CN111041082B

CN111041082B - Method for screening skin photoaging improving active substance by using skin photoaging target and skin photoaging improving active substance

Info

Publication number: CN111041082B
Application number: CN201811184736.3A
Authority: CN
Inventors: 蒋丹丹; 摩根·多斯桑托斯; 李慧
Original assignee: Jala Group Co
Current assignee: Shanghai Natural Hall Group Co ltd
Priority date: 2018-10-11
Filing date: 2018-10-11
Publication date: 2023-11-07
Anticipated expiration: 2038-10-11
Also published as: CN111041082A; WO2020073933A1

Abstract

The invention discloses a method for screening an active substance for improving skin photoaging by using a skin photoaging target and the active substance for improving skin photoaging, wherein microRNA in skin is used as a detection basis, and the microRNA is any one or a combination of more of hsa-miR-3161, hsa-miR-509-5p and hsa-miR-29b-1-5 p. The invention provides a new regulation and control target for developing the active ingredients of cosmetics for improving skin photoaging.

Description

Method for screening skin photoaging improving active substance by using skin photoaging target and skin photoaging improving active substance

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a screening method of a skin photoaging target, a method for screening an active substance by using the target and an active substance for improving skin photoaging.

Background

There is a growing concern in today's society about its own skin condition, and it is desirable to maintain a healthy, young state by skin care products, and beyond this desire, to produce a finely divided type of product that addresses different skin problems. Over time, every consumer is faced with skin aging problems, and thus anti-aging skin care products have been an important place in every major category of cosmetics.

Aging of skin is classified into endogenous aging, which is aging caused by genetic programming, and exogenous aging, which is aging caused by external influences such as ultraviolet irradiation, smoking, environmental pollution, etc. The human skin is inevitably exposed to solar radiation for a long time, and ultraviolet radiation in the sun is the most important factor directly causing skin aging, which is also called photoaging.

With the recent progress in epigenetic research, new knowledge is being given to the way human genes are expressed. The physiological and pathological conditions of human body are not simply determined by genes, and the epigenetic factors can also change the instructions of whether the genes are expressed or not and make the instructions stably inherited. The main mechanisms of epigenetic science include DNA methylation, histone modification, non-coding RNA and the like, wherein the non-coding RNA has very important and diverse regulatory functions, so the non-coding RNA has high attention in the fields of molecular biology, cell biology and physiology, and has extremely high research value. The present invention concerns a non-coding RNA called microRNA which has been of great interest in recent years.

micrornas were found in 1993 as endogenous small fragment RNAs that involved RNA interference: they are able to target messenger RNAs (mrnas) and cause them to degrade or terminate their translation. Micrornas such as these thus play a very important regulatory role in cells. Moreover, they form a population among the largest class of regulatory molecules. They are endogenous and originate from the primary micrornas encoded by the genome (pri-mirnas). To date, approximately 700 human micrornas have been identified. Their function and targets have not been fully explained or demonstrated.

micrornas are a class of small molecules that are ubiquitous in organisms, 17-27 nucleotides in length, and can specifically inhibit expression of a target gene by binding to complementary mRNA targets. Studies have shown that thousands of human protein-encoding genes are regulated by micrornas, suggesting that micrornas are the "master regulator" of many important biological processes.

The naming of microRNAs is based on the temporal order in which they were found, exemplified by hsa-miR-29b-1-5 p: hsa indicates that the species to which the molecule belongs is human, miR is an identification of mature microRNA, 29 is a serial number given in sequence when the family member to which the microRNA belongs is found or submitted to a public database, and "b", "-1" "-5p" respectively refer to the precursor sequence, genomic position and number of the 5' arm of the precursor for generating the mature microRNA.

There is currently limited research on skin miRNAs, especially on human skin.

Expressed miRNAs have been cloned on animal skin, initially in mice. They play an important role in the morphogenesis of the epidermis and the fur. In recent years, miRNAs have been found to be associated with increased coat mass in goats and sheep. Recently, miR203 among mirnas has been identified in the skin of mice, which plays an important role in the induction of epidermal differentiation by reducing the potential of cell proliferation. In humans, there are studies comparing the expression of miRNAs in normal skin with psoriasis skin and eczema skin. miR203 in mirnas is highly expressed in skin (relative to other organs) and is expressed only by keratinocytes. It has been shown to be a miRNA that is overexpressed in psoriatic skin.

