龟肉蛋白肽延缓果蝇衰老的作用及机制研究

吴俊豪; 王晶; KHOSETHYKUN; 覃川; 王倩倩; 余鹏; 冯凤琴

doi:10.13386/j.issn1002-0306.2021110057

龟肉蛋白肽延缓果蝇衰老的作用及机制研究

doi: 10.13386/j.issn1002-0306.2021110057

吴俊豪^{1, 2,},
王晶^{1, 2},
KHOSETHYKUN¹,
覃川¹,
王倩倩¹,
余鹏³,
冯凤琴^{1, 2, ,}

1.
浙江大学生物系统工程与食品科学学院，浙江杭州 310058
2.
浙江大学宁波研究院，浙江宁波 315100
3.
余姚市冷江鳖业有限公司，浙江宁波 315400

基金项目: 宁波市自然基金项目（20211JCGY010347）；余姚市农业农村和社会发展科技计划（2020NS03）；宁波市“科技创新2025”重大专项（2019B10060）。

详细信息

作者简介:
吴俊豪（1998−），男，硕士研究生，研究方向：生物活性肽，E-mail：wujunhaojon@zju.edu.cn

通讯作者:
冯凤琴（1964−），女，博士，教授，研究方向：食品功能配料及营养健康，E-mail：fengfq@zju.edu.cn

中图分类号: TS201.4
计量
- 文章访问数: 188
- HTML全文浏览量: 39
- PDF下载量: 27
- 被引次数: 0
出版历程
- 收稿日期: 2021-11-08
- 网络出版日期: 2022-06-26
- 刊出日期: 2022-08-26

Research on Anti-aging Function and Mechanism of Pond Turtle Protein-derived Peptides on Drosophila melanogaster

WU Junhao^{1, 2
,},
WANG Jing^{1, 2},
KHO Sethykun¹,
QIN Chuan¹,
WANG Qianqian¹,
YU Peng³,
FENG Fengqin^{1, 2
, ,}

1.
College of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou 310058, China
2.
Ningbo Research Institute, Zhejiang University, Ningbo 315100, China
3.
Yuyao Lengjiang Turtle Industry, Ningbo 315400, China

摘要

摘要: 目的：探究龟肉蛋白肽（pond turtle protein-derived peptides，PTPDP）延缓果蝇衰老的作用和机制。方法：酶解乌龟肉得到PTPDP，测定其营养成分、分子质量分布以及氨基酸组成；分别以0.2%、0.4%和0.8%的PTPDP喂养果蝇，通过生存实验和测定体内超氧化物歧化酶（superoxide dismutase，SOD）、过氧化氢酶（catalase，CAT）活性和丙二醛（malondialdehyde，MDA）含量来分析其抗衰老功能；通过测定0.8% PTPDP喂养的雌性果蝇抗氧化相关基因Sod1、Sod2、Cat和寿命相关基因mth、Rpn11的表达来探究其抗衰老机制。结果：龟肉酶解产物以1000 Da以下的小分子肽段为主，富含甘氨酸、谷氨酸和脯氨酸等具有抗氧化活性的氨基酸。生存实验和抗氧化活性实验中，0.8% PTPDP组雌雄果蝇的平均寿命分别显著延长了18.92%和9.37%（P<0.01)；SOD活性分别显著提高了7.13%和7.37%（P<0.05）；CAT活性分别极显著提高了42.14%和84.66%（P<0.01）；MDA含量分别显著下降了22.22%和23.08%（P<0.05）。基因表达结果显示，0.8% PTPDP喂养显著上调了抗氧化相关基因Sod1，Sod2（P<0.05）和Cat（P<0.01）的表达，同时对寿命相关基因mth和Rpn11（P<0.01）的表达也表现出显著的调节作用。结论：PTPDP通过影响果蝇抗氧化和寿命相关基因的表达，提高其体内抗氧化活性，延长果蝇寿命，有潜在的抗衰老功效。
- 龟肉蛋白肽 /
- 分子质量 /
- 氨基酸组成 /
- 寿命 /
- 抗氧化活性 /
- 基因表达
Abstract: Objective: To explore the anti-aging function and mechanism of pond turtle protein-derived peptides (PTPDP) on Drosophila melanogaster. Methods: PTPDP were obtained by enzymatic hydrolysis of turtle meat. Nutrients content, molecular weight distribution and amino acid composition of PTPDP were determined. D. melanogaster were fed with 0.2%, 0.4%, and 0.8% PTPDP. The activities of superoxide dismutase (SOD) and catalase (CAT), along with the content of malondialdehyde (MDA) were determined to analyze the anti-aging function of PTPDP. The anti-aging mechanism was explored by measuring the expression of antioxidant genes (Sod1, Sod2 and Cat) and lifespan-related genes (mth and Rpn11) in female D. melanogaster fed with 0.8% PTPDP. Results: The enzymatic hydrolysis products of turtle meat were mainly composed of small molecule peptides less than 1000 Da. PTPDP were rich in amino acids with antioxidant activity, such as glycine, glutamic acid and proline. In the survival experiment, the average lifespans of female and male D. melanogaster were significantly prolonged by 18.92% and 9.37% (P<0.01), respectively. In the antioxidant activity experiment, the SOD activity of female and male D. melanogaster increased significantly by 7.13% and 7.37% (P<0.05); the CAT activity of female and male D. melanogaster increased significantly by 42.14% and 84.66% (P<0.01); the MDA content of female and male D. melanogaster decreased significantly by 22.22% and 23.08% (P<0.05). In female D. melanogaster fed with 0.8% PTPDP, the expression of anti-oxidant related genes Sod1, Sod2 (P<0.05) and Cat (P<0.01) were significantly up-regulated, and genes (mth and Rpn11) related to lifespan also showed different degrees of regulation (P<0.01). Conclusion: PTPDP increase the antioxidant activity of D. melanogaster by affecting the expression of antioxidant genes and lifespan-related genes, thereby prolonging the lifespan of D. melanogaster. This mechanism implies that PTPDP has potential anti-aging effects.
- pond turtle protein-derived peptides /
- molecular mass /
- amino acid composition /
- life span /
- antioxidant activity /
- gene expression

