RESUMO
Both heart failure with preserved ejection fraction (HFpEF) and non-alcoholic fatty liver disease (NAFLD) develop due to metabolic dysregulation, has similar risk factors (e.g., insulin resistance, systemic inflammation) and are unresolved clinical challenges. Therefore, the potential link between the two disease is important to study. We aimed to evaluate whether NASH is an independent factor of cardiac dysfunction and to investigate the age dependent effects of NASH on cardiac function. C57Bl/6 J middle aged (10 months old) and aged mice (24 months old) were fed either control or choline deficient (CDAA) diet for 8 weeks. Before termination, echocardiography was performed. Upon termination, organ samples were isolated for histological and molecular analysis. CDAA diet led to the development of NASH in both age groups, without inducing weight gain, allowing to study the direct effect of NASH on cardiac function. Mice with NASH developed hepatomegaly, fibrosis, and inflammation. Aged animals had increased heart weight. Conventional echocardiography revealed normal systolic function in all cohorts, while increased left ventricular volumes in aged mice. Two-dimensional speckle tracking echocardiography showed subtle systolic and diastolic deterioration in aged mice with NASH. Histologic analyses of cardiac samples showed increased cross-sectional area, pronounced fibrosis and Col1a1 gene expression, and elevated intracardiac CD68+ macrophage count with increased Il1b expression. Conventional echocardiography failed to reveal subtle change in myocardial function; however, 2D speckle tracking echocardiography was able to identify diastolic deterioration. NASH had greater impact on aged animals resulting in cardiac hypertrophy, fibrosis, and inflammation.
Assuntos
Envelhecimento , Modelos Animais de Doenças , Ecocardiografia , Insuficiência Cardíaca , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica , Volume Sistólico , Animais , Envelhecimento/fisiologia , Hepatopatia Gordurosa não Alcoólica/fisiopatologia , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/patologia , Insuficiência Cardíaca/fisiopatologia , Insuficiência Cardíaca/etiologia , Volume Sistólico/fisiologia , Masculino , CamundongosRESUMO
Previously, the presence of a blood-myenteric plexus barrier and its disruption was reported in experimentally induced colitis via a macrophage-dependent process. The aim of this study is to reveal how myenteric barrier disruption and subsequent neuronal injury affects gut motility in vivo in a murine colitis model. We induced colitis with dextran sulfate sodium (DSS), with the co-administration of liposome-encapsulated clodronate (L-clodronate) to simultaneously deplete blood monocytes contributing to macrophage infiltration in the inflamed muscularis of experimental mice. DSS-treated animals receiving concurrent L-clodronate injection showed significantly decreased blood monocyte numbers and colon muscularis macrophage (MM) density compared to DSS-treated control (DSS-vehicle). DSS-clodronate-treated mice exhibited significantly slower whole gut transit time than DSS-vehicle-treated animals and comparable to that of controls. Experiments with oral gavage-fed Evans-blue dye showed similar whole gut transit times in DSS-clodronate-treated mice as in control animals. Furthermore, qPCR-analysis and immunofluorescence on colon muscularis samples revealed that factors associated with neuroinflammation and neurodegeneration, including Bax1, Hdac4, IL-18, Casp8 and Hif1a are overexpressed after DSS-treatment, but not in the case of concurrent L-clodronate administration. Our findings highlight that MM-infiltration in the muscularis layer is responsible for colitis-associated dysmotility and enteric neuronal dysfunction along with the release of mediators associated with neurodegeneration in a murine experimental model.
