Application of NMNH in anti-aging
By using 10 μg/ml of NMNH in zebrafish, the problem that the anti-aging effect of NMNH in cells was solved, and the effect of significantly improving NAD+ levels and delaying aging was achieved.
Patent Information
- Application Number
- CN202510389828.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-31
- Publication Date
- 2025-06-03
AI Technical Summary
There is no report on the concentration of NMNH that plays an anti-aging role in cells in the prior art, and there is no quantitative anti-aging application of NMNH in zebrafish.
10μg/ml NMNH was used for anti-aging applications of zebrafish. By transferring zebrafish to a culture medium containing NMNH and continuously processing to 72 hpf, it was used to improve the NAD+ level in the body and delay aging.
It significantly improved the NAD+ level in zebrafish, delayed the aging process, and proved that NMNH had significant anti-aging effects.
Smart Images
Figure CN120078795A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of biochemistry, and particularly relates to the application of NMNH in anti-aging. Background Art
[0002] The Chinese name of NMNH is "reduced nicotinamide mononucleotide" or "reduced β-nicotinamide mononucleotide", which is the reduced form of NMN. It is a new precursor for supplementing NAD+, and has a better NAD+-promoting effect than NMN and other biological functions such as increasing the antioxidant capacity of cells, reducing fat accumulation, reducing inflammatory responses, and inhibiting the growth of tumor cells. It is a health-promoting reagent with significant commercial potential.
[0003] According to existing research references, NAD+ is closely related to aging inhibition. The higher the level of NAD+ in the body, the more obvious the anti-aging effect. Hydrogen peroxide H 2 O 2 can induce the production of excessive reactive oxygen species (ROS) in zebrafish, damage biomacromolecules in cells, reduce the level of NAD+ in the body, lead to cell aging, and thus make the whole zebrafish show an aging state.
[0004] According to publicly available information, NMNH can increase the level of NAD+ in the body, significantly enhance mitochondrial function, and relieve cell aging. So far, there is no report on the concentration at which NMNH plays an anti-aging role in cells, and there is no quantitative anti-aging application of NMNH in zebrafish. Summary of the Invention
[0005] To solve the above technical problems, the present invention provides an application of NMNH in anti-aging, and the application of 10 μg / ml NMNH in anti-aging of zebrafish.
[0006] The application of NMNH in anti-aging, NMNH is used for anti-aging.
[0007] As a preferred technical solution, the use of NMNH in the preparation of health products for anti-aging.
[0008] As a preferred technical solution, the use of NMNH in the preparation of skin care products for anti-aging.
[0009] As a preferred technical solution, the use of NMNH in the preparation of drugs for anti-aging.
[0010] As a preferred technical solution, 10 μg / ml NMNH is used for anti-aging.
[0011] As a preferred technical solution, the use of 10 μg / ml NMNH in the preparation of skin care products for anti-aging.
[0012] As a preferred technical solution, the use of 10 μg / ml NMNH in the preparation of health care products for anti-aging.
[0013] As a preferred technical solution, the use of 10 μg / ml NMNH in the preparation of drugs for anti-aging.
[0014] As a preferred technical solution, NMNH is used for anti-aging in zebrafish.
[0015] As a preferred technical solution, 10 μg / ml NMNH is used for anti-aging in zebrafish. Beneficial effects
[0016] 1. The present invention firstly provides the use of 10 μg / ml NMNH for anti-aging.
[0017] 2. The present invention provides the use of 10 μg / ml NMNH for anti-aging in zebrafish, and its anti-aging effect is obvious. Description of the drawings
[0018] Figure 1 Relative fluorescence intensity graph.
[0019] Figure 2 Side view of zebrafish incubated for 30 minutes.
[0020] Figure 3 Side view of zebrafish incubated for 60 minutes.
[0021] Figure 4 Side view of zebrafish incubated for 120 minutes. Detailed implementation manners
[0022] In order to make the present invention easy to understand, the present invention will be described in detail below in conjunction with specific embodiments. However, before describing the present invention in detail, it should be understood that the present invention is not limited to the described specific implementation manners. It should also be understood that the terms used herein are only for describing specific implementation manners and do not represent restrictive.
