologia , Animais , Citocinas/metabolismo , Glicólise , Macrófagos/efeitos dos fármacos , Camundongos , Óxido Nítrico/metabolismo , Fenótipo , Células RAW 264.7 , Espécies Reativas de Oxigênio/metabolismoRESUMO
Oxidative stress plays a crucial role in pathogenesis of various cardiovascular diseases. Recent studies reported that isosteviol sodium (STVNa) harbor cardioprotective properties. Here, we explore the potential cardioprotective effect of STVNa on H2 O2 -induced oxidative stress on heart embryonic H9c2 cardiomyocytes and the underlying mechanism. We have found that STVNa pretreatment improved cell viability, nuclear morphology and prevented LDH release induced by oxidative stress. STVNa pretreatment also reduced production of reactive oxygen species, preserved mitochondrial function, restored biological antioxidant defense systems and prevented cell death. Western blotting analysis revealed that STVNa regulated the mitochondrial related pro- and anti-apoptotic protein (Bax and Bcl-2 respectively) levels, increased phosphorylation of Akt (ser473) and GSK-3ß (ser9) and promoted binding between HK-II and mitochondria under the normal or oxidative stress conditions. LY294002, a PI3K inhibitor, abolished cytoprotective effects of STVNa by inhibiting activation of Akt and GSK-3ß. Based on these findings, we conclude that STVNa protects H9c2 cells against oxidative stress by activating Akt/GSK-3ß signaling pathway, which, in turn, leads to recruitment of HK-II to mitochondria and regulating Bcl2/Bax levels.
Assuntos
Cardiotônicos/farmacologia , Diterpenos do Tipo Caurano/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cromonas/farmacologia , Glicogênio Sintase Quinase 3 beta/metabolismo , L-Lactato Desidrogenase/metabolismo , Mitocôndrias/metabolismo , Morfolinas/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Espécies Reativas de Oxigênio/metabolismoRESUMO
BACKGROUND: Isosteviol is a synthetic derivative of steviol glycosides with promising pharmacological properties and might find future use as a cardioprotective agent. OBJECTIVE: A simple LC-MS/MS technique was developed and validated for the bioanalysis of isosteviol in plasma and erythrocytes. This method was subsequently utilized for the in vitro assessment of isosteviol's partitioning into blood compartments of humans and rats. METHODS: Fresh blood samples from healthy humans and Wistar rats were equilibrated with 1, 10, and 30 µM isosteviol at 37⯰C in a shaking dry-bath. The levels of isosteviol in plasma and erythrocytes partitions were determined in these samples, after separation, at intervals over a 60-minute period. The data derived was used to estimate erythrocyte-to-plasma and blood-to-plasma coefficients. RESULTS: Mean erythrocyte-to-plasma partition coefficients (SD) after 60 minutes of equilibration were observed to be 0.039 (0.002) and 0.040 (0.003) in humans and rats, respectively. Derived values for the blood-to-plasma ratio (SD) were 0.576 (0.001) in humans and 0.543 (0.007) in rats, whereas plasma component binding was estimated to be more than 96%. CONCLUSIONS: The findings suggest that isosteviol preferentially partitions into plasma compartments in humans and rats. The significance of this profile for the efficacy, tissue uptake, and retention of isosteviol will have to be further studied.
