Application of beta-glucan in preparation of preparation for reducing expression quantity of related genes of skin extracellular matrix inflammation
By using transcriptomics screening and β-glucan treatment, the expression of inflammation-related genes in the extracellular matrix of skin cells is reduced, which solves the problem of insufficient detection effect in existing technologies and realizes the anti-inflammatory effect and rapid detection of cosmetics at the gene level.
Patent Information
- Application Number
- CN202511451601.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-30
- Publication Date
- 2026-01-13
AI Technical Summary
Current technologies lack the ability to detect the effects of β-glucan on skin extracellular matrix inflammation at the gene level, making it impossible to effectively develop agents that reduce the expression levels of skin extracellular matrix inflammation-related genes.
Transcriptomics was used to screen for skin extracellular matrix inflammation-related genes, and β-glucan was used to reduce the expression levels of these genes. The preparation methods included fermentation, centrifugation, and alcohol precipitation. The gene expression was then detected by combining specific primer combinations with the gene expression in cosmetics.
It significantly reduces the expression of inflammation-related genes in the skin extracellular matrix, providing a reference for cosmetic development. The detection effect is accurate and has rapid detection capability.
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Figure CN121313488A_ABST
Abstract
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 reduce the expression of inflammation-related genes in the extracellular matrix of skin cells. Background Technology
[0002] The process of skin inflammation is complex. When the skin is exposed to a "triggered" stimulus, such as ultraviolet (UV) radiation, irritants, or allergens, cells in the skin produce a variety of inflammatory cytokines and chemokines. These cytokines and chemokines bind to specific receptors on target cells and stimulate the production of other inflammatory mediators, which cause vasodilation and activate nerve cells. The expression of cytokines and chemokines by inflammatory cells also leads to the production of enzymes, free radicals, and other biological mediators that damage the skin. The end result of the initial triggering event is an amplified large inflammatory response that, while intended to help the skin resist infection by invading bacteria, actually causes considerable damage to the skin.
[0003] The most basic function of β-glucan is its immunomodulatory effect. β-glucan can stimulate the innate immune system, thereby significantly enhancing the body's ability to resist bacteria, fungi, and viruses. It can also stimulate the differentiation and activation of T lymphocytes by activating T cells, macrophages, and natural killer cells, influencing and activating complement pathway changes, ultimately enhancing the body's cellular and humoral immunity. β-glucan can also act as dietary fiber, increasing intestinal transit time and slowing down intestinal absorption.
[0004] Existing research and literature disclose numerous experimental methods for detecting the effects of β-glucan on the degree of inflammation in skin tissues. However, most of these detections are at the macroscopic level and lack gene-level biomarkers. Therefore, developing genes related to β-glucan and skin extracellular matrix inflammation has important application value. Summary of the Invention
[0005] To address the aforementioned technical problems, this invention provides the application of β-glucan in the preparation of formulations that reduce the expression levels of inflammation-related genes in the extracellular matrix of skin cells.
[0006] To achieve this objective, the present invention adopts the following technical solution:
[0007] In a first aspect, the present invention provides the application of β-glucan in the preparation of formulations that reduce the expression of inflammation-related genes in the extracellular matrix of skin cells, wherein the structure of β-glucan is formed by the connection of structural units;
[0008] The structural unit is Equation 1, where 150≤n≤2500;
[0009]
[0010] Formula 1.
[0011] The skin extracellular matrix inflammation-related genes include any one or a combination of at least two of the following: IL1A, IL1B, IL1R1, IL1RAP, IL6, NFKB2, FOSB, FOSL1, JUN, JUND, CCL2, PTGS2, MMP10, MMP12, MMP13, CXCL8, CD86, PLAU, GADD45A, TNFSF18, TNFRSF10B, TNFRSF12A, TNFRSF14, or TNFRSF21.
[0012] This invention uses transcriptomics to screen a series of genes related to skin extracellular matrix inflammation and demonstrates that the β-glucan in this invention can reduce the expression of these genes. Therefore, it can be used in the development and application of various cosmetic products with anti-skin extracellular matrix inflammation effects. Furthermore, the aforementioned series of genes can also serve as a reference for future evaluation of the ability of other raw materials or cosmetics to reduce skin extracellular matrix inflammation.
