β-胍基丙酸对果蝇寿命的影响及其机制

发布时间：2018-03-08 18:15

  本文选题：β-胍基丙酸　切入点：果蝇　出处：《华中科技大学》2015年博士论文　论文类型：学位论文

【摘要】：第一部分β-胍基丙酸对果蝇寿命的影响 目的：β一胍基丙酸(p-guanadinopropionic acid, β-GPA)是肌酸类似物,可以慢性激活AMPK,同时还可以增加线粒体酶的活性,改善线粒体功能。由于AMPK的慢性激活和线粒体功能的增强可以延缓衰老,延长寿命。因此,β-GPA极可能有抗衰老,延长寿命的作用。因此,本实验研究β-GPA对果蝇寿命的影响。 方法：在果蝇培养基中添加浓度分别为300、900和2700mM的β-GPA,观察果蝇寿命的变化；分别采用只含有琼脂的培养基来模拟饥饿环境,采用含有3MH202的培养基模拟强氧化环境,观察加入含有β-GPA的培养基饲养30天后,果蝇在这两种环境中的存活情况；采用羟胺法测定SOD活性,采用硫代巴比安酸(TBA)比色法测定MDA的含量,观察β-GPA对果蝇SOD活性和MDA含量的影响。 结果：(1)浓度为900和2700mM的β-GPA能够显著延长果蝇(雌性和雄性)寿命。900mM和2700mM的β-GPA分别将雌性果蝇寿命由60d延长至65d和68d(n=200,p0.001)；将雄性果蝇的寿命由55d延长至59d和60d(n=200,p0.001)。(2)浓度为900mM的β-GPA能够显著延长果蝇在饥饿环境下的存活时间。900mM的β-GPA将饥饿环境下雌性和雄性果蝇的寿命均从5d延长至7d(n=100,p0.001),增强了果蝇对应激的抵抗力。(3)浓度为900mM的β-GPA能够显著延长果蝇在强氧化环境下存活时间。900mM β-GPA将强氧化环境下雌性和雄性果蝇的寿命均从1d延长至2d(n=100,p0.001),并显著增加果蝇SOD活性,降低MDA含量,增强果蝇的抗氧化能力。 结论：β-GPA浓度依赖性的延长果蝇的寿命,且增加果蝇对不同应激的抵抗能力,增强其抗氧化能力。 第二部分β-胍基丙酸延长果蝇寿命的机制 目的：腺苷酸活化蛋白激酶(adenosine5'-monophosphate (AMP)-activated protein kinase, AMPK)的激活可以延缓衰老,延长寿命。p-GPA可以慢性激活AMPK,且后者可以通过调节哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin, mTOR)和unc-51样激酶(Unc-51like kinase1, ULK1/Atg1)的活性对自噬进行调控。最近研究发现,自噬和衰老,寿命密切相关。但尚不清楚β-GPA是否通过激活AMPK来增强自噬,从而发挥延长寿命的作用。本实验旨在对β-GPA延长果蝇寿命的分子机制进行探讨。 方法：Western blotting实验检测β-GPA对果蝇中AMPK磷酸化和自噬相关指标Atg5和p62的表达；分别采用药理学方法和RNA干扰技术降低果蝇中AMPK和自噬关键分子Atg1和Atg5的表达后,观察β-GPA对果蝇寿命的影响；采用药理学方法和分子生物学方法抑制AMPK后,β-GPA对自噬相关指标和与延长寿命作用密切相关蛋白的影响。 结果：(1)浓度为900和2700mM的β-GPA能够显著增加果蝇各个部分AMPK的活性。果蝇各部位AMPK的磷酸化水平分别为：头部：对照：100±13.54,900mM β-GPA:143.36±12.2,2700mMβ-GPA:147.13±8.54(n=6, p0.05);胸部：对照：100±15.49,900mM β-GPA:155.14±17.53,2700mM β-GPA:155.78±14.59(n=6,p0.05)；腹部：对照：100±13.35,900mM β-GPA:152.91±15.8,2700mM P-GPA:152.4±16.14(n=6, p0.05)。(2)浓度为900的β-GPA能够显著增加果蝇自噬水平：下调Atg5,降低果蝇自噬水平,P-GPA处理后使雌性果蝇寿命由62d下降至51d(n=200,p0.001),雄性果蝇寿命由59d减少至51d(n=200,p0.001),说明下调Atg5水平取消了P-GPA延长寿命的作用。(3)降低果蝇AMPK活性,β-GPA诱导的自噬增加作用消失,并且AMPK-RNAi使雌性果蝇寿命由63d下降至51d(n=200,p0.001),雄性果蝇寿命由57d减少至51d(n=200,p0.001),表明给予AMPK-RNAi阻碍了β-GPA延长果蝇寿命的作用,提示β-GPA的这些作用依赖于AMPK。(4)Atg1参与调节β-GPA诱导的自噬增加和寿命延长。(5) β-GPA可以通过激活AMPK抑制mTORC1的活性。 结论：β-GPA延长果蝇寿命作用是依赖于AMPK-Atg1-autophagy信号通路。
[Abstract]:Part 1 Effect of beta guanidinopropionic acid on lifespan of Drosophila melanogaster
Objective: beta guanidinopropionic acid (p-guanadinopropionic acid, P -GPA) is creatine analogs can be chronic activation of AMPK, but also can increase the activity of mitochondrial enzymes and improve mitochondrial function. Due to the enhancement of AMPK and chronic activation of mitochondrial function can delay aging, prolong life expectancy. Therefore, beta -GPA probably anti-aging to extend the life of the role. Therefore, this research studied the effect of beta -GPA on the lifespan of Drosophila melanogaster.
Methods: in Drosophila medium concentration were 300900 and 2700mM beta -GPA, to observe the change of the life span of Drosophila; using medium to simulate the hunger environment containing only agar medium containing 3MH202, the simulation of strong oxidizing environment, to observe the beta -GPA culture medium after 30 days of feeding, Drosophila survival in these two environments; SOD activity was determined by hydroxylamine method, using thio BABIAN acid (TBA) was measured by MDA colorimetry, observe the effect of -GPA on Drosophila beta SOD activity and MDA content.