At present, most of microRNA researches on skin are focused on diagnosis and treatment of serious skin diseases, and researches on physiological aging of skin are very limited.

In the analysis of the action mechanism of microRNA molecules with obvious difference, a target gene set of the microRNA molecules can be predicted by using a Validated Target Module module of a MiRWalk2.0 database, and the function and metabolic pathway enrichment analysis of the target gene set can be performed by using a biological information database DAVID v6.8, so that the microRNA molecules playing a key role, namely the microRNA markers of skin photoaging, can be obtained according to the weight of the target genes in the skin aging function. Finally, the correlation effect of the microRNA markers and the predicted target genes can be verified through RT-PCR experiments.

The discovery of the microRNA markers can be used for establishing an in-vitro biological model, checking the biological efficacy of chemical substances according to the expression quantity of the microRNA markers, and being applied to the development of cosmetic active ingredients.

Disclosure of Invention

In one aspect, the invention provides a method for developing an active substance for improving skin photoaging, which uses microRNA markers of skin photoaging as detection basis, so as to develop novel active efficacy components for cosmetics for improving skin photoaging.

In order to achieve the above purpose, the present invention adopts the following technical scheme:

and collecting a normal human skin sample irradiated by sunlight ultraviolet rays and prevented from being irradiated by the sunlight ultraviolet rays, and testing the microRNA expression profile by using a high-flux humanized microRNA chip. And comparing the difference of the skin microRNA expression profiles under different irradiation conditions, and screening and summarizing to obtain the microRNA marker for skin photoaging.

The microRNA marker for skin photoaging is characterized in that the marker is any one or a combination of more of hsa-miR-3161, hsa-miR-509-5p and hsa-miR-29b-1-5 p.

The hsa-miR-3161 sequence is shown as SEQ ID NO.1, the hsa-miR-509-5p sequence is shown as SEQ ID NO.2, the hsa-miR-29b-1-5p sequence is shown as SEQ ID NO.3, wherein,

SEQ ID NO.1 CUGAUAAGAACAGAGGCCCAGAU

SEQ ID NO.2 UACUGCAGACAGUGGCAAUCA

SEQ ID NO.3 GCUGGUUUCAUAUGGUGGUUUAGA

according to the bioinformatics analysis result, the target gene of the marker hsa-miR-3161 comprises TYRP1, the target gene of the hsa-miR-509-5p comprises HIF1A, and the target gene of the hsa-miR-29b-1-5p comprises TXNIP.

RT-PCR experiments prove that the marker hsa-miR-3161 is related to the expression of TYRP1 genes in skin, hsa-miR-509-5p is related to the expression of HIF1A genes in skin, and hsa-miR-29b-1-5p is related to the expression of TXNIP genes in skin.

The coding product of the TYRP1 gene (Entrez ID: 7306) is tyrosinase related protein 1, and has a promoting effect on melanin generation. The coded product of the HIF1A gene (Entrez ID: 3091) is hypoxia inducible factor 1A, and plays an important role in maintaining the homeostasis of the epidermis under oxidative stress. The coding product of the TXNIP gene (Entrez ID: 10628) is thioredoxin interaction protein, and can regulate oxidative stress pressure to melanocytes.

The markers are used for developing cosmetic active ingredients for improving skin photoaging. Specifically, normal human-derived skin cells are cultured in vitro, and the skin cells are treated with an active substance to be detected; harvesting skin cells after the culture is finished, performing semi-quantitative detection of microRNA RT-PCR by taking the marker as a target, and performing remarkable down-regulation on any one or more of the markers hsa-miR-509-5p and hsa-miR-29b-1-5pP<0.05 Or has obvious up-regulation function on the marker hsa-miR-3161P<0.05 The actives are useful in developing cosmetics for improving skin photoaging.

Compared with the prior art, the invention has the remarkable advantages that:

the method for detecting the efficacy of the active substance by taking the microRNA chip technology and the expression of the photo-aged skin microRNA as targets has the advantages of high efficiency, high specificity and high sensitivity, and is suitable for developing the active ingredient of the cosmetic for improving the photo-aging of the skin.

