Application of NMNH in light aging resistance

Abstract

The invention belongs to the technical field of biochemistry, and particularly relates to application of NMNH in light aging resistance. The invention provides application of NMNH in preparation of a skin care product for resisting light aging. The NMNH with the concentration of 0.0125 mg / mL-0. 025 mg / mL can be used for skin photoaging resistance, the photoaging resistance effect of the NMNH on human immortalized keratinocytes under the concentration of 0.025 mg / mL and 0.0125 mg / mL is improved by 132.10% and 180.39%, and the photoaging resistance effect is obvious.

Description

Technical Field

[0001] The present invention belongs to the technical field of biochemistry, and particularly relates to the application of NMNH in anti-photoaging. Background Art

[0002] NMNH, whose Chinese name is "reduced nicotinamide mononucleotide" or "reduced β-nicotinamide mononucleotide", is the reduced form of NMN, a new precursor for supplementing NAD+, with better NAD+-promoting effects than NMN and other biological functions such as increasing cellular antioxidant capacity, reducing fat accumulation, reducing inflammatory responses, and inhibiting the growth of tumor cells. It is a health-promoting reagent with significant commercial potential.

[0003] Ultraviolet rays penetrate the skin to varying degrees and interact with skin cells. Among ultraviolet rays, UVA (320 - 400 nm) can penetrate the epidermis to reach the dermis layer, mainly causing photoaging of skin cells. UVA radiation acts on melanocytes and other skin cells, such as keratinocytes, leading to DNA damage through oxidative stress and the generation of reactive oxygen species (ROS). ROS activates signal pathways related to cell and tissue growth, differentiation, aging, and photoaging. In addition, the ROS generated in human skin cells irradiated with UVA is the main cause of photoaging. Ultraviolet irradiation can also induce the expression of ROS production, resulting in skin inflammation and downregulation of SIRT1.

[0004] According to publicly available information, NMNH can increase the level of NAD+ in the body, significantly enhance mitochondrial function, and alleviate cell aging. So far, there has been no report on the anti-photoaging effect of NMNH on the skin, no report on the concentration of NMNH that plays an anti-photoaging role in the skin, and even less on the quantitative anti-photoaging application of NMNH in human immortalized keratinocytes. Summary of the Invention

[0005] The object of the present invention is to overcome the deficiencies of the prior art and provide the application of NMNH in anti-photoaging, especially the quantitative anti-photoaging application of NMNH in human immortalized keratinocytes.

[0006] The present invention provides an application of NMNH in anti-photoaging.

[0007] As a preferred technical solution, NMNH is used in the preparation of skin care products for anti-photoaging.

[0008] As a preferred technical solution, NMNH with a concentration of 0.0125 mg / mL - 0.025 mg / mL is used for skin anti-photoaging.

[0009] As a preferred technical solution, the use of NMNH with a concentration of 0.0125 mg / mL - 0.025 mg / mL in the preparation of anti-photoaging skin care products.

[0010] As a preferred technical solution, NMNH with a concentration of 0.0125 mg / mL or 0.025 mg / mL is used for anti-photoaging of the skin.

[0011] As a preferred technical solution, the use of NMNH with a concentration of 0.0125 mg / mL or 0.025 mg / mL in the preparation of anti-photoaging skin care products.

[0012] As a preferred technical solution, NMNH is used for anti-photoaging of human immortalized keratinocytes.

[0013] As a preferred technical solution, NMNH with a concentration of 0.0125 mg / mL - 0.025 mg / mL is used for anti-photoaging of human immortalized keratinocytes.

[0014] As a preferred technical solution, NMNH with a concentration of 0.0125 mg / mL or 0.025 mg / mL is used for anti-photoaging of human immortalized keratinocytes.

[0015] As a preferred technical solution, NMNH with a concentration of 0.0125 mg / mL is used for anti-photoaging of human immortalized keratinocytes.

[0016] The experimental results prove that NMNH has obvious anti-photoaging effects at concentrations of 0.025 mg / mL and 0.0125 mg / mL. Beneficial effects

[0017] 1. The present invention first applies NMNH to the anti-photoaging of the skin; 2. The present invention uses NMNH with a concentration of 0.0125 mg / mL - 0.025 mg / mL for anti-photoaging of the skin.