Results: (1) the concentration of 900 2700mM and beta -GPA could prolong the life of Drosophila melanogaster (male and female).900mM and 2700mM -GPA respectively in female Drosophila beta extended from 60d to 65D and 68D (n=200, p0.001); the male Drosophila lifespan was extended from 55D to 59D and 60d (n=200, p0.001). (2) the survival time of.900mM concentration of 900mM beta -GPA can significantly prolong Drosophila hunger in the environment of the beta -GPA hunger environment of female and male Drosophila life are extended from 5D to 7d (n=100, p0.001), enhanced resistance to stress in Drosophila. (3) the concentration of 900mM beta -GPA could significantly prolong the survival time of.900mM beta -GPA in strong oxidizing environment the strong oxidation environment of female and male Drosophila life are extended from 1D to 2D (n=100, p0.001), Drosophila and Drosophila significantly increased SOD activity, decreased the content of MDA, enhance the antioxidant ability of Drosophila melanogaster.
Conclusion: the concentration of beta -GPA can prolong the life of Drosophila melanogaster, and increase the resistance of Drosophila melanogaster to different stress and enhance its antioxidant capacity.
The second part mechanism bgpa extend the life of Drosophila
Objective: adenosine monophosphate activated protein kinase (adenosine5'-monophosphate (AMP) -activated protein kinase, AMPK) activation can delay aging, prolong the life of chronic.P-GPA can activate AMPK, and the latter can be adjusted by the mammalian target of rapamycin (mammalian target of rapamycin, mTOR) and UNC-51 (Unc-51like kinase1, ULK1/Atg1 like kinase) activity regulation of autophagy was. A recent study found that autophagy and aging is closely related to life. But it is not clear whether beta -GPA through activation of AMPK enhanced autophagy, which play a role. To extend the life of the molecular mechanism of this experiment is to extend the life of Drosophila beta on -GPA.
Methods: the expression of Western blotting beta -GPA detection experiment of Drosophila AMPK phosphorylation and autophagy related indicators of Atg5 and p62 respectively; expression by pharmacological method and RNA interference technology to reduce AMPK and autophagy in Drosophila key molecules Atg1 and Atg5, to observe the beta -GPA on Drosophila life influence; inhibition of AMPK by pharmacological and molecular methods biological methods, effects of beta -GPA related indicators on autophagy and closely related with the longevity protein.
Results: (1) the concentration of 900 2700mM and beta -GPA could significantly increase the activity of AMPK. Each part of the Drosophila flies in different parts of the phosphorylation level of AMPK are: head of control: 100 + 13.54900mM + 12.22700mM + -GPA:147.13 beta -GPA:143.36 beta 8.54 (n=6, P0.05); chest: control: 100 + 15.49900mM beta -GPA:155.14 + 17.532700mM + -GPA:155.78 beta 14.59 (n=6, P0.05); abdomen: control: 100 + 13.35900mM + 15.82700mM + P-GPA:152.4 -GPA:152.91 beta 16.14 (n=6, P0.05). (2) the concentration of 900 P -GPA can significantly increase the level of autophagy in Drosophila: Atg5, Drosophila reduced autophagy, after P-GPA treatment to female flies life decreased from 62D to 51D (n=200, p0.001), male Drosophila melanogaster decreased from 59D to 51D (n=200, p0.001), that lowered the level of Atg5 cancelled the life extension of the P-GPA effect. (3) reduce Drosophila AMPK activity, beta -GPA induced autophagy Increase the effect disappeared, and AMPK-RNAi make the female Drosophila life decreased from 63d to 51D (n=200, p0.001), male Drosophila melanogaster decreased from 57d to 51D (n=200, p0.001), suggested that AMPK-RNAi blocked beta -GPA extend the life of Drosophila, the role of beta -GPA depends on AMPK. (4) Atg1 is involved in the regulation of beta -GPA induced autophagy and increase life expectancy. (5) beta -GPA can activate AMPK to inhibit mTORC1 activity.
Conclusion: the effect of beta -GPA on prolonging the life span of Drosophila is dependent on the AMPK-Atg1-autophagy signaling pathway.

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R965

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