Application of beta-glucan in preparation of preparation for improving expression quantity of skin extracellular matrix cell proliferation related genes

Abstract

The invention relates to application of beta-glucan in preparation of a preparation for improving the expression quantity of skin extracellular matrix cell proliferation related genes. The structure of the beta-glucan is formed by connecting structural units; the structural unit is shown in the formula 1, and n is larger than or equal to 150 and smaller than or equal to 2500. The invention also provides a biomarker, the biomarker is a gene related to skin extracellular matrix cell proliferation, and the gene comprises any one or a combination of at least two of CCNF, VEGFA, AURKA, CDCA2, CDCA8, CDCA3, PLK1, CENPA, CENPL, MKI67, SALL4, EZH1, JDP2, PBK, BUB1B, KIF15, KIF18B, KIF20A, KIF2C, KIFC1, RIT1, PIMREG, PSAT1, FZD2, FZD4, NFIL3 or FN1.

Description

Technical Field

[0001] This invention relates to the field of cosmetic raw material efficacy technology, and in particular to the application of β-glucan in the preparation of formulations that enhance the expression of genes related to the proliferation of skin extracellular matrix cells. Background Technology

[0002] Cells are the largest organs in the human body, and human aging can be considered, to a certain extent, cellular aging. During skin aging, keratinocytes and fibroblasts age, the epidermis thins and deteriorates, and the dermis produces less collagen and elastin, leading to wrinkles, increased skin elasticity, and a loss of elasticity. Therefore, one of the key manifestations of cellular aging is the loss of cell proliferation capacity.

[0003] The skin covers the body's surface and is the body's first line of defense against external harm. Since the skin is in direct contact with the outside world, it is highly susceptible to damage from trauma, burns, and other processes. Besides acute trauma, many common diseases can slow down or prevent wound healing, and wound healing time is closely related to susceptibility to infection, duration of pain, length of hospital stay, and scarring rate.

[0004] Under normal circumstances, some tissues and cells are constantly consumed, age, and die, and are continuously replenished by the division and proliferation of the same type of cells; this is called physiological regeneration, such as the shedding and renewal of the epidermis. This regeneration is closely related to cell proliferation; the stronger the cell proliferation capacity, the more effective it is in anti-aging and skin repair. Currently, some methods to enhance cell proliferation capacity involve using substances containing purine and pyrimidine nucleic acids, such as CN1646078A, or using substances like collagen to improve cell proliferation, such as CN116370340A. However, the activity of these substances is environment-dependent and easily reduced by environmental factors.

[0005] Therefore, there is an urgent need for a substance that can significantly enhance cell proliferation and provide biomarkers that can effectively detect cell proliferation effects. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides the application of β-glucan in the preparation of formulations that enhance the expression of genes related to the proliferation of extracellular matrix cells in skin cells.

[0007] To achieve this objective, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides the application of β-glucan in the preparation of formulations that enhance the expression of genes related to the proliferation of extracellular matrix cells of skin cells, wherein the structure of β-glucan is formed by the connection of structural units;

[0009] The structural unit is Equation 1, where 150≤n≤2500;

[0010]

[0011] Formula 1.

[0012] Preferably, the extracellular matrix cell proliferation-related genes of the skin include any one or a combination of at least two of CCNF, VEGFA, AURKA, CDCA2, CDCA8, CDCA3, PLK1, CENPA, CENPL, MKI67, SALL4, EZH1, JDP2, PBK, BUB1B, KIF15, KIF18B, KIF20A, KIF2C, KIFC1, RIT1, PIMREG, PSAT1, FZD2, FZD4, NFIL3, or FN1.

[0013] This invention uses transcriptomics to screen a series of genes related to the proliferation of extracellular matrix cells (ESCs) in the skin, and demonstrates that the β-glucan in this invention can enhance the expression of these genes. Therefore, it can be used in the development and application of various cosmetic products that promote ESC proliferation. Furthermore, the aforementioned gene series can also serve as a reference for future evaluation of other raw materials or cosmetics that enhance the proliferative capacity of ESCs.

[0014] Preferably, the formulation includes a drug or a cosmetic.

[0015] Preferably, the cosmetic includes any one or a combination of at least two of the following: face cream, lotion, gel, toner, serum, face mask, eye cream, aerosol cleansing foam, spray, shower gel, or facial cleanser.

[0016] Preferably, the concentration of β-glucan in the formulation is 0.5%-5%. For example, it can be 0.5%, 1%, 2%, 3%, 4%, or 5%, etc.