[0018] 3. The present invention uses NMNH with a concentration of 0.0125 mg / mL - 0.025 mg / mL for anti-photoaging of human immortalized keratinocytes, and the anti-photoaging effect is improved by 132.10% - 180.39%, and its anti-photoaging effect is obvious. Brief description of the drawings

[0019] Figure 1 Graph of the results of detecting the cell viability of the test samples Figure 2 Histogram of the results of the relative gene expression levels Figure 3 Cell detection graph of sample group 1 Figure 4Cell detection diagram of sample group 2 Figure 5 Cell detection diagram of sample group 3 Figure 6 Cell detection diagram of blank control group Figure 7 Cell detection diagram of model control group Specific implementation manners

[0020] 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 specific implementation manners described. It should also be understood that the terms used herein are only for describing the specific implementation manners and do not indicate restrictive.

[0021] 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.

[0022] I. Main experimental reagents NMNH (BONTAC, BT22-CYSC006) Human immortalized keratinocytes (HaCaT) DMEM medium (LOT: 2537078) Fetal bovine serum (LOT: BC20230111) Trypsin (LOT: 2523119) PBS buffer (LOT: 2309011) TRIzol reagent (LOT: 041223230824).

[0023] II. Main experimental instruments Biological safety cabinet (SafeFast Classic 212 A) Carbon dioxide incubator (SFEGROW 188 PRO) Inverted phase contrast microscope (MI52-N) Microplate reader (SpectraMax 340PC384) Analytical balance (FA2204N) Flow cytometer (JIMBIO iCytal S1) Cell phototoxicity irradiator (LUYOR-3450) Quantitative PCR instrument (ABIPRISM,7500 Sequence Detection System).

[0024] Test Example 1 Zero Adjustment Group Add 100 μl of cell culture medium to a 96-well cell culture plate, and place the 96-well plate in a carbon dioxide incubator at 37 °C and 5% CO 2 , and culture for 24 hours in an environment with a humidity > 90%. Discard the supernatant, add medium containing MTT to each well, and incubate in a carbon dioxide incubator at 37 °C and 5% CO 2 , and incubate for 3 hours in an environment with a humidity > 90%. After incubation, discard the supernatant, add 100 μl of dimethyl sulfoxide (DMSO) to each well, and measure the absorbance at a wavelength of 570 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0025] Test Example 2 Blank Control Group Inoculate the cell suspension at 100 μl / well into a 96-well cell culture plate, and place it in a carbon dioxide incubator at 37 °C and 5% CO 2 , and culture for 18 - 24 hours in an environment with a humidity > 90%. Add 100 μl of cell culture medium to each well, and place the 96-well plate in a carbon dioxide incubator at 37 °C and 5% CO 2 , and culture for 24 hours in an environment with a humidity > 90%. Discard the supernatant, add medium containing MTT to each well, and incubate in a carbon dioxide incubator at 37 °C and 5% CO 2 , and incubate for 3 hours in an environment with a humidity > 90%. After incubation, discard the supernatant, add 100 μl of dimethyl sulfoxide (DMSO) to each well, and measure the absorbance at a wavelength of 570 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0026] Test Example 3 Sample Group 1 Inoculate the cell suspension at 100 μl / well into a 96-well cell culture plate, and place it in a carbon dioxide incubator at 37 °C and 5% CO 2 , and culture for 18 - 24 hours in an environment with a humidity > 90%. Add 100 μl of cell culture medium containing 50.00 mg / mL NMNH to each well, and place the 96-well plate in a carbon dioxide incubator at 37 °C and 5% CO 2 , and culture for 24 hours in an environment with a humidity > 90%. Discard the supernatant, add medium containing MTT to each well, and incubate in a carbon dioxide incubator at 37 °C and 5% CO 2 , and incubate for 3 hours in an environment with a humidity > 90%. After incubation, discard the supernatant, add 100 μl of dimethyl sulfoxide (DMSO) to each well, and measure the absorbance at a wavelength of 570 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0027] Test Example 4 Sample Group 2 Inoculate the cell suspension at 100 μl / well into a 96-well cell culture plate, and place it in a carbon dioxide incubator at 37 °C and 5% CO 2, culture for 18 - 24 hours in an environment with humidity > 90%. Add 100 μl of cell culture medium containing 15.82 mg / mL NMNH to each well, and place the 96 - well plate in a carbon dioxide incubator at 37 °C, 5% CO 2 , culture for 24 hours in an environment with humidity > 90%. Discard the supernatant, add medium containing MTT to each well, and place it in a carbon dioxide incubator at 37 °C, 5% CO 2 , incubate for 3 hours in an environment with humidity > 90%. After the incubation, discard the supernatant, add 100 μl of dimethyl sulfoxide (DMSO) to each well, and measure the absorbance at a wavelength of 570 nm using an enzyme - linked immunosorbent assay (ELISA) reader.

