Anti-aging Effect of Ginseng Extract and Its Mechanism on Drosophila melanogaster
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摘要: 目的：基于果蝇模式生物，探讨人参提取物对果蝇衰老的延缓作用及其机制。方法：通过高效液相色谱法分析人参提取物中人参皂苷成分组成；将野生型Canton-S果蝇随机分成对照组及人参提取物组，通过寿命实验及检测摄食量、甘油三酯水平、繁殖能力、运动能力，以及在饥饿、热应激以及氧化应激条件下果蝇的存活时间来分析人参提取物的抗衰老能力；通过聚合酶链式反应测定腺苷酸活化蛋白激酶（AMP-activated protein kinase，AMPK）通路及哺乳动物雷帕霉素靶蛋白（mammalian target of rapamycin，mTOR）通路相关基因转录水平。结果：人参提取物中含13种人参皂苷成分。与对照组相比，7.5 mg/mL人参提取物对果蝇的延寿率达9.72%，且不显著影响其摄食量、甘油三酯水平以及产卵量（P>0.05），能够显著提高果蝇的攀爬能力（P<0.05或P<0.01），可显著增加果蝇抵抗压力应激能力（P<0.05或P<0.01）。与对照组相比，人参提取物的摄入显著提高了AMPKα、FoxO、Sirt1、LKB1和CaMKII的转录水平（P<0.05或P<0.01），显著降低HDAC4和SREBP的转录水平（P<0.05）、提高ATP含量和ATP/ADP比值（P<0.05或P<0.01）；显著降低mTOR通路相关基因PI3K、TORC、4E-BP和S6K的转录水平（P<0.05或P<0.01），极显著提高Atg1、Atg5、Atg8a和Atg8b的转录水平（P<0.01）。结论：人参提取物可通过激活AMPK通路并抑制mTOR通路延缓果蝇衰老。Abstract: Objective: Based on the model organism of Drosophila melanogaster, the anti-aging effect of ginseng extract and its mechanism on Drosophila melanogaster were investigated. Methods: The composition of ginsenosides in ginseng extract were analyzed by HPLC. Wild Canton-S Drosophila melanogaster were randomly divided into control group and ginseng extract group. The life span test, food intake, triglyceride level, reproductive ability, exercise ability, and the survival time of Drosophila melanogaster under starvation, heat stress and oxidative stress were applied to analyze the anti-aging function of ginseng extract. The transcription levels of genes related to AMP-activated protein kinase (AMPK) pathway and mammalian target of rapamycin (mTOR) pathway were determined by polymerase chain reaction. Results: Ginseng extract contained 13 ginsenosides. Compared with the control group, 7.5 mg/mL ginseng extract could prolong the life span of Drosophila melanogaster by 9.72%, and had no significant effect on the food intake, triglyceride level and fecundity (P>0.05), significantly improved the climbing ability (P<0.05 or P<0.01), and increased the ability to resist stress (P<0.05 or P<0.01). Compared with the control group, the intake of ginseng extract significantly increased the transcript levels of AMPKα, FoxO, Sirt1, LKB1 and CaMKII (P<0.05 or P<0.01), decreased the transcript levels of HDAC4 and SREBP (P<0.05), and enhanced ATP content and ATP/ADP ratio (P<0.05 or P<0.01). In addition, ginseng extract significantly decreased the transcription levels of mTOR-related genes PI3K, TORC, 4E-BP and S6K (P<0.05 or P<0.01), and significantly increased the transcription levels of Atg1, Atg5, Atg8a and Atg8b (P<0.01). Conclusion: Ginseng extract could delay the aging of Drosophila melanogaster by activating AMPK pathway and inhibiting mTOR pathway.