[0023] Unless otherwise defined, all terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the implementation of the present invention, the preferred methods and materials are now described.
[0024] Experimental instruments Artificial climate incubator (RGX-70ES), microscope (Braun), stereomicroscope fluorescence microscope.
[0025] Experimental consumables 60 mm culture dish, 90 mm culture dish, six-well plate, 3 mL plastic dropper, 15 mL EP tube, etc.
[0026] Experimental reagents 60×E3 solution (NaCl 17.4 g / L; CaCl•2H 2 O 2.9 g / L; KCl 0.8 g / L; MgCl 2 •6H 2 O 4.89 g / L); DMSO (CAS: 67-68-5); Methylene blue (CAS: 7220-79-3); NMNH (BT22-CYSC006); NAD+ red fluorescent probe (50 ug), etc.
[0027] Test system: Wild-type AB strain zebrafish.
[0028] Example 1 Sample treatment group for 30 minutes Select 10 healthy adult wild-type (AB) zebrafish, and obtain fertilized embryos according to the conventional breeding method. Cultivate them at a water temperature of 28 ± 1 °C, pH 7.0 - 7.5, and keep the water quality clean until 6 hpf. Use the culture solution containing H 2 O 2 (500 μM) for 24 hours to induce the establishment of an aging model. Transfer the zebrafish larvae to the culture solution containing 10 μg / ml NMNH and continue to treat until 72 hpf.
[0029] Fix the zebrafish larvae with 4% paraformaldehyde for 2 - 4 hours, then rinse them 3 times with PBS for 5 minutes each time. Then permeabilize them with PBS solution containing 0.2% Triton X-100 for 15 - 20 minutes, and rinse them 3 times with PBS for 5 minutes each time. Add the treated zebrafish larvae to the NAD+ red fluorescent probe solution and incubate them at a constant temperature of 32 °C for 30 minutes. After the incubation, wash away the unbound probe with PBS.
[0030] Fluorescence detection and analysis: Observe the labeled zebrafish larvae through a fluorescence microscope and take pictures for recording. Use image analysis software (Imagel) to quantitatively analyze the fluorescence intensity in the pictures. Select the same area for measurement for each sample, and take the average value as the fluorescence intensity value of the sample at the corresponding incubation time, so as to represent the NAD+ level in the zebrafish larvae.
[0031] Example 2 Sample treatment group for 60 minutes Select 10 healthy adult wild-type (AB) zebrafish, and obtain fertilized embryos according to the conventional breeding method. Cultivate them at a water temperature of 28 ± 1 °C, pH 7.0 - 7.5, and keep the water quality clean until 6 hpf. Use the culture solution containing H 2 O 2In a culture medium (500 μM), treat for 24 hours to induce the establishment of an aging model. Transfer zebrafish larvae to a culture medium containing 10 μg / ml NMNH and continue the treatment until 72 hpf.
[0032] Fix zebrafish larvae with 4% paraformaldehyde for 2 - 4 hours, then rinse with PBS 3 times, 5 minutes each time. Next, permeabilize with a PBS solution containing 0.2% Triton X - 100 for 15 - 20 minutes, and then rinse with PBS 3 times, 5 minutes each time. Add the treated zebrafish larvae to the NAD+ red fluorescent probe solution and incubate at a constant temperature of 32 °C for 60 minutes. After incubation, wash away the unbound probe with PBS.
[0033] Fluorescence detection and analysis: Observe the labeled zebrafish larvae through a fluorescence microscope and take pictures for recording. Use image analysis software (Imagel) to quantitatively analyze the fluorescence intensity in the pictures. Select the same area for measurement in each sample, and take the average value as the fluorescence intensity value of the sample at the corresponding incubation time, which represents the NAD+ level in zebrafish larvae.
