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1.
Sci Rep ; 13(1): 436, 2023 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-36624121

RESUMO

We aimed to explore whether superfluous sympathetic activity affects myoblast differentiation, fusion, and myofiber types using a continuous single-dose isoprenaline exposure model in vitro and to further confirm the role of distinct NFATs in ISO-mediated effects. Compared with delivery of single and interval single, continuous single-dose ISO most obviously diminished myotube size while postponing myoblast differentiation/fusion in a time- and dose-dependent pattern, accompanied by an apparent decrease in nuclear NFATc1/c2 levels and a slight increase in nuclear NFATc3/c4 levels. Overexpression of NFATc1 or NFATc2, particularly NFATc1, markedly abolished the inhibitory effects of ISO on myoblast differentiation/fusion, myotube size and Myh7 expression, which was attributed to a remarkable increase in the nuclear NFATc1/c2 levels and a reduction in the nuclear NFATc4 levels and the associated increase in the numbers of MyoG and MEF2C positive nuclei within more than 3 nuclei myotubes, especially in MEF2C. Moreover, knockdown of NFATc3 by shRNA did not alter the inhibitory effect of ISO on myoblast differentiation/fusion or myotube size but partially recovered the expression of Myh7, which was related to the slightly increased nuclear levels of NFATc1/c2, MyoG and MEF2C. Knockdown of NFATc4 by shRNA prominently increased the number of MyHC +, MyoG or MEF2C + myoblast cells with 1 ~ 2 nuclei, causing fewer numbers and smaller myotube sizes. However, NFATc4 knockdown further deteriorated the effects of ISO on myoblast fusion and myotube size, with more than 5 nuclei and Myh1/2/4 expression, which was associated with a decrease in nuclear NFATc2/c3 levels. Therefore, ISO inhibited myoblast differentiation/fusion and myotube size through the NFAT-MyoG-MEF2C signaling pathway.


Assuntos
Fibras Musculares Esqueléticas , Transdução de Sinais , Isoproterenol/farmacologia , Isoproterenol/metabolismo , Diferenciação Celular , Fibras Musculares Esqueléticas/metabolismo , Mioblastos/metabolismo , RNA Interferente Pequeno/metabolismo
2.
Int J Mol Sci ; 24(1)2023 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-36614209

RESUMO

Physiologically, ß-adrenoceptors are major regulators of lipid metabolism, which may be reflected in alterations in lipid droplet dynamics. ß-adrenoceptors have also been shown to participate in breast cancer carcinogenesis. Since lipid droplets may be seen as a hallmark of cancer, the present study aimed to investigate the role of ß-adrenoceptors in the regulation of lipid droplet dynamics in MCF-7 breast cancer cells. Cells were treated for up to 72 h with adrenaline (an endogenous adrenoceptor agonist), isoprenaline (a non-selective ß-adrenoceptor agonist) and salbutamol (a selective ß2-selective agonist), and their effects on lipid droplets were evaluated using Nile Red staining. Adrenaline or isoprenaline, but not salbutamol, caused a lipid-accumulating phenotype in the MCF-7 cells. These effects were significantly reduced by selective ß1- and ß3-antagonists (10 nM atenolol and 100 nM L-748,337, respectively), indicating a dependence on both ß1- and ß3-adrenoceptors. These effects were dependent on the cAMP signalling pathway, involving both protein kinase A (PKA) and cAMP-dependent guanine-nucleotide-exchange (EPAC) proteins: treatment with cAMP-elevating agents (forskolin or 8-Br-cAMP) induced lipid droplet accumulation, whereas either 1 µM H-89 or 1 µM ESI-09 (PKA or EPAC inhibitors, respectively) abrogated this effect. Taken together, the present results demonstrate the existence of a ß-adrenoceptor-mediated regulation of lipid droplet dynamics in breast cancer cells, likely involving ß1- and ß3-adrenoceptors, revealing a new mechanism by which adrenergic stimulation may influence cancer cell metabolism.


Assuntos
Gotículas Lipídicas , Neoplasias , Humanos , Isoproterenol/farmacologia , Células MCF-7 , Proteínas Quinases Dependentes de AMP Cíclico , Agonistas Adrenérgicos beta/farmacologia , Receptores Adrenérgicos beta , Albuterol/farmacologia , Epinefrina , Fatores de Troca do Nucleotídeo Guanina , Antagonistas Adrenérgicos beta/farmacologia
3.
Int J Mol Med ; 51(2)2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-36579657

RESUMO

CXCR4 is a seven­transmembrane­spanning Gi­coupled receptor for the SDF­1 chemokine and plays a critical role in cardiovascular development and post­injury repair. However, the specific role of CXCR4 in cardiomyocytes is incompletely understood. It was hypothesized that CXCR4 activation in cardiomyocytes antagonizes ß­adrenoceptor/Gs signaling­induced cardiac dysfunction. Cardiomyocyte­specific CXCR4 knockout (CXCR4­CMKO) mice were generated by crossing CXCR4fl/fl and MHC­Cre+/­ mice. Their cardiac structure and function in the basal state are equivalent to that of the control MHC­Cre+/­ littermates until at least 4 months old. However, following continuous subcutaneous administration of isoproterenol (Iso) via an osmotic mini­pump, the ventricular myocardial contractility, dilation, cardiomyocyte apoptosis, and interstitial fibrosis are worse in CXCR4­CMKO mice than in MHC­Cre+/­ littermates. In the cultured H9C2 cardiomyocytes, SDF­1 treatment markedly attenuated Iso­induced apoptosis and reduction in phospho­Akt, and this protective effect was lost by knockdown of CXCR4 or by co­treatment with Gi inhibitors. In conclusion, CXCR4 promotes cardiomyocyte survival and heart function during ß­adrenergic stress.