[0028] Test Example 5, Sample Group 3 Inoculate the cell suspension at 100 μl / well into a 96 - well cell culture plate, and place it in a carbon dioxide incubator at 37 °C, 5% CO 2 , culture for 18 - 24 hours in an environment with humidity > 90%. Add 100 μl of cell culture medium containing 5.01 mg / mL NMNH to each well, and place the 96 - well plate in a carbon dioxide incubator at 37 °C, 5% CO 2 , culture for 24 hours in an environment with humidity > 90%. Discard the supernatant, add medium containing MTT to each well, and place it in a carbon dioxide incubator at 37 °C, 5% CO 2 , incubate for 3 hours in an environment with humidity > 90%. After the incubation, discard the supernatant, add 100 μl of dimethyl sulfoxide (DMSO) to each well, and measure the absorbance at a wavelength of 570 nm using an enzyme - linked immunosorbent assay (ELISA) reader.

[0029] Test Example 6, Sample Group 4 Inoculate the cell suspension at 100 μl / well into a 96 - well cell culture plate, and place it in a carbon dioxide incubator at 37 °C, 5% CO 2 , culture for 18 - 24 hours in an environment with humidity > 90%. Add 100 μl of cell culture medium containing 1.58 mg / mL NMNH to each well, and place the 96 - well plate in a carbon dioxide incubator at 37 °C, 5% CO 2 , culture for 24 hours in an environment with humidity > 90%. Discard the supernatant, add medium containing MTT to each well, and place it in a carbon dioxide incubator at 37 °C, 5% CO 2 , incubate for 3 hours in an environment with humidity > 90%. After the incubation, discard the supernatant, add 100 μl of dimethyl sulfoxide (DMSO) to each well, and measure the absorbance at a wavelength of 570 nm using an enzyme - linked immunosorbent assay (ELISA) reader.

[0030] Test Example 7, Sample Group 5 Inoculate the cell suspension at 100 μl / well into a 96 - well cell culture plate, and place it in a carbon dioxide incubator at 37 °C, 5% CO 2, culture for 18 - 24 hours in an environment with humidity > 90%. Add 100 ul of cell culture medium containing 0.50 mg / mL NMNH to each well, and place the 96 - well plate in a carbon dioxide incubator at 37 °C with 5% CO 2 , culture for 24 hours in an environment with humidity > 90%. Discard the supernatant, add medium containing MTT to each well, and place it in a carbon dioxide incubator at 37 °C with 5% CO 2 , incubate for 3 hours in an environment with humidity > 90%. After incubation, discard the supernatant, add 100 ul of dimethyl sulfoxide (DMSO) to each well, and measure the absorbance at a wavelength of 570 nm using an enzyme - linked immunosorbent assay (ELISA) reader.

[0031] Test Example 8, Sample Group 6 Inoculate the cell suspension at 100 ul / well into a 96 - well cell culture plate, and place it in a carbon dioxide incubator at 37 °C with 5% CO 2 , culture for 18 - 24 hours in an environment with humidity > 90%. Add 100 ul of cell culture medium containing 0.16 mg / mL NMNH to each well, and place the 96 - well plate in a carbon dioxide incubator at 37 °C with 5% CO 2 , culture for 24 hours in an environment with humidity > 90%. Discard the supernatant, add medium containing MTT to each well, and place it in a carbon dioxide incubator at 37 °C with 5% CO 2 , incubate for 3 hours in an environment with humidity > 90%. After incubation, discard the supernatant, add 100 ul of dimethyl sulfoxide (DMSO) to each well, and measure the absorbance at a wavelength of 570 nm using an enzyme - linked immunosorbent assay (ELISA) reader.