HTML全文

图 1 雌性（A）和雄性（B）果蝇的生存曲线

Figure 1. The survival curves of female (A) and male (B) D. melanogaster

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图 2 0.8% PTPDP喂养的雌性果蝇基因表达

Figure 2. Gene expressions of female D. melanogaster fed by 0.8% PTPDP

注：*表示与对照组相比差异显著，P<0.05；**表示与对照组相比差异极显著，P<0.01。

下载: 全尺寸图片幻灯片

表 1 梯度洗脱时间

Table 1. Gradient elution time

时间（min）	流速（mL/min）	流动相A（%）	流动相B（%）
0	1	100	0
13	1	93	7
23	1	77	23
29	1	65	35
35	1	60	40
40	1	0	100
45	1	0	100
47	1	100	0
50	1	100	0

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表 2 引物序列

Table 2. Primer sequences

基因	上游引物	下游引物
rp49	GCTAAGCTGTCGCACAAATG	TGTGCACCAGGAACTTCTTG
Sod1	ACCGACTCCAAGATTACGCTCT	GTTGCCCGTTGACTTGCTC
Sod2	CCACATCAACCACACCATCT	CAGTTTGCCCGACTTCTTGT
Cat	TACGAGCAGGCCAAGAAGTT	ACCTTGTACGGGCAGTTCAC
mth	CGTCTTTCTGGTGGCTGACT	CGTGGATTTAGGATGGTTCC
Rpn11	CATTTGGAGGAGAAGGTGGA	GCGATTTGTGGGTATGAAGG

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表 3 龟肉蛋白肽的营养成分

Table 3. Nutrient content of PTPDP

营养成分	龟肉蛋白肽
水分（%）	4.04±0.05
灰分（%）	3.11±0.08
蛋白含量（%）	86.49±1.17
酸溶蛋白含量（%）	85.98±1.02

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表 4 龟肉蛋白肽分子量分布

Table 4. Molecular weight distribution of PTPDP

分子量（Da）	含量（%）
<180	11.32±0.20
180~500	60.39±1.01
500~1000	22.99±0.77
>1000	5.29±0.08