Assuntos
Ácido Clodrônico , Colite , Camundongos , Animais , Ácido Clodrônico/farmacologia , Colite/induzido quimicamente , Inflamação , Macrófagos , Colo , Sulfato de Dextrana/toxicidade , Camundongos Endogâmicos C57BL , Modelos Animais de DoençasRESUMO
The identification of novel drug targets is needed to improve the outcomes of heart failure (HF). G-protein-coupled receptors (GPCRs) represent the largest family of targets for already approved drugs, thus providing an opportunity for drug repurposing. Here, we aimed (i) to investigate the differential expressions of 288 cardiac GPCRs via droplet digital PCR (ddPCR) and bulk RNA sequencing (RNAseq) in a rat model of left ventricular pressure-overload; (ii) to compare RNAseq findings with those of ddPCR; and (iii) to screen and test for novel, translatable GPCR drug targets in HF. Male Wistar rats subjected to transverse aortic constriction (TAC, n = 5) showed significant systolic dysfunction vs. sham operated animals (SHAM, n = 5) via echocardiography. In TAC vs. SHAM hearts, RNAseq identified 69, and ddPCR identified 27 significantly differentially expressed GPCR mRNAs, 8 of which were identified using both methods, thus showing a correlation between the two methods. Of these, Prostaglandin-F2α-receptor (Ptgfr) was further investigated and localized on cardiomyocytes and fibroblasts in murine hearts via RNA-Scope. Antagonizing Ptgfr via AL-8810 reverted angiotensin-II-induced cardiomyocyte hypertrophy in vitro. In conclusion, using ddPCR as a novel screening method, we were able to identify GPCR targets in HF. We also show that the antagonism of Ptgfr could be a novel target in HF by alleviating cardiomyocyte hypertrophy.
Assuntos
Insuficiência Cardíaca , Masculino , Ratos , Camundongos , Animais , Ratos Wistar , Insuficiência Cardíaca/genética , Miócitos Cardíacos , Reação em Cadeia da Polimerase , HipertrofiaRESUMO
Interleukin-1ß (IL-1ß) is a key mediator of non-alcoholic steatohepatitis (NASH), a chronic liver disease, and of systemic inflammation-driven aging. IL-1ß contributes to cardio-metabolic decline, and may promote hepatic oncogenic transformation. Therefore, IL-1ß is a potential therapeutic target in these pathologies. We aimed to investigate the hepatic and cardiac effects of an IL-1ß targeting monoclonal antibody in an aged mouse model of NASH. 24 months old male C57Bl/6J mice were fed with control or choline deficient (CDAA) diet and were treated with isotype control or anti-IL-1ß Mab for 8 weeks. Cardiac functions were assessed by conventional-and 2D speckle tracking echocardiography. Liver samples were analyzed by immunohistochemistry and qRT-PCR. Echocardiography revealed improved cardiac diastolic function in anti-IL-1ß treated mice with NASH. Marked hepatic fibrosis developed in CDAA-fed group, but IL-1ß inhibition affected fibrosis only at transcriptomic level. Hepatic inflammation was not affected by the IL-1ß inhibitor. PCNA staining revealed intensive hepatocyte proliferation in CDAA-fed animals, which was not influenced by neutralization of IL-1ß. IL-1ß inhibition increased hepatic expression of Pd-1 and Ctla4, while Pd-l1 expression increased in NASH. In conclusion, IL-1ß inhibition improved cardiac diastolic function, but did not ameliorate features of NASH; moreover, even promoted hepatic immune checkpoint expression, with concomitant NASH-related hepatocellular proliferation.
Assuntos
Hepatopatia Gordurosa não Alcoólica , Masculino , Camundongos , Animais , Hepatopatia Gordurosa não Alcoólica/patologia , Interleucina-1beta/metabolismo , Fígado/metabolismo , Cirrose Hepática/patologia , Modelos Animais de Doenças , Fibrose , Camundongos Endogâmicos C57BLRESUMO
BACKGROUND AND PURPOSE: Immune checkpoint inhibitors (ICI), such as anti-PD-1 monoclonal antibodies, have revolutionized cancer therapy by enhancing the cytotoxic effects of T-cells against tumours. However, enhanced T-cell activity also may cause myocarditis and cardiotoxicity. Our understanding of the mechanisms of ICI-induced cardiotoxicity is limited. Here, we aimed to investigate the effect of PD-1 inhibition on cardiac function and explore the molecular mechanisms of ICI-induced cardiotoxicity. EXPERIMENTAL APPROACH: C57BL6/J and BALB/c mice were treated with isotype control or anti-PD-1 antibody. Echocardiography was used to assess cardiac function. Cardiac transcriptomic changes were investigated by bulk RNA sequencing. Inflammatory changes were assessed by qRT-PCR and immunohistochemistry in heart, thymus, and spleen of the animals. In follow-up experiments, anti-CD4 and anti-IL-17A antibodies were used along with PD-1 blockade in C57BL/6J mice. KEY RESULTS: Anti-PD-1 treatment led to cardiac dysfunction and left ventricular dilation in C57BL/6J mice, with increased nitrosative stress. Only mild inflammation was observed in the heart. However, PD-1 inhibition resulted in enhanced thymic inflammatory signalling, where Il17a increased most prominently. In BALB/c mice, cardiac dysfunction was not evident, and thymic inflammatory activation was more balanced. Inhibition of IL-17A prevented anti-PD-1-induced cardiac dysfunction in C57BL6/J mice. Comparing myocardial transcriptomic changes in C57BL/6J and BALB/c mice, differentially regulated genes (Dmd, Ass1, Chrm2, Nfkbia, Stat3, Gsk3b, Cxcl9, Fxyd2, and Ldb3) were revealed, related to cardiac structure, signalling, and inflammation. CONCLUSIONS: PD-1 blockade induces cardiac dysfunction in mice with increased IL-17 signalling in the thymus. Pharmacological inhibition of IL-17A treatment prevents ICI-induced cardiac dysfunction.