In one aspect, the invention relates to a method of screening for a skin photoaging target comprising the steps of:

step 1: collecting a plurality of skin samples, and grouping according to whether the skin collecting parts are daily irradiated by ultraviolet rays in sunlight;

step 2: extracting total RNA from the plurality of skin samples;

step 3: hybridizing the total RNA extraction sample with a human microRNA chip;

step 4: scanning the chip, and obtaining a microRNA expression profile through data processing;

step 5: data processing to obtain differential microRNA;

step 6: and carrying out functional and metabolic pathway enrichment analysis on the target gene set of the differential microRNA.

The preferred scheme of the invention is carried out sequentially according to the sequence of the steps 1-6. However, it will be obvious to those skilled in the art that the order of several steps may be modified according to the actual situation, in which the same function is achieved by the similar means of the present invention, and the same effect is achieved.

In one or more embodiments of the invention, the differential microRNA is selected from one or more of hsa-miR-3161, hsa-miR-509-5p and hsa-miR-29b-1-5p in a skin cell.

In one or more embodiments of the invention, the skin cells are selected from dermal fibroblasts, or epidermal melanocytes, or epidermal keratinocytes.

In one aspect, the invention relates to a method for screening an active substance for improving skin photoaging with a skin photoaging target, screening and detecting with one or more than one differential microRNA selected from skin cells as a target, wherein the differential microRNA(s) are selected from one or more of hsa-miR-3161, hsa-miR-509-5p and hsa-miR-29b-1-5p in skin cells.

In one or more embodiments of the invention, the screening of the differential microRNA(s) comprises the steps of:

step 1: collecting a plurality of skin samples, and grouping the skin collecting parts according to whether the skin collecting parts are irradiated by ultraviolet rays in sunlight or not;

step 2: extracting total RNA from the plurality of skin samples;

step 3: hybridizing the total RNA extraction sample with a human microRNA chip;

step 5: data processing to obtain differential microRNA;

In one or more embodiments of the invention, the skin cells are selected from one or more of dermal fibroblasts, epidermal keratinocytes, and epidermal melanocytes.

In one or more embodiments of the invention, modulation of the target of skin cells by the active ingredient is determined by RT-PCR methods. Preferably, the RT-PCR method is carried out by expressing the target in an amount of 2 ^-∆Ct Measured by the weight of the sample. More preferably, the determination result of the RT-PCR method is determined by: whether the expression level of the skin cell target treated by the active ingredient is significantly reduced compared with the control groupP<0.05）。

In one or more embodiments of the invention, the type of application of the active agent for improving skin photoaging obtained by screening is a skin external agent. Preferably, the skin external agent may be selected from a facial care product, a make-up product, a hair care product, a body care product, and the like.

In another aspect, the invention also relates to an active agent capable of improving skin photoaging, the active agent treated sample being compared to an untreated control sample, the expression level of the target(s) being determined by RT-PCR method to 2 using one or more selected from hsa-miR-3161, hsa-miR-509-5p and hsa-miR-29b-1-5p in skin cells as target ^-∆Ct To measure whether any one or more values of hsa-miR-509-5p and hsa-miR-29b-1-5p of the sample treated with the active matter are significantly reduced compared with the control groupP<0.05 Or whether hsa-miR-3161 is significantly upregulatedP<0.05). PreferablyThe active substances are selected from one or more of rose honey, bamboo rice extract, flavanone derivatives, cudrania tricuspidata extract, sandy plant mixture, snow chrysanthemum extract, bamboo juice extract, peach gum solution, peony extract microemulsion, aristolochia praecox extract, aromatic wood extract, phellinus linteus polysaccharide extract, compound seed extract, compound flower extract, prinsepia utilis royle extract, saccharomyces cerevisiae extract, ginseng seed extract, herba polygoni multiflori extract, peony seed extract, gentian extract, limonum extract, milk seed extract, green spine leaf extract, composition containing pomegranate extract, composition containing snow ginseng extract, composition containing snow chrysanthemum extract, rosa davurica extract, garden balsam extract, scindapsus aureus extract and acacia extract.

Drawings

FIG. 1 is a cluster heatmap of differential microRNAs of normal human skin samples screened by high-throughput chips and exposed to and protected from sunlight ultraviolet.

FIG. 2 is an experimental result of verifying the correlation between hsa-miR-3161 and TYRP1 gene expression using an RT-PCR experiment.