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表 5 龟肉蛋白肽的氨基酸组成

Table 5. Amino acids composition of PTPDP

氨基酸名称	含量（mg/g）	含量（%）
天冬氨酸	59.86±0.97	8.04±0.26
谷氨酸	109.47±3.54	14.70±0.23
丝氨酸	20.95±0.51	2.81±0.04
甘氨酸	117.59±7.63	15.79±0.87
组氨酸	9.17±0.47	1.23±0.07
精氨酸	31.28±0.81	4.20±0.05
苏氨酸	22.08±0.35	2.96±0.01
丙氨酸	55.99±0.42	7.52±0.16
脯氨酸	72.45±1.54	9.73±0.05
酪氨酸	20.55±0.70	2.76±0.10
缬氨酸	35.88±0.66	4.82±0.10
甲硫氨酸	14.21±0.75	1.91±0.13
异亮氨酸	52.87±2.29	7.10±0.28
亮氨酸	48.94±1.96	6.57±0.24
苯丙氨酸	22.87±1.33	3.07±0.16
赖氨酸	50.61±0.53	6.80±0.05

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表 6 不同剂量龟肉蛋白肽对雌、雄果蝇寿命的影响

Table 6. Effects of different doses of PTPDP on the lifespan of female and male D. melanogaster

组别	半数死亡时间（d）		平均寿命（d）		平均最高寿命（d）		平均寿命延长率（%）
组别	雌	雄	雌	雄	雌	雄	雌	雄
对照组	36	48	36.40±0.97	46.18±0.72	60.31±0.64	63.41±0.61	−	−
低剂量组	36	48	38.54±1.01	46.49±0.82	63.21±0.51^*	63.62±0.60	5.87±0.13	0.67±0.05
中剂量组	39	51	39.76±1.06	47.76±0.80	65.57±0.59^**	65.57±0.63	9.24±0.17	3.43±0.12
高剂量组	45^**	51	43.29±1.04^**	50.51±0.78^**	69.00±0.93^**	68.33±0.82^*	18.92±0.23	9.37±0.17
注：表示各剂量组与对照组相比差异显著，P<0.05；*表示各剂量组与对照组相比差异极显著，P<0.01，表7～表9同。

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表 7 不同剂量龟肉蛋白肽对果蝇体内SOD活性的影响

Table 7. Effects of different doses of PTPDP on SOD activity of D. melanogaster

组别	雌果蝇SOD活性（U/mg prot）		雄果蝇SOD活性（U/mg prot）
组别	20 d	40 d	20 d	40 d
对照组	264.71±12.85	261.68±8.58	358.33±5.84	328.71±10.32
低剂量组	282.44±9.11	257.49±4.65	364.18±13.93	329.97±8.90
中剂量组	294.24±3.37^*	283.59±7.15^*	390.90±7.08^*	333.45±2.91
高剂量组	315.30±14.33^**	280.35±3.52^*	385.16±10.76^*	352.93±5.21^*

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表 8 不同剂量龟肉蛋白肽对雌、雄果蝇体内CAT活性的影响

Table 8. Effects of different doses of PTPDP on CAT activity of female and male D. melanogaster

组别	雌果蝇CAT活性（U/mg prot）		雄果蝇CAT活性（U/mg prot）
组别	20 d	40 d	20 d	40 d
对照组	57.03±3.22	63.49±4.56	117.25±9.19	85.33±6.49
低剂量组	82.14±12.16^*	81.24±5.36^*	114.19±5.04	94.41±7.91
中剂量组	97.08±4.86^**	88.32±3.48^**	121.38±11.41	93.07±7.48
高剂量组	113.59±3.99^**	90.31±9.77^**	144.28±5.89^*	157.57±3.58^**

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表 9 不同剂量龟肉蛋白肽对雌、雄果蝇体内MDA含量的影响

Table 9. Effects of different doses of PTPDP on MDA content of female and male D. melanogaster

组别	雌果蝇MDA含量（nmol/mg prot）		雄果蝇MDA含量（nmol/mg prot）
组别	20 d	40 d	20 d	40 d
对照组	0.33±0.04	0.36±0.01	0.25±0.02	0.26±0.02
低剂量组	0.32±0.04	0.36±0.04	0.24±0.01	0.23±0.04
中剂量组	0.24±0.01	0.33±0.03	0.22±0.02	0.19±0.01^*
高剂量组	0.21±0.03^*	0.28±0.02^*	0.19±0.04^*	0.20±0.02^*

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