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1.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 37(3): 240-245, 2021 Mar.
Artigo em Chinês | MEDLINE | ID: mdl-33766232

RESUMO

Objective To study the role of long non-coding RNA growth arrest specific transcript 5 (lncGAS5) in the autophagy of hepatocytes induced by homocysteine (Hcy). Methods HL7702 human hepatocyte cells were cultured in vitro and divided into control group and Hcy group. Western blotting was used to detect the expression levels of microtubule-associated protein 1 light chain 3B (LC3B) and P62. The cells were transfected with mRFP-GFP-LC3 adenovirus to observe the autophagy flow with laser scanning confocal microscope. Real-time quantitative PCR was performed to detect the expression level of lncGAS5. lncGAS5 small interfering RNA (si-lncGAS5) and negative control small interfering RNA (si-NC) were transfected into the cells. After the transfected cells were treated with Hcy, the changes of LC3B, P62 and autophagy flow were analyzed with the above methods. Results Compared with the control group, the LC3BII/LC3BI ratio increased and the expression of P62 protein decreased in the Hcy group. When the lever of Hcy lifted, the number of autophagosomes and autolysosomes and the expression of lncGAS5 increased in the cells. After knock-down of lncGAS5, the ratio of LC3BII/LC3BI decreased and the expression of P62 increased. Moreover, the number of autophagosomes and autolysosomes were reduced in the cells. Conclusion lncGAS5 can promote the autophagy of hepatocytes induced by Hcy.


Assuntos
RNA Longo não Codificante , Autofagia/genética , Hepatócitos , Homocisteína/farmacologia , Humanos , RNA Longo não Codificante/genética , RNA Nucleolar Pequeno
2.
Int J Mol Med ; 45(4): 1081-1090, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32124965

RESUMO

Pirfenidone (PFD) is an anti­fibrotic agent that is clinically used in the treatment of idiopathic pulmonary fibrosis. PFD has been shown to exert protective effects against damage to orbital fibroblasts, endothelial cells, liver cells and renal proximal tubular cells; however, its effect on myocardial cell apoptosis remains unclear. The present study aimed to characterize the effects of PFD on homocysteine (Hcy)­induced cardiomyocyte apoptosis and investigated the underlying mechanisms. H9C2 rat cardiomyocytes were pre­treated with PFD for 30 min followed by Hcy exposure for 24 h. The effects of PFD on cell cytotoxicity were evaluated by CCK­8 assay. The apoptosis rate of each group was determined by flow cytometry. The protein and mRNA levels of connexin 43 (Cx43), Bax, B­cell lymphoma­2 (Bcl­2) and caspase­3 were measured by western blot analysis and reverse transcription­quantitative PCR, respectively. The present results demonstrated that the apoptotic rate increased following Hcy exposure, whereas the apoptotic rate significantly decreased following PFD pre­treatment. Furthermore, the ratio of Bax/Bcl2 was upregulated following Hcy exposure, and Hcy upregulated the expression levels of cleaved caspase­3 and Cx43. Notably, these effects were prevented by PFD. Additionally, the effects of PFD were inhibited by the Cx43 agonist, AAP10. In summary, the findings of the present study demonstrate that PFD protects H9C2 rat cardiomyocytes against Hcy­induced apoptosis by modulating the Cx43 signaling pathway.


Assuntos
Apoptose/efeitos dos fármacos , Conexina 43/metabolismo , Homocisteína/farmacologia , Miócitos Cardíacos/metabolismo , Piridonas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Linhagem Celular , Miócitos Cardíacos/patologia , Ratos
3.
Int J Mol Sci ; 21(5)2020 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-32150828

RESUMO

High homocysteine is routinely observed in diabetic patients, and this non-protein amino acid is considered as an independent risk factor for diabetic retinopathy. Homocysteine biosynthesis from methionine forms S-adenosyl methionine (SAM), which is a major methyl donor critical in DNA methylation. Hyperhomocysteinemia is implicated in increased oxidative stress and activation of MMP-9, and in diabetic retinopathy, the activation of MMP-9 facilitates capillary cell apoptosis. Our aim was to investigate the mechanism by which homocysteine activates MMP-9 in diabetic retinopathy. Human retinal endothelial cells, incubated with/without 100 µM homocysteine, were analyzed for MMP-9 and its tissue inhibitor Timp1 expressions and interactions, and ROS levels. Timp1 and MMP-9 promoters were analyzed for methylated and hydroxymethylated cytosine levels (5mC and 5hmC respectively) by the DNA capture method, and DNA- methylating (Dnmt1) and hydroxymethylating enzymes (Tet2) binding by chromatin immunoprecipitation. The results were confirmed in retinal microvessels from diabetic rats receiving homocysteine. Homocysteine supplementation exacerbated hyperglycaemia-induced MMP-9 and ROS levels and decreased Timp1 and its interactions with MMP-9. Homocysteine also aggravated Dnmts and Tets activation, increased 5mC at Timp1 promoter and 5hmC at MMP-9 promoter, and suppressed Timp1 transcription and activated MMP-9 transcription. Similar results were obtained from retinal microvessels from diabetic rats receiving homocysteine. Thus, hyperhomocysteinemia in diabetes activates MMP-9 functionally by reducing Timp1-MMP-9 interactions and transcriptionally by altering DNA methylation-hydroxymethylation of its promoter. The regulation of homocysteine could prevent/slow down the development of retinopathy and prevent their vision loss in diabetic patients.


