Application of reagent for detecting aging-related metabolites in preparation of products for detecting aging of mammals

Abstract

The invention relates to the technical field of biomedicine, and particularly discloses application of a reagent for detecting aging-related metabolites in preparation of a product for detecting aging of mammals. The senescence-related metabolite is Pen itrem a and / or UDP-N-acetyl glucosamine (UDP-N-acetyl glucosamine). By detecting the content of the Pen itrem a and / or the UDP-N-acetyl glucosamine, young individuals and senescence individuals are distinguished, and the senescence degree is detected.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to the use of a reagent for detecting aging-related metabolites in the preparation of a product for detecting mammalian aging. Background Art

[0002] With the acceleration of the global population aging trend and the significant improvement of public health awareness, the market demand for anti-aging products and services has shown an unprecedented growth trend. As a key means to meet people's pursuit of higher quality health management needs, accurate aging detection technology is leading the vigorous development of the anti-aging industry. This technology can capture the gradual decline of physiological functions and metabolic capacity of the human body during the aging process, and then warn of a series of potential health risks, such as cardiovascular disease, diabetes, osteoporosis and cognitive decline.

[0003] Through aging testing, individuals can obtain detailed data about their aging process, physiological function status, and metabolic efficiency. These data become the scientific cornerstone for developing personalized health management plans, including customized nutrition plans, targeted exercise guidance, and drug intervention when necessary. Although existing detection technologies, such as telomere length analysis and DNA methylation pattern detection, have initially revealed the biological mysteries of aging, in the implementation process, they involve DNA extraction and real-time fluorescence quantitative PCR, which requires relatively professional knowledge and instruments. The operation process is time-consuming and requires a clean and pollution-free operating environment, making it difficult to popularize to the public. Therefore, it is necessary to develop new ways to detect aging. Summary of the invention

[0004] In order to develop the use of metabolite detection reagents in preparing products for detecting and distinguishing aging, the present invention provides the use of reagents for detecting aging-related metabolites in preparing products for detecting aging in mammals. The present invention achieves the distinction between young and aging individuals and detects the degree of aging by detecting the content of Penitrema and / or UDP-N-acetylglucosamine.

[0005] The present invention provides the use of a reagent for detecting aging-related metabolites in the preparation of a product for detecting mammalian aging, wherein the aging-related metabolites are Penitram a and / or UDP-N-acetylglucosamine.

[0006] The present invention can distinguish young and old individuals and detect the degree of aging by detecting the content of Penitram a and / or UDP-N-acetylglucosamine. The content of Penitram a and / or UDP-N-acetylglucosamine in the serum of old dogs over 11 years old is significantly higher than that of Penitram a and / or UDP-N-acetylglucosamine in the serum of young dogs under 6 years old.

[0007] Furthermore, the reagents for detecting aging-related metabolites include water, ammonium acetate, ammonia water, acetic acid, methanol and acetonitrile.

[0008] Furthermore, for mammalian dogs, the content of Peniteram a and / or UDP-N-acetylglucosamine in the serum of dogs over 11 years old is significantly higher than that of Peniteram a and / or UDP-N-acetylglucosamine in the serum of dogs under 6 years old. The content of Peniteram a in the serum of dogs over 11 years old is greater than 10 ng / ml, and the content of UDP-N-acetylglucosamine is greater than 20 ng / ml.

[0009] Furthermore, the product is a detection reagent.

[0010] Furthermore, the product is a diagnostic kit.

[0011] Furthermore, the diagnostic kit also includes water, ammonium acetate, ammonia water, acetic acid, methanol and acetonitrile.

[0012] The present invention also provides the use of aging-related metabolites in constructing a mammalian cell aging model, wherein the aging-related metabolites are Penitram a and / or UDP-N-acetylglucosamine.

[0013] Furthermore, a mammalian cell aging model was constructed by adding Peniteram a and / or UDP-N-acetylglucosamine during the culture of mesenchymal stem cells from young dogs under 6 years old and acting for 24h to 26h.

