RESUMO
EndMT plays an important role in the relationship between endothelial dysfunction and atherosclerosis. This work will elucidate the biofunction induced by miR-449a and lipid rafts in EndMT and development of atherosclerosis. The differential miRNA expression between atherosclerotic plaques and normal arteries were analyzed. The luciferase activities of AdipoR2 3' UTR treated with miR-449a were determined. ECs were dealt with miR-449a mimics or inhibitors, then cell proliferation and migration were assessed. Moreover, the expression of AdipoR2 and mesenchymal cell markers were analyzed. The influences of lipid rafts related to reciprocity between E-cadherin and AdipoR2 on TNF-α-induced damage in ECs were investigated. ApoE KO diabetic mice were used to explore the potential roles of miR-449a on atherosclerosis. Our results indicated that compared with normal arteries, 17 miRNAs were upregulated and 3 miRNAs were down-regulated in atherosclerotic plaques. The relative expression of miR-449a in plaques was significantly higher than that in normal arteries. MiR-449a suppressed AdipoR2 expression, additionally its interaction protein E-cadherin in ECs. MiR-449a enhanced expression of mesenchymal cell markers, induced cell proliferation and migration of ECs, regulated the interaction between E-cadherin and AdipoR2 interceded by lipid rafts. The miR-449a antagomir could protect against the development process of atherosclerosis in ApoE KO diabetic mice. In conclusion, miR-449a targeted to AdipoR2, and was a crucial mediator of EndMT and atherosclerosis in ECs through regulating E-cadherin bindability with AdipoR2 in lipid rafts. These results suggested that aim to lipid rafts and miR-449a in chronic EC inflammation response, was a feasible therapy strategy for atherosclerosis.
Assuntos
Aterosclerose/metabolismo , Caderinas/metabolismo , Transição Epitelial-Mesenquimal/fisiologia , Microdomínios da Membrana/metabolismo , MicroRNAs/biossíntese , Receptores de Adiponectina/biossíntese , Animais , Aterosclerose/patologia , Caderinas/antagonistas & inibidores , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Humanos , Masculino , Microdomínios da Membrana/patologia , Camundongos , Camundongos Knockout , Receptores de Adiponectina/antagonistas & inibidoresRESUMO
Effective drugs and strategies for treating type 2 diabetes mellitus (2DM) are urgently required. The aim of the present study was to elucidate the mechanism underlying microRNA (miR)68353p regulation of adiponectin receptor 1 (AdipoR1) expression and the miR68353p/AdipoR1 signaling pathway in pancreatic islet cells. In addition, the potential antidiabetes effect of miR68353p on insulin secretion was investigated. Luciferase activity analysis was performed to evaluate how miR68353p targets the 3'untranslated region of AdipoR1. The SU.86.86 and MIN6 cell lines were cocultured with or without miR68353p inhibitors or mimics, and the insulin secretory functions of these cell lines were then determined. Luciferase reporter analysis revealed that AdipoR1 was a direct target of miR68353p. In addition, miR68353p overexpression suppressed the mRNA and protein expression levels of AdipoR1 in the SU.86.86 and MIN6 cell lines. Furthermore, miR68353p exerted negative effects on insulin secretion in SU.86.86 and MIN6 cells, which were mediated by regulating AdipoR1 expression. AdipoR1 was a direct target of miR68353p; therefore, inhibition of AdiopR1 expression may reduce insulin secretion and may be considered a key regulator of insulin secretion. The results of the present study suggested that targeting AdipoR1 with miR68353p inhibitors may be a potential strategy for promoting glucosestimulated insulin secretion, and thereby, may be an effective treatment for type 2DM.
