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1.
J Cell Mol Med ; 24(5): 3139-3148, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31970902

RESUMO

Macrophage activation participates in the pathogenesis of pulmonary inflammation. As a coenzyme, vitamin B6 (VitB6) is mainly involved in the metabolism of amino acids, nucleic acids, glycogen and lipids. We have previously reported that activation of AMP-activated protein kinase (AMPK) produces anti-inflammatory effects both in vitro and in vivo. Whether VitB6 via AMPK activation prevents pulmonary inflammation remains unknown. The model of acute pneumonia was induced by injecting mice with lipopolysaccharide (LPS). The inflammation was determined by measuring the levels of interleukin-1 beta (IL-1ß), IL-6 and tumour necrosis factor alpha (TNF-α) using real time PCR, ELISA and immunohistochemistry. Exposure of cultured primary macrophages to VitB6 increased AMP-activated protein kinase (AMPK) Thr172 phosphorylation in a time/dose-dependent manner, which was inhibited by compound C. VitB6 downregulated the inflammatory gene expressions including IL-1ß, IL-6 and TNF-α in macrophages challenged with LPS. These effects of VitB6 were mirrored by AMPK activator 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR). However, VitB6 was unable to inhibit LPS-induced macrophage activation if AMPK was in deficient through siRNA-mediated approaches. Further, the anti-inflammatory effects produced by VitB6 or AICAR in LPS-treated macrophages were abolished in DOK3 gene knockout (DOK3-/- ) macrophages, but were enhanced in macrophages if DOK3 was overexpressed. In vivo studies indicated that administration of VitB6 remarkably inhibited LPS-induced both systemic inflammation and acute pneumonia in wild-type mice, but not in DOK3-/- mice. VitB6 prevents LPS-induced acute pulmonary inflammation in mice via the inhibition of macrophage activation.

2.
Clin Pharmacol Ther ; 105(1): 201-209, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-29672839

RESUMO

Development of nitrate tolerance is a major drawback to nitrate therapy. Prostacyclin (PGI2) is a powerful vasodilator produced from prostaglandin (PGH2) by prostacyclin synthase (PGIS) in endothelial cells. This study aimed to determine the role of PGIS S-nitrosylation in nitrate tolerance induced by nitroglycerin (GTN). In endothelial cells, GTN increased PGIS S-nitrosylation and disturbed PGH2 metabolism, which were normalized by mutants of PGIS cysteine 231/441 to alanine (C231/441A). Clearance of nitric oxide by carboxy-PTIO or inhibition of S-nitrosylation by N-acetyl-cysteine decreased GTN-induced PGIS S-nitrosylation. Enforced expression of mutated PGIS with C231/441A markedly abolished GTN-induced PGIS S-nitrosylation and nitrate cross-tolerance in Apoe-/- mice. Inhibition of cyclooxygenase 1 by aspirin, supplementation of PGI2 by beraprost, and inhibition of PGIS S-nitrosylation by N-acetyl-cysteine improved GTN-induced nitrate cross-tolerance in rats. In patients, increased PGIS S-nitrosylation was associated with nitrate tolerance. In conclusion, GTN induces nitrate cross-tolerance through PGIS S-nitrosylation at cysteine 231/441.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Tolerância a Medicamentos/fisiologia , Oxirredutases Intramoleculares/metabolismo , Nitratos/metabolismo , Óxido Nítrico/metabolismo , Nitroglicerina/farmacologia , Idoso , Idoso de 80 Anos ou mais , Sequência de Aminoácidos , Animais , Bovinos , Cricetinae , Sistema Enzimático do Citocromo P-450/genética , Relação Dose-Resposta a Droga , Feminino , Células Endoteliais da Veia Umbilical Humana , Humanos , Oxirredutases Intramoleculares/genética , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Ratos , Ratos Sprague-Dawley , Vasodilatadores/farmacologia
3.
J Mol Med (Berl) ; 96(5): 403-412, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29502204