Assuntos
Metilação de DNA , Diabetes Mellitus Experimental/complicações , Retinopatia Diabética/tratamento farmacológico , Regulação da Expressão Gênica , Homocisteína/farmacologia , Metaloproteinase 1 da Matriz/química , Inibidor Tecidual de Metaloproteinase-1/antagonistas & inibidores , Animais , Apoptose , Células Cultivadas , Retinopatia Diabética/etiologia , Retinopatia Diabética/metabolismo , Retinopatia Diabética/patologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Masculino , Metaloproteinase 1 da Matriz/genética , Metaloproteinase 1 da Matriz/metabolismo , Estresse Oxidativo , Ratos , Ratos Wistar , Vasos Retinianos/efeitos dos fármacos , Vasos Retinianos/metabolismo , Vasos Retinianos/patologia , Inibidor Tecidual de Metaloproteinase-1/genética , Inibidor Tecidual de Metaloproteinase-1/metabolismo
4.
Molecules ; 25(1)2020 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-31948056

RESUMO

Memory impairment has been shown to be associated with glutamate (Glu) excitotoxicity, homocysteine (Hcy) accumulation, and oxidative stress. We hypothesize that Glu and Hcy could damage neuronal cells, while astaxanthin (ATX) could be beneficial to alleviate the adverse effects. Using PC12 cell model, we showed that Glu and Hcy provoked a huge amount of reactive oxygen species (ROS) production, causing mitochondrial damage at EC50 20 and 10 mm, respectively. The mechanisms of action include: (1) increasing calcium influx; (2) producing ROS; (3) initiating lipid peroxidation; (4) causing imbalance of the Bcl-2/Bax homeostasis; and (5) activating cascade of caspases involving caspases 12 and 3. Conclusively, the damages caused by Glu and Hcy to PC12 cells can be alleviated by the potent antioxidant ATX.


Assuntos
Ácido Glutâmico/farmacologia , Homocisteína/farmacologia , Animais , Apoptose/efeitos dos fármacos , Cálcio/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Fármacos Neuroprotetores/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Células PC12 , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos , Espécies Reativas de Oxigênio/metabolismo , Xantofilas/farmacologia
5.
Mol Med Rep ; 21(1): 371-378, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31746381

RESUMO

As a novel anti­inflammatory cytokine of the interleukin (IL)­1 family, IL­37 protects the human body from diseases characterized by excessive inflammation. The pathologic process of hyperhomocysteinemia (hHcy) is accompanied by persistent inflammation. However, little is known regarding the role of IL­37 in hHcy. In the present study, the levels of cytokines including IL­37, IL­1ß, IL­6 and tumor necrosis factor­α in the supernatant were detected by ELISA. mRNA and protein expression were detected by Reverse transcription­quantitative PCR and western blotting, respectively. LDH level was determined by ELISA and the cell viability was detected through CCK­8 kit. In the present study, mean serum IL­37 levels of patients with hHcy were 32.3% lower than those of controls (P<0.01). In peripheral blood mononuclear cells (PBMCs) from patients with hHcy, mean IL­37 mRNA expression was 73.5% lower (P<0.01) and IL­37 protein expression was 77.7% lower compared with that of healthy controls (P<0.01). Furthermore, the results demonstrated that exogenous homocysteine (Hcy) stimulation markedly downregulated the mRNA and protein expression levels of IL­37 in PBMCs in vitro. In 293T cells, overexpression of IL­37 restored the cell viability impaired by Hcy, and reduced the release of lactate dehydrogenase and the proinflammatory cytokines IL­1ß, IL­6 and tumor necrosis factor­α. In conclusion, IL­37 was downregulated by Hcy in vivo and in vitro, and IL­37 exhibited a protective role against cell injury induced by Hcy.


Assuntos
Homocisteína/metabolismo , Hiper-Homocisteinemia/sangue , Inflamação/sangue , Interleucina-1/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Homocisteína/farmacologia , Homocisteína/toxicidade , Humanos , Hidroliases/sangue , Hiper-Homocisteinemia/induzido quimicamente , Hiper-Homocisteinemia/complicações , Hiper-Homocisteinemia/genética , Inflamação/induzido quimicamente , Inflamação/complicações , Inflamação/genética , Interleucina-1/sangue , Interleucina-1beta/sangue , Interleucina-6/sangue , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/sangue , Fator de Necrose Tumoral alfa/sangue , Fator de Necrose Tumoral alfa/genética
6.
Gene ; 725: 144143, 2020 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-31629816

RESUMO

Atherosclerosis is a common cardiovascular disorder and is characterized by damage of endothelial cells, cell inflammation, hyper-proliferation of vascular smooth muscle cells and the accumulation of extracellular lipids and fibrous tissues. In this study, we firstly examined the expression level of long intergenic non-protein coding RNA, regulator of reprogramming (linc-ROR) in homocysteine (Hcy)-stimulated human aortic smooth muscle cells (HASMCs), and then looked into the potential molecular signaling axis of linc-ROR in regulating the proliferation and migration of HASMCs. Hcy promoted HASMC proliferation and up-regulated linc-ROR expression. Functional studies showed that linc-ROR exerted enhanced actions on the proliferation and migration of HASMCs. In addition, linc-ROR acted as a competing endogenous RNA for miR-195-5p and repressed the miR-195-5p expression in HASMCs. Linc-ROR was up-regulated the miR-195-3p was down-regulated in the plasma from CAD patients when compared to normal controls. Furthermore, fibroblast growth factor 2 (FGF2) was identified as a target of miR-195-5p and was negatively regulated by miR-195-5p in HASMCs. The rescue experiments revealed that linc-ROR-mediated HASMC proliferation and migration may be via regulating miR-195-5p/FGF2 axis. Linc-ROR inhibition blocked the miR-195-5p/FGF2 signaling in Hcy-treated HASMCs, and this effect may also involve in the miR-195-5p/FGF2 axis. To summarize, the data of the present study identified the up-regulation of linc-ROR in Hcy-stimulated HASMCs, and further mechanistic functional studies revealed that linc-ROR promoted HASMC proliferation and migration via regulating miR-195-5p/FGF2 axis. The present study provided the novel actions of linc-ROR in regulating HASMC proliferation and migration, which may be related to the pathophysiology of atherosclerosis.