[0014] Furthermore, the concentration of Penitram a added is 9 μM to 11 μM, and the concentration of UDP-N-acetylglucosamine added is 9 μM to 11 μM.

[0015] Furthermore, Penitram a was added at a concentration of 10 μM, and UDP-N-acetylglucosamine was added at a concentration of 10 μM.

[0016] Furthermore, the aging-related metabolites Penitram a and / or UDP-N-acetylglucosamine are used to prepare products that enlarge cells and promote cell morphology swelling, increase the expression of Caspase, and reduce the level of mitochondrial membrane potential.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The use of the reagent for detecting aging-related metabolites Peniteram a and / or UDP-N-acetylglucosamine provided by the present invention in the preparation of a product for detecting aging of mammals can achieve the distinction between young and aging individuals and detect the degree of aging by detecting the content of Peniteram a and / or UDP-N-acetylglucosamine. The content of Peniteram a and / or UDP-N-acetylglucosamine in the serum of aging dogs over 11 years old is significantly higher than that of Peniteram a and / or UDP-N-acetylglucosamine in the serum of young dogs under 6 years old.

[0019] The present invention collects blood through minimally invasive methods, performs rapid serum separation, and can quickly obtain aging evaluation through mass spectrometry analysis. The present invention does not require the collection of important tissues and organs of the body, and has the significant advantages of being fast, efficient, safe, and simple. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0021] Figure 1 To identify 51 age-related metabolites with increasing age trends;

[0022] In the figure, A shows the trend of metabolites of cluster group 1 with increasing age;

[0023] B is the changing trend of metabolites in cluster group 2 with increasing age;

[0024] C is the changing trend of metabolites of cluster group 3 with increasing age;

[0025] D is the changing trend of metabolites of cluster group 4 with increasing age;

[0026] E is the changing trend of metabolites of cluster group 5 with increasing age;

[0027] F is the changing trend of metabolites in cluster group 6 with increasing age.

[0028] Figure 2 Results of KEGG enrichment analysis of metabolites in six age-related cluster groups.

[0029] Figure 3 The intensities of metabolites that increase or decrease with age and their KEGG enrichment are shown.

[0030] Figure 4Volcano plot of differential metabolites screened during the aging process.

[0031] Figure 5 The results of Giemsa staining after stem cells were treated with Penitram a and UDP-N-acetylglucosamine;

[0032] In the figure, A is a Giemsa-stained cell image, and from top to bottom, it represents the Giemsa-stained cell images of the control group without any treatment, and the stem cells treated with Penitram a and UDP-N-acetylglucosamine;

[0033] B is a statistical chart of the nuclear-cytoplasmic ratio of stem cells after treatment with Penitram a and UDP-N-acetylglucosamine.

[0034] Figure 6 The changes of mitochondrial membrane potential level after stem cells were treated with Penitram a and UDP-N-acetylglucosamine;

[0035] Figure 6 In the middle, A is the horizontal flow cytometric graph of mitochondrial membrane potential after stem cells were treated with Peniteram a;

[0036] B is a statistical diagram of changes in mitochondrial membrane potential levels after stem cells were treated with Peniteram a;

[0037] C is the horizontal flow cytometric graph of mitochondrial membrane potential after stem cells were treated with UDP-N-acetylglucosamine;

[0038] D is a statistical graph showing changes in mitochondrial membrane potential levels after stem cells were treated with UDP-N-acetylglucosamine.

[0039] Figure 7 The changes of Casepase3 level after stem cells were treated with Penitram a and UDP-N-acetylglucosamine;

[0040] Figure 7 In the middle, A is the horizontal flow cytometric graph of Caspase3 after stem cells were treated with Penitram a;

[0041] B is a statistical chart showing the changes in Caspase3 levels after stem cells were treated with Peniteram a;

[0042] C is a horizontal flow cytometric diagram of Caspase3 after stem cells were treated with UDP-N-acetylglucosamine;

[0043] D is a statistical graph showing the changes in Caspase3 levels after stem cells were treated with UDP-N-acetylglucosamine.