Assuntos
Insuficiência Cardíaca , Miócitos Cardíacos , Camundongos , Animais , Miócitos Cardíacos/metabolismo , Isoproterenol/farmacologia , Insuficiência Cardíaca/metabolismo , Morte Celular , Miocárdio , Apoptose , Camundongos Knockout , Remodelação Ventricular/fisiologia
4.
Circ Res ; 132(2): e59-e77, 2023 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-36583384

RESUMO

BACKGROUND: PKA (protein kinase A)-mediated phosphorylation of cardiac RyR2 (ryanodine receptor 2) has been extensively studied for decades, but the physiological significance of PKA phosphorylation of RyR2 remains poorly understood. Recent determination of high-resolution 3-dimensional structure of RyR2 in complex with CaM (calmodulin) reveals that the major PKA phosphorylation site in RyR2, serine-2030 (S2030), is located within a structural pathway of CaM-dependent inactivation of RyR2. This novel structural insight points to a possible role of PKA phosphorylation of RyR2 in CaM-dependent inactivation of RyR2, which underlies the termination of Ca2+ release and induction of cardiac Ca2+ alternans. METHODS: We performed single-cell endoplasmic reticulum Ca2+ imaging to assess the impact of S2030 mutations on Ca2+ release termination in human embryonic kidney 293 cells. Here we determined the role of the PKA site RyR2-S2030 in a physiological setting, we generated a novel mouse model harboring the S2030L mutation and carried out confocal Ca2+ imaging. RESULTS: We found that mutations, S2030D, S2030G, S2030L, S2030V, and S2030W reduced the endoplasmic reticulum luminal Ca2+ level at which Ca2+ release terminates (the termination threshold), whereas S2030P and S2030R increased the termination threshold. S2030A and S2030T had no significant impact on release termination. Furthermore, CaM-wild-type increased, whereas Ca2+ binding deficient CaM mutant (CaM-M [a loss-of-function CaM mutation with all 4 EF-hand motifs mutated]), PKA, and Ca2+/CaMKII (CaM-dependent protein kinase II) reduced the termination threshold. The S2030L mutation abolished the actions of CaM-wild-type, CaM-M, and PKA, but not CaMKII, in Ca2+ release termination. Moreover, we showed that isoproterenol and CaM-M suppressed pacing-induced Ca2+ alternans and accelerated Ca2+ transient recovery in intact working hearts, whereas CaM-wild-type exerted an opposite effect. The impact of isoproterenol was partially and fully reversed by the PKA inhibitor N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide and the CaMKII inhibitor N-[2-[N-(4-chlorocinnamyl)-N-methylaminomethyl]phenyl]-N-(2-hydroxyethyl)-4-methoxybenzenesulfonamide individually and together, respectively. S2030L abolished the impact of CaM-wild-type, CaM-M, and N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide-sensitive component, but not the N-[2-[N-(4-chlorocinnamyl)-N-methylaminomethyl]phenyl]-N-(2-hydroxyethyl)-4-methoxybenzenesulfonamide-sensitive component, of isoproterenol.


Assuntos
Canal de Liberação de Cálcio do Receptor de Rianodina , Serina , Camundongos , Animais , Humanos , Isoproterenol/farmacologia , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Serina/metabolismo , Serina/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Calmodulina/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Isoquinolinas/farmacologia , Sulfonamidas/farmacologia , Cálcio/metabolismo , Miócitos Cardíacos/metabolismo , Retículo Sarcoplasmático/metabolismo
5.
Am J Physiol Heart Circ Physiol ; 324(2): H229-H240, 2023 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-36563015

RESUMO

Despite the many advantages of isoproterenol (Iso)-induced models of cardiomyopathy, the extant literature suggests that the reproducibility of the Iso-induced stress cardiomyopathy phenotype varies considerably depending on the dose of Iso used, the mode of administration of Iso (subcutaneous vs. intraperitoneal), and the species of the animal that is being studied. Recently, we have shown that a single injection of Iso into female C57BL/6J mice provokes transient myocardial injury that is characterized by a brisk release of troponin I within 1 h, as well as a self-limited myocardial inflammatory response that is associated with increased myocardial tissue edema, inferoapical regional left ventricular (LV) wall motion abnormalities, and a transient decrease in global LV function, which were completely recovered by day 7 after the Iso injection (i.e., stress-induced reversible cardiomyopathy). Here we expand upon this initial report in this model by demonstrating important sexually dimorphic differences in the response to Iso-induced tissue injury, the ensuing myocardial inflammatory response, and changes in LV structure and function. We also provide information with respect to enhancing the reproducibility in this model by optimizing animal welfare during the procedure. The acute Iso-induced myocardial injury model provides a low-cost, relatively high-throughput small-animal model that mimics human disease (e.g., Takotsubo cardiomyopathy). Given that the model can be performed in different genetic backgrounds, as well as different experimental conditions, the acute Iso injury model should provide the cardiovascular community with a valuable nonsurgical animal model for understanding the myocardial response to tissue injury.NEW & NOTEWORTHY The present study highlights the importance of sexual dimorphism with respect to isoproterenol injury, as well as the importance of animal handling and welfare to obtain reproducible results from investigator to investigator. Based on serial observations of animal recovery (locomotor activity and grooming behavior), troponin I release, and inflammation, we identified that the method used to restrain the mice for the intraperitoneal injection was the single greatest source of variability in this model.