[0017] In a second aspect, the present invention provides a method for preparing the β-glucan described in the first aspect, the method comprising: inoculating Schizophyllum commune into a fermentation medium for fermentation culture, centrifuging after fermentation, and then performing alcohol precipitation followed by washing to obtain the β-glucan.

[0018] Preferably, the fermentation medium contains glucose, tryptone, yeast extract, magnesium sulfate, potassium dihydrogen phosphate, vitamin B1, and folic acid.

[0019] Preferably, the inoculum amount of *Schizophyllum commune* is 5-15%. For example, it can be 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15%.

[0020] Preferably, the fermentation culture temperature is 25-30℃, the time is 40-60 h, and the rate is 100-200 r / min. The 25-30℃ can be, for example, 25℃, 26℃, 27℃, 28℃, 29℃, or 30℃. The 40-60 h can be, for example, 40 h, 45 h, 50 h, 55 h, or 60 h. The 100-200 r / min can be, for example, 100 r / min, 120 r / min, 140 r / min, 160 r / min, 180 r / min, or 200 r / min.

[0021] Preferably, the fermentation medium contains 0.5%-5% glucose, 0.5%-3% tryptone, 0.05%-2% yeast extract, 0.01%-1% magnesium sulfate, 0.05%-0.5% potassium dihydrogen phosphate, 10-20 mg / L vitamin B1, and 1-10 mg / L folic acid. The 0.5%-5% can be, for example, 0.5%, 1%, 2%, 3%, 4%, or 5%. The 0.5%-3% can be, for example, 0.5%, 1%, 1.5%, 2%, 2.5%, or 3%. The 0.05%-2% can be, for example, 0.05%, 0.1%, 0.2%, 0.4%, 0.6%, 0.8%, 1%, 1.2%, 1.4%, 1.6%, 1.8%, or 2%. The 0.01%-1% can be, for example, 0.01%, 0.1%, 0.2%, 0.4%, 0.6%, 0.8%, or 1%. The 0.05%-0.5% can be, for example, 0.05%, 0.06%, 0.08%, 0.1%, 0.2%, 0.3%, 0.4%, or 0.5%. The 10-20 mg / L can be, for example, 10 mg / L, 12 mg / L, 14 mg / L, 16 mg / L, 18 mg / L, or 20 mg / L. The 1-10 mg / L can be, for example, 1 mg / L, 2 mg / L, 4 mg / L, 6 mg / L, 8 mg / L, or 10 mg / L.

[0022] Preferably, the alcohol precipitation involves mixing the fermentation product with 1-3 times its volume of anhydrous ethanol and then filtering. The 1-3 times volume can be, for example, 1, 1.5, 2, 2.5, or 3 times.

[0023] Thirdly, the present invention provides the application of products for detecting the expression level of biomarkers in evaluating the ability of cosmetics or their raw materials to enhance the proliferation of skin extracellular matrix cells. The biomarkers are skin extracellular matrix cell proliferation-related genes, which include any one or a combination of at least two of CCNF, VEGFA, AURKA, CDCA2, CDCA8, CDCA3, PLK1, CENPA, CENPL, MKI67, SALL4, EZH1, JDP2, PBK, BUB1B, KIF15, KIF18B, KIF20A, KIF2C, KIFC1, RIT1, PIMREG, PSAT1, FZD2, FZD4, NFIL3, or FN1.

[0024] Preferably, the product comprises any one or a combination of at least two of the following: primer combinations, reagents, or detection models.

[0025] Fourthly, the present invention provides a primer combination for detecting the expression level of a biomarker, characterized in that the primer combination comprises:

[0026] The upstream primer for amplifying CCNF is shown in SEQ ID NO.1, and the downstream primer is shown in SEQ ID NO.2.

[0027] SEQ ID NO.1:CCCCGAAGATGTGCTCTTTCA.

[0028] SEQ ID NO. 2: GCCTTCATGTAGAGGTAGGCT.

[0029] The upstream primer for amplifying VEGFA is shown in SEQ ID NO.3, and the downstream primer is shown in SEQ ID NO.4.

[0030] SEQ ID NO. 3: AGGGCAGAATCATCACGAAGT.

[0031] SEQ ID NO.4: AGGGTCTCGATTGGATGGCA.

[0032] The upstream primer for amplifying AURKA is shown in SEQ ID NO.5, and the downstream primer is shown in SEQ ID NO.6.