FIG. 3 is an experimental result of verifying the correlation between hsa-miR-509-5p and HIF1A gene expression using an RT-PCR experiment.

FIG. 4 is an experimental result of verifying the correlation between hsa-miR-29b-1-5p and TXNIP gene expression using an RT-PCR experiment.

FIG. 5 shows the results of an experiment for detecting the efficacy of cosmetic actives using RT-PCR with skin photoaged microRNA markers as targets.

Detailed Description

For a better understanding of the present invention, reference will now be made to the accompanying examples and drawings, in which, however, the practice of the invention is not limited thereto. The experimental procedures, not specifically identified in the examples below, are conventional procedures and conditions well known to those skilled in the art or are carried out according to the commercial specifications.

Example 1: high throughput microRNA chip expression profile for detecting normal human skin samples irradiated with and protected from solar ultraviolet light

1. Obtaining skin samples

Fresh skin samples were purchased from Shanghai core Biotechnology Inc., skin sample donors were healthy women between ages 30-40 years old, born in Shanghai and living in Shanghai, excluding the donors of stop menstruation, pregnancy, smoking, drinking for a total of 16 cases.

Wherein the skin irradiated with solar ultraviolet rays is obtained from eyelid skin obtained by eyelid cosmetic surgery, and the average age of the group is (33.5+ -2.8) years. Wherein the skin protected from ultraviolet rays of sunlight is obtained from chest or abdomen skin obtained by abdominal plastic surgery, and the average age of the group is (34.8+ -3.5) years.

Specifically, the skin tissue is collected after the regular plastic surgery, and the skin tissue is treated as soon as possible. Shearing skin tissue to a thickness of not more than 0.5cm, shearing the skin tissue into pieces of 0.5cm x 1cm x 1cm (thickness x length x width), placing the sheared pieces of small tissue into sterile cryopreservation tubes, and adding 2ml RNAlater (Sigma company) into each tube to submerge the tissue for preservation; and placing 1 piece of cut skin tissue into each freezing tube, and storing in a deep low-temperature refrigerator at-80 ℃.

2. Total RNA extraction of skin samples

And extracting total RNA of the sample by adopting a mirVana ™ miRNA Isolation Kit without phenol reagent (Ambion company) special for extracting miRNA of common tissues and cells according to a standard operation flow provided by a manufacturer, and detecting the total RNA obtained by extraction by Agilent Bioanalyzer 2100 (Agilent technologies company) after passing electrophoresis quality inspection.

3. High-flux microRNA chip detection expression profile

The database was derived from microRNA database miRBase V21.0 version using Agilent Human miRNA chips covering 2549 human related micrornas. After dephosphorylation, denaturation and ligation of the total RNA samples, chip hybridization was performed. And after the washing is finished, scanning the chip, reading data, and normalizing to obtain the skin irradiated by sunlight ultraviolet rays and the microRNA chip expression profile of the skin which is not irradiated by the sunlight ultraviolet rays.

Example 2: screening for differential micrornas that induce solar uv-irradiated and solar uv-irradiated skin

Differential expression of micrornas in solar uv-irradiated and solar uv-irradiated skin samples was studied using an agilmicrorna R analysis kit that processes test data based on a limma linear model. The analysis result is shown in a cluster heat diagram of FIG. 1, the color level distribution in the diagram intuitively represents the expression condition of microRNAs in different samples in a graphic form, the deeper the color is, the more obvious the expression difference of the microRNAs is, and the white region indicates that the expression of the microRNAs is not obviously different. From fig. 1, it can be seen that there is a significant difference in microRNA expression levels between skin samples exposed to and protected from solar uv light. Several microRNA molecules with particularly significant differences are shown in table 1.

TABLE 1 in the skin which was screened by the chip experiment and was exposed to and protected from the sunlight ultraviolet rays

microRNA molecules with significant differences

Example 3: bioinformatics analysis to obtain microRNA markers and target genes for skin photoaging

The "Validated Target Module" module of the MiRWalk2.0 database is used for predicting target genes of differential microRNAs of skin samples irradiated by sunlight ultraviolet rays and prevented from being irradiated by the sunlight ultraviolet rays, and a group of prediction gene sets are generated through the step.