Assuntos
Cardiotoxicidade , Cardiopatias , Camundongos , Animais , Cardiotoxicidade/etiologia , Inibidores de Checkpoint Imunológico/efeitos adversos , Interleucina-17 , Camundongos Endogâmicos C57BL , Inflamação/complicaçõesRESUMO
BACKGROUND: Cardiac cell lines and primary cells are widely used in cardiovascular research. Despite increasing number of publications using these models, comparative characterization of these cell lines has not been performed, therefore, their limitations are undetermined. We aimed to compare cardiac cell lines to primary cardiomyocytes and to mature cardiac tissues in a systematic manner. METHODS AND RESULTS: Cardiac cell lines (H9C2, AC16, HL-1) were differentiated with widely used protocols. Left ventricular tissue, neonatal primary cardiomyocytes, and human induced pluripotent stem cell-derived cardiomyocytes served as reference tissue or cells. RNA expression of cardiac markers (e.g. Tnnt2, Ryr2) was markedly lower in cell lines compared to references. Differentiation induced increase in cardiac- and decrease in embryonic markers however, the overall transcriptomic profile and annotation to relevant biological processes showed consistently less pronounced cardiac phenotype in all cell lines in comparison to the corresponding references. Immunocytochemistry confirmed low expressions of structural protein sarcomeric alpha-actinin, troponin I and caveolin-3 in cell lines. Susceptibility of cell lines to sI/R injury in terms of viability as well as mitochondrial polarization differed from the primary cells irrespective of their degree of differentiation. CONCLUSION: Expression patterns of cardiomyocyte markers and whole transcriptomic profile, as well as response to sI/R, and to hypertrophic stimuli indicate low-to-moderate similarity of cell lines to primary cells/cardiac tissues regardless their differentiation. Low resemblance of cell lines to mature adult cardiac tissue limits their potential use. Low translational value should be taken into account while choosing a particular cell line to model cardiomyocytes.
Assuntos
Células-Tronco Pluripotentes Induzidas , Miócitos Cardíacos , Animais , Biomarcadores/metabolismo , Diferenciação Celular/genética , Linhagem Celular , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Miócitos Cardíacos/metabolismo , Fenótipo , TranscriptomaRESUMO
BACKGROUND & AIMS: Neuroinflammation in the gut is associated with many gastrointestinal (GI) diseases, including inflammatory bowel disease. In the brain, neuroinflammatory conditions are associated with blood-brain barrier (BBB) disruption and subsequent neuronal injury. We sought to determine whether the enteric nervous system is similarly protected by a physical barrier and whether that barrier is disrupted in colitis. METHODS: Confocal and electron microscopy were used to characterize myenteric plexus structure, and FITC-dextran assays were used to assess for presence of a barrier. Colitis was induced with dextran sulfate sodium, with co-administration of liposome-encapsulated clodronate to deplete macrophages. RESULTS: We identified a blood-myenteric barrier (BMB) consisting of extracellular matrix proteins (agrin and collagen-4) and glial end-feet, reminiscent of the BBB, surrounded by a collagen-rich periganglionic space. The BMB is impermeable to the passive movement of 4 kDa FITC-dextran particles. A population of macrophages is present within enteric ganglia (intraganglionic macrophages [IGMs]) and exhibits a distinct morphology from muscularis macrophages, with extensive cytoplasmic vacuolization and mitochondrial swelling but without signs of apoptosis. IGMs can penetrate the BMB in physiological conditions and establish direct contact with neurons and glia. Dextran sulfate sodium-induced colitis leads to BMB disruption, loss of its barrier integrity, and increased numbers of IGMs in a macrophage-dependent process. CONCLUSIONS: In intestinal inflammation, macrophage-mediated degradation of the BMB disrupts its physiological barrier function, eliminates the separation of the intra- and extra-ganglionic compartments, and allows inflammatory stimuli to access the myenteric plexus. This suggests a potential mechanism for the onset of neuroinflammation in colitis and other GI pathologies with acquired enteric neuronal dysfunction.