Assuntos
Cardiomiopatias , Modelos Animais de Doenças , Animais , Feminino , Humanos , Camundongos , Isoproterenol/farmacologia , Camundongos Endogâmicos C57BL , Reprodutibilidade dos Testes , Troponina I
6.
Biochem Biophys Res Commun ; 636(Pt 1): 33-40, 2022 12 25.
Artigo em Inglês | MEDLINE | ID: mdl-36332480

RESUMO

BACKGROUND: Inflammation can contribute to the initiation and progression of atrial fibrillation (AF), and pinocembrin can suppress downstream inflammatory cytokine production by inhibiting the inflammation pathway. In our previous studies, pinocembrin was also beneficial in ameliorating cardiac arrhythmia in different models of rats, such as depression, myocardial infarction, and heart failure. This study aims to investigate the effect of pinocembrin on the susceptibility to AF in isoproterenol-induced rats. METHODS: Rats were randomly divided into four groups. Pinocembrin was injected through the tail vein. Isoproterenol was treated by intraperitoneal injection for one week (5 mg/kg/day). We evaluated the susceptibility to AF by atrial electrophysiological experiments. Masson staining was used to evaluate the fibrosis area. The protein levels of connexin (Cx) 40, Cav1.2, Kv4.2, collagen I, collagen III, α-SMA, transforming growth factor (TGF)-ß, NLRP3, caspase 1, and interleukin (IL)-1ß were detected by western blot. RESULTS: Our data demonstrated that pinocembrin could prolong the atrial effective refractory period (ERP) and action potential duration (APD), and decrease AF inducibility. Isoproterenol increased the expression of Cav1.2 and Kv4.2 ion channels whereas pinocembrin could alleviate this change. Pinocembrin could reduce the fibrosis area, fibrosis-related protein collagen I, collagen III, α-SMA, and TGF-ß and upregulate gap junction protein Cx40. In addition, pinocembrin reduced the expression of NLRP3, caspase 1, and IL-1ß. CONCLUSIONS: Our study indicated that pinocembrin was beneficial to alleviate atrial electrical remodeling and fibrosis. Accompanied the downregulation of ion channels and upregulation of gap junction protein Cx40. Pinocembrin may produce these effects by inhibiting the NLRP3 pathway.


Assuntos
Fibrilação Atrial , Remodelamento Atrial , Ratos , Animais , Fibrilação Atrial/induzido quimicamente , Fibrilação Atrial/tratamento farmacológico , Fibrilação Atrial/metabolismo , Isoproterenol/farmacologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Caspase 1/metabolismo , Ratos Sprague-Dawley , Átrios do Coração , Fibrose , Conexinas/metabolismo , Inflamação/patologia , Colágeno Tipo I/metabolismo , Canais Iônicos/metabolismo , Modelos Animais de Doenças
7.
PLoS One ; 17(11): e0275258, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36327230

RESUMO

Cardiac fibrosis is a common cause of most cardiovascular diseases. Leonurine, an alkaloid from Herba leonuri, had been indicated to treat cardiovascular diseases due to its cardioprotective effects. Recently, pyroptosis, a programmed form of cell death that releases inflammatory factors, has been shown to play an important role in cardiovascular diseases, especially cardiac fibrosis. This study examined the novel mechanism by which leonurine protects against cardiac fibrosis. In rats with isoprenaline-induced cardiac fibrosis, leonurine inhibited the expression of proteins related to pyroptosis and improved cardiac fibrosis. In vitro, leonurine inhibited the expression of proteins related to pyroptosis and fibrosis. Additionally, leonurine regulated the TGF-ß/Smad2 signalling pathway and inhibited pyroptosis to protect cardiomyocytes and improve cardiac fibrosis. Therefore, leonurine might improve cardiac fibrosis induced by isoprenaline by inhibiting pyroptosis via the TGF-ß/Smad2 signalling pathway.


Assuntos
Doenças Cardiovasculares , Miócitos Cardíacos , Animais , Ratos , Doenças Cardiovasculares/patologia , Fibrose , Isoproterenol/farmacologia , Miócitos Cardíacos/metabolismo , Piroptose , Fator de Crescimento Transformador beta/metabolismo , Transdução de Sinais
8.
Molecules ; 27(22)2022 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-36431969

RESUMO

One of the most common diseases affecting people and leading to high morbidity is kidney injury. The alleviation of inflammation and apoptosis is considered a potential therapeutic approach for kidney injury. Sophocarpine (SOP), a tetracyclic quinolizidine alkaloid, exhibits various beneficial biological properties. To investigate the effects of SOP on isoproterenol (ISO)-induced kidney injury, we randomly divided mice into four groups: Control, ISO, ISO+SOP (20 mg/kg) and ISO+SOP (40 mg/kg). SOP was administered intraperitoneally to the mice over two weeks, accompanied by intraperitoneal stimulation of ISO (10 mg/kg) for another four weeks. After the mice were sacrificed, several methods such as ELISA, staining (H&E, TUNEL, DHE and Masson) and Western blotting were applied to detect the corresponding indicators. The kidney injury serum biomarkers SCr and BUN increased after the ISO challenge, while this effect was reversed by treatment with SOP. Pathological changes induced by ISO were also reversed by treatment with SOP in the staining. The inflammatory cytokines IL-ß, IL-6, TNF-α, MCP-1 and NLRP3 increased after the challenge with ISO, while they were decreased by treatment with SOP. The apoptotic proteins cleaved-caspase-3 and Bax increased, while Bcl-2 decreased, after the challenge with ISO, and these effects were reversed by treatment with SOP. The antioxidant proteins SOD-1 and SOD-2 decreased after being stimulated by ISO, while they increased after the treatment with SOP. The fibrotic proteins collagen I, collagen III, α-SMA, fibronectin, MMP-2 and MMP-9 increased after the challenge with ISO, while they decreased after the treatment with SOP. We further discovered that the TLR-4/NF-κB and TGF-ß1/Smad3 signaling pathways were suppressed, while the Nrf2/HO-1 signaling pathway was activated. In summary, SOP could alleviate ISO-induced kidney injury by inhibiting inflammation, apoptosis, oxidative stress and fibrosis. The molecular mechanisms were suppression of the TLR-4/NF-κB and TGF-ß1/Smad3 signaling pathways and activation of the Nrf2/HO-1 signaling pathway, indicating that SOP might serve as a novel therapeutic strategy for kidney injury.