The functional and metabolic pathway enrichment analysis was performed on this predicted gene set using the bioinformatic database DAVID v 6.8. The enrichment analysis is based on 5 annotation class modules, including a gene ontology (go_term), KEGG pathway, BIOCARTA pathway, interPRO database, and up_keywors database. The results of a partial analysis are shown in Table 2, listing 3 functional or metabolic pathway notes with higher enrichment.

TABLE 2 functional and metabolic pathway enrichment analysis results of differential microRNA target genes

Among the functions and metabolic pathways listed in Table 2, the encoded product of TYRP1 gene is tyrosinase-related protein 1, which has a promoting effect on melanin production; the coded product of the HIF1A gene is hypoxia inducible factor 1A, and plays an important role in maintaining the homeostasis of the epidermis under oxidative stress; the coding product of TXNIP gene is thioredoxin interactive protein, which can regulate the oxidative stress pressure of melanocytes. These functions are all related to the stress response caused by ultraviolet irradiation of skin cells, so that microRNA molecules hsa-miR-3161, hsa-miR-509-5p and hsa-miR-29b-1-5p for regulating these genes are considered to have potential application as markers of skin photoaging.

Example 4: RT-PCR experiments verify the association between microRNA markers and predicted target genes

1. In vitro monolayer culture of skin cells

Primary isolation of epidermal keratinocytes and epidermal melanocytes was performed in a sterile environment using a commercially available fresh facial skin sample of a 47 year old female donor. Skin was sterilized 1 time each with iodophor and 75% alcohol, washed with PBS, subcutaneous adipose tissue and blood vessels were cut with scissors, the skin was cut into 0.5cm x 0.5cm pieces, and then digested with Dispase II (Roche Co.) at 4℃for 15 hours, and dermis and epidermis layers were separated. The epidermis was digested with 0.05% pancreatin (Invitrogen) for 13 min and then split into two portions, centrifuged at 1000rpm for 10min, and the supernatant was discarded. A portion was resuspended in K-FSM medium (Invitrogen) and then seeded 162cm ² In a square bottle, placing the square bottle in a cell incubator at 37 ℃ and 5% CO ₂ Culturing under saturated humidity. And (5) harvesting the cells for freezing and storing when the cell layers grow to 70% -80% confluence, and establishing an epidermal keratinocyte library. Another part of the cells was treated with Melanocyte medium M (Promocel)l) after the culture solution is resuspended, inoculated to 162cm ² In a square bottle, placing the square bottle in a cell incubator at 37 ℃ and 5% CO ₂ Culturing under saturated humidity. And (5) harvesting the cells for freezing and storing when the cell layers grow to 70% -80% confluence, and establishing an epidermal melanocyte library.

2. Increasing or decreasing the content of specific microRNA molecules in skin cells by cell transfection

Epidermal keratinocytes or epidermal melanocytes prepared in vitro monolayer culture at 300,000 cells/cm ² Is inoculated into a culture dish with the diameter of 100mm, and is added with a corresponding culture medium for culture until the cells grow to 80% confluence, and can be used for testing the correlation experiment between the microRNA markers and the predicted target genes.

In the embodiment, a cell transfection technology is adopted to introduce a specific microRNA mimic (pre-miR) into skin cells, the content of the microRNA in the skin cells is artificially increased, and then the corresponding content change of the predicted target genes is inspected through an RT-PCR experiment. Or transferring a specific microRNA inhibitor (anti-miR) into skin cells by adopting a cell transfection technology, artificially reducing the content of the microRNA in the skin cells, and then detecting the predicted expression quantity change of the target genes.

In the case of hsa-miR-3161, the content of hsa-miR-3161 in skin after irradiation of sunlight ultraviolet rays is reduced, so that a transfection reagent Lipofectamine RNAimax (Invitrogen) is particularly adopted in the embodiment, and a specific inhibitor anti-miR-3161 (Thermo) of hsa-miR-3161 of 50 nM is introduced into melanocytes, and simultaneously anti-miR ™ miRNA Inhibitor Negative Control (Thermo) of 50 nM is introduced as a negative control.