Assuntos
Colite/etiologia , Colite/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Plexo Mientérico/citologia , Plexo Mientérico/metabolismo , Animais , Biomarcadores , Colite/patologia , Modelos Animais de Doenças , Suscetibilidade a Doenças , Sistema Nervoso Entérico/imunologia , Sistema Nervoso Entérico/metabolismo , Matriz Extracelular , Imunofluorescência , Imuno-Histoquímica , Imunofenotipagem , Camundongos , Plexo Mientérico/ultraestrutura , Neuroglia/metabolismo , Neuroglia/ultraestrutura , Doenças Neuroinflamatórias/etiologia , Doenças Neuroinflamatórias/metabolismo , Doenças Neuroinflamatórias/patologia , Infiltração de NeutrófilosRESUMO
AIMS: Interleukin-1ß (IL-1ß) is an important pathogenic factor in cardiovascular diseases including chronic heart failure (HF). The CANTOS trial highlighted that inflammasomes as primary sources of IL-1 ß are promising new therapeutic targets in cardiovascular diseases. Therefore, we aimed to assess inflammasome activation in failing hearts to identify activation patterns of inflammasome subtypes as sources of IL-1ß. METHODS AND RESULTS: Out of the four major inflammasome sensors tested, expression of the inflammasome protein absent in melanoma 2 (AIM2) and NLR family CARD domain-containing protein 4 (NLRC4) increased in human HF regardless of the aetiology (ischaemic or dilated cardiomyopathy), while the NLRP1/NALP1 and NLRP3 (NLR family, pyrin domain containing 1 and 3) inflammasome showed no change in HF samples. AIM2 expression was primarily detected in monocytes/macrophages of failing hearts. Translational animal models of HF (pressure or volume overload, and permanent coronary artery ligation in rat, as well as ischaemia/reperfusion-induced HF in pigs) demonstrated activation pattern of AIM2 similar to that of observed in end-stages of human HF. In vitro AIM2 inflammasome activation in human Tohoku Hospital Pediatrics-1 (THP-1) monocytic cells and human AC16 cells was significantly reduced by pharmacological blockade of pannexin-1 channels by the clinically used uricosuric drug probenecid. Probenecid was also able to reduce pressure overload-induced mortality and restore indices of disease severity in a rat chronic HF model in vivo. CONCLUSIONS: This is the first report showing that AIM2 and NLRC4 inflammasome activation contribute to chronic inflammation in HF and that probenecid alleviates chronic HF by reducing inflammasome activation. The present translational study suggests the possibility of repositioning probenecid for HF indications.