Assuntos
Alcaloides , Fator 2 Relacionado a NF-E2 , Camundongos , Animais , Isoproterenol/farmacologia , Fator 2 Relacionado a NF-E2/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , NF-kappa B/metabolismo , Receptor 4 Toll-Like/metabolismo , Estresse Oxidativo , Fibrose , Alcaloides/farmacologia , Alcaloides/uso terapêutico , Alcaloides/metabolismo , Apoptose , Inflamação/induzido quimicamente , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Rim/metabolismo
9.
Shanghai Kou Qiang Yi Xue ; 31(3): 225-231, 2022 Jun.
Artigo em Chinês | MEDLINE | ID: mdl-36204947

RESUMO

PURPOSE: To investigate the effect of tension on the inflammatory response of human periodontal ligament cells (PDLCs) induced by isoproterenol (ISO) and its molecular mechanism. METHODS: Human PDLCs were cultured in vitro and stimulated with a certain concentration of ISO(0.01, 0.1, 1 µmol/L) for 24 h. Cyclic tensile strain with different degrees of elongation (5%, 10% and 15%) were applied. The expression of IL-1ß and IL-6 mRNA in PDLCs was detected by real-time quantitative PCR(RT-qPCR). The protein expression of p-PERK, PERK, p-eIF2α, eIF2α and ATF4 related to ER stress was detected by Western blot. The expression of PERK gene in PDLCs was knocked down by cell transfection technique, and the expression of IL-1ß and IL-6 mRNA in PDLCs with low expression of PERK was detected by RT-qPCR under the stimulation of ISO and low magnitude tension. Statistical analysis was conducted with SPSS 22.0 software package. RESULTS: ISO induction could significantly up-regulate the IL-1ß and IL-6 mRNA expression in PDLCs(P<0.05). Compared with the control group, the expression of IL-1ß and IL-6 mRNA in PDLCs induced by ISO was inhibited by low magnitude tension, and the difference was statistically significant(P<0.05). Western blot results showed that low magnitude tension could inhibit the ISO-stimulated phosphorylation of PERK and eIF2α and the expression of ATF4(P<0.05). Compared with the negative control group, IL-1ß and IL-6 mRNA expression was decreased in the ISO-stimulated PDLCs silenced by PERK gene. CONCLUSIONS: Tension with 5% degrees of elongation may inhibit ISO-stimulated periodontal inflammatory response through endoplasmic reticulum stress pathway.


Assuntos
Interleucina-6 , Ligamento Periodontal , Células Cultivadas , Estresse do Retículo Endoplasmático , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , Isoproterenol/metabolismo , Isoproterenol/farmacologia , Ligamento Periodontal/metabolismo , RNA Mensageiro/metabolismo
10.
J Cell Mol Med ; 26(21): 5414-5425, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36201630

RESUMO

Prevalence of heart failure (HF) continues to rise over time and is a global difficult problem; new drug targets are urgently needed. In recent years, pyroptosis is confirmed to promote cardiac remodelling and HF. Echinacoside (ECH) is a natural phenylethanoid glycoside and is the major active component of traditional Chinese medicine Cistanches Herba, which is reported to possess powerful anti-oxidation and anti-inflammatory effects. In addition, we previously reported that ECH reversed cardiac remodelling and improved heart function, but the effect of ECH on pyroptosis has not been studied. So, we investigated the effects of ECH on cardiomyocyte pyroptosis and the underlying mechanisms. In vivo, we established HF rat models induced by isoproterenol (ISO) and pre-treated with ECH. Indexes of heart function, pyroptotic marker proteins, ROS levels, and the expressions of NOX2, NOX4 and ER stress were measured. In vitro, primary cardiomyocytes of neonatal rats were treated with ISO and ECH; ASC speckles and caspase-1 mediated pyroptosis in cardiomyocytes were detected. Hoechst/PI staining was also used to evaluate pyroptosis. ROS levels, pyroptotic marker proteins, NOX2, NOX4 and ER stress levels were all tested. In vivo, we found that ECH effectively inhibited pyroptosis, down-regulated NOX2 and NOX4, decreased ROS levels, suppressed ER stress and improved heart function. In vitro, ECH reduced cardiomyocyte pyroptosis and suppressed NADPH/ROS/ER stress. We concluded that ECH inhibited cardiomyocyte pyroptosis and improved heart function via suppressing NADPH/ROS/ER stress.