[0013] Preferably, the formulation includes a drug or a cosmetic.
[0014] 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.
[0015] 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.
[0016] 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.
[0017] Preferably, the fermentation medium contains glucose, tryptone, yeast extract, magnesium sulfate, potassium dihydrogen phosphate, vitamin B1, and folic acid.
[0018] 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%.
[0019] 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.
[0020] 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.
[0021] 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.
[0022] 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 maintain the anti-inflammatory capacity of the skin extracellular matrix. The biomarkers are skin extracellular matrix inflammation-related genes, including any one or a combination of at least two of IL1A, IL1B, IL1R1, IL1RAP, IL6, NFKB2, FOSB, FOSL1, JUN, JUND, CCL2, PTGS2, MMP10, MMP12, MMP13, CXCL8, CD86, PLAU, GADD45A, TNFSF18, TNFRSF10B, TNFRSF12A, TNFRSF14, or TNFRSF21.
[0023] Preferably, the product comprises any one or a combination of at least two of the following: primer combinations, reagents, or detection models.
[0024] Fourthly, the present invention provides a primer combination for detecting the expression level of a biomarker, the primer combination comprising:
[0025] The upstream primer for amplifying IL1A is shown in SEQ ID NO.1, and the downstream primer is shown in SEQ ID NO.2.
[0026] SEQ ID NO. 1: TGGTAGTAGCAACCAACGGGA.
[0027] SEQ ID NO. 2: ACTTTGATTGAGGGCGTCATTC.
[0028] The upstream primer for amplifying IL1B is shown in SEQ ID NO.3, and the downstream primer is shown in SEQ ID NO.4.
[0029] SEQ ID NO. 3: ATGATGGCTTATTACAGTGGCAA.
[0030] SEQ ID NO. 4: GTCGGAGATTCGTAGCTGGA.
[0031] The upstream primer for amplifying IL1R1 is shown in SEQ ID NO.5, and the downstream primer is shown in SEQ ID NO.6.
[0032] SEQ ID NO. 5:ATGAAATTGATGTTCGTCCCTGT.
[0033] SEQ ID NO. 6: ACCACGCAATAGTAATGTCCTG.
[0034] The upstream primer for amplifying IL1RAP is shown in SEQ ID NO.7, and the downstream primer is shown in SEQ ID NO.8.
[0035] SEQ ID NO. 7: ACACTTCTGTGGTGTGTAGTGA.
[0036] SEQ ID NO. 8: TGGTGTCTAGTCCCCAGTCAT.
[0037] The upstream primer for amplifying IL6 is shown in SEQ ID NO.9, and the downstream primer is shown in SEQ ID NO.10.
[0038] SEQ ID NO.9: ACTCACCTCTTCAGAACGAATTG.
[0039] SEQ ID NO. 10: CCATCTTTGGAAAGGTTCAGGTTG.
[0040] The upstream primer for amplifying NFKB2 is shown in SEQ ID NO.11, and the downstream primer is shown in SEQ ID NO.12.
[0041] SEQ ID NO. 11: ATGGAGAGTTGCTACAACCCA.
[0042] SEQ ID NO. 12: CTGTTCCAGATCACCAGGTA.
[0043] The upstream primer for amplifying FOSB is shown in SEQ ID NO.13, and the downstream primer is shown in SEQ ID NO.14.
[0044] SEQ ID NO. 13: GCTGCAAGATCCCCTACGAAG.
[0045] SEQ ID NO. 14: ACGAAGAAGTGTACGAAGGGTT.
[0046] The upstream primer for amplifying FOSL1 is shown in SEQ ID NO.15, and the downstream primer is shown in SEQ ID NO.16.
[0047] SEQ ID NO. 15: CAGGCGGAGACTGACAAACTG.
[0048] SEQ ID NO. 16: TCCTTCCGGGATTTTGCAGAT.
[0049] The upstream primer for amplifying JUN is shown in SEQ ID NO.17, and the downstream primer is shown in SEQ ID NO.18.
[0050] SEQ ID NO. 17: TCCAAGTGCCGAAAAAGGAAG.
[0051] SEQ ID NO. 18: CGAGTTCTGAGCTTTCAAGGT.