Assuntos
Proteínas Adaptadoras de Sinalização CARD/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ligação a DNA/metabolismo , Insuficiência Cardíaca/metabolismo , Inflamassomos/metabolismo , Miócitos Cardíacos/metabolismo , Receptores de Superfície Celular/metabolismo , Adolescente , Adulto , Idoso , Animais , Proteínas Adaptadoras de Sinalização CARD/genética , Proteínas Adaptadoras de Sinalização CARD/imunologia , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/imunologia , Estudos de Casos e Controles , Conexinas/antagonistas & inibidores , Conexinas/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/imunologia , Modelos Animais de Doenças , Feminino , Insuficiência Cardíaca/tratamento farmacológico , Insuficiência Cardíaca/imunologia , Insuficiência Cardíaca/fisiopatologia , Humanos , Inflamassomos/imunologia , Masculino , Pessoa de Meia-Idade , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/imunologia , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/metabolismo , Probenecid/farmacologia , Ratos Wistar , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/imunologia , Transdução de Sinais , Sus scrofa , Células THP-1 , Função Ventricular Esquerda , Adulto JovemRESUMO
BACKGROUND: The prevalence of non-alcoholic steatohepatitis (NASH) rapidly increases with associated metabolic disorders such as dyslipidemia; therefore, NASH is now considered an independent risk factor of cardiovascular diseases. NASH displays sex-linked epidemiological, phenotypical, and molecular differences; however, little is known about the background of these sex-specific differences on the molecular level. OBJECTIVES: We aimed to assess sex-specific differences in the expression of inflammatory and fibrotic genes, as well as in cholesterol metabolism, focusing on the expression of Pcsk9 in several tissues in a mouse model of NASH that shows the typical features of the human condition. METHODS AND RESULTS: We fed 10-months-old male and female C57Bl/6J mice with a NASH-inducing CDAA or corresponding control diet for 8 weeks. We found that, compared to the control male mice baseline, hepatic Pcsk9 expression as well as serum PCSK9 level was significantly higher in females, and both circulating PCSK9 level and the hepatic Pcsk9 gene were markedly decreased in female mice during NASH development. Histological analysis revealed that male and female mice develop a similar degree of steatosis; however, fibrosis was more pronounced in males upon CDAA diet feeding. Strikingly, female mice have higher hepatic expression of the pro-inflammatory cytokines (Il1b, Ifng), and increased IL-1ß cleavage by the NLRP3 inflammasome, and a decrease in Clec4f+ resident Kupffer cell population in comparison to males in the CDAA-fed groups. CONCLUSION: This is the first demonstration that there are critical sex-specific differences during NASH development in middle-aged mice regarding inflammation, fibrosis, and cholesterol metabolism and that changes in PCSK9 and IL-1ß are likely important contributors to sex-specific changes during the transition to NASH.
RESUMO
AIM: Acute myocardial infarction and subsequent post-infarction heart failure are among the leading causes of mortality worldwide. The endocannabinoid system has emerged as an important modulator of cardiovascular disease, however the role of endocannabinoid metabolic enzymes in heart failure is still elusive. Herein, we investigated the endocannabinoids and their metabolic enzymes in ischemic end-stage failing human hearts and non-failing controls. METHODS AND RESULTS: Quantitative real-time PCR, targeted lipidomics, and activity-based protein profiling (ABPP) enabled assessment of the endocannabinoids and their metabolic enzymes in ischemic end-stage failing human hearts and non-failing controls. Based on lipidomic analysis, two subgroups were identified within the ischemic heart failure group; the first similar to control hearts and the second with decreased levels of the endocannabinoid 2-arachidonoyl-glycerol (2-AG) and drastically increased levels of the endocannabinoid anandamide (AEA), other N-acylethanolamines (NAEs) and free fatty acids. The altered lipid profile was accompanied by strong reductions in the activity of 13 hydrolases, including the 2-AG hydrolytic enzyme monoacylglycerol lipase (MGLL). CONCLUSIONS: Our findings suggest the presence of different biological states within the ischemic heart failure group, based on alterations in the lipid and hydrolase activity profiles. In addition, this study demonstrates that ABPP is a valuable tool to rapidly analyze enzyme activity in clinical samples with potential for novel drug and biomarker discovery.
Assuntos
Endocanabinoides/metabolismo , Insuficiência Cardíaca/metabolismo , Hidrolases/metabolismo , Isquemia Miocárdica/metabolismo , Adulto , Feminino , Humanos , Lipidômica , Masculino , Pessoa de Meia-Idade , Infarto do Miocárdio/metabolismo , ProteômicaRESUMO
Imidazoline receptors (IRs) have been recognized as promising targets in the treatment of numerous diseases; and moxonidine and rilmenidine, agonists of I1-IRs, are widely used as antihypertensive agents. Some evidence suggests that IR ligands may induce anti-inflammatory effects acting on I1-IRs or other molecular targets, which could be beneficial in patients with inflammatory bowel disease (IBD). On the other hand, several IR ligands may stimulate also alpha2-adrenoceptors, which were earlier shown to inhibit, but in more recent studies to rather aggravate colitis. Hence, this study aimed to analyse for the first time the effect of various I1-IR ligands on intestinal inflammation. Colitis was induced in C57BL/6 mice by adding dextran sulphate sodium (DSS) to the drinking water for 7 days. Mice were treated daily with different IR ligands: moxonidine and rilmenidine (I1-IR agonists), AGN 192403 (highly selective I1-IR ligand, putative antagonist), efaroxan (I1-IR antagonist), as well as with the endogenous IR agonists agmatine and harmane. It was found that moxonidine and rilmenidine at clinically relevant doses, similarly to the other IR ligands, do not have a significant impact on the macroscopic and histological signs of DSS-evoked inflammation. Likewise, colonic myeloperoxidase and serum interleukin-6 levels remained unchanged in response to these agents. Thus, our study demonstrates that imidazoline ligands do not influence significantly the severity of DSS-colitis in mice and suggest that they probably neither affect the course of IBD in humans. However, the translational value of these findings needs to be verified with other experimental colitis models and human studies.