[0032] Test Example 9, Sample Group 7 Inoculate the cell suspension at 100 ul / well into a 96 - well cell culture plate, and place it in a carbon dioxide incubator at 37 °C with 5% CO 2 , culture for 18 - 24 hours in an environment with humidity > 90%. Add 100 ul of cell culture medium containing 0.05 mg / mL NMNH to each well, and place the 96 - well plate in a carbon dioxide incubator at 37 °C with 5% CO 2 , culture for 24 hours in an environment with humidity > 90%. Discard the supernatant, add medium containing MTT to each well, and place it in a carbon dioxide incubator at 37 °C with 5% CO 2 , incubate for 3 hours in an environment with humidity > 90%. After incubation, discard the supernatant, add 100 ul of dimethyl sulfoxide (DMSO) to each well, and measure the absorbance at a wavelength of 570 nm using an enzyme - linked immunosorbent assay (ELISA) reader.

[0033] Test Example 10, Sample Group 8 Inoculate the cell suspension at 100 ul / well into a 96 - well cell culture plate, and place it in a carbon dioxide incubator at 37 °C with 5% CO 2, culture for 18 - 24 hours in an environment with humidity > 90%. Add 100 ul of cell culture medium containing 0.02 mg / mL NMNH to each well, and place the 96 - well plate in a carbon dioxide incubator at 37 °C, 5% CO 2 , culture for 24 hours in an environment with humidity > 90%. Discard the supernatant, add medium containing MTT to each well, and place it in a carbon dioxide incubator at 37 °C, 5% CO 2 , incubate for 3 hours in an environment with humidity > 90%. After incubation, discard the supernatant, add 100 ul of dimethyl sulfoxide (DMSO) to each well, and measure the absorbance at a wavelength of 570 nm using an enzyme - linked immunosorbent assay (ELISA) reader.

[0034] Calculation of cell viability: Cell viability (%) = (OD of the test sample group - OD of the test zero - adjustment group) ÷ (OD of the test blank control group - OD of the test zero - adjustment group) × 100% Experimental results, cell viability, and MTT test results are shown in the following table.

[0035]

[0036] Example 1, Sample Group 1 Inoculate the cell suspension at 2 mL / well into a 6 - well cell culture plate, place it in a carbon dioxide incubator at 37 °C, 5% CO 2 , culture for 18 - 24 hours in an environment with humidity > 90%. Add 2 mL of medium containing 0.025 mg / mL NMNH, and expose the cells for 24 hours. After completion, wash the well plate with PBS, and perform UVA irradiation treatment. After irradiation, add 2 mL of medium containing 0.025 mg / mL NMNH, and incubate for another 4 hours. Wash twice with 1 mL of PBS. After washing, add 1 mL of Trizol reagent to each well and pipette to fully lyse the cells. Extract RNA, reverse - transcribe it into cDNA, and then perform fluorescence quantitative PCR detection.

[0037] Example 2, Sample Group 2 Inoculate the cell suspension at 2 mL / well into a 6 - well cell culture plate, place it in a carbon dioxide incubator at 37 °C, 5% CO 2 , culture for 18 - 24 hours in an environment with humidity > 90%. Add 2 mL of medium containing 0.0125 mg / mL NMNH, and expose the cells for 24 hours. After completion, wash the well plate with PBS, and perform UVA irradiation treatment. After irradiation, add 2 mL of medium containing 0.0125 mg / mL NMNH, and incubate for another 4 hours. Wash twice with 1 mL of PBS. After washing, add 1 mL of Trizol reagent to each well and pipette to fully lyse the cells. Extract RNA, reverse - transcribe it into cDNA, and then perform fluorescence quantitative PCR detection.

[0038] Comparative Example 1, Sample Group 3 Inoculate the cell suspension into a 6-well cell culture plate at 2 mL / well, and place it in a carbon dioxide incubator at 37 °C, 5% CO 2 , and culture for 18 - 24 hours in an environment with a humidity > 90%. Add 2 mL of medium containing 0.05 mg / mL NMNH, and expose the cells for 24 hours. After completion, wash the well plate with PBS, and perform UVA irradiation treatment. After the irradiation is completed, add 2 mL of medium containing 0.05 mg / mL NMNH, and incubate again for 4 hours. Wash twice with 1 mL of PBS. After the washing is completed, add 1 mL of Trizol reagent to each well and pipette to fully lyse the cells. Extract RNA, reverse transcribe it into cDNA, and then perform fluorescence quantitative PCR detection.