[0033] SEQ ID NO. 5: GAGGTCCAAAACGTGTTCTCG.

[0034] SEQ ID NO. 6: ACAGGATGAGGTACACTGGTTG.

[0035] The upstream primer for amplifying CDCA2 is shown in SEQ ID NO.7, and the downstream primer is shown in SEQ ID NO.8.

[0036] SEQ ID NO. 7: TGCCGAATTACCTCCTAATCCT.

[0037] SEQ ID NO. 8: TGCTCTACGGTTACTGTGGAAA.

[0038] The upstream primer for amplifying CDCA8 is shown in SEQ ID NO.9, and the downstream primer is shown in SEQ ID NO.10.

[0039] SEQ ID NO. 9: GAAGGGCAGTAGTCGGGTG.

[0040] SEQ ID NO. 10: TCACGGTCGAAGTCTTTCAGA.

[0041] The upstream primer for amplifying CDCA3 is shown in SEQ ID NO.11, and the downstream primer is shown in SEQ ID NO.12.

[0042] SEQ ID NO. 11: AAGAGCGTCCCAGTCACAC.

[0043] SEQ ID NO. 12: CCAGCACTAGGTGAACGGG.

[0044] The upstream primer for amplifying PLK1 is shown in SEQ ID NO.13, and the downstream primer is shown in SEQ ID NO.14.

[0045] SEQ ID NO. 13: AAAGAGATCCCGGAGGTCCTA.

[0046] SEQ ID NO. 14: GGCTGCGGTGAATGGATATTTC.

[0047] The upstream primer for amplifying CENPA is shown in SEQ ID NO.15, and the downstream primer is shown in SEQ ID NO.16.

[0048] SEQ ID NO. 15: GACGCCTATTCTCTCACCTTA.

[0049] SEQ ID NO. 16: GTTGCACATCCTTTGGGAAGA.

[0050] The upstream primer for amplifying CENPL is shown in SEQ ID NO.17, and the downstream primer is shown in SEQ ID NO.18.

[0051] SEQ ID NO. 17: CACCAGAGTCAACTCCTAGTGC.

[0052] SEQ ID NO. 18:TCTGCTTCCTGACCGATTCTAA.

[0053] The upstream primer for amplifying MKI67 is shown in SEQ ID NO.19, and the downstream primer is shown in SEQ ID NO.20.

[0054] SEQ ID NO. 19: ACGCCTGGTTACTATCAAAAGG.

[0055] SEQ ID NO. 20: CAGACCCATTTACTTGTGTTGGA.

[0056] The upstream primer for amplifying SALL4 is shown in SEQ ID NO.21, and the downstream primer is shown in SEQ ID NO.22.

[0057] SEQ ID NO. 21: AGCACATCAACTCGGAGGAG.

[0058] SEQ ID NO. 22: CATTCCTGGGTGGTTCACTG.

[0059] The upstream primer for amplifying EZH1 is shown in SEQ ID NO.23, and the downstream primer is shown in SEQ ID NO.24.

[0060] SEQ ID NO. 23: ATGCGACTTCGACAACTTAAACG.

[0061] SEQ ID NO. 24: GGCTTCATGACTGAACAGGTT.

[0062] The upstream primer for amplifying JDP2 is shown in SEQ ID NO.25, and the downstream primer is shown in SEQ ID NO.26.

[0063] SEQ ID NO. 25: CCCAGCCCGTGAAAAGTGA.

[0064] SEQ ID NO. 26: CGGTGTCGGTTCAGCATCA.

[0065] The upstream primer for amplifying PBK is shown in SEQ ID NO.27, and the downstream primer is shown in SEQ ID NO.28.

[0066] SEQ ID NO. 27: CCAAACATTGTTGGTTATCGTGC.

[0067] SEQ ID NO. 28: GGCTGGCTTTATATCGTTCTTCT.

[0068] The upstream primer for amplifying BUB1B is shown in SEQ ID NO.29, and the downstream primer is shown in SEQ ID NO.30.

[0069] SEQ ID NO. 29: AAATGACCCTCTGGATGTTTGG.

[0070] SEQ ID NO. 30: GCATAAACGCCCTAATTTAAGCC.

[0071] The upstream primer for amplifying KIF15 is shown in SEQ ID NO.31, and the downstream primer is shown in SEQ ID NO.32.

[0072] SEQ ID NO. 31: AAAACTGAGTTACGCAGCGTG.