RESUMO

Diabetes mellitus is one of risk factors of cardiovascular diseases including atherosclerosis. Whether and how diabetes promotes the formation of unstable atherosclerotic plaque is not fully understood. Here, we show that streptozotocin-induced type 1 diabetes reduced collagen synthesis, leading to the formation of unstable atherosclerotic plaque induced by collar placement around carotid in apolipoprotein E knockout (Apoe-/-) mice. These detrimental effects of hyperglycemia on plaque stability were reversed by metformin in vivo without altering the levels of blood glucose and lipids. Mechanistically, we found that high glucose reduced the phosphorylated level of AMP-activated protein kinase alpha (AMPKα) and the transcriptional activity of activator protein 2 alpha (AP-2α), increased the expression of miR-124 expression, and downregulated prolyl-4-hydroxylase alpha 1 (P4Hα1) protein expression and collagen biosynthesis in cultured vascular smooth muscle cells. Importantly, these in vitro effects produced by high glucose were abolished by AMPKα pharmacological activation or adenovirus-mediated AMPKα overexpression. Further, adenovirus-mediated AMPKα gain of function remitted the process of diabetes-induced plaque destabilization in Apoe-/- mice injected with streptozotocin. Administration of metformin enhanced pAP-2α level, reduced miR-124 expression, and increased P4Hα1 and collagens in carotid atherosclerotic plaque in diabetic Apoe-/- mice. We conclude that streptozotocin-induced toxic diabetes promotes the formation of unstable atherosclerotic plaques based on the vulnerability index in Apoe-/- mice, which is related to the inactivation of AMPKα/AP-2α/miRNA-124/P4Hα1 axis. Clinically, targeting AMPKα/AP-2α/miRNA-124/P4Hα1 signaling should be considered to increase the plaque stability in patients with atherosclerosis. KEY MESSAGES: Hyperglycemia reduced collagen synthesis, leading to the formation of unstable atherosclerotic plaque induced by collar placement around carotid in apolipoprotein E knockout mice. Hyperglycemia destabilizes atherosclerotic plaque in vivo through an AMPKα/AP-2α/miRNA-124/P4Hα1-dependent collagen synthesis. Metformin functions as a stabilizer of atherosclerotic plaque to reduce acute coronary accent.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , MicroRNAs/metabolismo , Placa Aterosclerótica/metabolismo , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Fator de Transcrição AP-2/metabolismo , Animais , Colágeno Tipo I/metabolismo , Colágeno Tipo II/metabolismo , Hipoglicemiantes/farmacologia , Masculino , Metformina/farmacologia , Camundongos Knockout para ApoE , Miócitos de Músculo Liso/metabolismo
4.
Oncotarget ; 8(9): 14294-14305, 2017 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-28179583

RESUMO

We have previously reported that activation of AMP-activated kinase alpha 2 (AMPKα2) by nicotine or angiotensin II (AngII) instigates formation of abdominal aortic aneurysms (AAA) in Apoe-/- mice. Statins, used to treat hyperlipidemia widely, activate AMPK in vascular cells. We sought to examine the effects of pravastatin on AAA formation and uncover the molecular mechanism. The AAA model was induced by AngII and evaluated by incidence, elastin degradation, and maximal abdominal aortic diameter in Apoe-/- mice. The phosphorylated levels of AMPKα2 and activator protein 2 alpha (AP-2α) were examined in cultured vascular smooth muscle cells (VSMCs) or in mice. We observed that pravastatin (50 mg/kg/day, 8 weeks) remarkably increased the AngII-induced AAA incidence in mice. In VSMCs, pravastatin increased the levels of pAMPK, pAP-2α, and MMP2 in both basal and AngII-stressed conditions, which were abolished by tempol and compound C. Pravastatin-upregulated MMP2 was abrogated by AMPKα2 or AP-2α siRNA. Lentivirus-mediated gene silence of AMPKα2 or AP-2α abolished pravastatin-worsened AAA formations in AngII-infused Apoe-/- mice. Clinical investigations demonstrated that both AMPKα2 and AP-2α phosphorylations were increased in AAA patients or human subjects taking pravastatin. In conclusion, pravastatin promotes AAA formation through AMPKα2-dependent AP-2α activations.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Angiotensina II/efeitos adversos , Aneurisma da Aorta Abdominal/etiologia , Apolipoproteínas E/fisiologia , Regulação da Expressão Gênica/efeitos dos fármacos , Pravastatina/efeitos adversos , Fator de Transcrição AP-2/metabolismo , Animais , Anticolesterolemiantes/farmacologia , Aneurisma da Aorta Abdominal/metabolismo , Aneurisma da Aorta Abdominal/patologia , Western Blotting , Células Cultivadas , Modelos Animais de Doenças , Humanos , Masculino , Camundongos , Camundongos Knockout , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Fosforilação , Transdução de Sinais
5.
Appl Biochem Biotechnol ; 182(1): 411-427, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-27878744