Assuntos
Fator 2 de Crescimento de Fibroblastos/metabolismo , MicroRNAs/metabolismo , RNA Longo não Codificante/metabolismo , Aorta/citologia , Aorta/efeitos dos fármacos , Aorta/metabolismo , Aterosclerose/genética , Aterosclerose/metabolismo , Aterosclerose/patologia , Autofagia/fisiologia , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/fisiologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Fator 2 de Crescimento de Fibroblastos/genética , Fator 2 de Crescimento de Fibroblastos/fisiologia , Homocisteína/farmacologia , Humanos , MicroRNAs/genética , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , RNA Longo não Codificante/biossíntese , RNA Longo não Codificante/genética , Transdução de Sinais
7.
Spectrochim Acta A Mol Biomol Spectrosc ; 225: 117516, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31518754

RESUMO

We describe a sensitive turn-on fluorescent assay for antioxidants by using fluorescence-tunable graphene quantum dots (GQDs). GQDs exhibited strong fluorescence without dopamine (DA). DA could self-polymerize to a thin polydopamine (PDA) film on the surface of GQDs under alkaline environment, resulting in the fluorescence quenching of GQDs via fluorescence resonance energy transfer (FRET). However, the self-polymerization of DA could be effectively inhibited in the presence of antioxidants including glutathione (GSH), ascorbic acid (AA), cysteine (Cys), and homocysteine (Hcys). Thus, the fluorescence of GQDs restored. The "turn-on" sensing of antioxidants could be achieved with high sensitivity. The detection limit for GSH, AA, Cys, and Hcys could be achieved as low as 2.4 nM, 1.5 nM, 4.2 nM, and 4.4 nM, respectively. Finally, the GQDs@PDA system was applied for monitoring cerebral antioxidants in rat brain microdialysates. This work promises new opportunities to evaluate antioxidant capacity in physiological and pathological fields.


Assuntos
Antioxidantes/análise , Dopamina/química , Pontos Quânticos/química , Antioxidantes/farmacologia , Ácido Ascórbico/análise , Ácido Ascórbico/farmacologia , Química Encefálica , Cisteína/análise , Cisteína/farmacologia , Estudos de Viabilidade , Fluorescência , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/química , Glutationa/análise , Glutationa/farmacologia , Grafite , Homocisteína/análise , Homocisteína/farmacologia , Indóis/química , Limite de Detecção , Microdiálise , Polimerização/efeitos dos fármacos , Polímeros/química , Pontos Quânticos/ultraestrutura
8.
J Biol Chem ; 294(51): 19465-19474, 2019 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-31628194

RESUMO

One of the main characteristics of atherosclerosis is vascular calcification, which is linked to adverse cardiovascular events. Increased homocysteine (Hcy), a feature of hyperhomocysteinemia, is correlated with advanced vascular calcification and phenotypic switching of vascular smooth muscle cells (VSMCs). Oxidative stress and high phosphate levels also induce VSMC calcification, suggesting that the Krüppel-like factor 4 (KLF4) signaling pathway may also contribute to vascular calcification. In this study, we investigated this possibility and the role and mechanisms of Hcy in vascular calcification. We found that in atherosclerotic apolipoprotein E-deficient (ApoE-/-) mice, Hcy significantly increases vascular calcification in vivo, as well as VSMC calcification in vitro Of note, the Hcy-induced VSMC calcification was correlated with elevated KLF4 levels. Hcy promoted KLF4 expression in calcified atherosclerotic lesions in vivo and in calcified VSMCs in vitro shRNA-mediated KLF4 knockdown blocked the Hcy-induced up-regulation of runt-related transcription factor 2 (RUNX2) and VSMC calcification. RUNX2 inhibition abolished Hcy-induced VSMC calcification. Using ChIP analysis, we demonstrate that KLF4 interacts with RUNX2, an interaction promoted by Hcy stimulation. Our experiments also revealed that the KLF4 knockdown attenuates Hcy-induced RUNX2 transactivity, indicating that KLF4 is important in modulating RUNX2 transactivity. These findings support a role for Hcy in regulating vascular calcification through a KLF4-RUNX2 interaction and indicate that Hcy-induced, enhanced RUNX2 transactivity increases VSMC calcification. These insights reveal possible opportunities for developing interventions that prevent or manage vascular calcification.