[0034] Example 3 Sample treatment group 120 minutes Select 10 healthy adult wild - type (AB) zebrafish and obtain fertilized embryos according to the conventional breeding method. Culture them at a water temperature of 28 ± 1 °C, pH 7.0 - 7.5, and keep the water quality clean until 6 hpf. Use a culture medium containing H 2 O 2 In a culture medium (500 μM), treat for 24 hours to induce the establishment of an aging model. Transfer zebrafish larvae to a culture medium containing 10 μg / ml NMNH and continue the treatment until 72 hpf.
[0035] Fix zebrafish larvae with 4% paraformaldehyde for 2 - 4 hours, then rinse with PBS 3 times, 5 minutes each time. Next, permeabilize with a PBS solution containing 0.2% Triton X - 100 for 15 - 20 minutes, and then rinse with PBS 3 times, 5 minutes each time. Add the treated zebrafish larvae to the NAD+ red fluorescent probe solution and incubate at a constant temperature of 32 °C for 120 minutes. After incubation, wash away the unbound probe with PBS.
[0036] Fluorescence detection and analysis: Observe the labeled zebrafish larvae through a fluorescence microscope and take pictures for recording. Use image analysis software (Imagel) to quantitatively analyze the fluorescence intensity in the pictures. Select the same area for measurement in each sample, and take the average value as the fluorescence intensity value of the sample at the corresponding incubation time, which represents the NAD+ level in zebrafish larvae.
[0037] Control 1 Model control group 30 minutes Select 10 healthy adult wild-type (AB) zebrafish, and obtain fertilized embryos according to the conventional breeding method. Cultivate them at a water temperature of 28 ± 1 °C, pH 7.0 - 7.5, and keep the water quality clean until 6 hpf. Use a culture solution containing H 2 O 2 (500 μM) to treat for 24 hours to induce the establishment of an aging model. The zebrafish larvae continue to be cultured in normal culture solution and continuously treated until 72 hpf.
[0038] Fix the zebrafish larvae with 4% paraformaldehyde for 2 - 4 hours, then rinse them 3 times with PBS, 5 minutes each time. Then permeabilize them with a PBS solution containing 0.2% Triton X-100 for 15 - 20 minutes, and rinse them 3 times with PBS, 5 minutes each time. Add the treated zebrafish larvae to the NAD+ red fluorescent probe solution and incubate at a constant temperature of 32 °C for 30 minutes. After incubation, wash away the unbound probe with PBS.
[0039] Fluorescence detection and analysis: Observe the labeled zebrafish larvae through a fluorescence microscope and take pictures for recording. Use image analysis software (Imagel) to quantitatively analyze the fluorescence intensity in the pictures. Select the same area for measurement for each sample, and take the average value as the fluorescence intensity value of the sample at the corresponding incubation time, so as to represent the NAD+ level in the zebrafish larvae.
[0040] Model control group of Comparative Example 2 for 60 minutes Select 10 healthy adult wild-type (AB) zebrafish, and obtain fertilized embryos according to the conventional breeding method. Cultivate them at a water temperature of 28 ± 1 °C, pH 7.0 - 7.5, and keep the water quality clean until 6 hpf. Use a culture solution containing H 2 O 2 (500 μM) to treat for 24 hours to induce the establishment of an aging model. The zebrafish larvae continue to be cultured in normal culture solution and continuously treated until 72 hpf.
[0041] Fix the zebrafish larvae with 4% paraformaldehyde for 2 - 4 hours, then rinse them 3 times with PBS, 5 minutes each time. Then permeabilize them with a PBS solution containing 0.2% Triton X-100 for 15 - 20 minutes, and rinse them 3 times with PBS, 5 minutes each time. Add the treated zebrafish larvae to the NAD+ red fluorescent probe solution and incubate at a constant temperature of 32 °C for 60 minutes. After incubation, wash away the unbound probe with PBS.
[0042] Fluorescence detection and analysis: Observe the labeled zebrafish larvae under a fluorescence microscope and take pictures for recording. Use image analysis software (ImageJ) to quantitatively analyze the fluorescence intensity in the pictures. Select the same area for measurement in each sample, and take the average value as the fluorescence intensity value of the sample at the corresponding incubation time, which represents the NAD+ level in the zebrafish larvae.