Assuntos
Colite/tratamento farmacológico , Colite/metabolismo , Sulfato de Dextrana/toxicidade , Receptores de Imidazolinas/metabolismo , Imidazolinas/metabolismo , Imidazolinas/uso terapêutico , Animais , Colite/induzido quimicamente , Feminino , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Resultado do TratamentoRESUMO
It has been hypothesized that α2-adrenoceptors (α2-ARs) may be involved in the pathomechanism of colitis; however, the results are conflicting because both aggravation and amelioration of colonic inflammation have been described in response to α2-AR agonists. Therefore, we aimed to analyze the role of α2-ARs in acute murine colitis. The experiments were carried out in wild-type, α2A-, α2B-, and α2C-AR knockout (KO) C57BL/6 mice. Colitis was induced by dextran sulfate sodium (DSS, 2%); alpha2-AR ligands were injected i.p. The severity of colitis was determined both macroscopically and histologically. Colonic myeloperoxidase (MPO) and cytokine levels were measured by enzyme-linked immunosorbent assay and proteome profiler array, respectively. The nonselective α2-AR agonist clonidine induced a modest aggravation of DSS-induced colitis. It accelerated the disease development and markedly enhanced the weight loss of animals, but did not influence the colon shortening, tissue MPO levels, or histologic score. Clonidine induced similar changes in α2B- and α2C-AR KO mice, whereas it failed to affect the disease activity index scores and caused only minor weight loss in α2A-AR KO animals. In contrast, selective inhibition of α2A-ARs by BRL 44408 significantly delayed the development of colitis; reduced the colonic levels of MPO and chemokine (C-C motif) ligand 3, chemokine (C-X-C motif) ligand 2 (CXCL2), CXCL13, and granulocyte-colony stimulating factor; and elevated that of tissue inhibitor of metalloproteinases-1. In this work, we report that activation of α2-ARs aggravates murine colitis, an effect mediated by the α2A-AR subtype, and selective inhibition of these receptors reduces the severity of gut inflammation.
Assuntos
Antagonistas de Receptores Adrenérgicos alfa 2/farmacologia , Colite/induzido quimicamente , Colite/tratamento farmacológico , Sulfato de Dextrana/farmacologia , Intestinos/efeitos dos fármacos , Receptores Adrenérgicos alfa 2/metabolismo , Antagonistas de Receptores Adrenérgicos alfa 2/uso terapêutico , Animais , Clonidina/farmacologia , Clonidina/uso terapêutico , Colite/metabolismo , Colite/fisiopatologia , Ingestão de Líquidos/efeitos dos fármacos , Feminino , Técnicas de Inativação de Genes , Imidazóis/farmacologia , Imidazóis/uso terapêutico , Mucosa Intestinal/metabolismo , Intestinos/patologia , Isoindóis/farmacologia , Isoindóis/uso terapêutico , Locomoção/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Receptores Adrenérgicos alfa 2/deficiência , Receptores Adrenérgicos alfa 2/genéticaRESUMO
BACKGROUND: Allyphenyline, a novel α2-adrenoceptor (AR) ligand, has been shown to selectively activate α2C-adrenoceptors (AR) and 5HT1A receptors, but also to behave as a neutral antagonist of α2A-ARs. We exploited this unique pharmacological profile to analyze the role of α2C-ARs and 5HT1A receptors in the regulation of gastric mucosal integrity and gastrointestinal motility. METHODS: Gastric injury was induced by acidified ethanol in Wistar rats. Mucosal catalase and superoxide dismutase levels were measured by assay kits. The effect of allyphenyline on electrical field stimulation (EFS)-induced fundic and colonic contractions was determined in C57BL/6 mice. RESULTS: Intracerebroventricularly injected allyphenyline (3 and 15 nmol/rat) dose dependently inhibited the development of mucosal damage, which was antagonized by ARC 239 (α2B/C-AR and 5HT1A receptor antagonist), (S)-WAY 100135 (selective 5HT1A receptor antagonist), and JP-1302 (selective α2C-AR antagonist). This protection was accompanied by significant elevation of mucosal catalase and superoxide dismutase levels. Allyphenyline (10(-9)-10(-5) M) also inhibited EFS-induced fundic contractions, which was antagonized by ARC 239 and (S)-WAY 100135, but not by JP-1302. Similar inhibition was observed in the colon; however, in this case only ARC 239 reduced this effect, while neither selective inhibition of α2C-ARs and 5HT1A receptors nor genetic deletion of α2A- and α2B-ARs influenced it. CONCLUSIONS: Activation of both central α2C-ARs and 5HT1A receptors contributes to the gastroprotective action of allyphenyline in rats. Its inhibitory effect on fundic contractions is mediated by 5HT1A receptors, but neither α2-ARs nor 5HT1A receptors take part in its inhibitory effect on colonic contractility in mice.