Assuntos
Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Hiper-Homocisteinemia/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Miócitos de Músculo Liso/citologia , Calcificação Vascular/metabolismo , Animais , Aterosclerose/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Feminino , Homocisteína/farmacologia , Fatores de Transcrição Kruppel-Like/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout para ApoE , Músculo Liso Vascular/citologia , Estresse Oxidativo , Fosfatos/sangue , RNA Interferente Pequeno/metabolismo , Calcificação Vascular/genética
9.
Cell Signal ; 64: 109394, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31472244

RESUMO

Cellular Senescence is associated with organismal aging and related pathologies. Previously, we reported that plasminogen activator inhibitor-1 (PAI-1) is an essential mediator of senescence and a potential therapeutic target for preventing aging-related pathologies. In this study, we investigate the efficacies of PAI-1 inhibitors in both in vitro and in vivo models of homocysteine (Hcy)-induced cardiovascular aging. Elevated Hcy, a known risk factor of cardiovascular diseases, induces endothelial senescence as evidenced by increased senescence-associated ß-Gal positivity (SA-ß-Gal), flattened cellular morphology, and cylindrical appearance of cellular nuclei. Importantly, inhibition of PAI-1 by small molecule inhibitors reduces the number of SA-ß-Gal positive cells, normalizes cellular morphology and nuclear shape. Furthermore, while Hcy induces the levels of senescence regulators PAI-1, p16, p53 and integrin ß3, and suppresses catalase expression, treatment with PAI-1 inhibitors blocks the Hcy-induced stimulation of senescence cadres, and reverses the Hcy-induced suppression of catalase, indicating that PAI-1 specific small molecule inhibitors are efficient to prevent Hcy-induced cellular senescence. Our in vivo study shows that the levels of integrin ß3, a recently identified potential regulator of cellular senescence, and its interaction with PAI-1 are significantly elevated in Hcy-treated heart tissues. In contrast, Hcy suppresses antioxidant gene regulator Nrf2 expression in hearts. However, co-treatment with PAI-1 inhibitor completely blocks the stimulation of Hcy-induced induction of integrin ß3 and reverses Nrf2 expression. Collectively these in vitro and in vivo studies indicate that pharmacological inhibition of PAI-1 improves endothelial and cardiac health by suppressing the pro-senescence effects of hyperhomocysteinemia through suppression of Hcy-induced master regulators of cellular senescence PAI-1 and integrin ß3. Therefore, PAI-1 inhibitors are promising drugs for amelioration of hyperhomocysteinemia-induced vascular aging and aging-related disease.


Assuntos
Senescência Celular/efeitos dos fármacos , Homocisteína/farmacologia , Piperazinas/farmacologia , Inibidor 1 de Ativador de Plasminogênio/fisiologia , para-Aminobenzoatos/farmacologia , Células A549 , Animais , Células Endoteliais da Veia Umbilical Humana , Humanos , Integrina beta3/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fator 2 Relacionado a NF-E2/metabolismo
10.
Cell Death Dis ; 10(8): 561, 2019 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-31332165

RESUMO

The elevated level of the amino acid metabolite homocysteine (Hcy) is known as a risk factor for ischemic stroke. The molecular mechanisms responsible for neurotoxicity of Hcy remain largely unknown in ischemic brains. The previous studies have shown that Hcy decreases the proliferation and viability of neural stem cells (NSCs) in vivo and in vitro. Autophagy is required for the maintenance of NSCs homeostasis. In the current study, we hypothesized that the toxic effect of Hcy on NSCs may involve the changes in autophagy level following cerebral ischemia/reperfusion injury. The results showed that Hcy reduced cell viability, increased LDH release, and induced nonapoptotic cell death in primary NSCs exposed to oxygen-glucose deprivation)/reoxygenation (OGD/R). Treatment with autophagy inhibitor 3-methyladenine (3MA) partly reversed the decrease in the viability and prevented LDH release triggered by Hcy combined with OGD/R. Increased punctate LC3 dots co-localizing with Nestin-stained NSCs were also observed in the subventricular zone of Hcy-treated MCAO animals, which were partially blocked by 3MA. In vitro studies further revealed that Hcy induced the formation of autophagosomes, markedly increased the expression of the autophagic markers and decreased p-ERK, p-PI3K, p-AKT, and p-mTOR levels. In addition, MHY1485, an activator of mTOR, reduced Hcy-induced increase in LC3 and Beclin 1 protein levels, meanwhile ERK and PI3K activators (TPA, curcumin for ERK and IGF-1 for PI3K, respectively) enhanced Hcy-triggered mTOR inhibition in OGD/R NSCs. Our findings suggest that Hcy may cause excessive autophagy by downregulation of both PI3K-AKT- and ERK- dependent mTOR signaling, thereby facilitates the toxicity of Hcy on NSCs in ischemic brains.


Assuntos
Autofagia/efeitos dos fármacos , Isquemia Encefálica/metabolismo , Homocisteína/farmacologia , Células-Tronco Neurais/metabolismo , Acidente Vascular Cerebral/metabolismo , Adenina/análogos & derivados , Adenina/farmacologia , Animais , Morte Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo
11.
Nutr Metab Cardiovasc Dis ; 29(8): 856-864, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31272869

