红景天和阿魏酸激活不同年龄小鼠自噬和热休克抵抗相关基因的表达

O. Demianchuk
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The levels of messenger ribonucleic acid (mRNA) were assessed using AriaMx real-time polymerase chain reaction (RT-PCR) instrument (Agilent). Ribonucleic acid was purified using the Monarch Miniprep kit (New England BioLabs (NEB), T2010), complementary deoxyribonucleic acid synthesis was performed using the ProtoScript II kit (NEB, E6560), and quantitative RT-PCR (qRT-PCR) was performed using the Luna Universal kit (NEB, E3003). The expression of genes ATG5 (an autophagy marker), HSPB8 (a small heat shock protein, an FoxO target), UCP2 (uncoupling protein 2, a senescence marker), CDKN2 (cell cycle regulator, a senescence marker), PDK2 and PDK4 (pyruvate dehydrogenase kinases 2 and 4, regulators of oxidative metabolism), and TFEB (transcription factor EB, a transcriptional regulator of autophagy) was evaluated. Results. Livers of young mice that consumed food supplemented with either sodium ferulate or R. rosea extract had 3.2-fold and 3.6-fold higher levels of mRNA of the small heat shock protein HspB8 than control mice, respectively. In old mice, the levels of mRNA for this protein were 3.3-fold higher in mice reared on the diet containing R. rosea extract as compared with the control. However, there was no significant difference between control mice and those that consumed ferulate-supplemented food. In young mice, ferulate and R. rosea extract induced synthesis of mRNA of PDK4 by 4.3 and 6.6 times from the control level, respectively. Ferulate and R. rosea extract also affected the levels of mRNA of ATG5 and PDK2 in the livers of old mice. The levels of PDK2 were 1.5-fold higher in the livers of mice that consumed ferulate-supplemented food than in control mice. Conclusions. 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引用次数: 0