[0044] Figure 8 The results of β-galactosidase staining, an indicator of aging, after stem cells were treated with Penitram a and UDP-N-acetylglucosamine;

[0045] In the figure, A is a β-galactosidase stained cell image, and from top to bottom, it represents the β-galactosidase stained cell images of the control group without any treatment, Penitram a and stem cells treated with UDP-N-acetylglucosamine;

[0046] B is a statistical graph of the β-galactosidase positive cell ratio after stem cells were treated with Peniteram a and UDP-N-acetylglucosamine.

[0047] Fig. 9 The results of immunofluorescence staining of Lamin B1, an aging indicator, after stem cells were treated with Penitram a and UDP-N-acetylglucosamine;

[0048] In the figure, A is a Lamin B1 immunofluorescence staining cell image, and from top to bottom, it shows the Lamin B1 immunofluorescence staining results of the control group without any treatment, Penitram a and stem cells treated with UDP-N-acetylglucosamine;

[0049] B is a statistical graph of the average immunofluorescence staining intensity of Lamin B1 after stem cells were treated with Penitreme a and UDP-N-acetylglucosamine.

[0050] Fig.10 The comparison of Penirtrem a / Udp-n-acetylglucosamine content in serum of dogs under 6 years old and dogs over 11 years old;

[0051] In the figure, A is a comparison of the levels of Penirtrem a in the serum of dogs under 6 years old and dogs over 11 years old;

[0052] B is a comparison of the Udp-n-acetylglucosamine content in the serum of dogs under 6 years old and dogs over 11 years old. DETAILED DESCRIPTION

[0053] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified, and the materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0054] Example 1: Identification of aging-related metabolites.

[0055] Canine serum metabolomics data acquisition and analysis.

[0056] 1. Experimental Methods

[0057] 1. Obtaining canine serum

[0058] According to the degree of aging of the dogs, at least one female dog aged 6 or 12 was used. All dogs were in good condition and had no abnormalities. Serum was obtained from each dog. The specific steps are as follows:

[0059] (1) Collect 5 ml of fresh blood from the cephalic vein of the forelimb using a blood collection needle and place it in a 5 mL centrifuge tube. After blood collection, place it in a 37°C water bath for 1 hour and then immediately centrifuge it at 5000 rpm / 20 min. The supernatant obtained is canine serum.

[0060] (2) The serum was transferred to 4°C, and an appropriate amount of serum was added to a precooled methanol / acetonitrile / water solution (2:2:1, v / v). The serum was vortexed, sonicated at low temperature for 30 min, allowed to stand at -20°C for 10 min, and centrifuged at 14,000 g for 20 min at 4°C. The supernatant was vacuum dried and added with 100 μL of acetonitrile-water solution (acetonitrile: water = 1:1, v / v) for reconstitution during mass spectrometry analysis. The serum was vortexed, centrifuged at 14,000 g for 15 min at 4°C, and the supernatant was used as a sample for analysis.

[0061] 2. Chromatography-mass spectrometry analysis

[0062] (1) Chromatographic conditions

[0063] The samples were separated by Vanquish LC ultra-high performance liquid chromatography system (UHPLC) HILIC column; column temperature 25℃; flow rate 0.3mL / min; injection volume 2μL; mobile phase composition A: water + 25mM ammonium acetate + 25mM ammonia, B: acetonitrile; gradient elution program is as follows: 0---1.5min, 98% B; 1.5---12min, B linearly changes from 98% to 2%; 12---14min, B is maintained at 2%; 14---14.1min, B linearly changes from 2% to 98%; 14.1--17min, B is maintained at 98%; the samples were placed in the 4℃ autosampler during the entire analysis process. In order to avoid the influence of fluctuations in instrument detection signals, the samples were analyzed continuously in random order. QC samples were inserted into the sample queue to monitor and evaluate the stability of the system and the reliability of the experimental data.