RESUMO

BACKGROUND AND AIM: Increased homocysteine (Hcy) is associated with coronary artery disease (CAD). Hcy increases reactive oxygen species (ROS) via NADPH oxidases (Nox), reducing acetylcholine-mediated vasorelaxation. We aimed to determine if putative Nox2 inhibitors prevent Hcy-impaired acetylcholine-mediated vasorelaxation. METHODS AND RESULTS: New Zealand White rabbit and wild-type (C57BL/6) and Nox2-/- (NOX) mice aortic rings were mounted in organ baths. Rabbit rings were incubated with either apocynin (10 µM), gp91ds-tat (GP, 1 µM) or PhoxI2 (1 µM) and mice rings GP (1 µM) only. Some rabbit rings were incubated with 3 mM Hcy, before pre-contraction, followed by dose-response relaxation to acetylcholine (ACh; 0.01µM-10µM). In rabbit rings treated with Hcy and GP, O2‾ donor pyrogallol (1 µM) or Akt activator SC79 (1 µM) was added 5 min before ACh. Mice rings were used to compare Nox2 deletion to normal acetylcholine-mediated relaxation. In rabbits, Hcy reduced acetylcholine-mediated relaxation vs. control (p < 0.0001). Treatment + Hcy reduced relaxation compared with treatment alone (p < 0.0001). Pyrogallol and SC79 reversed the response of GP + Hcy (p = 0.0001). In mice, Nox2 deletion reduced acetylcholine-mediated vasorelaxation. Rabbit tissue analysis revealed that Hcy reduced eNOS phosphorylation at Thr495 and increased eNOS phosphorylation at Ser1177; no further alteration at Thr495 was observed with GP. In contrast, GP prevented increased phosphorylation at Ser1177. CONCLUSIONS: Apocynin, GP and PhoxI2 worsens acetylcholine-mediated vascular relaxation in rabbit aorta, which is supported by results from mouse Nox2 deletion data. These inhibitors worsen Hcy-induced vascular dysfunction, suggesting that current putative Nox2 inhibitors might not be useful in treating HHcy.


Assuntos
Acetilcolina/farmacologia , Aorta/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Homocisteína/farmacologia , NADPH Oxidase 2/antagonistas & inibidores , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologia , Acetofenonas/farmacologia , Animais , Aorta/enzimologia , Glicoproteínas/farmacologia , Técnicas In Vitro , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , NADPH Oxidase 2/genética , NADPH Oxidase 2/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Fosforilação , Coelhos , Serina , Treonina
12.
J Biol Chem ; 294(29): 11154-11165, 2019 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-31167782

RESUMO

Homocysteine, a metabolite of the methionine cycle, is a known agonist of N-methyl-d-aspartate receptor (NMDAR), a glutamate receptor subtype and is involved in NMDAR-mediated neurotoxicity. Our previous findings have shown that homocysteine-induced, NMDAR-mediated neurotoxicity is facilitated by a sustained increase in phosphorylation and activation of extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK MAPK). In the current study, we investigated the role GluN1/GluN2A-containing functional NMDAR (GluN2A-NMDAR) and GluN1/GluN2B-containing functional NMDAR (GluN2B-NMDAR) in homocysteine-induced neurotoxicity. Our findings revealed that exposing primary cortical neuronal cultures to homocysteine leads to a sustained low-level increase in intracellular Ca2+ We also showed that pharmacological inhibition of GluN2A-NMDAR or genetic deletion of the GluN2A subunit attenuates homocysteine-induced increase in intracellular Ca2+ Our results further established the role of GluN2A-NMDAR in homocysteine-mediated sustained ERK MAPK phosphorylation and neuronal cell death. Of note, the preferential role of GluN2A-NMDAR in homocysteine-induced neurotoxicity was distinctly different from glutamate-NMDAR-induced excitotoxic cell death that involves overactivation of GluN2B-NMDAR and is independent of ERK MAPK activation. These findings indicate a critical role of GluN2A-NMDAR-mediated signaling in homocysteine-induced neurotoxicity.


Assuntos
Cálcio/metabolismo , Morte Celular/efeitos dos fármacos , Homocisteína/farmacologia , Neurônios/efeitos dos fármacos , Receptores de N-Metil-D-Aspartato/metabolismo , Animais , Ativação Enzimática , Feminino , Transporte de Íons , Camundongos , Camundongos Knockout , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Neurônios/citologia , Fosforilação , Gravidez , Ratos , Ratos Sprague-Dawley , Receptores de N-Metil-D-Aspartato/genética
13.
Calcif Tissue Int ; 105(4): 446-457, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31250042

RESUMO

Homocysteine (Hcy) increases oxidation and inflammation; however, the mechanism of Hcy-induced bone fragility remains unclear. Because selective estrogen modulators (SERMs) have an anti-oxidative effect, SERMs may rescue the Hcy-induced bone fragility. We aimed to examine whether oxidative stress and pro-inflammatory cytokines such as interleukin (IL)-1ß and IL-6 are involved in the Hcy-induced apoptosis of osteocytes and whether bazedoxifene (BZA) inhibits the detrimental effects of Hcy. We used mouse osteocyte-like cell lines MLO-Y4-A2 and Ocy454. Apoptosis was examined by DNA fragmentation ELISA and TUNEL staining, and gene expression was evaluated by real-time PCR. Hcy 5 mM significantly increased expressions of NADPH oxidase (Nox)1, Nox2, IL-1ß, and IL-6 as well as apoptosis in MLO-Y4-A2 cells. Nox inhibitors, diphenyleneiodonium chloride and apocynin, significantly suppressed Hcy-induced IL-1ß and IL-6 expressions. In contrast, an IL-1ß receptor antagonist and an IL-6 receptor monoclonal antibody had no effects on Hcy-induced Nox1 and Nox2 expressions, but significantly rescued Hcy-induced apoptosis. BZA (1 nM-1 µM) and 17ß estradiol 100 nM significantly rescued Hcy-induced apoptosis, while an estrogen receptor blocker ICI 182,780 reversed the effects of BZA and 17ß estradiol. BZA also rescued Hcy-induced apoptosis of Ocy454 cell, and ICI canceled the effect of BZD. Moreover, BZA significantly ameliorated Hcy-induced expressions of Nox1, Nox2, IL-1ß, and IL-6, and ICI canceled the effects of BZA on their expressions. Hcy increases apoptosis through stimulating Nox 1 and Nox 2-IL-1ß and IL-6 expressions in osteocyte-like cells. BZA inhibits the detrimental effects of Hcy on osteocytes via estrogen receptor.