[0073] SEQ ID NO. 32: AGTTGCGAATACAGATTCCTGAG.

[0074] The upstream primer for amplifying KIF18B is shown in SEQ ID NO.33, and the downstream primer is shown in SEQ ID NO.34.

[0075] SEQ ID NO. 33: GCTGCAAGTAGTGGTACGGG.

[0076] SEQ ID NO. 34: CCTCAGGGTTAAACACCAGCA.

[0077] The upstream primer for amplifying KIF20A is shown in SEQ ID NO.35, and the downstream primer is shown in SEQ ID NO.36.

[0078] SEQ ID NO. 35: TGCTGTCCGATGACGATGTC.

[0079] SEQ ID NO. 36: AGGTTCTTGCGTACCACAGAC.

[0080] The upstream primer for amplifying KIF2C is shown in SEQ ID NO.37, and the downstream primer is shown in SEQ ID NO.38.

[0081] SEQ ID NO. 37: CTGTTTCCCGGTCTCGCTATC.

[0082] SEQ ID NO. 38: AGAAGCTGTAAGAGTTCTGGGT.

[0083] The upstream primer for amplifying KIFC1 is shown in SEQ ID NO.39, and the downstream primer is shown in SEQ ID NO.40.

[0084] SEQ ID NO. 39: GGTGCAACGACCAAAATTACC.

[0085] SEQ ID NO. 40: GGGTCCTGTCTTCTTGGAAAC.

[0086] The upstream primer for amplifying RIT1 is shown in SEQ ID NO.41, and the downstream primer is shown in SEQ ID NO.42.

[0087] SEQ ID NO. 41: TTCATCAGCCACCGATTCCC.

[0088] SEQ ID NO. 42: GCAGGCTCATCATCAATACGGA.

[0089] The upstream primer for amplifying PIMREG is shown in SEQ ID NO.43, and the downstream primer is shown in SEQ ID NO.44.

[0090] SEQ ID NO. 43: CGCTCAGCTAAGAGTGCTTTGG.

[0091] SEQ ID NO. 44: TGCCCTTCTGTGCTCTCTCTT.

[0092] The upstream primer for amplifying PSAT1 is shown in SEQ ID NO.45, and the downstream primer is shown in SEQ ID NO.46.

[0093] SEQ ID NO. 45: TGCCGCACTCAGTGTTGTTAG.

[0094] SEQ ID NO. 46: GCAATTCCCGCACAAGATTCT.

[0095] The upstream primer for amplifying FZD2 is shown in SEQ ID NO.47, and the downstream primer is shown in SEQ ID NO.48.

[0096] SEQ ID NO. 47: GTGCCATCCTATCTCAGCTACA.

[0097] SEQ ID NO. 48: CTGCATGTCTACCAAGTACGTG.

[0098] The upstream primer for amplifying FZD4 is shown in SEQ ID NO.49, and the downstream primer is shown in SEQ ID NO.50.

[0099] SEQ ID NO. 49: CCTCCGGCTACAACGTGACC.

[0100] SEQ ID NO. 50: TGCACATTGGCACATAAACAGA.

[0101] The upstream primer for amplifying NFIL3 is shown in SEQ ID NO.51, and the downstream primer is shown in SEQ ID NO.52.

[0102] SEQ ID NO. 51: AAAATGCAGACCGTCAAAAAGGA.

[0103] SEQ ID NO. 52: TGACACTTCCGTTAAAGCAGAAT.

[0104] The upstream primer for amplifying FN1 is shown in SEQ ID NO.53, and the downstream primer is shown in SEQ ID NO.54.

[0105] SEQ ID NO. 53: CGGTGGCTGTCAGTCAAAG.

[0106] SEQ ID NO. 54: AAACCTCGGCTTCCTCCATAA.

[0107] Compared with the prior art, the present invention has at least the following beneficial effects:

[0108] 1. This invention discovers that the prepared β-glucan has the effect of enhancing cell proliferation. Through transcriptomics, a series of genes related to the proliferation of skin extracellular matrix cells were screened, and it was found that β-glucan can significantly increase the expression level of the above genes, proving that this β-glucan can be used for the development and application of various cosmetic products that promote the proliferation of skin extracellular matrix cells.