RESUMO

The aims of our study were to evaluate the effects of Saccharomyces boulardii (S. boulardii) on deoxynivalenol (DON)-induced injury in porcine alveolar macrophage cells (PAMCs) and to explore the underlying mechanisms. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, flow cytometric analysis, ELISA, qRT-PCR, and western blot were performed to assess whether S. boulardii could prevent DON-induced injury by p38 mitogen-activated protein kinase (p38 MAPK) signal pathway. The results showed that pretreatment with 8 µM DON could decrease the viability of PAMC and significantly increase the apoptosis rate of PAMC, whereas S. boulardii could rescue apoptotic PAMC cells induced by DON. Further experiments revealed that S. boulardii effectively reversed DON-induced cytotoxicity via downregulating the expression of TNF-α, IL-6, and IL-lß. In addition, S. boulardii significantly alleviated DON-induced phosphorylation and mRNA expression of p38 and further increased the expression of apoptosis regulation genes Bcl-xl and Bcl-2 and inhibited the activation of Bax. Our results suggest that S. boulardii could suppress DON-induced p38 MAPK pathway activation and reduce the expression of downstream inflammatory cytokines, as well as promote the expression of anti-apoptotic genes to inhibit apoptosis induced by DON in PAMC.


Assuntos
Macrófagos Alveolares/efeitos dos fármacos , Fatores de Proteção , Saccharomyces boulardii/metabolismo , Transdução de Sinais , Tricotecenos/toxicidade , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Técnicas de Cocultura , Regulação da Expressão Gênica , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Macrófagos Alveolares/citologia , Macrófagos Alveolares/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Saccharomyces boulardii/crescimento & desenvolvimento , Suínos , Tricotecenos/antagonistas & inibidores , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Proteína bcl-X/genética , Proteína bcl-X/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
6.
Clin Exp Pharmacol Physiol ; 44(1): 114-122, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27757983

RESUMO

Poly (ADP-ribose) polymerase (PARP) plays an important role in endothelial dysfunction, leading to atherogenesis and vascular-related diseases. However, whether PARP regulates nitric oxide (NO), a key regulator of endothelial function, is unclear so far. We investigated whether inhibition of PARP-1, the most abundant PARP isoform, prevents atherogenesis by regulating NO production and tried to elucidate the possible mechanisms involved in this phenomenon. In apolipoprotein E-deficient (apoE-/- ) mice fed a high-cholesterol diet for 12 weeks, PARP-1 inhibition via treatment with 3,4-dihydro-54-(1-piperindinyl) butoxy-1(2H)-isoquinoline (DPQ) or PARP-1 gene knockout reduced aortic atherosclerotic plaque areas (49% and 46%, respectively). Both the groups showed restored NO production in mouse aortas with reduced arginase II (Arg II) expression compared to that in the controls. In mouse peritoneal macrophages and aortic endothelial cells (MAECs), PARP-1 knockout resulted in lowered Arg II expression. Moreover, phosphorylation of endothelial NO synthase (eNOS) was preserved in the aortas and MAECs when PARP-1 was inhibited. Reduced NO production in vitro due to PARP-1 deficiency could be restored by treating the MAECs with oxidized low-density lipoprotein treatment, but this effect could not be achieved with peritoneal macrophages, which was likely due to a reduction in the expression of induced NOS expression. Our findings indicate that PARP-1 inhibition may attenuate atherogenesis by restoring NO production in endothelial cells and thus by reducing Arg II expression and consequently arginase the activity.