Assuntos
Insuficiência Cardíaca , Miócitos Cardíacos , Ratos , Animais , Miócitos Cardíacos/metabolismo , Isoproterenol/farmacologia , Piroptose , Espécies Reativas de Oxigênio/metabolismo , NADP/metabolismo , Remodelação Ventricular , Glicosídeos/farmacologia , Insuficiência Cardíaca/induzido quimicamente , Insuficiência Cardíaca/tratamento farmacológico , Insuficiência Cardíaca/metabolismo
11.
J Physiol Pharmacol ; 73(3)2022 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-36302532

RESUMO

Acute cardiac pathologies represent one of the leading causes of death, while iron metabolism is recognized to be implicated in reactive oxygen species production, lipid peroxidation, and inflammation. The aim of the present study was to assess iron chelation effects in isoproterenol (ISO) induced acute cardiac stress. We divided male Wistar rats into preventive and secondary treatment groups, with the active arm consisting in deferiprone (DFP), a lipid permeable chelator. Mortality of ISO was 10-18.18% in both preventive and secondary groups. We analyzed serum and myocardial tissue parameters of inflammation, iron dynamics, and lipid peroxidation, accompanied by ultramicroscopy, histological, and ultrasound-derived parameters of left ventricular function. Results reveal that ISO-mediated lipid peroxidation and inflammation are alleviated by administration of DFP, with negligible effect on systemic ferroregulation dynamics and global ventricular function (as assessed by ultrasound). DFP administration after cardiovascular stress is associated with a decrease in lipid peroxidation and inflammation, without an improvement in gross left ventricular parameters.


Assuntos
Isoflurofato , Miocárdio , Ratos , Masculino , Animais , Peroxidação de Lipídeos , Isoproterenol/farmacologia , Isoproterenol/metabolismo , Isoflurofato/metabolismo , Isoflurofato/farmacologia , Ratos Wistar , Miocárdio/metabolismo , Quelantes de Ferro/farmacologia , Quelantes de Ferro/uso terapêutico , Quelantes de Ferro/metabolismo , Ferro/metabolismo , Inflamação/induzido quimicamente , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Estresse Oxidativo
12.
Virulence ; 13(1): 1614-1630, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-36121102

RESUMO

To investigate the role of adrenergic signalling (AS) in the host immune response and Porphyromonas gingivalis virulence, we compared norepinephrine (NE) and isoproterenol (ISO) responses in Galleria mellonella. P. gingivalis infection was evaluated by survival; humoral immune responses (i.e. melanization and cecropin and gloverin mRNA expression); cellular immune responses (i.e. haemocyte count, nodulation by histology); and P. gingivalis recovery (CFU/mL). P. gingivalis was cultivated in the presence of ISO (PgISO) or NE and injected into the larvae for survival evaluation. Finally, we co-injected ISO and PgISO to evaluate the concomitant effects on the immune response and bacterial virulence. None of the ligands were toxic to the larvae; ISO increased haemocyte number, even after P. gingivalis infection, by mobilizing sessile haemocytes in a ß-adrenergic-specific manner, while NE showed the opposite effect. ISO treatment reduced larval mortality and the number of recovered bacteria, while NE increased mortality and showed no effect on bacterial recovery. ISO and NE had similar effects on melanization and decreased the expression of cecropin. Although co-cultivation with NE and ISO increased the gene expression of bacterial virulence factors in vitro, only the injection of PgISO increased larval death, which was partially reversed by circulating ISO. Therefore, α- and ß-adrenergic signalling had opposite effects after P. gingivalis infection. Ultimately, the catecholamine influence on the immune response overcame the effect of more virulent strains. The effect of AS directly on the pathogen found in vitro did not translate to the in vivo setting.


Assuntos
Cecropinas , Mariposas , Adrenérgicos , Animais , Imunidade Inata , Isoproterenol/farmacologia , Larva/microbiologia , Norepinefrina/farmacologia , Porphyromonas gingivalis , RNA Mensageiro , Virulência , Fatores de Virulência
13.
Sci Rep ; 12(1): 15831, 2022 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-36138030

RESUMO

Inflammatory cytokines and non-esterified fatty acids (NEFAs) are obesity-linked factors that disturb insulin secretion. The aim of this study was to investigate whether pancreatic adipose tissue (pWAT) is able to generate a NEFA/cytokine overload within the pancreatic environment and as consequence to impact on insulin secretion. Pancreatic fat is a minor fat depot, therefore we used high-fat diet (HFD) feeding to induce pancreatic steatosis in mice. Relative Adipoq and Lep mRNA levels were higher in pWAT of HFD compared to chow diet mice. Regardless of HFD, Adipoq and Lep mRNA levels of pWAT were at least 10-times lower than those of epididymal fat (eWAT). Lipolysis stimulating receptors Adrb3 and Npr1 were expressed in pWAT and eWAT, and HFD reduced their expression in eWAT only. In accordance, HFD impaired lipolysis in eWAT but not in pWAT. Despite expression of Npr mRNA, lipolysis was stimulated solely by the adrenergic agonists, isoproterenol and adrenaline. Short term co-incubation of islets with CD/HFD pWAT did not alter insulin secretion. In the presence of CD/HFD eWAT, glucose stimulated insulin secretion only upon isoproterenol-induced lipolysis, i.e. in the presence of elevated NEFA. Isoproterenol augmented Il1b and Il6 mRNA levels both in pWAT and eWAT. These results suggest that an increased sympathetic activity enhances NEFA and cytokine load of the adipose microenvironment, including that of pancreatic fat, and by doing so it may alter beta-cell function.