For hsa-miR-509-5p, the hsa-miR-509-5p content in skin is increased after solar ultraviolet irradiation, so that 5pM of hsa-miR-509-5p mimic Pre-miR-509-5p (Thermo) is introduced into keratinocytes by using a transfection reagent in this example, and 5pM of Pre-miR ™ miRNA Precursor Negative Control #1 (Thermo) is introduced as a negative control.

For hsa-miR-29b-1-5p, the hsa-miR-29b-1-5p content in skin is increased after irradiation of sunlight ultraviolet rays, so that 5pM of hsa-miR-29b-1-5p mimic Pre-miR-29b-1-5p (Thermo) is introduced into melanocytes by using a transfection reagent in the example, and 5pM of Pre-miR ™ miRNA Precursor Negative Control #1 (Thermo) is introduced as a negative control.

Cells were harvested 48h after transfection, total RNA was extracted using mirVana miRNA isolation kit (Ambion) and frozen at-80℃for RT-PCR reactions.

3. Primer design for RT-PCR reactions

The relevance between the change of the skin photoaging microRNA marker and the target gene expression level is verified through an RT-PCR experiment, the RT-PCR of the experimental microRNA takes U6 as an internal reference gene, and the RT-PCR of the gene to be detected takes 18S as the internal reference gene. The primers for each of the test gene and the reference gene were as follows:

TYRP1 sense strand: 5 'TCTCAATGGCGAGTGGTCTGTG' (SEQ ID NO. 4)

TYRP1 antisense strand: 5 'CCTGTGGTTCAGGAAGACGTTG' (SEQ ID NO. 5)

HIF1A sense strand: 5 'TATGAGCCAGAAGAACTTTTAGGC' (SEQ ID NO. 6)

HIF1A antisense strand: 5 'CACCTCTTTTGGCAAGCATCCTG' (SEQ ID NO. 7)

TXNIP sense strand: 5 'CAGCAGTGCAAACAGACTTCGG' (SEQ ID NO. 8)

TXNIP antisense strand: 5 'CTGAGGAAGCTCAAAGCCGAAC' (SEQ ID NO. 9)

U6：5’ CAAGGATGACACGCAAATTGG 3’（SEQ ID NO.10）

18S sense strand: 5 'TCTGTGATGCCCTTAGATGTCC' (SEQ ID NO. 11)

18S antisense strand: 5 'AATGGGGTTCAACGGGTTAC' (SEQ ID NO. 12)

The presence of non-specificity of amplified fragment sequences of interest was avoided by BLAST analysis (https:// BLAST. Ncbi. Nlm. Nih. Gov/BLAST. Cgi) after the primer design was completed. Primers were synthesized by Shanghai Biotechnology Co.

4. Reverse transcription

RNA was removed from the-80℃refrigerator, thawed at 4℃and then the reaction solution High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) was prepared in a 0.2 ml PCR tube, which was then incubated at 37℃for 15min, denatured at 98℃for 5min, and incubated at 4 ℃.

5. SYBR Green qPCR

The PCR tube was placed in a PCR apparatus for reaction, incubated at 50℃for 2min, then at 95℃for 10min, followed by 40 cycles: 95 ℃ for 15 seconds; 60 ℃,1min, and finally adding a dissolution curve.

6. Data processing

After transfection of microRNA inhibitor, mimetic or negative control, the change in skin photoaging microRNA marker content and the change in expression level of target gene were compared with the percentage value of the untransfected group level, and the data results are shown in Table 3. As shown in FIG. 2, when the content of hsa-miR-3161 in melanocytes is reduced, the expression of TYRP1 gene is increased; as shown in FIG. 3, when hsa-miR-509-5p levels are elevated in keratinocytes, HIF1A gene expression is decreased; as shown in FIG. 4, when the hsa-miR-29b-1-5p content in melanocytes was decreased, the expression of TXNIP gene was decreased. The verification experiment result of MicroRNA markers proves that the MicroRNA markers can be used as the regulatory factors of skin photoaging related genes.

TABLE 3 expression level of skin photoaging microRNA markers on target genes

Example 5: efficacy of detecting cosmetic active substances by taking microRNA markers of skin photoaging as targets

1. In vitro culture of epidermal melanocytes

Isolation and in vitro culture of epidermal melanocytes as described in example 4 at 10,000 cells/cm ² Is seeded into 6-well cell culture plates.