[0064] (2) Q Exactive series mass spectrometry conditions

[0065] Q Exactive series mass spectrometer was used to collect primary and secondary spectra of samples.

[0066] The samples were separated by Vanquish LC ultra-high performance liquid chromatography (UHPLC) system and analyzed by mass spectrometry using a Q Exactive series mass spectrometer (Thermo), using electrospray ionization (ESI) positive and negative ion modes for detection.

[0067] The ESI source and mass spectrometer setting parameters are as follows: nebulizer gas auxiliary heating gas 1 (Gas1): 60, auxiliary heating gas 2 (Gas2): 60, curtain gas (CUR): 30psi, ion source temperature: 600℃, spray voltage (ISVF) ±5500V (positive and negative modes); primary mass-to-charge ratio detection range: 80-1200Da, resolution: 60000, scan accumulation time: 100ms, secondary adopts segmented acquisition method, the scan range is 70-1200Da, secondary resolution: 30000, scan accumulation time: 50ms, dynamic exclusion time: 4s.

[0068] Acetonitrile was purchased from Fisher Chemical, product number 4L A955-4. Ammonium acetate was purchased from Sigma, product number 73594. Methanol was purchased from Fisher, product number A456-4. Ammonia was purchased from Fisher, product number A470-500.

[0069] 3. Data analysis process

[0070] The raw data were converted into .mzXML format by ProteoWizard, and then peak alignment, retention time correction and peak area extraction were performed using XCMS software. The data extracted by XCMS were first subjected to metabolite structure identification and data preprocessing, then experimental data quality evaluation, and finally data analysis.

[0071] 2. Experimental Results

[0072] In order to explore the dynamic changes of serum metabolites during canine aging, the present invention used a correlation test to calculate the Spearman correlation coefficient between each metabolite and age. The present invention only retained those metabolites that were significantly correlated with age (p<0.05), resulting in 51 age-related metabolites. Subsequently, the present invention used the fuzzy C-means clustering (FCM) algorithm provided by the R package Mfuzz to cluster these metabolites according to their intensity distribution in different age groups. The metabolites were divided into 6 groups, respectively recorded as Cluster Group 1, Cluster Group 2, Cluster Group 3, Cluster Group 4, Cluster Group 5 and Cluster Group 6. Among them, Cluster Group 1, Cluster Group 4 and Cluster Group 5 represent metabolites that decrease with age, while Cluster Group 2, Cluster Group 3 and Cluster Group 6 represent metabolites that increase with age (such as Figure 1 ). Afterwards, the KEGG database was used to perform functional enrichment analysis on each group ( Figure 2 and Figure 3 ). Enrichment analysis of these metabolites showed that ABC transporters and insulin resistance are key markers of aging. In addition, pyrimidine metabolism gradually increases with age, while sterol metabolism gradually decreases. In order to study the changes in mammalian aging, the present invention compared the metabolites that changed significantly and screened and identified two metabolites, Penitrem A and UDP-N-acetylglucosamine, which increased significantly with age (such as Figure 4 ).

[0073] Example 2: Application of mammalian aging-related metabolite Penitram a / Udp-n-acetylglucosamine in aging.

[0074] The present invention collects mesenchymal stem cells from young dogs in clinical practice, takes cells without any addition of culture medium components as a control group, and adds Penitram a and Udp-n-acetylglucosamine during the culture process, which promotes the occurrence of aging. Giemsa staining shows that Penitram A and Udp-n-acetylglucosamine cause cell enlargement and morphological swelling, which is consistent with the trend of aging ( Figure 5 Both metabolites enhanced CASPASE3 expression and decreased mitochondrial membrane potential levels, indicating their promoting effects on aging ( Figure 6 and Figure 7 β-galactosidase staining confirmed the association of PenitremeA and Udp-n-acetylglucosamine with aging ( Figure 8 ).