[0109] 2. Experimental verification of this invention shows that the expression levels of the cell proliferation genes screened above are significantly increased in inflammatory and damaged cells, indicating that they can also serve as a reference for future evaluation of other raw materials or cosmetics that enhance the proliferative capacity of skin extracellular matrix cells. This invention has developed a series of primers for skin extracellular matrix cell proliferation-related genes. The detection results of these primers are accurate, facilitating rapid and accurate detection of the expression capacity of related genes in the future. Attached Figure Description

[0110] Figure 1 This is a heatmap for transcriptomics analysis. Detailed Implementation

[0111] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. However, the following examples are merely simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention is determined by the claims.

[0112] Example 1

[0113] This embodiment describes the preparation of β-glucan.

[0114] *Schizophyllum commune* was activated by inoculation onto agar slant culture medium and cultured at 28°C for 6 days until the mycelium completely covered the slant. The slant culture was then transferred to liquid culture medium and cultured for 2 days to obtain a seed culture. A fermentation medium was then prepared, comprising the following components: 2% glucose, 1% tryptone, 0.5% yeast extract, 0.05% magnesium sulfate, 0.1% potassium dihydrogen phosphate, 15 mg / L vitamin B1, and 5 mg / L folic acid, pH 6.0. 10% of the *Schizophyllum commune* inoculum was inoculated into the above medium and fermented at 28°C and 160 r / min for 50 h.

[0115] The fermentation product was centrifuged to remove the bacterial cells, yielding the fermentation product. Then, twice the volume of anhydrous ethanol was added for alcohol precipitation. The mixture was filtered, and the precipitated crude polysaccharide was collected. This was washed multiple times with anhydrous ethanol and then freeze-dried under vacuum to obtain β-glucan (SPG). 100 mg of solid SPG was weighed and dissolved completely in 100 mL of ultrapure water to prepare a 1 mg / mL SPG working solution. This solution was stored at 4°C for future use.

[0116] Example 2

[0117] This embodiment uses transcriptomics sequencing to study the effects of β-glucan on the skin in Example 1.

[0118] In this embodiment, four models were established using human fibroblasts (BJ cells): an experimental group, a drug-treated group, an inflammation group, and a repair group. The experimental group was supplemented with 2% ultrapure water; the drug-treated group was supplemented with 2% β-glucan prepared in Example 1; the inflammation group was supplemented with lipopolysaccharide (LPS) to a final concentration of 10 μg / mL to induce an inflammation model; and the repair group was supplemented with lipopolysaccharide (LPS) to a final concentration of 10 μg / mL to induce an inflammation model, along with 2% β-glucan prepared in Example 1. After 24 h of culture, the cell pellet was collected and sent to BGI Genomics for transcriptomics sequencing.

[0119] Transcriptomics sequencing results were obtained. In the comparison between the inflammation group and the control group, results with p-values ​​less than 0.05 and differential values ​​(log2 fold change > 0.25) were selected to identify a batch of genes that could serve as inflammatory biomarkers. The selected genes are as follows: CCNF, VEGFA, AURKA, CDCA2, CDCA8, CDCA3, PLK1, CENPA, CENPL, MKI67, SALL4, EZH1, JDP2, PBK, BUB1B, KIF15, KIF18B, KIF20A, KIF2C, KIFC1, RIT1, PIMREG, PSAT1, FZD2, FZD4, NFIL3, and FN1.

[0120] The genes selected in this invention, including CCNF, AURKA, PLK1, the CDCA series, CENPA, CENPL, BUB1B, and MKI67, constitute the core regulatory network of cell cycle and mitosis. They directly drive cell proliferation by coordinating key processes such as G2 / M phase transition, spindle assembly, and chromosome segregation. This proliferation process is precisely controlled by gene expression programs set by transcription factors such as SALL4, EZH1, JDP2, and NFIL3, as well as epigenetic regulators, and is supported by intracellular signal transduction mediated by KIF family kinases and kinases such as PBK and RIT1. Crucially, a dynamic interaction exists between cell proliferation and the extracellular matrix (ECM) microenvironment: FN1 (fibronectin), a core component of the ECM, not only provides a structural scaffold and adhesion signal for proliferation, but its remodeling process is also profoundly influenced by VEGFA-induced angiogenesis and the Wnt signaling pathway mediated by FZD2 / FZD4 receptors; while metabolic genes such as PSAT1 provide the material basis for ECM remodeling and rapid cell proliferation by supporting biosynthesis. These genes form a synergistic network in function, and their aberrant expression disrupts the balance between ECM homeostasis and cell proliferation, thus playing a key role in pathological processes such as tissue repair, fibrosis, and tumor progression.