Assuntos
Ácidos Graxos não Esterificados , Lipólise , Tecido Adiposo/metabolismo , Adrenérgicos/metabolismo , Agonistas Adrenérgicos/metabolismo , Animais , Citocinas/metabolismo , Dieta Hiperlipídica/efeitos adversos , Epinefrina/metabolismo , Epinefrina/farmacologia , Ácidos Graxos não Esterificados/metabolismo , Glucose/metabolismo , Interleucina-6/metabolismo , Isoproterenol/metabolismo , Isoproterenol/farmacologia , Lipólise/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/metabolismo
14.
Molecules ; 27(18)2022 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-36144661

RESUMO

This present study aimed to delineate Rumex hastatus D. Don crude extract (Rh.Cr), n-Hexane, ethyl acetate, aqueous fractions (Rh.n-Hex, Rh.ETAC, Rh.Aq) and rutin for antidiarrheal, antisecretory effects, anti-spasmodic, gastrointestinal transient time, anti H. pylori, antiulcer effects, and toxicology. The preliminary phytochemical analysis of Rumex hastatus showed different phytoconstituents and shows different peaks in GC-MC chromatogram. Rumex hastatus crude extract (Rh.Cr), fractions, and rutin attributed dose-dependent (50-300 mg/kg) protection (0-100%) against castor oil-induced diarrhea and dose-dependently inhibited intestinal fluid secretions in mice. They decreased the distance traversed by charcoal in the gastrointestinal transit model in rats. In rabbit jejunum preparations, Rh.Cr and Rh.ETAC caused a concentration-dependent relaxation of both spontaneous and K+ (80 mM)-induced contractions at a similar concentration range, whereas Rh.n-Hex, rutin, and verapamil were relatively potent against K+-induced contractions and shifted the Ca2+ concentration-response curves (CRCs) to the right, Rh.Cr (0.3-1 mg/mL) and Rh.ETAC (0.1-0.3 mg/mL) shifted the isoprenaline-induced inhibitory CRCs to the left. Rh.n-Hex, Rh.ETAC and rutin showed anti-H. pylori effect, also shows an inhibitory effect against H+/K+-ATPase. Rumex hastatus showed gastroprotective and antioxidant effects. Histopathological evaluation showed improvement in cellular architecture and a decrease in the expression of inflammatory markers such as, cyclooxygenase (COX-2), tumor necrosis factor (TN,F-α) and phosphorylated nuclear factor kappa B (p-NFƙB), validated through immunohistochemistry and ELISA techniques. In RT-PCR it decreases H+/K+-ATPase mRNA levels. Rumex hastatus was found to be safe to consume up to a dose of 2000 mg/kg in a comprehensive toxicity profile. Docking studies revealed that rutin against H+/K+-ATPase pump and voltage-gated L-type calcium channel showed E-values of -8.7 and -9.4 Kcal/mol, respectively. MD simulations Molecular Mechanics Poisson Boltzmann surface area and molecular mechanics Generalized Born surface area (MMPBSA/GBSA) findings are consistent with the in-vitro, in-vivo and docking results.


Assuntos
Gastroenteropatias , Rumex , Adenosina Trifosfatases , Animais , Antidiarreicos/química , Antioxidantes/farmacologia , Canais de Cálcio Tipo L , Hidróxido de Cálcio , Óleo de Rícino , Carvão Vegetal/farmacologia , Ciclo-Oxigenase 2 , Gastroenteropatias/tratamento farmacológico , Isoproterenol/farmacologia , Jejuno , Camundongos , NF-kappa B/farmacologia , Parassimpatolíticos/farmacologia , Compostos Fitoquímicos/farmacologia , Extratos Vegetais/química , Extratos Vegetais/farmacologia , RNA Mensageiro , Coelhos , Ratos , Rumex/química , Rutina/farmacologia , Fatores de Necrose Tumoral , Verapamil/farmacologia , Óxido de Zinco
15.
Molecules ; 27(18)2022 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-36144682

RESUMO

Chronic stress has been reported to stimulate the release of catecholamines, including norepinephrine (NE) and epinephrine (E), which promote cancer progression by activating the adrenergic receptor (AR). Although previous studies showed that ß2-AR mediated chronic stress-induced tumor growth and metastasis, the underlying mechanism has not been fully explored. In this study, we aimed to investigate the molecular mechanism by which ß2-AR exerts a pro-metastatic function in hepatocarcinoma (HCC) cells and breast cancer (BC) cells. Our results showed that Hep3B human HCC cells and MDA-MB-231 human BC cells exhibited the highest ADRB2 expression among diverse HCC and BC cell lines. NE, E, and isoprenaline (ISO), adrenergic agonists commonly increased the migration and invasion of Hep3B cells and MDA-MB-231 cells. The phosphorylation level of Src was significantly increased by E/NE. Dasatinib, a Src kinase inhibitor, blocked E/NE-induced migration and invasion, indicating that AR agonists enhanced the mobility of cancer cells by activating Src. ADRB2 knockdown attenuated E/NE-induced Src phosphorylation, as well as the metastatic ability of cancer cells, suggesting the essential role of ß2-AR. Taken together, our results demonstrate that chronic stress-released catecholamines promoted the migration and invasion of HCC cells and BC cells via ß2-AR-mediated Src activation.


Assuntos
Receptores Adrenérgicos beta 2 , Transdução de Sinais , Agonistas Adrenérgicos , Catecolaminas , Linhagem Celular Tumoral , Dasatinibe , Epinefrina/farmacologia , Humanos , Isoproterenol/farmacologia , Processos Neoplásicos , Norepinefrina/metabolismo , Norepinefrina/farmacologia , Receptores Adrenérgicos beta 2/genética , Quinases da Família src/genética , Quinases da Família src/metabolismo
16.
Eur J Pharmacol ; 933: 175263, 2022 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-36100128