RESUMO
In mouse fetal liver, hepatoblasts, sinusoidal endothelial cells and macrophages (or erythroblastic islands) promote differentiation and proliferation of hematopoietic cells through cell-cell interactions and secretion of cytokines and extracellular matrix factors. Until now, we have had little knowledge of the hematopoietic cytokines or extracellular matrix mRNAs expressed in hepatic stellate cells. Using p75 neurotrophin receptor (p75NTR) to mark this cell population, we sorted 12.5, 14.5 and 16.5 dpc hepatic stellate cells and analyzed expression of cytokines and extracellular matrix mRNAs. Among cytokines, insulin-like growth factor 2 (Igf2) was highly expressed at all three stages analyzed. The extracellular matrix molecule fibronectin (Fn1) was highly expressed in 12.5 dpc cells, whereas vitronectin (Vtn) was highly expressed in 14.5 and 16.5 dpc hepatic stellate cells. Among liver cells, Igf2 was predominantly expressed in hepatoblast-like cells at all three stages examined, suggesting that hepatoblast-like cells are an essential part of the niche that maintains homeostasis of hematopoietic cells in embryonic mouse liver. Defining these expression patterns could facilitate our understanding of cross talk between cytokine and extracellular matrix molecules in hepatic stellate cells and benefit research in developmental hematopoiesis as well as the study of liver biology.
Assuntos
Citocinas/metabolismo , Fibronectinas/metabolismo , Células Estreladas do Fígado/metabolismo , Fator de Crescimento Insulin-Like II/metabolismo , RNA Mensageiro/metabolismo , Vitronectina/metabolismo , Animais , Citocinas/genética , Matriz Extracelular/metabolismo , Fibronectinas/genética , Fator de Crescimento Insulin-Like II/genética , Fígado/citologia , Fígado/embriologia , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos ICR , RNA Mensageiro/genética , Vitronectina/genéticaRESUMO
It is an intriguing approach to target the ecto-5'-nucleotidase CD73 to confer synergetic beneficial survival in cancer patients, along with clinically established immunotherapy targets. In this study, a fully human, subnanomolar affinity CD73 antibody IBI325 was developed using the yeast display platform. Compared with Oleclumab, IBI325 was equivalent in hCD73 affinity and more potent in cell-bound and soluble CD73 enzymatic inhibition, and no hook effects were observed. Correspondingly, adenosine monophosphate-mediated immune suppression was reversed by IBI325, and significant T cell proliferation and release of cytokines were observed. Also, IBI325 enhanced the T cell recall response by inducing interferon-γ secretion. The antitumor efficacy of IBI325 was investigated in a hPBMC-reconstituted NOG mouse model, and a hCD73 knock-in mouse model. Consequently, IBI325 induced a significant tumor regression by inducing intratumoral immune cell expansion, and a combo therapy of IBI325 and aPD-1 was superior in efficacy than aCD73 or aPD-1 monotherapy. Additionally, the binding epitopes of CD73 to IBI325 were distinct from previously reported aCD73 therapeutics. IBI325 displayed acceptable pharmacokinetics and sufficient tolerable safety profiles to support clinical development. In conclusion, the pharmacology, pharmacokinetics, and toxicity profiles of IBI325 with complete CD73 inhibition were characterized, and encouraging preclinical outcomes were reported.
Assuntos
Antineoplásicos , Neoplasias , Camundongos , Animais , Humanos , 5'-Nucleotidase , Monofosfato de Adenosina/metabolismo , Neoplasias/tratamento farmacológico , ImunoterapiaRESUMO
AIMS: Individuals with nonalcoholic hepatosteatosis (NAFLD) have a worse atherogenic lipoprotein profile and are susceptible to cardiovascular diseases. The MEK-ERK signaling cascades are central regulators of the levels of LDL receptor (LDLR), a major determinant of circulating cholesterol. It is elusive how hepatic steatosis contributes to dyslipidemia, especially hypercholesterolemia. MAIN METHODS: The effects of BChE on signaling pathways were determined by immunoblotting in a BChE knockout hepatocyte cell line. DiI-LDL probe was used to explore the effect of BChE expression on LDL internalization. Co-immunoprecipitation and LC-MS were used to explore the interacting proteins with BChE. Finally, a hepatocyte-restricted BChE silencing mouse model was established by AAV8-Tbg-shRNA, and the hypercholesterolemia was induced by 65% kcal% high-fat, high-sucrose diet feeding. MAIN FINDINGS: Here we demonstrate that butyrylcholinesterase (BChE) governs the LDL receptor levels and LDL uptake capacity through the MEK-ERK signaling cascades to promote Ldlr transcription. BChE interacts and co-localizes with PRMT5, a protein methylation modifier controlling the ERK signaling. PRMT5 regulates LDLR-dependent LDL uptake and is a substrate of chaperone-mediated autophagy (CMA). BChE deficiency induces the PRTM5 degradation dependent on CMA activity, possibly through facilitating the HSC70 (Heat shock cognate 71 kDa) recognition of PRMT5. Remarkably, in vivo hepatocyte-restricted BChE silencing reduces plasma cholesterol levels substantially. In contrast, the BChE knockout mice are predisposed to hypercholesterolemia. SIGNIFICANCE: Taken together, these findings outline a regulatory role for the BChE-PRMT5-ERK-LDLR axis in hepatocyte cholesterol metabolism, and suggest that targeting liver BChE is an effective therapeutic strategy to treat hypercholesterolemia.