Assuntos
Agonistas de Receptores Adrenérgicos alfa 2/farmacologia , Compostos Alílicos/farmacologia , Motilidade Gastrointestinal/efeitos dos fármacos , Imidazolinas/farmacologia , Receptores Adrenérgicos alfa 2/metabolismo , Agonistas do Receptor de Serotonina/farmacologia , Compostos Alílicos/química , Animais , Colo/efeitos dos fármacos , Colo/fisiologia , Imidazolinas/química , Masculino , Estrutura Molecular , Ratos , Ratos WistarRESUMO
Agmatine (decarboxylated arginine) exerts cytoprotective action in several tissues, such as in the brain, heart or kidneys, but there is still controversy over the effects of agmatine on the gastric mucosa. The aim of the present study was to reveal the potential gastroprotective action of agmatine by using an acid-independent ulcer model to clarify which receptors and peripheral factors are involved in it. Gastric mucosal damage was induced by acidified ethanol. Mucosal levels of calcitonin gene-related peptide (CGRP) and somatostatin were determined by radioimmunoassay. For analysis of gastric motor activity the rubber balloon method was used. It was found that agmatine given intracerebroventricularly (i.c.v., 0.044-220 nmol/rat) significantly inhibited the development of ethanol-induced mucosal damage, while in the case of intraperitoneal injection (0.001-50mg/kg i.p.) it had only a minor effect. The central gastroprotective action of agmatine was completely antagonized by mixed alpha2-adrenoceptor and imidazoline I1 receptor antagonists (idazoxan, efaroxan), but only partially by yohimbine (selective alpha2-adrenoceptor antagonist) and AGN 192403 (selective I1 receptor ligand, putative antagonist). It was also inhibited by the non-selective opioid-receptor antagonist naloxone and the selective δ-opioid receptor antagonist naltrindole, but not by ß-funaltrexamine and nor-Binaltorphimine (selective µ- and κ-opioid receptor antagonists, respectively). Furthermore, the effect of agmatine was antagonized by bilateral cervical vagotomy and by pretreatment with indomethacin and NG-nitro-l-arginine. Agmatine also reversed the ethanol-induced reduction of gastric mucosal CGRP and somatostatin content, but did not have any significant effect on gastric motor activity. These results indicate that agmatine given centrally induces gastric cytoprotection, which is mediated by central imidazoline I1 receptors, alpha2-adrenoceptors and δ-opioid receptors. Activation of these receptors induces the release of different mucosal protective factors, such as NO, prostaglandins, CGRP and somatostatin by a vagal-dependent mechanism. Alterations of gastric motility are not likely to contribute to the observed protective effect.