Assuntos
Apoptose/efeitos dos fármacos , Indóis/farmacologia , Interleucina-1beta/efeitos dos fármacos , Osteócitos/efeitos dos fármacos , Animais , Linhagem Celular , Homocisteína/farmacologia , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Camundongos , NADPH Oxidases/efeitos dos fármacos , Osteócitos/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos
14.
Nitric Oxide ; 90: 15-28, 2019 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-31146011

RESUMO

Neuro-inflammation plays a critical role in hyperhomocysteinemia (HHcy)-associated neurodegenerative disorders. Hydrogen sulfide (H2S) has been suggested as an endogenous neuromodulator and potent anti-inflammatory molecule. In present study, we have investigated the effect of NaHS supplementation (a H2S source) on inflammatory response in animals subjected to HHcy. NaHS adminstration restored the decreased levels of H2S and polysulfides with a concomitant increase in the activity of cystathionase (CSE) and cystathionine ß-synthase (CBS) in the brain regions of HHcy animals. NaHS supplementation reduced the expression of glial fibrillary acidic protein (GFAP) and ionized calcium binding adaptor molecule 1 (Iba1) suggesting attenuation of astrocyte and microglia activation in HHcy animals. Tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) were decreased in the cortex and hippocampus of HHcy animals following NaHS supplementation. Moreover, NaHS supplementation also decreased the TNF-α, IL-6 and MCP-1 in the serum of HHcy animals. NaHS supplementation reduced nitrite levels, 3-nitrotyrosine (3-NT) modified proteins and inducible nitric oxide synthase (iNOS) in the cortex and hippocampus of HHcy animals. However, NaHS administration increased endothelial nitric oxide synthase (eNOS) expression in brain regions of Hcy treated animals. Expression of platelet endothelial cell adhesion molecule (PECAM) was decreased in the microvessels from HHcy animals supplemented with NaHS. Furthermore, HHcy-induced memory deficits assessed by Morris water maze and novel object recognition test were reversed by NaHS administration. Taken together, the findings suggest that NaHS supplementation ameliorates Hcy-induced glia mediated inflammatory response and cognitive deficits. Therefore, H2S may be a novel therapeutic molecule to treat HHcy associated neurological disorders and neuro-inflammatory conditions.


Assuntos
Homocisteína/antagonistas & inibidores , Sulfeto de Hidrogênio/farmacologia , Inflamação/tratamento farmacológico , Neuroglia/efeitos dos fármacos , Animais , Homocisteína/farmacologia , Inflamação/metabolismo , Masculino , Neuroglia/metabolismo , Ratos , Ratos Sprague-Dawley
15.
Cell Signal ; 61: 66-77, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31085234

RESUMO

Cellular damage and accumulation of extracellular matrix (ECM) protein in the glomerulo-interstitial space are the signatures of chronic kidney disease (CKD). Hyperhomocysteinemia (HHcy), a high level of homocysteine (Hcy) is associated with CKD and further contributes to kidney damage. Despite a large number of studies, the signalling mechanism of Hcy-mediated cellular damage and ECM remodelling in kidney remains inconclusive. Hcy metabolizes to produce hydrogen sulphide (H2S), and a number of studies have shown that H2S mitigates the adverse effect of HHcy in a variety of diseases involving several signalling molecules, including forkhead box O (FOXO) protein. FOXO is a group of transcription factor that includes FOXO1, which plays important roles in cell growth and proliferation. On the other hand, a cell survival factor, Akt regulates FOXO under normal condition. However, the involvement of Akt/FOXO1 pathway in Hcy-induced mesangial cell damage remains elusive, and whether H2S plays any protective roles has yet to be clearly defined. We treated mouse mesangial cells with or without H2S donor, GYY4137 and FOXO1 inhibitor, AS1842856 in HHcy condition and determined the involvement of Akt/FOXO1 signalling cascades. Our results indicated that Hcy inactivated Akt and activated FOXO1 by dephosphorylating both the signalling molecules and induced FOXO1 nuclear translocation followed by activation of the FOXO1 transcription factor. These led to the induction of cellular apoptosis and synthesis of excessive ECM protein, in part, due to increased ROS production, loss of mitochondrial membrane potential (ΔΨm), reduction in intracellular ATP concentration, increased MMP-2, -9, -14 mRNA and protein expression, and Col I, IV and fibronectin protein expression. Interestingly, GYY4137 or AS1842856 treatment prevented these changes by modulating Akt/FOXO1 axis in HHcy. We conclude that GYY4137 and/or AS1842856 mitigates HHcy induced mesangial cell damage and ECM remodelling by regulating Akt/FOXO1 pathway.