Assuntos
Butirilcolinesterase/deficiência , Hepatócitos/metabolismo , Hipercolesterolemia/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Receptores de LDL/metabolismo , Transcrição Gênica/fisiologia , Sequência de Aminoácidos , Animais , Butirilcolinesterase/genética , Tetracloreto de Carbono/toxicidade , Células Hep G2 , Humanos , Hipercolesterolemia/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Hepatopatia Gordurosa não Alcoólica/induzido quimicamente , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/metabolismo , Proteína-Arginina N-Metiltransferases/genética , Receptores de LDL/genéticaRESUMO
Inflammatory bowel diseases (IBDs) constitute a group of chronic intestinal conditions prominently featuring deranged metabolism. Effective pharmacological treatments for IBDs are lacking. Isosteviol sodium (STV-Na) exhibits anti-inflammatory activity and may offer therapeutic benefits in chronic colitis. However, the associated mechanism remains unclear. This study is aimed at exploring the therapeutic effects of STV-Na against chronic colitis in terms of metabolic reprogramming and macrophage polarization. Results show that STV-Na attenuated weight loss and colonic pathological damage and restored the hematological and biochemical parameters in chronic colitis mice models. STV-Na also restored intestinal permeability by increasing the goblet cell numbers, which was accompanied by lowered plasma lipopolysaccharide and diamine oxidase levels. Metabolomic analysis highlighted 102 candidate biomarkers and 5 vital pathways that may be crucial in the potential pharmacological mechanism of STV-Na in regulating intestinal inflammation and oxidative stress. These pathways were glycerophospholipid metabolism, phenylalanine metabolism, phenylalanine, tyrosine and tryptophan biosynthesis, the pentose phosphate pathway, and phosphonate and phosphinate metabolism. Furthermore, STV-Na significantly decreased M1 macrophage polarization in the spleen and colon. The mRNA and protein levels of IL-1ß, TNF-α, and NF-κB/p65 in colonic tissue from the colitis mice were decreased after the STV-Na treatment. Overall, STV-Na could alleviate chronic colitis by suppressing oxidative stress and inflammation levels, reprogramming the metabolic profile, inhibiting macrophage polarization, and suppressing the NF-κB/p65 signaling pathway. STV-Na remains a promising candidate drug for treating IBDs.