RESUMO

Obesity is an important risk factor for diabetes mellitus (DM) which is a major global health problem. Electro-mechanical dysfunction has been extensively described in diabetic heart and cardiovascular complications are an important cause of mortality and morbidity in diabetic patients. OBJECTIVES: To examine the effects of Isoprenaline (ISO) in obesity and diabesity on ventricular myocyte shortening and Ca2+ transport in Zucker fatty (ZF), Zucker diabetic fatty (ZDF) in comparison to Zucker lean (ZL) rats. METHODS: Myocyte shortening and intracellular Ca2+ were investigated with video imaging and fluorescence photometry, respectively. RESULTS: The amplitude of Isoprenaline stimulated shortening was significantly (p < 0.05) decreased in ZDF and ZF compared to ZL myocytes. The amplitude of Isoprenaline stimulated Ca2+ transient was also significantly (p < 0.05) reduced in ZF compared to ZL and modestly reduced in ZDF compared to ZL myocytes. Mean Isoprenaline stimulated time to peak along with time to half relaxation of shortening were unchanged in ZDF and ZF compared to ZL myocytes. Mean Isoprenaline stimulated time to peak Ca2+ transient was significantly shortened in ZF compared to ZL myocytes. Time to half decay of the Ca2+ transient was considerably prolonged in ZDF compared to ZL myocytes. Amplitude of Isoprenaline stimulated caffeine-evoked Ca2+ transients were significantly reduced in ZDF and ZF in comparison to ZL myocytes. CONCLUSION: Isoprenaline was less effective at generating an increase in the amplitude of shortening in ZDF and ZF in comparison to ZL myocytes and defects in Ca2+ signaling, and in particular SR Ca2+ transport, might partly underlie these abnormalities.


Assuntos
Diabetes Mellitus Tipo 2 , Diabetes Mellitus , Animais , Cafeína , Isoproterenol/farmacologia , Miócitos Cardíacos/fisiologia , Obesidade , Ratos , Ratos Zucker
17.
Int J Mol Sci ; 23(17)2022 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-36077457

RESUMO

Hyperactive poly(ADP-ribose) polymerases (PARP) promote ischemic heart failure (IHF) after myocardial infarction (MI). However, the role of tankyrases (TNKSs), members of the PARP family, in pathogenesis of IHF remains unknown. We investigated the expression and activation of TNKSs in myocardium of IHF patients and MI rats. We explored the cardioprotective effect of TNKS inhibition in an isoproterenol-induced zebrafish HF model. In IHF patients, we observed elevated TNKS2 and DICER and concomitant upregulation of miR-34a-5p and miR-21-5p in non-infarcted myocardium. In a rat MI model, we found augmented TNKS2 and DICER in the border and infarct areas at the early stage of post-MI. We also observed consistently increased TNKS1 in the border and infarct areas and destabilized AXIN in the infarct area from 4 weeks onward, which in turn triggered Wnt/ß-catenin signaling. In an isoproterenol-induced HF zebrafish model, inhibition of TNKS activity with XAV939, a TNKSs-specific inhibitor, protected against ventricular dilatation and cardiac dysfunction and abrogated overactivation of Wnt/ß-catenin signaling and dysregulation of miR-34a-5p induced by isoproterenol. Our study unravels a potential role of TNKSs in the pathogenesis of IHF by regulating Wnt/ß-catenin signaling and possibly modulating miRNAs and highlights the pharmacotherapeutic potential of TNKS inhibition for prevention of IHF.


Assuntos
Insuficiência Cardíaca , MicroRNAs , Tanquirases , Animais , Dilatação , Insuficiência Cardíaca/tratamento farmacológico , Isoproterenol/farmacologia , MicroRNAs/genética , Ratos , Tanquirases/antagonistas & inibidores , Tanquirases/metabolismo , Via de Sinalização Wnt , Peixe-Zebra/metabolismo , beta Catenina/metabolismo
18.
J Nat Prod ; 85(9): 2192-2198, 2022 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-35983865

RESUMO

Previously, we isolated 2R,3S,15R-calofolic acids (CAs) from Calophyllum scriblitifolium bark, which showed vasorelaxant activity on phenylephrine (PE)-precontracted rat aortic rings. Although the effect was suggested to be induced via an extracellular Ca2+-independent manner and mainly acts on vascular smooth muscle, the exact mechanism of action of CAs remained unclear. Thus, this study investigated the detailed mechanism of calofolic acid-A (CA-A) induced vasorelaxation in an aortic ring specimen using rat vascular smooth muscle cells (VSMCs). The levels of PE-induced phosphorylation on MLC Ser19 decreased in VSMCs pretreated with CA-A. CA-A also decreased the phosphorylation of MYPT1 Thr696 and MYPT1 Thr853. On the other hand, CA-A increased the PE-induced phosphorylation of MYPT1 Ser695 and MYPT1 Ser668, which are reported to be phosphorylated by a cAMP-dependent protein kinase (PKA). CA-A slightly increased PKA substrate phosphorylation in a concentration-dependent manner. Furthermore, CA-A enhanced isoproterenol (ISO)-induced cAMP accumulation and PKA substrate phosphorylation. Treatment with PI-3 kinase (PI3K) inhibitor, LY294002, enhanced ISO-induced cAMP accumulation and PKA substrate phosphorylation in the same manner as CA-A treatment. Furthermore, CA-A was found to directly inhibit PI3K enzyme activity in a dose-dependent manner. Taken together, the present study indicated that CA-A induces vasorelaxation through an indirectly activated PKA-MYPT1 pathway caused by inhibition of PI3K activity.