[0121] The expression levels of the above genes in the transcriptome sequencing of each group were summarized, and the specific results are shown in Table 1. The expression levels of the above genes were then plotted as heatmaps using the GeoDio Cloud bioinformatics tool. Figure 1 As shown.

[0122] Table 1

[0123]

[0124] The sequencing results above show that the expression levels of the aforementioned cell proliferation-related genes significantly decreased after LPS induction, indicating a decline in cell proliferation capacity in the inflammation model. Furthermore, by adding the β-glucan prepared according to this invention to the inflammation model, the expression levels of the aforementioned genes were significantly increased compared to the inflammation group, demonstrating that β-glucan can significantly promote the expression of these cell proliferation genes. Similarly, by adding the β-glucan prepared according to this invention to the validation model, the expression levels of the aforementioned genes were significantly increased compared to the inflammation group, demonstrating that β-glucan can significantly promote the expression of these cell proliferation genes.

[0125] The expression levels of the aforementioned genes were calculated, as shown in Table 2. Compared to the control group, the LPS-induced inflammation model showed a significant decrease in the expression levels of many cell proliferation-related genes. The repair group, formed after the addition of β-glucan, significantly promoted the expression of these genes compared to the inflammation group. However, comparing the repair group with the control group revealed virtually no change in the expression levels of cell proliferation-related genes. This demonstrates the promoting effect of β-glucan on cell proliferation-related genes.

[0126] Table 2

[0127]

[0128] Example 3

[0129] Effects of different β-glucans on cell proliferation genes

[0130] This embodiment investigates the effects of β-glucan prepared in Example 1, commercially available β-glucan 1, and commercially available β-glucan 2 on cell proliferation-related genes.

[0131] LPS was added to BJ cells to a final concentration of 10 μg / mL to induce an inflammation model. Then, 2% of β-glucan prepared in Example 1, commercially available β-glucan 1, and commercially available β-glucan 2 were added respectively. After culturing for 24 h, the cell pellet was collected, RNA was extracted and reverse transcribed into cDNA, and qPCR was performed for detection. The genes detected were CCNF, VEGFA, AURKA, CDCA2, CDCA8, CDCA3, PLK1, CENPA, CENPL, MKI67, SALL4, EZH1, JDP2, PBK, BUB1B, KIF15, KIF18B, KIF20A, KIF2C, KIFC1, RIT1, PIMREG, PSAT1, FZD2, FZD4, NFIL3, and FN1. The primers used for qPCR detection were SEQ ID NO.1-SEQ ID NO.54. After detection, the promotion rates of β-glucan, commercially available β-glucan 1, and commercially available β-glucan 2 on cell proliferation genes were obtained. The calculation formula was: promotion rate (Log2FC) = Log2(gene expression level in the β-glucan repair group / gene expression level in the inflammation model group). The results are shown in Table 3.

[0132] Table 3

[0133]

[0134] Example 4

[0135] This embodiment performs ROC curve verification.

[0136] In this embodiment, ROC curve analysis was performed on all 27 cell proliferation-related genes screened in Example 2 to verify their sensitivity and specificity as biomarkers for diagnosing cell proliferation capacity in an inflammatory model.

[0137] In this embodiment, human fibroblasts (BJ cells) were used to prepare samples. Lipopolysaccharide (LPS) was used in the inflammation group to induce an inflammation model at a final concentration of 10 μg / mL. Thirty replicate biological samples were prepared for both the control and untreated groups. The expression levels of all 27 genes were detected using qPCR, with primer sequences identical to those in Example 3. The gene ΔCt values ​​obtained by qPCR were used as diagnostic variables. ROC curves were plotted for each gene using GraphPad Prism 9.0 software, and the AUC (area under the curve), sensitivity at the optimal critical value, and specificity were calculated. The ROC curve analysis results are shown in Table 4. The AUC values ​​of all 27 genes were greater than 0.7, with 24 genes having AUC values ​​greater than 0.8, indicating that this set of biomarkers has extremely high reliability in distinguishing inflammatory states.

[0138] The results above show that the 27 genes screened in this invention have excellent diagnostic performance and can be used as reliable biomarkers to evaluate the cell proliferation-promoting and repairing effects of cosmetic raw materials or products.

[0139] Table 4

[0140]

[0141] Example 5

[0142] This embodiment undergoes biological verification.

[0143] This embodiment verifies whether the β-glucan (SPG) prepared in Example 1 can effectively inhibit the expression of inflammatory genes in other cell types different from human fibroblasts (BJ), in order to demonstrate the universality of its anti-inflammatory effect.