Assuntos
Colite/patologia , Diterpenos do Tipo Caurano/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Metaboloma/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Animais , Doença Crônica , Colite/induzido quimicamente , Colite/tratamento farmacológico , Colo/efeitos dos fármacos , Colo/metabolismo , Colo/patologia , Sulfato de Dextrana/toxicidade , Diterpenos do Tipo Caurano/uso terapêutico , Glicerofosfolipídeos/metabolismo , Interleucina-1beta/sangue , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos/citologia , Macrófagos/metabolismo , Masculino , Metabolômica , Camundongos , Camundongos Endogâmicos C57BL , Via de Pentose Fosfato , Fenilalanina/metabolismo , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismoRESUMO
Butyrylcholinesterase (BChE) is detected in plaques preferentially in Alzheimer's disease (AD) and may be associated with stress disorders. However, the physiological function of BChE in the central nervous system remains to be further investigated. BChE knockout (KO) mice and wild-type (WT) mice with orally or intranasal administration of (R)-bambuterol were used to explore the effect of BChE on behavior changes. (R)-bambuterol is a specific and reversible inhibitor of BChE. The behavior changes were evaluated and compared among 3-10 month old mice. Our finding showed that BChE KO and (R)-bambuterol administration enhanced episodic memory, including fear conditioning memory and fear extinction memory in fear conditioning and fear extinction test. BChE KO and (R)-bambuterol administered mice rescued age-related spatial memory and general activity in the water maze test and open field test. The brain metabolomics were imaged using a desorption electrospray ionization mass spectrometry imaging (DESI-MSI). The image of DESI-MS demonstrated that glutamine content increased in the brain of BChE KO mice. In conclusion, this study found that inhibition of BChE ameliorated episodic and spatial memories. This study also suggested that (R)-bambuterol as a BChE inhibitor has the potential application in the treatment of post-traumatic stress disorder (PTSD) and early cognitive decline.
RESUMO
PURPOSE: Inflammatory bowel diseases (IBDs) are global health problems that are associated with immune regulation, but clinical IBDs treatment is currently inadequate. Effective preventive or therapeutic methods for immune disorders rely on controlling the function of immune cells. Isosteviol sodium (STV-Na) has antioxidant activity, but the therapeutic effect of STV-Na against IBD remain undocumented. Herein, we investigated the therapeutic effect of STV-Na in mice models with IBDs. METHODS: Mice received 3.5% DSS for 7 days to establish IBD models. Intraperitoneal STV-Na was given 2 days before DSS and lasted for 9 days. Commercially available drugs used in treating IBDs (5-aminosalicylic acid, dexamethasone, and infliximab) were used as positive controls. Samples were collected 7 days after colitis induction. Histopathological score, biochemical parameters, molecular biology methods, and metabolomics were used for evaluating the therapeutic effect of STV-Na. RESULTS: Our data revealed that STV-Na could significantly alleviate colon inflammation in mice with colitis. Specifically, STV-Na treatment improved body weight loss, increased colon length, decreased histology scores, and restored the hematological parameters of mice with colitis. The untargeted metabolomics analysis revealed that metabolic profiles were restored by STV-Na treatment. Furthermore, STV-Na therapy suppressed the number of CD68 macrophages and F4/80 cell infiltration. And STV-Na suppressed M1 and M2 macrophage numbers along with the mRNA expressions of proinflammatory cytokines. Moreover, STV-Na administration increased the number of regulatory T (Treg) cells while decreasing Th1/Th2/Th17 cell counts in the spleen. Additionally, STV-Na treatment restored intestinal barrier disruption in DSS-triggered colitis tissues by ameliorating the TJ proteins, increasing goblet cell proportions, and mucin protein production, and decreasing the permeability to FITC-dextran, which was accompanied by decreased plasma LPS and DAO contents. CONCLUSION: These results indicate that STV-Na can ameliorate colitis by modulating immune responses along with metabolic reprogramming, and could therefore be a promising therapeutic strategy for IBDs.