[0039] Comparative Example 2, Blank Control Group Inoculate the cell suspension into a 6-well cell culture plate at 2 mL / well, and place it in a carbon dioxide incubator at 37 °C, 5% CO 2 , and culture for 18 - 24 hours in an environment with a humidity > 90%. Add 2 mL of medium, and expose the cells for 24 hours. After completion, wash the well plate with PBS. Add 2 mL of medium, and incubate again for 4 hours. Wash twice with 1 mL of PBS. After the washing is completed, add 1 mL of Trizol reagent to each well and pipette to fully lyse the cells. Extract RNA, reverse transcribe it into cDNA, and then perform fluorescence quantitative PCR detection.

[0040] Comparative Example 3, Model Control Group Inoculate the cell suspension into a 6-well cell culture plate at 2 mL / well, and place it in a carbon dioxide incubator at 37 °C, 5% CO 2 , and culture for 18 - 24 hours in an environment with a humidity > 90%. Add 2 mL of medium, and expose the cells for 24 hours. After completion, wash the well plate with PBS, and perform UVA irradiation treatment. After the irradiation is completed, add 2 mL of medium, and incubate again for 4 hours. Wash twice with 1 mL of PBS. After the washing is completed, add 1 mL of Trizol reagent to each well and pipette to fully lyse the cells. Extract RNA, reverse transcribe it into cDNA, and then perform fluorescence quantitative PCR detection.

[0041] The experimental data is normalized with respect to the blank control group, and statistical analysis is performed using the t-test method. Compared with the model control group, significance is indicated by *, P-value < 0.05 is indicated by *, P-value < 0.01 is indicated by **, and P-value < 0.001 is indicated by ***. If the SIRT1 gene is significantly downregulated in the model control group compared to the blank control group, it indicates that the modeling is successful; if the SIRT1 gene is significantly upregulated in the sample group compared to the model control group, it indicates that the sample has an anti-photoaging effect.

[0042]

[0043] The relative expression levels of the SIRT1 gene are shown in the following table.

[0044]

[0045] Experimental conclusion: Compared with the blank control group, the relative expression level of the SIRT1 gene in the model control group was significantly down-regulated, proving that the aging model was successfully established; at the concentrations of 0.025 mg / mL and 0.0125 mg / mL of NMNH, the relative expression level of the SIRT1 gene was significantly up-regulated, and the promotion rate of the relative expression level of the gene was 132.10% - 180.39%, showing an anti-photoaging effect.

Claims

1. The application of NMNH in anti-photoaging is characterized by: NMNH is used for skin anti-photoaging.

2. The use of NMNH in anti-photoaging according to claim 1, characterized in that: Use of NMNH in preparing anti-photoaging skin care products.

3. The use of NMNH in anti-photoaging according to claim 1, characterized in that: NMNH at a concentration of 0.0125 mg / mL-0.025 mg / mL is used for skin anti-photoaging.

4. The use of NMNH in anti-photoaging according to claim 3, characterized in that: Use of NMNH with a concentration of 0.0125 mg / mL-0.025 mg / mL in preparing anti-photoaging skin care products.

5. The use of NMNH in anti-photoaging according to claim 3, characterized in that: NMNH at a concentration of 0.0125 mg / mL or 0.025 mg / mL is used for skin anti-photoaging.

6. The use of NMNH in anti-photoaging according to claim 5, characterized in that: Use of NMNH at a concentration of 0.0125 mg / mL or 0.025 mg / mL in the preparation of anti-photoaging skin care products.

7. The use of NMNH in anti-photoaging according to claim 1, characterized in that: NMNH is used to protect human immortalized keratinocytes against photoaging.

8. The use of NMNH in anti-photoaging according to claim 7, characterized in that: NMNH at a concentration of 0.0125 mg / mL-0.025 mg / mL was used to protect human immortalized keratinocytes against photoaging.

9. The use of NMNH in anti-photoaging according to claim 8, characterized in that: NMNH at a concentration of 0.0125 mg / mL or 0.025 mg / mL was used to protect human immortalized keratinocytes against photoaging.

10. The use of NMNH in anti-photoaging according to claim 9, characterized in that: NMNH at a concentration of 0.0125 mg / mL was used to protect human immortalized keratinocytes against photoaging.

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