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Key words:
- ginseng extract /
- Drosophila melanogaster /
- anti-aging /
- AMPK /
- mTOR
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图 1 HPLC法测定人参提取物中人参皂苷组成
Figure 1. Determination of ginsenosides in ginseng extract by HPLC
注：A：标准品；B：人参提取物。
图 3 人参提取物对果蝇摄食量及甘油三酯含量的影响
Figure 3. Effect of ginseng extract on the food intake and triglyceride content of Drosophila melanogaster
注：与对照组相比，ns表示差异不显著P>0.05；图4同。
图 4 人参提取物对果蝇繁殖能力的影响
Figure 4. Effect of ginseng extract on reproductive ability of Drosophila melanogaster
图 5 人参提取物对果蝇运动能力的影响
Figure 5. Effect of ginseng extract on athletic ability of Drosophila melanogaster
图 6 人参提取物对果蝇应激能力的影响
Figure 6. Effect of ginseng extract on stress ability of Drosophila melanogaster
注：a：饥饿应激；b：热应激；c：氧化应激。
图 7 人参提取物对AMPKα、FoxO、Sirt1、HDAC4、SREBP、LKB1和CaMKII转录水平的影响
Figure 7. Effect of ginseng extract on transcription levels of AMPKα, FoxO, Sirt1, HDAC4, SREBP, LKB1 and CaMKII
图 8 人参提取物对ATP含量和ATP/ADP比值的影响
Figure 8. Effect of ginseng extract on ATP content and ATP/ADP ratio
图 9 人参提取物对PI3K、TORC、4E-BP、S6K、Atg1
、Atg5、Atg8a和Atg8b转录水平的影响 Figure 9. Effect of ginseng extract on transcription levels of PI3K, TORC, 4E-BP, S6K, Atg1
, Atg5, Atg8a and Atg8b 表 1 流动相梯度
Table 1. Gradient of mobile phase
时间（min） 流动相A（%） 流动相B（%） 0~30 19 81 30~40 19~29 81~71 40~70 29 71 70~80 29~32 71~68 80~90 32~42 68~58 90~105 42~60 58~40 105~113 60 40 
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表 2 引物序列
Table 2. The sequences of primers
基因名称 上游序列 下游序列 GAPDH 5'-CTCGACTCACGGTCGTTTCA-3' 5'-GGTGATCTTCTGGCCGTTCA-3' AMPKα 5'-GGCCATCGCTTACCATCTGA-3' 5'-ATCGATGATTGGGGAACGGG-3' FoxO 5'-GCGCCTCTACTTTGATAGGAC-3' 5'-TATTGATGTCCAGCAGGCCG-3' Sirt1 5'-AACCAGGTGCCAGACACTAC-3' 5'-GTTGAGGCCAGAATTTCCGC-3' HDAC4 5'-CGAATGGCACAACGACAACG-3' 5'-GTTCGGTTCGGATGCTGTCT-3' SREBP 5'-TGGCTTCTACCAAGTGCCAG-3' 5'-CAAGAGCTGTTGCGTTGGAC-3' LKB1 5'-ATACGCCACGACACTTGGTT-3' 5'-ACCGTGGAGTTGCGGTATTT-3' CaMKII 5'-CTCGCAGTGCTCTTCAGCTA-3' 5'-TTTGCAGTTTTTACCAAGAAC-3' PI3K 5'-CCTAATCTGCCTGTTGCCCA-3' 5'-ACTGAGTCGCTTCGTTTCGT-3' TORC 5'-GCGCCTCTACTTTGATAGGAC-3' 5'-TATTGATGTCCAGCAGGCCG-3' 4E-BP 5'-ACCCTCTACTCCACCACTCC-3' 5'-GGAGTTTGGCTCAATGGGGA-3' S6K 5'-CATGCATTTGGAGCGTGAGG-3' 5'-TCCTTGCACAGTCCGAAGTC-3' Atg1 5'-GCCAGCTCCATCGAAAATAACC-3' 5'-GCGGCGCAGCAGGCACAG-3' Atg5 5'-GCCCCTGCGACTTCACTATCC-3' 5'-CCATTAAATCGGCCAAACTCTTCT-3' Atg8a 5'-CCAATACAAGGAGGAGCAC-3' 5'-AGGAAGTAGAACTGACCGAC-3' Atg8b 5'-AATGTGATCCCACCGACATC-3' 5'-TTGAGCGAGTAGTGCCAATG-3'
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表 3 人参提取物中各人参皂苷含量
Table 3. Contents of ginsenosides in ginseng extract
人参皂苷 含量（mg/g） Rg1 0.1282 Re 0.2066 Rf 0.0503 Rg2 0.0319 Rh1 0.0072 Ro 0.0071 Rb1 0.2472 Rc 0.3198 Rb2 0.2230 Rb3 0.0360 Rd 0.1616 Rg3（S） 0.0149 Rh2（S） 0.0006
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