Assuntos
Agmatina/farmacologia , Citoproteção/efeitos dos fármacos , Citoproteção/fisiologia , Mucosa Gástrica/efeitos dos fármacos , Antagonistas Adrenérgicos alfa/farmacologia , Agmatina/administração & dosagem , Animais , Peptídeo Relacionado com Gene de Calcitonina/análise , Etanol/toxicidade , Mucosa Gástrica/patologia , Motilidade Gastrointestinal/efeitos dos fármacos , Região Hipotalâmica Lateral/efeitos dos fármacos , Receptores de Imidazolinas/antagonistas & inibidores , Receptores de Imidazolinas/fisiologia , Injeções Intraventriculares , Masculino , Antagonistas de Entorpecentes/farmacologia , Ratos Wistar , Receptores Adrenérgicos alfa 2/fisiologia , Receptores Opioides delta/fisiologia , Somatostatina/análise , Úlcera Gástrica/induzido quimicamente , Úlcera Gástrica/fisiopatologia , VagotomiaRESUMO
The aim of the present study was to analyze whether angiotensin II via the endocannabinoid system can induce gastric mucosal protection, since transactivation of cannabinoid CB1 receptors by angiotensin AT1 receptor in CHO cells was described. Experimental ulcer was induced by acidified ethanol given orally in male Wistar rats, CB1(+/+) wild type and CB1(-/-) knockout mice. The compounds were administered intracerebroventricularly. It was found, that 1. Angiotensin II inhibited the ethanol-induced gastric lesions (11.9-191pmol); the effect of angiotensin II (191pmol) was inhibited by the CB1 receptor inverse agonist AM 251 (1.8nmol) and the inhibitor of diacylglycerol lipase (DAGL), tetrahydrolipstatin (0.2nmol). 2. Angiotensin II exerted gastroprotection in wild type, but not in CB1(-/-) mice. 3. The gastroprotective effect of angiotensin II (191pmol) was reduced by atropine (1mg/kg i.v.) and bilateral cervical vagotomy. In conclusion, stimulation of central angiotensin AT1 receptors via activation of cannabinoid CB1 receptors induces gastroprotection in a DAGL-dependent and vagus-mediated mechanism.
Assuntos
Angiotensina II/farmacologia , Receptor Tipo 1 de Angiotensina/genética , Receptor CB1 de Canabinoide/genética , Úlcera Gástrica/tratamento farmacológico , Estômago/efeitos dos fármacos , Animais , Atropina/farmacologia , Células CHO , Cricetulus , Etanol , Mucosa Gástrica/metabolismo , Regulação da Expressão Gênica , Injeções Intraventriculares , Lactonas/farmacologia , Lipase Lipoproteica/antagonistas & inibidores , Lipase Lipoproteica/genética , Lipase Lipoproteica/metabolismo , Masculino , Camundongos , Camundongos Knockout , Orlistate , Piperidinas/farmacologia , Pirazóis/farmacologia , Ratos , Ratos Wistar , Receptor Tipo 1 de Angiotensina/metabolismo , Receptor CB1 de Canabinoide/agonistas , Receptor CB1 de Canabinoide/metabolismo , Transdução de Sinais , Estômago/patologia , Úlcera Gástrica/induzido quimicamente , Úlcera Gástrica/metabolismo , Úlcera Gástrica/patologia , Vagotomia , Nervo VagoRESUMO
Several lines of evidence suggest that imidazoline receptors mediate various physiological processes. It is rather difficult, however, to distinguish the imidazoline receptor-mediated effects from the alpha2-adrenoceptor-mediated ones due to the reasonable affinity of most imidazoline ligands for the alpha2-adrenoceptors. In the present study the effects of different imidazoline ligands were tested on the electrical field stimulation (EFS)-induced gastric contractions in wild-type (WT), alpha2A-, alpha2B- and alpha2C-adrenoceptor knockout (KO) mice in order to analyze, whether imidazoline I1 and I2 receptors take part in the regulation of gastric motor activity. Clonidine, moxonidine and rilmenidine inhibited the EFS-induced gastric contractions in a concentration dependent manner in WT, alpha2B- and alpha2C-adrenoceptor KO mice, whereas they had no or only weak effect in alpha2A-adrenoceptor KO mice. Their effects in WT mice were inhibited by idazoxan and BRL 44408, but not by ARC 239, AGN 192403 and BU 224. The endogenous imidazoline receptor ligand agmatine failed to affect the EFS-induced contractions, while harmane (an other endogenous imidazoline receptor ligand) and 2-BFI (a selective imidazoline I2 receptor agonist) exerted a slight effect in both WT and alpha2A-adrenoceptor KO mice, but this was not reversible by idazoxan, AGN 192403 and BU 224. It can be concluded, that the inhibitory effect of the tested imidazoline compounds on cholinergic gastric contractions is mediated mainly by alpha2A-adrenoceptors. Although at higher concentrations other receptors may also contribute to their effects, the lack of inhibition by AGN 192403 and BU 224 suggests that these are not imidazoline I1 and I2 receptors.