Assuntos
Calophyllum , Proteínas Quinases Dependentes de AMP Cíclico , Músculo Liso Vascular , Fosfatidilinositol 3-Quinases , Inibidores de Fosfoinositídeo-3 Quinase , Vasodilatação , Vasodilatadores , Animais , Cálcio/metabolismo , Calophyllum/química , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Isoproterenol/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/enzimologia , Fenilefrina/metabolismo , Fenilefrina/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase/química , Inibidores de Fosfoinositídeo-3 Quinase/farmacologia , Fosforilação , Casca de Planta/química , Ratos , Vasodilatadores/química , Vasodilatadores/farmacologia
19.
J Dairy Sci ; 105(10): 8426-8438, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-35965124

RESUMO

Sustained lipolysis and insulin resistance increase the risk of metabolic dysfunction in dairy cows during the transition period. Proinflammatory cytokines are key regulators of adipose tissue metabolism in nonruminants, but biological functions of these molecules in ruminants are not well known. Thus, the objective of this study was to investigate whether tumor necrosis factor-α (TNF-α) could affect insulin sensitivity and lipolysis in bovine adipocytes as well as the underlying mechanisms. Bovine adipocytes (obtained from the omental and mesenteric adipose depots) isolated from 5 Holstein female calves (1 d old) with similar body weight (median: 36.9 kg, range: 35.5-41.2 kg) were differentiated and used for (1) treatment with different concentrations of TNF-α (0, 0.1, 1, or 10 ng/mL) for 12 h; (2) pretreatment with 10 µM lipolytic agonist isoproterenol (ISO) for 3 h, followed by treatment with or without 10 ng/mL TNF-α for 12 h; and (3) pretreatment with the c-Jun N-terminal kinase (JNK) inhibitor SP600125 (20 µM for 2 h) and nuclear factor kappa B (NF-κB) inhibitor BAY 11-7082 (10 µM for 1 h) followed by treatment with or without 10 ng/mL TNF-α for 12 h. The TNF-α increased glycerol content in supernatant, decreased triglyceride content and insulin-stimulated phosphorylation of protein kinase B suggesting activation of lipolysis and impairment of insulin sensitivity. The TNF-α reduced cell viability, upregulated mRNA abundance of Caspase 3 (CASP3), an apoptosis marker, and increased activity of Caspase 3. In addition, increased phosphorylation of NF-κB and JNK, upregulation of mRNA abundance of interleukin-6 (IL-6), TNFA, and suppressor of cytokine signaling 3 (SOCS3) suggested that TNF-α activated NF-κB and JNK signaling pathways. Furthermore, ISO plus TNF-α-activated NF-κB and JNK signaling pathway to a greater extent than TNF-α alone. Combining TNF-α and ISO aggravated TNF-α-induced apoptosis, insulin insensitivity and lipolysis. In the absence of TNF-α, inhibition of NF-κB and JNK did not alter glycerol content in supernatant, triglyceride content or insulin-stimulated phosphorylation of protein kinase B. In the presence of TNF-α, inhibition of NF-κB and JNK alleviated TNF-α-induced apoptosis, insulin insensitivity and lipolysis. Overall, TNF-α impairs insulin sensitivity and induces lipolysis and apoptosis in bovine adipocytes, which may be partly mediated by activation of NF-κB and JNK. Thus, the data suggested that NF-κB and JNK are potential therapeutic targets for alleviating lipolysis dysregulation and insulin resistance in adipocytes.


Assuntos
Doenças dos Bovinos , Resistência à Insulina , Insulinas , Adipócitos/metabolismo , Animais , Caspase 3/metabolismo , Bovinos , Doenças dos Bovinos/metabolismo , Feminino , Glicerol/metabolismo , Interleucina-6/metabolismo , Isoproterenol/farmacologia , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Lipólise , NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/metabolismo , Triglicerídeos/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
20.
J Biol Chem ; 298(9): 102362, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-35963431

RESUMO

The activity of protein phosphatase 2A (PP2A) is determined by the expression and localization of the regulatory B-subunits. PP2A-B56α is the dominant isoform of the B'-family in the heart. Its role in regulating the cardiac response to ß-adrenergic stimulation is not yet fully understood. We therefore generated mice deficient in B56α to test the functional cardiac effects in response to catecholamine administration versus corresponding WT mice. We found the decrease in basal PP2A activity in hearts of KO mice was accompanied by a counter-regulatory increase in the expression of B' subunits (ß and γ) and higher phosphorylation of sarcoplasmic reticulum Ca2+ regulatory and myofilament proteins. The higher phosphorylation levels were associated with enhanced intraventricular pressure and relaxation in catheterized KO mice. In contrast, at the cellular level, we detected depressed Ca2+ transient and sarcomere shortening parameters in KO mice at basal conditions. Consistently, the peak amplitude of the L-type Ca2+ current was reduced and the inactivation kinetics of ICaL were prolonged in KO cardiomyocytes. However, we show ß-adrenergic stimulation resulted in a comparable peak amplitude of Ca2+ transients and myocellular contraction between KO and WT cardiomyocytes. Therefore, we propose higher isoprenaline-induced Ca2+ spark frequencies might facilitate the normalized Ca2+ signaling in KO cardiomyocytes. In addition, the application of isoprenaline was associated with unchanged L-type Ca2+ current parameters between both groups. Our data suggest an important influence of PP2A-B56α on the regulation of Ca2+ signaling and contractility in response to ß-adrenergic stimulation in the myocardium.


Assuntos
Adrenérgicos , Proteína Fosfatase 2 , Adrenérgicos/metabolismo , Adrenérgicos/farmacologia , Animais , Cálcio/metabolismo , Isoproterenol/farmacologia , Camundongos , Camundongos Knockout , Contração Miocárdica , Miócitos Cardíacos/metabolismo , Fosforilação , Proteína Fosfatase 2/genética , Proteína Fosfatase 2/metabolismo , Retículo Sarcoplasmático/metabolismo
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