Assuntos
Apoptose/efeitos dos fármacos , Matriz Extracelular/metabolismo , Proteína Forkhead Box O1/metabolismo , Homocisteína/farmacologia , Sulfeto de Hidrogênio/metabolismo , Células Mesangiais/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Linhagem Celular , Matriz Extracelular/efeitos dos fármacos , Proteína Forkhead Box O1/antagonistas & inibidores , Hiper-Homocisteinemia/tratamento farmacológico , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Morfolinas/farmacologia , Compostos Organotiofosforados/farmacologia , Quinolonas/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos
16.
Ann Hepatol ; 18(4): 633-639, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31078441

RESUMO

INTRODUCTION AND OBJECTIVES: Liver cirrhosis is characterized by increased intrahepatic resistance, splanchnic vasodilation/angiogenesis, and formation of portosystemic collateral vessels. Collaterals can cause lethal complications such as gastroesophageal variceal hemorrhage. Homocysteine is linked to vascular dysfunction and angiogenesis and higher levels have been reported in cirrhotic patients. It is also known that folic acid supplementation reverses the effects of homocysteine. However, the treatment effect in cirrhosis has yet to be investigated. MATERIAL AND METHODS: Liver cirrhosis was induced in Sprague-Dawley rats with common bile duct ligation (CBDL). The CBDL rats randomly received (1) vehicle; (2) dl-homocysteine thiolactone (1g/kg/day); (3) dl-homocysteine thiolactone plus folic acid (100mg/kg/day); or (4) folic acid. On the 29th day, hemodynamic parameters, liver and renal biochemistry, protein expressions of proangiogenic factors, mesenteric vascular density and portosystemic shunting were evaluated. RESULTS: In the cirrhotic rats, homocysteine increased mesenteric vascular density and the severity of shunting. It also up-regulated the protein expressions of mesenteric vascular endothelial growth factor (VEGF) and phosphorylated-endothelial nitric oxide synthase (p-eNOS). These effects were reversed by folic acid treatment (P<0.05). CONCLUSION: Folic acid ameliorated the adverse effects of homocysteine in the cirrhotic rats, which may be related to down-regulation of the VEGF-NO signaling pathway.


Assuntos
Circulação Colateral/efeitos dos fármacos , Ácido Fólico/farmacologia , Homocisteína/análogos & derivados , Cirrose Hepática/fisiopatologia , Neovascularização Patológica/induzido quimicamente , Sistema Porta/efeitos dos fármacos , Circulação Esplâncnica/efeitos dos fármacos , Complexo Vitamínico B/farmacologia , Animais , Ducto Colédoco , Hemodinâmica/efeitos dos fármacos , Homocisteína/farmacologia , Ligadura , Cirrose Hepática/complicações , Neovascularização Patológica/etiologia , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Óxido Nítrico Sintase Tipo III/efeitos dos fármacos , Óxido Nítrico Sintase Tipo III/metabolismo , Fosforilação , Sistema Porta/patologia , Ratos , Fator A de Crescimento do Endotélio Vascular/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/metabolismo
17.
J Cell Mol Med ; 23(7): 4611-4626, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31104361

RESUMO

It is well-established that homocysteine (Hcy) is an independent risk factor for atherosclerosis. Hcy can promote vascular smooth muscle cell (VSMC) proliferation, it plays a key role in neointimal formation and thus contribute to arteriosclerosis. However, the molecular mechanism on VSMCs proliferation underlying atherosclerosis is not well elucidated. Mitofusin-2 (MFN2) is an important transmembrane GTPase in the mitochondrial outer membrane and it can block cells in the G0/G1 stage of the cell cycle. To investigate the contribution of aberrant MFN2 transcription in Hcy-induced VSMCs proliferation and the underlying mechanisms. Cell cycle analysis revealed a decreased proportion of VSMCs in G0/G1 and an increased proportion in S phase in atherosclerotic plaque of APOE-/- mice with hyperhomocystinaemia (HHcy) as well as in VSMCs exposed to Hcy in vitro. The DNA methylation level of MFN2 promoter was obviously increased in VSMCs treated with Hcy, leading to suppressed promoter activity and low expression of MFN2. In addition, we found that the expression of c-Myc was increased in atherosclerotic plaque and VSMCs treated with Hcy. Further study showed that c-Myc indirectly regulates MFN2 expression is duo to the binding of c-Myc to DNMT1 promoter up-regulates DNMT1 expression leading to DNA hypermethylation of MFN2 promoter, thereby inhibits MFN2 expression in VSMCs treated with Hcy. In conclusion, our study demonstrated that Hcy-induced hypermethylation of MFN2 promoter inhibits the transcription of MFN2, leading to VSMCs proliferation in plaque formation, and the increased binding of c-Myc to DNMT1 promoter is a new and relevant molecular mechanism.


Assuntos
Aterosclerose/genética , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , GTP Fosfo-Hidrolases/genética , Homocisteína/farmacologia , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/patologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , Transcrição Genética , Animais , Aterosclerose/patologia , Sequência de Bases , Proliferação de Células/efeitos dos fármacos , GTP Fosfo-Hidrolases/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Camundongos Endogâmicos C57BL , Modelos Biológicos , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Placa Aterosclerótica/patologia , Regiões Promotoras Genéticas/genética , Ligação Proteica/efeitos dos fármacos , Transcrição Genética/efeitos dos fármacos
18.
Physiol Int ; 106(1): 29-38, 2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30888218