[0075] Lamin B1 is a member of the lamin family. Its main function is to maintain the integrity of the cytoskeleton and participate in cell proliferation and aging by affecting chromosome distribution, gene expression and DNA damage repair. Lamin B1 staining confirmed the effects of Penitram A and Udp-n-acetylglucosamine on cytoskeleton integrity in aging ( Fig. 9 ).

[0076] Example 3: Clinical application of mammalian aging-related metabolite Penitram a / Udp-n-acetylglucosamine.

[0077] The present invention collects serum from dogs under 6 years old and over 11 years old, adds pre-cooled methanol / acetonitrile / water solution (2:2:1, v / v), vortexes to mix, performs low-temperature ultrasound for 30 minutes, stands at -20°C for 10 minutes, centrifuges at 14000g at 4°C for 20 minutes, takes the supernatant and vacuum dries it, adds 100 μL of acetonitrile water solution (acetonitrile: water = 1:1, v / v) for re-dissolution during mass spectrometry analysis, vortexes, centrifuges at 14000g at 4°C for 15 minutes, and takes the supernatant as a sample for injection analysis. Compared with dogs under 6 years old, the content of Penirtrem a / Udp-n-acetylglucosamine in the serum of dogs over 11 years old is significantly increased ( Fig.10 The mass spectrometry results of Penirtrem a / Udp-n-acetylglucosamine in the serum of dogs under 6 years old and dogs over 11 years old confirmed that the increase in the content of Penirtrem a / Udp-n-acetylglucosamine in aging mammals can serve as a marker of aging.

[0078] Although preferred embodiments of the present invention have been described, additional changes and modifications may occur to these embodiments once those skilled in the art are aware of the basic inventive concepts.

[0079] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. Use of a reagent for detecting aging-related metabolites in the preparation of a product for detecting aging in mammals, characterized in that: The aging-related metabolites are Penitram a and / or UDP-N-acetylglucosamine.

2. Use of the reagent for detecting aging-related metabolites according to claim 1 in preparing a product for detecting aging in mammals, characterized in that: The reagents for detecting aging-related metabolites also include water, ammonium acetate, ammonia water, acetic acid, methanol and acetonitrile.

3. Use of the reagent for detecting aging-related metabolites according to claim 1 in preparing a product for detecting aging in mammals, characterized in that: The levels of Penitram a and / or UDP-N-acetylglucosamine in the serum of dogs over 11 years old were significantly higher than those in the serum of dogs under 6 years old.

4. Use of the reagent for detecting aging-related metabolites according to claim 1 in preparing a product for detecting aging in mammals, characterized in that: The product is a detection reagent.

5. Use of the reagent for detecting aging-related metabolites according to claim 1 in preparing a product for detecting aging in mammals, characterized in that: The product is a diagnostic kit.

6. Application of aging-related metabolites in the construction of mammalian cell aging models, characterized in that: The aging-related metabolite is Penitram a and / or UDP-N-acetylglucosamine as described in claim 1.

7. The use of aging-related metabolites in constructing a mammalian cell aging model according to claim 6, characterized in that: A mammalian cell aging model was constructed by adding Penitram a and / or UDP-N-acetylglucosamine during the culture of mesenchymal stem cells from young dogs under 6 years old and allowing the cells to act for 24 to 26 hours.

8. The use of aging-related metabolites in constructing a mammalian cell aging model according to claim 7, characterized in that: The concentration of penitrem a added was 9 μM to 11 μM, and the concentration of UDP-N-acetylglucosamine added was 9 μM to 11 μM.

9. The use of aging-related metabolites in constructing a mammalian cell aging model according to claim 8, characterized in that: Penitrem a was added at a concentration of 10 μM, and UDP-N-acetylglucosamine was added at a concentration of 10 μM.

10. The use of aging-related metabolites in constructing a mammalian cell aging model according to claim 9, characterized in that: The aging-related metabolites Penitram a and / or UDP-N-acetylglucosamine are used to prepare products that enlarge cells and promote swelling of cell morphology, increase the expression of CASPASE3, and reduce the level of mitochondrial membrane potential.

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