RESUMO
Ischemia heart disease, one of the lethal cardiovascular diseases, irreversibly impairs cardiac function and is recognized as the primary risk factor for mortality in industrialized countries. The myocardial ischemia treatment still faces a considerable degree of increasing unmet needs. Isosteviol sodium (STVNa) and its derivatives have been proven to effectively alleviate metabolic diseases, hypertension, and heart hypertrophy. Little is known about how STVNa confers the cardioprotective effect during acute myocardial ischemia (AMI). In the present study, a rat model of acute ST-segment-elevation myocardial ischemia by left anterior descending (LAD) ligation was established. Compared to the AMI model group, STVNa administration (4 mg/kg, twice a day) well preserved left ventricle function by ejection fraction (45.10 ± 10.39 vs. 73.64 ± 13.15, p = 0.0013) and fractional shortening (22.94 ± 6.28 vs. 44.00 ± 11.05, p = 0.0017). Further analysis shows that high-dose STVNa (4 mg/kg) significantly improved the hemodynamics in AMI rats, with LVSP (88.25 ± 12.78 vs 99.75 ± 5.10, p = 0.018), max dP/dt (2978.45 ± 832.46 vs 4048.56 ± 827.23, p = 0.096), LVEDP (19.88 ± 2.00 vs 22.26 ± 3.21, p = 0.04) and left ventricular relaxation time constant (Tau) (0.030 ± 0.006 vs 0.021 ± 0.004, p = 0.021). Mechanically, STVNa administration retained the myocardial levels of phosphorylated AMPK, and CPT1b. Moreover, STVNa significantly increased the total energy expenditure, and reduced fatty acid accumulation through mitochondrial oxidative phosphorylation, which was supported by the indirect calorimetry and cellular energy analysis. Taken together, these findings suggest that STVNa is a potential cardioprotection agent for ischemic cardiomyopathy, likely through improving energy homeostasis, left ventricular hemodynamics, and heart function.
Assuntos
Diterpenos do Tipo CauranoRESUMO
Cholinesterases act as bio scavengers to clear organophosphorus (OP) compounds and prodrugs. The butyrylcholinesterase (BChE) gene has been found in several types of teleost fish but this gene has yet to be identified in cyprinid fish. Indeed, BChE homologs have not been found in the zebrafish (Danio rerio) genomic database. Here, we demonstrate that BChE activity is present in zebrafish, in line with other groups' findings. Using in-gel native-PAGE enzymatic activity staining and LC-MS/MS technique, an atypical BChE-like protein was identified in zebrafish. The si:ch211-93f2.1 gene was cloned, and His-tagged recombinant protein was expressed using the Pichia yeast system. The purified protein (molecular weight ~ 180 kDa) showed BChE activity, and degraded acetylcholinesterase (ACh) at a higher rate than BCh. However, phylogram analysis shows that this novel cholinesterase shared an evolutionary origin with carboxylic esterase rather than BChE. The zebrafish BChE-like protein shares structural characteristics with cholinesterase and carboxylesterase. The 2-arachidonoylglycerol (2-AG), nicosulfuron, and triacetin exhibited a higher binding affinity to the zebrafish BChE-like protein than BCh and ACh. With the identification of BChE-like protein in zebrafish, this study could shed light on the origin of BChE and may contribute towards the development of a BChE knockout zebrafish model for sensitive drug or toxin screening.
Assuntos
Hidrolases de Éster Carboxílico , Clonagem Molecular , Proteínas de Peixe-Zebra , Peixe-Zebra , Animais , Hidrolases de Éster Carboxílico/biossíntese , Hidrolases de Éster Carboxílico/química , Hidrolases de Éster Carboxílico/genética , Domínios Proteicos , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/biossíntese , Proteínas de Peixe-Zebra/química , Proteínas de Peixe-Zebra/genéticaRESUMO
Isosteviol has been reported to reverse hypertrophy and related inflammatory responses in in vitro models representative of cardiac muscle cells. The disposition of isosteviol is, however, characterized by secondary peaks and long plasma residence time despite reports of a relatively short half-life in liver fractions. The present study describes a compartmental approach to modelling the secondary peaks characteristic of isosteviol's concentration-time data in Sprague-Dawley rats. Oral (4 mg/kg) and intravenous (4 mg/kg) doses of isosteviol were administered to male and female Sprague-Dawley rats. Plasma samples collected between 0 and 72 h, and total bile secreted in 24 h, were analysed for isosteviol content with LC-MS/MS techniques. The disposition of isosteviol was, thereafter, described with a structural model that accounted for the sampling, liver and biliary secretion compartments, with a gap-time characterizing the accumulation and subsequent emptying of isosteviol for re-absorption. The half-life of isosteviol following oral dosing was about 103% greater in female rats than in the male, and the model-derived area under the concentration-time curve (AUC) in 72 h was about 756% greater in female animals than in males. Following the administration of intravenous doses of isosteviol, half-life and AUC in 24 h were about 332% and 595%, respectively, higher in female rats than in males. Isosteviol equivalent secreted into bile over 24 h accounted for about 94% of orally administered dose in male rats, and about 59% of oral dose in females. These findings show a differential systemic removal of isosteviol in Sprague-Dawley rats, likely explainable by gender-related differences in the glucuronidation-capacity of isosteviol.