RESUMO

PURPOSE: We previously found that homocysteine (Hcy)-induced apoptosis in endothelial cells coincided with increased NADPH oxidase (NOX) activity. In addition, in ischemic endothelial cells present in the heart, we showed that loss of serine protease dipeptidyl peptidase IV (DPP4) expression was correlated with induction of tissue factor (TF) expression. Since Hcy can initiate thrombosis through the induction of TF expression, in this study, we evaluated whether the inverse relation of TF and DPP4 is also Hcy-dependent and whether NOX-mediated reactive oxygen species (ROS) is playing a role herein. METHODS: Human umbilical vein endothelial cells (HUVECs) were incubated with 2.5 mM Hcy for 3 and 6 h. The effects of Hcy on DPP4 and TF expression and NOX2/p47phox-mediated nitrotyrosine (ROS) production were studied using digital-imaging microscopy. RESULTS: In HUVECs, high levels of Hcy showed a significant increase of TF expression and a concomitant loss of DPP4 expression after 6 h. In addition, NOX subunits NOX2 and p47phox were also significantly increased after 6 h of Hcy incubation and coincided with nitrotyrosine (ROS) expression. Interestingly, inhibition of NOX-mediated nitrotyrosine (ROS) with the use of apocynin not only reduced these effects, but also counteracted the effects of Hcy on TF and DPP4 expression. CONCLUSION: These results indicate that the inverse relation of TF and DPP4 in endothelial cells is also Hcy-dependent and related to NOX activity.


Assuntos
Dipeptidil Peptidase 4/metabolismo , Homocisteína/farmacologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , NADPH Oxidases/metabolismo , Tromboplastina/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Oxirredução , Espécies Reativas de Oxigênio/metabolismo
19.
J Cell Physiol ; 234(10): 18602-18614, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-30912146

RESUMO

Homocysteine (Hcy) is detrimental to bone health in a mouse model of diet-induced hyperhomocysteinemia (HHcy). However, little is known about Hcy-mediated osteoblast dysfunction via mitochondrial oxidative damage. Hydrogen sulfide (H2 S) has potent antioxidant, anti-inflammatory, and antiapoptotic effects. In this study, we hypothesized that the H2 S mediated recovery of osteoblast dysfunction by maintaining mitochondrial biogenesis in Hcy-treated osteoblast cultures in vitro. MC3T3-E1 osteoblastic cells were exposed to Hcy treatment in the presence or absence of an H2 S donor (NaHS). Cell viability, osteogenic differentiation, reactive oxygen species (ROS) production were determined. Mitochondrial DNA copy number, adenosine triphosphate (ATP) production, and oxygen consumption were also measured. Our results demonstrated that administration of Hcy increases the intracellular Hcy level and decreases intracellular H2 S level and expression of the cystathionine ß-synthase/Cystathionine γ-lyase system, thereby inhibiting osteogenic differentiation. Pretreatment with NaHS attenuated Hcy-induced mitochondrial toxicity (production of total ROS and mito-ROS, ratio of mitochondrial fission (DRP-1)/fusion (Mfn-2)) and restored ATP production and mitochondrial DNA copy numbers as well as oxygen consumption in the osteoblast as compared with the control, indicating its protective effects against Hcy-induced mitochondrial toxicity. In addition, NaHS also decreased the release of cytochrome c from the mitochondria to the cytosol, which induces cell apoptosis. Finally, flow cytometry confirmed that NaHS can rescue cells from apoptosis induced by Hcy. Our studies strongly suggest that NaHS has beneficial effects on mitochondrial toxicity, and could be developed as a potential therapeutic agent against HHcy-induced mitochondrial dysfunction in cultured osteoblasts in vitro.


Assuntos
Homocisteína/farmacologia , Sulfeto de Hidrogênio/farmacologia , Mitocôndrias/patologia , Osteoblastos/patologia , Animais , Apoptose/efeitos dos fármacos , Calcificação Fisiológica/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteogênese/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo
20.
Methods Mol Biol ; 1866: 13-26, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30725404

RESUMO

Many different types of cancer cells have been shown to be methionine (MET) dependent. Cancer cells, unlike normal cells, grow poorly or not at all when MET is restricted. Cancer cells have an elevated requirement for exogenous MET for growth, despite high levels of endogenous synthesis. This requirement reflects increased utilization of MET by cancer cells, analogous to increased utilization glucose by cancer cells (Warburg effect). To answer the critical question of whether MET-dependent cancer cells synthesize normal amounts of MET, we determined the levels of MET, S-adenosylmethionine (AdoMET), and S-adenosylhomocysteine (AdoHCY) that were synthesized by MET-dependent cancer cells under conditions of MET restriction. We demonstrated that MET-dependent cells synthesize a normal amount of endogenously synthesized MET but are still deficient in AdoMET. In contrast, exogenously supplied MET results in normal AdoMET levels. The ratio of AdoMET to AdoHCY is low in MET-dependent cells growing in MET-restricted medium but is normal when MET is supplied. Under conditions of MET restriction, the low AdoMET/AdoHCY ratio probably limits proliferation of MET-dependent cancer cells. The amount of free MET is also low in MET-dependent cancer cells under MET restriction. The elevated MET requirement for cancer cells may be due to enhanced overall rates of transmethylation compared to normal human cells. Thus, MET-dependent cancer cells have low levels of free MET, low levels of AdoMET, and elevated levels of AdoHCY under conditions of MET restriction probably due to overuse of MET for transmethylation reactions ("Hoffman effect"), thereby blocking cellular proliferation.


Assuntos
Metionina/metabolismo , Neoplasias/metabolismo , 5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Homocisteína/farmacologia , Humanos , Metionina/deficiência , Metilação , Neoplasias/enzimologia , Neoplasias/patologia , S-Adenosil-Homocisteína/metabolismo , S-Adenosilmetionina/metabolismo
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