Assuntos
Aorta/metabolismo , Arginase/metabolismo , Aterosclerose/metabolismo , Regulação para Baixo/fisiologia , Óxido Nítrico/biossíntese , Poli(ADP-Ribose) Polimerase-1/deficiência , Animais , Aterosclerose/induzido quimicamente , Aterosclerose/prevenção & controle , Células Cultivadas , Colesterol na Dieta/efeitos adversos , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos
7.
Mol Cell Endocrinol ; 392(1-2): 163-72, 2014 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-24887517

RESUMO

Epithelial-to-mesenchymal transition (EMT) plays an important role in renal interstitial fibrosis (RIF) with diabetic nephropathy (DN). Smad7 (a inhibitory smad), a downstream signaling molecules of TGF-ß1, represses the EMT. The physiological function of miR-21 is closely linked to EMT and RIF. However, it remained unclear whether miR-21 over-expression affected TGF-ß1-induced EMT by regulating smad7 in DN. In this study, real-time RT-PCR, cell transfection, luciferase reporter gene assays, western blot and confocal microscope were used, respectively. Here, we found that miR-21 expression was upregulated by TGF-ß1 in time- and concentration -dependent manner. Moreover, miR-21 over-expression enhanced TGF-ß1-induced EMT(upregulation of a-SMA and downregulation of E-cadherin) by directly down-regulating smad7/p-smad7 and indirectly up-regulating smad3/p-smad3, accompanied by the decrease of Ccr and the increase of col-IV, FN, the content of collagen fibers, RTBM, RTIAW and ACR. Meantime, the siRNA experiment showed that smad7 can directly regulate a-SMA and E-cadherin expression. More importantly, miR-21 inhibitor can not only inhibit EMT and fibrosis but also ameliorate renal structure and function. In conclusion, our results demonstrated that miR-21 overexpression can contribute to TGF-ß1-induced EMT by inhibiting target smad7, and that targeting miR-21 may be a better alternative to directly suppress TGF-ß1-mediated fibrosis in DN.


Assuntos
Nefropatias Diabéticas/patologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Rim/patologia , MicroRNAs/metabolismo , Proteína Smad7/metabolismo , Fator de Crescimento Transformador beta1/farmacologia , Actinas/metabolismo , Animais , Sequência de Bases , Biomarcadores/metabolismo , Caderinas/metabolismo , Linhagem Celular , Nefropatias Diabéticas/genética , Regulação para Baixo/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Humanos , Rim/efeitos dos fármacos , Rim/ultraestrutura , Masculino , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Dados de Sequência Molecular , Fosforilação/efeitos dos fármacos , Proteína Smad3/genética , Proteína Smad3/metabolismo , Proteína Smad7/genética , Regulação para Cima/efeitos dos fármacos
8.
Am J Physiol Renal Physiol ; 306(5): F486-95, 2014 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-24370587

RESUMO

Diabetic nephropathy (DN) is one of the most important diabetic microangiopathies. The epithelial-to-mesenchymal transition (EMT) plays an important role in DN. The physiological role of microRNA-21 (miR-21) was closely linked to EMT. However, it remained elusive whether tongxinluo (TXL) ameliorated renal structure and function by regulating miR-21-induced EMT in DN. This study aimed to determine the effect of TXL on miR-21-induced renal tubular EMT and to explore the relationship between miR-21 and TGF-ß1/smads signals. Real-time RT-PCR, cell transfection, in situ hybridization (ISH), and laser confocal microscopy were used, respectively. Here, we revealed that TXL dose dependently lowered miR-21 expression in tissue, serum, and cells. Overexpression of miR-21 can enhance α-smooth muscle actin (SMA) expression and decrease E-cadherin expression by upregulating smad3/p-smad3 expression and downregulating smad7 expression. Interestingly, TXL also increased E-cadherin expression and decreased α-SMA expression by regulating miR-21 expression. More importantly, TXL decreased collagen IV, fibronectin, glomerular basement membrane, glomerular area, and the albumin/creatinine ratio, whereas it increased the creatinine clearance ratio. The results demonstrated that TXL ameliorated renal structure and function by regulating miR-21-induced EMT, which was one of the mechanisms to protect against DN, and that miR-21 may be one of the therapeutic targets for TXL in DN.


Assuntos
Nefropatias Diabéticas/tratamento farmacológico , Medicamentos de Ervas Chinesas/uso terapêutico , Transição Epitelial-Mesenquimal/efeitos dos fármacos , MicroRNAs/metabolismo , Caderinas/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem Celular , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/metabolismo , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Humanos
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