2. Detection of the Effect of cosmetic actives on skin photoaging microRNA markers

After 3 days of melanocyte growth, cells were cultured using Melanocyte medium M medium containing cosmetic active, which in this example was nicotinamide, at a concentration of 50 μm in the medium, and cells were still cultured in the control group using conventional Melanocyte medium M2 medium.

After the cells were treated with the active substance for 48 hours, the cells were subjected to an accumulated dose of 2J/cm by means of a UV irradiation apparatus (VILBER LOURMAT/BioSun) ² While retaining cells that have not been treated with the active and have not been subjected to UV irradiation as controls. The cells were further cultured for 24 hours.

3. RT-PCR (reverse transcription-polymerase chain reaction) detection of expression of microRNA (ribonucleic acid) marker for skin photoaging

The cells were harvested to extract total RNA, and the changes in hsa-miR-3161 and hsa-miR-29b-1-5p, and the changes in TYRP1 and TXNIP gene expression in the cells were detected, and the specific methods for RT-PCR experiments and data analysis were as described in example 4. As shown in FIG. 5, the content of hsa-miR-3161 in the melanocytes which are not subjected to nicotinamide treatment is reduced to 23.9%, the content of hsa-miR-29b-1-5p is increased to 159.5%, the corresponding TYRP1 expression level is increased to 124.4%, and the TXNIP is reduced to 36.2%. And after the melanocyte subjected to nicotinamide pretreatment is irradiated by UV, the content of hsa-miR-3161 is reduced to 78.4%, the content of hsa-miR-29b-1-5p is increased to 116.5%, and the corresponding TYRP1 expression level is 104.2%, and the content of TXNIP is 89.5%. Therefore, nicotinamide can relieve the regulation and control level of hsa-miR-3161 and hsa-miR-29b-1-5p by ultraviolet irradiation, reduce the change of TYRP1 and TXNIP expression levels, and is beneficial to improving the application potential of skin photoaging phenomenon.

The above examples illustrate only a few embodiments of the present invention and are not intended to limit the invention, and those skilled in the art will be able to make various equivalent modifications or substitutions without departing from the spirit of the invention, which fall within the scope of the present invention, and the appended claims should be construed.

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Claims

1. A method for screening an active substance for improving skin photoaging by using a skin photoaging target, which is characterized by screening and detecting by using differential microRNAs selected from skin cells as targets, wherein the differential microRNAs are hsa-miR-3161, hsa-miR-509-5p and hsa-miR-29b-1-5p in the skin cells; the hsa-miR-3161 sequence is CUGAUAAGAACAGAGGCCCAGAU, the hsa-miR-509-5p sequence is UACUGCAGACAGUGGCAAUCA, and the hsa-miR-29b-1-5p sequence is GCUGGUUUCAUAUGGUGGUUUAGA;

the modulation of the target of the skin cells by the active ingredient is determined by RT-PCR methods; the RT-PCR method uses the expression quantity of the target to be 2 ^-ΔCt To measure; the determination result judgment standard of the RT-PCR method is as follows: the active ingredient has significant downregulation (P) on any one or more of the markers hsa-miR-509-5P, hsa-miR-29b-1-5P<0.05 And has a significant upregulation (P) on the marker hsamiR-3161<0.05)。

2. The method of claim 1, wherein the type of application of the active is a skin external agent.

3. The method of claim 2, wherein the skin external agent is selected from the group consisting of: one or more of a facial care product, a make-up product, a hair care product, a body care product.

4. A method according to any one of claims 1 to 3, wherein the active is selected from: rose honey, bamboo rice extract, flavanone derivatives, cudrania tricuspidata extract, a sandy plant mixture, a coreopsis tinctoria extract, a succus Bambusae extract, a peach gum solution, a peony extract microemulsion, a praecox extract, a rosewood extract, a Phellinus linteus polysaccharide extract, a compound seed extract, a compound flower extract, a prinsepia utilis royle extract, a saccharomyces cerevisiae extract, a ginseng seed extract, a herba cistanches extract, a peony seed extract, a gentian extract, a limonum extract, a milk seed extract, a green spine leaf extract, a composition containing a pomegranate extract, a composition containing a snow ginseng extract, a composition containing a coreopsis tinctoria extract, a rose hip extract, a impatiens balsamina extract, a green scindan extract, a acacia extract, or a composition selected from one or more of them.