摘要

我们研究的目的是测试玫瑰红提取物和阿魏酸是否激活年轻和老年小鼠肝脏中FoxO靶点、能量代谢和自噬调节因子的表达,以及玫瑰红提取物与阿魏酸对所研究基因的影响在多大程度上一致。方法。C57BL/6J小鼠在22±2°С、50-60%湿度和12/12小时光/暗周期下饲养。所有组均采用标准食物(脂肪含量为4.8%,蛋白质含量为21.8%,纤维含量为3.9%)饲养。实验组在处死前12周饮水,补充阿魏酸钠或玫瑰红。调节阿魏酸盐和玫瑰红的量,以在24小时内为小鼠提供每100g重量4mg含酚物质。我们测试了三个月大(“幼”)和十二个月大的雄性(“老”)。信使核糖核酸(mRNA)的水平使用AriaMx实时聚合酶链式反应(RT-PCR)仪器(安捷伦)进行评估。使用Monarch Miniprep试剂盒(新英格兰生物实验室(NEB),T2010)纯化核糖核酸,使用ProtoScript II试剂盒(NEB,E6560)进行互补脱氧核糖核酸合成,并使用Luna Universal试剂盒(NEX,E3003)进行定量RT-PCR(qRT-PCR)。评估基因ATG5(自噬标记物)、HSPB8(小热休克蛋白,FoxO靶点)、UCP2(解偶联蛋白2,衰老标记物),CDKN2(细胞周期调节因子,衰老标记)、PDK2和PDK4(丙酮酸脱氢酶激酶2和4,氧化代谢调节因子)以及TFEB(转录因子EB,自噬转录调节因子)的表达。后果食用补充有阿魏酸钠或玫瑰红提取物的食物的年轻小鼠的肝脏中,小热休克蛋白HspB8的mRNA水平分别是对照小鼠的3.2倍和3.6倍。在老年小鼠中,与对照组相比,在含有玫瑰红提取物的饮食中饲养的小鼠中,这种蛋白质的mRNA水平高3.3倍。然而,对照小鼠和食用添加了阿魏酸的食物的小鼠之间没有显著差异。在年轻小鼠中,阿魏酸和玫瑰红提取物诱导PDK4 mRNA的合成分别是对照水平的4.3倍和6.6倍。Ferulate和R.rosea提取物也影响老年小鼠肝脏中ATG5和PDK2的mRNA水平。摄入阿魏酸补充食物的小鼠肝脏中PDK2的水平是对照小鼠的1.5倍。结论。玫瑰红提取物及其活性成分之一阿魏酸都能促进HSPB8和PDK4基因mRNA水平的增加,HSPB8基因和PDK4分别编码小热休克蛋白和丙酮酸脱氢酶激酶4。在老年小鼠中,玫瑰红促进HSPB8、ATG5、PDK2和PDK4的表达。因此,阿魏酸和玫瑰红通过激活热休克反应和自噬对基因表达产生类似的影响,并通过促进PDK2和PDK4的表达抑制线粒体代谢。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
RHODIOLA ROSEA AND FERULIC ACID ACTIVATE EXPRESSION OF GENES RELATED TO AUTOPHAGY AND RESISTANCE TO HEAT SHOCK IN MICE OF DIFFERENT AGE
The aim of our study was testing whether R. rosea extract and ferulic acid activate expression of targets of FoxO, regulators of energy metabolism and autophagy in livers of young and old mice, and to what extent the effects of R. rosea extract and ferulic acid on the genes studied coincide. Methods. . C57BL/6J mice were reared at 22 ± 2 °С, 50-60% humidity, and 12/12 hour light/dark cycle. All groups were reared on a standard chow (4,8% fats, 21,8% protein, and 3,9% fibre). Experimental groups consumed water, supplemented with either sodium ferulate or R. rosea during 12 weeks prior sacrificing. The amounts of ferulate and R. rosea were adjusted to provide 4 mg of phenol-containing substances per 100 g weight, for a mouse, for 24 hours. We tested three-month-old (“young”) and twelve-month-old males (“old”). The levels of messenger ribonucleic acid (mRNA) were assessed using AriaMx real-time polymerase chain reaction (RT-PCR) instrument (Agilent). Ribonucleic acid was purified using the Monarch Miniprep kit (New England BioLabs (NEB), T2010), complementary deoxyribonucleic acid synthesis was performed using the ProtoScript II kit (NEB, E6560), and quantitative RT-PCR (qRT-PCR) was performed using the Luna Universal kit (NEB, E3003). The expression of genes ATG5 (an autophagy marker), HSPB8 (a small heat shock protein, an FoxO target), UCP2 (uncoupling protein 2, a senescence marker), CDKN2 (cell cycle regulator, a senescence marker), PDK2 and PDK4 (pyruvate dehydrogenase kinases 2 and 4, regulators of oxidative metabolism), and TFEB (transcription factor EB, a transcriptional regulator of autophagy) was evaluated. Results. Livers of young mice that consumed food supplemented with either sodium ferulate or R. rosea extract had 3.2-fold and 3.6-fold higher levels of mRNA of the small heat shock protein HspB8 than control mice, respectively. In old mice, the levels of mRNA for this protein were 3.3-fold higher in mice reared on the diet containing R. rosea extract as compared with the control. However, there was no significant difference between control mice and those that consumed ferulate-supplemented food. In young mice, ferulate and R. rosea extract induced synthesis of mRNA of PDK4 by 4.3 and 6.6 times from the control level, respectively. Ferulate and R. rosea extract also affected the levels of mRNA of ATG5 and PDK2 in the livers of old mice. The levels of PDK2 were 1.5-fold higher in the livers of mice that consumed ferulate-supplemented food than in control mice. Conclusions. Both, R. rosea extract and one of its active components – ferulic acid – promote increasing in the levels of mRNA for genes HSPB8 and PDK4, coding for small heat shock protein and pyruvate dehydrogenase kinase 4, respectively. In old mice, R. rosea promote expression of HSPB8, ATG5, PDK2, and PDK4. Thus, ferulic acid and R. rosea exert similar effects on gene expression by supposed activation of heat shock response and autophagy, and concomitant inhibition of mitochondrial metabolism via boosting expression of PDK2 and PDK4.
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