Assuntos
Diterpenos do Tipo Caurano/farmacocinética , Animais , Bile/metabolismo , Circulação Êntero-Hepática , Feminino , Glucuronídeos/metabolismo , Masculino , Ratos , Ratos Sprague-Dawley , Caracteres SexuaisRESUMO
The development, differentiation, and maturation of hematopoietic cells are regulated by the intrinsic and extrinsic regulation. Intrinsic activity is affected by cell autonomous gene expression and extrinsic factors originate from the so-called niche surrounding the hematopoietic cells. It remains unclear why the hematopoietic sites are shifted during embryogenesis. Flow cytometry and immunohistochemistry enable us to study embryonic regulation of hematopoietic niche in the mouse embryo.
Assuntos
Diferenciação Celular , Embrião de Mamíferos/citologia , Desenvolvimento Embrionário , Células-Tronco Hematopoéticas/citologia , Nicho de Células-Tronco/fisiologia , Animais , CamundongosRESUMO
Isosteviol is a lead compound whose cardioprotective property has been partly explained by its regulation of ion channels and interference with signalling pathways in the metabolism of some fatty acids. This study determined the metabolic stability of isosteviol in human liver microsomes and H9c2 cell line, and the identity of its metabolites in human and rat liver fractions. Isosteviol was largely unmetabolized in H9c2 cells and in NADPH-only supplemented human liver fractions, suggesting a very limited contribution of phase I biotransformation to its hepatic clearance. The in vitro half-life of isosteviol in UDPGA-only supplemented medium was observed to be 24.9 min with an estimated intrinsic clearance of 0.349 mL/min/kg in man. Analysis by LC-MS/MS and Q-tof showed that isosteviol is mainly metabolised to its acyl-ß-D-glucuronide in humans and rats. Mono-hydroxy-isosteviol and dihydroisosteviol were also identified. Rat liver fraction, however, generated dihydroxy-isosteviol in addition to two mono-hydroxy derivatives. Further studies confirmed that dihydroisosteviol is subsequently biotransformed to its acyl-ß-D-glucuronide in man and rat. These findings suggest that future studies of the efficacy and toxicity of isosteviol might have to consider xenobiotics that alter the glucuronidation pathways significantly in man.
Assuntos
Cardiotônicos/metabolismo , Diterpenos do Tipo Caurano/metabolismo , Microssomos Hepáticos/metabolismo , Animais , Biotransformação , Técnicas de Cultura de Células , Linhagem Celular , Estabilidade de Medicamentos , Glucuronídeos/metabolismo , Humanos , NADP/metabolismo , Ratos , Especificidade da EspécieRESUMO
During mammalian embryogenesis, hematopoietic stem and progenitor cells (HSPCs) originate from mesoderm-derived endothelial cells in the aorta-gonad-mesonephros (AGM) region and placenta (PL). Later, HSPCs expand in fetal liver (FL) and migrate to bone marrow (BM) shortly before birth. Understanding global transcriptional regulation governing HSPC emergence from embryonic stem/induced pluripotent stem cells is necessary to devise clinical applications, such as novel transplantation approaches. In this study, to assess transcriptional dynamics during development, we performed cap analysis of gene expression on 10 developmental murine HSPC populations isolated from the AGM region, PL, FL, and BM and identified 15,681 transcripts across HSPC ontogeny. We performed microarray analysis of AGM-derived HSPCs at 9.5 and 10.5 days postcoitum (dpc) and identified 40 differentially expressed genes, 23 confirmed as significantly changed by real-time polymerase chain reaction. We conclude that a transcriptional switch point occurs in HSPC ontogeny between 9.5 and 10.5 dpc in the AGM region.