[0144] This embodiment uses immortalized human keratinocytes (HaCaT cells) for experiments. Each cell group includes a control group, an inflammation model group, and an SPG repair group. In the inflammation model group, LPS was added to a final concentration of 10 μg / mL to induce an inflammation model; in the SPG repair group, LPS was first added to induce inflammation, followed by 2% of the SPG solution prepared in Example 1. After culturing for 24 h, total RNA was extracted from the cells, and the expression changes of all 27 cell proliferation-related genes were detected using the same qPCR primers as in Example 3. The log2 fold change of the SPG repair group relative to the inflammation model group was calculated.

[0145] The effects of SPG on the expression of cell proliferation-related genes in different cell models are shown in Table 5. In HaCaT cells, SPG significantly inhibited the low expression of cell proliferation-related genes induced by LPS.

[0146] Table 5

[0147]

[0148] The SPG prepared in this invention significantly inhibited the low expression of LPS-induced cell proliferation-related genes in human keratinocytes (HaCaT), and its inhibitory trend was highly consistent with its effect in BJ cells. This result fully demonstrates that the cell proliferation-promoting effect of SPG has universality across cell types and is not limited to a specific human fibroblast model, providing a solid experimental basis for its wide application in cosmetics.

[0149] In summary, this invention has found that the prepared β-glucan can enhance cell proliferation. Through transcriptomics, a series of genes related to the proliferation of skin extracellular matrix cells were screened, and it was found that β-glucan can significantly increase the expression level of the above genes, proving that this β-glucan can be used for the development and application of various cosmetic products that promote the proliferation of skin extracellular matrix cells.

[0150] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. The application of β-glucan in the preparation of formulations that enhance the expression levels of genes related to the proliferation of extracellular matrix cells in skin cells, characterized in that, The structure of the β-glucan is formed by the connection of structural units; The structural unit is Equation 1, where 150≤n≤2500; Formula 1.

2. The application of the β-glucan according to claim 1 in the preparation of formulations that enhance the expression of genes related to the proliferation of extracellular matrix cells in skin cells, characterized in that, The extracellular matrix cell proliferation-related genes of the skin include any one or a combination of at least two of the following: CCNF, VEGFA, AURKA, CDCA2, CDCA8, CDCA3, PLK1, CENPA, CENPL, MKI67, SALL4, EZH1, JDP2, PBK, BUB1B, KIF15, KIF18B, KIF20A, KIF2C, KIFC1, RIT1, PIMREG, PSAT1, FZD2, FZD4, NFIL3, or FN1.

3. The use of β-glucan according to any one of claims 1 or 2 in the preparation of formulations that enhance the expression of genes related to the proliferation of extracellular matrix cells in skin cells, characterized in that, The preparation includes pharmaceuticals or cosmetics; Preferably, the cosmetic includes any one or a combination of at least two of the following: face cream, lotion, gel, toner, serum, face mask, eye cream, aerosol cleansing foam, spray, shower gel, or facial cleanser.

4. The use of β-glucan according to any one of claims 1-3 in the preparation of formulations that enhance the expression of genes related to the proliferation of extracellular matrix cells in skin cells, characterized in that, The concentration of β-glucan in the formulation is 0.5%-5%.

5. The method for preparing β-glucan according to any one of claims 1-4, characterized in that, The preparation method includes: inoculating Schizophyllum commune into a fermentation medium for fermentation culture, centrifuging after fermentation, and then washing after alcohol precipitation to obtain the product.

6. The method for preparing β-glucan according to claim 5, characterized in that, The fermentation medium contains glucose, tryptone, yeast extract, magnesium sulfate, potassium dihydrogen phosphate, vitamin B1, and folic acid. Preferably, the inoculum amount of *Schizophyllum commune* is 5-15%; Preferably, the fermentation culture is carried out at a temperature of 25-30℃ for 40-60 h and at a rate of 100-200 r / min.

7. The method for preparing β-glucan according to claim 5, characterized in that, The fermentation medium contains 0.5%-5% glucose, 0.5%-3% tryptone, 0.05%-2% yeast extract, 0.01%-1% magnesium sulfate, 0.05%-0.5% potassium dihydrogen phosphate, 10-20 mg / L vitamin B1 and 1-10 mg / L folic acid; Preferably, the alcohol precipitation involves mixing 1-3 times the volume of anhydrous ethanol with the fermentation product and then filtering.