[0043] Model control group of Comparative Example 3, 120 minutes Select 10 healthy adult wild-type (AB) zebrafish, and obtain fertilized embryos according to the conventional breeding method. Culture them at a water temperature of 28 ± 1 °C and a pH of 7.0 - 7.5 until 6 hpf while keeping the water quality clean. Use the culture solution containing H 2 O 2 (500 μM) to treat for 24 hours to induce the establishment of an aging model. The zebrafish larvae continue to be cultured in the normal culture solution and continuously treated until 72 hpf.
[0044] Fix the zebrafish larvae with 4% paraformaldehyde for 2 - 4 hours, then rinse them 3 times with PBS, 5 minutes each time. Then permeabilize them with a PBS solution containing 0.2% Triton X-100 for 15 - 20 minutes, and rinse them 3 times with PBS, 5 minutes each time. Add the NAD+ red fluorescence probe solution to the treated zebrafish larvae and incubate them at a constant temperature of 32 °C for 120 minutes. After the incubation, wash away the unbound probe with PBS.
[0045] Fluorescence detection and analysis: Observe the labeled zebrafish larvae under a fluorescence microscope and take pictures for recording. Use image analysis software (ImageJ) to quantitatively analyze the fluorescence intensity in the pictures. Select the same area for measurement in each sample, and take the average value as the fluorescence intensity value of the sample at the corresponding incubation time, which represents the NAD+ level in the zebrafish larvae.
[0046] Data analysis: Use statistical software (SPSS) to analyze the data. Perform repeated measures analysis of variance on the fluorescence intensity of each group at different incubation times to observe the effect of the incubation time on the fluorescence intensity. P < 0.05 indicates that the difference is statistically significant.
[0047] Detection results: The relative fluorescence intensity of the sample treatment group in Example 1 after incubating for 30 minutes is 0.40, the relative fluorescence intensity of the sample treatment group in Example 2 after incubating for 60 minutes is 0.58, and the relative fluorescence intensity of the sample treatment group in Example 3 after incubating for 120 minutes is 0.62; the relative fluorescence intensity of the model control group in Comparative Example 1 after incubating for 30 minutes is 0.31, the relative fluorescence intensity of the model control group in Comparative Example 2 after incubating for 60 minutes is 0.35, and the relative fluorescence intensity of the model control group in Comparative Example 3 after incubating for 120 minutes is 0.39.
[0048] It can be seen from the detection results that after treating zebrafish with NMNH, as time goes on, the fluorescence intensity of the stain in the zebrafish body continues to increase, indicating that the NAD+ level in the zebrafish body continues to increase after NMNH treatment, and the effect of delaying aging is obvious. NMNH has a significant anti-aging effect.
Claims
1. The application of NMNH in anti-aging, characterized in that: NMNH is used for anti-aging.
2. The use of NMNH in anti-aging according to claim 1, characterized in that: Use of NMNH in preparing anti-aging health products.
3. The use of NMNH in anti-aging according to claim 1, characterized in that: Use of NMNH in preparing anti-aging skin care products.
4. The use of NMNH in anti-aging according to claim 1, characterized in that: Use of NMNH in preparing anti-aging medicines.
5. The use of NMNH in anti-aging according to claim 1, characterized in that: 10 μg / ml NMNH was used for anti-aging.
6. The use of NMNH in anti-aging according to claim 5, characterized in that: Use of 10 μg / ml NMNH in preparing anti-aging skin care products.
7. The use of NMNH in anti-aging according to claim 5, characterized in that: Use of 10 μg / ml NMNH in the preparation of anti-aging health products.
8. The use of NMNH in anti-aging according to claim 5, characterized in that: Use of 10 μg / ml NMNH in the preparation of anti-aging drugs.
9. The use of NMNH in anti-aging according to claim 1, characterized in that: NMNH is used for anti-aging in zebrafish.
10. The use of NMNH in anti-aging according to claim 9, characterized in that: 10 μg / ml NMNH was used for anti-aging in zebrafish.