Assuntos
Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Transcrição Gênica , Animais , Aorta/citologia , Separação Celular , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Gônadas/citologia , Mesonefro/citologia , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Análise de Sequência com Séries de Oligonucleotídeos , Capuzes de RNA/metabolismo , Reprodutibilidade dos TestesRESUMO
The aorta-gonad-mesonephros (AGM) region contains intra-aortic clusters (IACs) thought to have acquired hematopoietic stem cell (HSC) potential in vertebrate embryos. To assess extrinsic regulation of IACs in the AGM region, we employed mouse embryos harboring a Sall1-GFP reporter gene, which allows identification of mesonephros cells based on GFP expression. Analysis of AGM region tissue sections confirmed mesonephros GFP expression. Mesonephric cells sorted at E10.5 expressed mRNA encoding Csf1, a hematopoietic cytokine, and corresponding protein, based on real-time PCR and immunocytochemistry, respectively. Further analysis indicated that some IACs express the CSF1 receptor, CSF1R. Expression of Cebpa and Irf8 mRNAs was higher in CSF1R-positive IACs, whereas that of Cebpε and Gfi1 mRNAs was lower relative to CSF1R-negative IACs, suggesting that CSF1/CSF1R signaling functions in IAC myeloid differentiation by modulating expression of these transcription factors. Colony formation assays using CSF1R-positive IACs revealed increased numbers of myeloid colonies in the presence of CSF1. Analysis using an intra-cellular signaling array indicated the greatest fold increase of Cleaved Caspase-3 in AGM cells in the presence of CSF1. Immunohistochemistry revealed that Cleaved Caspase-3 is primarily expressed in IACs in the AGM region, and incubation of IACs with CSF1 up-regulated Cleaved Caspase-3. Overall, our findings suggest that CSF1 secreted from mesonephros accelerates IAC myeloid differentiation in the AGM region, possibly via Caspase-3 cleavage.
Assuntos
Aorta/metabolismo , Aorta/fisiologia , Diferenciação Celular/fisiologia , Fator Estimulador de Colônias de Macrófagos/metabolismo , Mesonefro/metabolismo , Células Mieloides/fisiologia , Animais , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Caspase 3/metabolismo , Fatores Reguladores de Interferon/metabolismo , Mesonefro/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Células Mieloides/metabolismo , RNA Mensageiro/metabolismo , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/metabolismo , Transdução de Sinais/fisiologia , Fatores de Transcrição/metabolismoRESUMO
Fetal spleen is a major hematopoietic site prior to initiation of bone marrow hematopoiesis. Morphologic analysis suggested erythropoietic activity in fetal spleen, but it remained unclear how erythropoiesis was regulated. To address this question, we performed flow cytometric analysis and observed that the number of spleen erythroid cells increased 18.6-fold from 16.5 to 19.5 days post-coitum (dpc). Among erythropoietic cytokines, SCF and IGF-1 were primarily expressed in hematopoietic, endothelial and mesenchymal-like fetal spleen cells. Cultures treated with SCF and/or IGF-1R inhibitors showed significantly decreased CD45-c-Kit-CD71+/-Ter119+ erythroid cells and downregulated Gata1, Klf1 and ß-major globin expression. Administration of these inhibitors to pregnant mice significantly decreased the number of CD45-c-Kit-CD71+/-Ter119+ cells and downregulated ß-major globin gene expression in embryos derived from these mice. We conclude that fetal spleen is a major erythropoietic site where endothelial and mesenchymal-like cells primarily accelerate erythropoietic activity through SCF and IGF-1 secretion.