Assuntos
RNA Mensageiro/genética , Receptores de Adiponectina/metabolismo , Regiões 3' não Traduzidas/genética , Western Blotting , Linhagem Celular Tumoral , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Regulação Neoplásica da Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Insulina/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , MicroRNAs/fisiologia , RNA Interferente Pequeno/genética , Receptores de Adiponectina/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologiaRESUMO
The urine excretion of L-carnitine (LC), acetyl-L-carnitine (ALC) and propionyl-Lcarnitine (PLC) and their relations with the antioxidant activities are presently unknown. Liquid L-carnitine (2.0 g) was administered orally as a single dose in 12 healthy subjects. Urine concentrations of LC, ALC and PLC were detected by HPLC. Superoxide dismutase (SOD), total antioxidative capacity (T-AOC), malondialdehyde (MDA) and nitrogen monoxidum (NO) activities were measured by spectrophotometric methods. The 0~2 h, 2~4 h, 4~8 h, 8~12 h, 12~24 h excretion of LC was 53.13±31.36 µmol, 166.93±76.87 µmol, 219.92±76.30 µmol, 100.48±23.89 µmol, 72.07±25.77 µmol, respectively. The excretion of ALC was 29.70±14.43 µmol, 80.59±32.70 µmol, 109.85±49.21 µmol, 58.65±18.55 µmol, and 80.43±35.44 µmol, respectively. The urine concentration of PLC was 6.63±4.50 µmol, 15.33±12.59 µmol, 15.46±6.26 µmol, 13.41±11.66 µmol and 9.67±7.92 µmol, respectively. The accumulated excretion rate of LC was 6.1% within 24h after its administration. There was also an increase in urine concentrations of SOD and T-AOC, and a decrease in NO and MDA. A positive correlation was found between urine concentrations of LC and SOD (r = 0.8277) or T-AOC (r = 0.9547), and a negative correlation was found between urine LC excretions and NO (r = -0.8575) or MDA (r = 0.7085). In conclusion, a single oral LC administration let to a gradual increase in urine L-carnitine excretion which was associated with an increase in urine antioxidant enzymes and the total antioxidant capacities. These data may be useful in designing therapeutic regimens of LC or its analogues in the future.
A excreção urinária de L-carnitina (LC), acetil-L-carnitina (ALC) e propionil-L-carnitine (PLC) e as suas relações com as atividades antioxidantes são presentemente desconhecidos. Líquido de L-carnitina (2,0 g) foi administrada por via oral como uma dose única em 12 indivíduos saudáveis. As concentrações urinárias de LC, PLC e ALC foram detectados por HPLC. Atividades superóxido dismutase (SOD), a capacidade antioxidante total (T-AOC), malondialdeído (MDA) e óxido nítrico (NO) foram medidas por métodos espectrofotométricos. O 0~2 h, 2~4 h, 4~8 h, 8~12 h, 12~24 h excreção de LC foi 53,13±31.36 µmol, 166,93±76.87 µmol, 219,92±76.30 µmol, 100,48±23.89 µmol, 72,07±25.77 µmol, respectivamente. A excreηão de ALC foi 29,70±14.43 µmol, 80,59±32.70 µmol, 109,85±49.21 µmol, 58,65±18.55 µmol, e 80,43±35.44 µmol, respectivamente. A concentraηão de urina de PLC foi 6,63±4.50 µmol, 15,33±12.59 µmol, 15,46±6.26 µmol, 13,41±11.66 µmol e 9,67±7.92 µmol, respectivamente. A taxa de excreηão acumulada de LC foi de 6,1% 24 horas após sua administração. Houve também um aumento nas concentrações de urina de SOD e T-COA e diminuição de NO e de MDA. Correlação positiva foi encontrada entre as concentrações de urina de LC e SOD (r = 0,8277) ou T-AOC (r = 0,9547) e correlação negativa entre a excreção de LC e NO (r = -0,8575) ou MDA (r = 0,7085). Em conclusão, a administração oral única de LC leva ao aumento gradual na excreção urinária de L-carnitina, que foi associada com o aumento das enzimas antioxidantes na urina e as capacidades antioxidantes totais. Estes dados podem ser úteis no futuro para o planejamento de esquemas terapêuticos de LC ou os seus análogos, no futuro.