8. The application of products used to detect the expression level of biomarkers in evaluating the ability of cosmetics or their raw materials to enhance the proliferation of skin extracellular matrix cells, characterized in that, The biomarker is a skin extracellular matrix cell proliferation-related gene, which includes any one or a combination of at least two of the following: CCNF, VEGFA, AURKA, CDCA2, CDCA8, CDCA3, PLK1, CENPA, CENPL, MKI67, SALL4, EZH1, JDP2, PBK, BUB1B, KIF15, KIF18B, KIF20A, KIF2C, KIFC1, RIT1, PIMREG, PSAT1, FZD2, FZD4, NFIL3, or FN1.

9. The application according to claim 8, characterized in that, The product includes any one or a combination of at least two of the following: primer combinations, reagents, or detection models.

10. A primer combination for detecting the expression level of a biomarker, characterized in that, The primer combination includes: The upstream primer for amplifying CCNF is shown in SEQ ID NO.1, and the downstream primer is shown in SEQ ID NO.2; The upstream primer for amplifying VEGFA is shown in SEQ ID NO.3, and the downstream primer is shown in SEQ ID NO.4; The upstream primer for amplifying AURKA is shown in SEQ ID NO.5, and the downstream primer is shown in SEQ ID NO.6; The upstream primer for amplifying CDCA2 is shown in SEQ ID NO.7, and the downstream primer is shown in SEQ ID NO.8; The upstream primer for amplifying CDCA8 is shown in SEQ ID NO.9, and the downstream primer is shown in SEQ ID NO.10; The upstream primer for amplifying CDCA3 is shown in SEQ ID NO.11, and the downstream primer is shown in SEQ ID NO.12; The upstream primer for amplifying PLK1 is shown in SEQ ID NO.12, and the downstream primer is shown in SEQ ID NO.14; The upstream primer for amplifying CENPA is shown in SEQ ID NO.15, and the downstream primer is shown in SEQ ID NO.16; The upstream primer for amplifying CENPL is shown in SEQ ID NO.17, and the downstream primer is shown in SEQ ID NO.18; The upstream primer for amplifying MKI67 is shown in SEQ ID NO.19, and the downstream primer is shown in SEQ ID NO.20; The upstream primer for amplifying SALL4 is shown in SEQ ID NO.21, and the downstream primer is shown in SEQ ID NO.22; The upstream primer for amplifying EZH1 is shown in SEQ ID NO.23, and the downstream primer is shown in SEQ ID NO.24; The upstream primer for amplifying JDP2 is shown in SEQ ID NO.25, and the downstream primer is shown in SEQ ID NO.26; The upstream primer for amplifying PBK is shown in SEQ ID NO.27, and the downstream primer is shown in SEQ ID NO.28; The upstream primer for amplifying BUB1B is shown in SEQ ID NO.29, and the downstream primer is shown in SEQ ID NO.30; The upstream primer for amplifying KIF15 is shown in SEQ ID NO.31, and the downstream primer is shown in SEQ ID NO.32; The upstream primer for amplifying KIF18B is shown in SEQ ID NO.33, and the downstream primer is shown in SEQ ID NO.34; The upstream primer for amplifying KIF20A is shown in SEQ ID NO.35, and the downstream primer is shown in SEQ ID NO.36; The upstream primer for amplifying KIF2C is shown in SEQ ID NO.37, and the downstream primer is shown in SEQ ID NO.38; The upstream primer for amplifying KIFC1 is shown in SEQ ID NO.39, and the downstream primer is shown in SEQ ID NO.40; The upstream primer for amplifying RIT1 is shown in SEQ ID NO.41, and the downstream primer is shown in SEQ ID NO.42; The upstream primer for amplifying PIMREG is shown in SEQ ID NO.43, and the downstream primer is shown in SEQ ID NO.44; The upstream primer for amplifying PSAT1 is shown in SEQ ID NO.45, and the downstream primer is shown in SEQ ID NO.46; The upstream primer for amplifying FZD2 is shown in SEQ ID NO.47, and the downstream primer is shown in SEQ ID NO.48; The upstream primer for amplifying FZD4 is shown in SEQ ID NO.49, and the downstream primer is shown in SEQ ID NO.50; The upstream primer for amplifying NFIL3 is shown in SEQ ID NO.51, and the downstream primer is shown in SEQ ID NO.52; The upstream primer for amplifying FN1 is shown in SEQ ID NO.53, and the downstream primer is shown in SEQ ID NO.54.

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