[0052] The upstream primer for amplifying JUND is shown in SEQ ID NO.19, and the downstream primer is shown in SEQ ID NO.20.
[0053] SEQ ID NO. 19: TCATCATCCAGTCCAACGGG.
[0054] SEQ ID NO. 20: TTCTGCTTGTGTAAATCCTCCAG.
[0055] The upstream primer for amplifying CCL2 is shown in SEQ ID NO.21, and the downstream primer is shown in SEQ ID NO.22.
[0056] SEQ ID NO. 21: CAGCCAGATGCAATCAATGCC.
[0057] SEQ ID NO. 22: TGGAATCCTGAACCCACTTCT.
[0058] The upstream primer for amplifying PTGS2 is shown in SEQ ID NO.23, and the downstream primer is shown in SEQ ID NO.24.
[0059] SEQ ID NO. 23: CTGGCGCTCAGCCATACAG.
[0060] SEQ ID NO. 24: CGCACTTATACTGGTCAAATCCC.
[0061] The upstream primer for amplifying MMP10 is shown in SEQ ID NO.25, and the downstream primer is shown in SEQ ID NO.26.
[0062] SEQ ID NO. 25: TGCTCTGCCTATCCTCTGAGT.
[0063] SEQ ID NO. 26: TCACATCCTTTTCGAGGTTGTAG.
[0064] The upstream primer for amplifying MMP12 is shown in SEQ ID NO.27, and the downstream primer is shown in SEQ ID NO.28.
[0065] SEQ ID NO. 27: CATGAACCGTGAGGATGTTGA.
[0066] SEQ ID NO. 28: GCATGGGCTAGGATTCCACC.
[0067] The upstream primer for amplifying MMP13 is shown in SEQ ID NO.29, and the downstream primer is shown in SEQ ID NO.30.
[0068] SEQ ID NO. 29: ACTGAGAGGCTCCGAGAAATG.
[0069] SEQ ID NO. 30: GAACCCCGCATCTTGGCTT.
[0070] The upstream primer for amplifying CXCL8 is shown in SEQ ID NO.31, and the downstream primer is shown in SEQ ID NO.32.
[0071] SEQ ID NO. 31: TTTTGCCAAGGAGTGCTAAAGA.
[0072] SEQ ID NO. 32: AACCCTCTGCACCCAGTTTTC.
[0073] The upstream primer for amplifying CD86 is shown in SEQ ID NO.33, and the downstream primer is shown in SEQ ID NO.34.
[0074] SEQ ID NO. 33: CTGCTCATCTATACACGGTTACC.
[0075] SEQ ID NO. 34: GGAAACGTCGTACAGTTCTGTG.
[0076] The upstream primer for amplifying PLAU is shown in SEQ ID NO.35, and the downstream primer is shown in SEQ ID NO.36.
[0077] SEQ ID NO. 35: GGGAATGGTCACTTTTACCGAG.
[0078] SEQ ID NO. 36: GGGCATGGTACGTTTGCTG.
[0079] The upstream primer for amplifying GADD45A is shown in SEQ ID NO.37, and the downstream primer is shown in SEQ ID NO.38.
[0080] SEQ ID NO. 37: GAGAGCAGAAGACCGAAAGGA.
[0081] SEQ ID NO. 38: CACAACACCACGTTATCGGG.
[0082] The upstream primer for amplifying TNFSF18 is shown in SEQ ID NO.39, and the downstream primer is shown in SEQ ID NO.40.
[0083] SEQ ID NO. 39: AGTGGCTCCCAATGCAAACTA.
[0084] SEQ ID NO. 40: TATACAGCCGCACCTCAAAAG.
[0085] The upstream primer for amplifying TNFRSF10B is shown in SEQ ID NO.41, and the downstream primer is shown in SEQ ID NO.42.
[0086] SEQ ID NO. 41: ATGGAACAACGGGGACAGAAC.
[0087] SEQ ID NO. 42: CTGCTGGGGAGCTAGGTCT.
[0088] The upstream primer for amplifying TNFRSF12A is shown in SEQ ID NO.43, and the downstream primer is shown in SEQ ID NO.44.
[0089] SEQ ID NO. 43:TCTGAGCCTGACCTTCGTGCTG.
[0090] SEQ ID NO. 44: GGCACATTGTCACTGGATCAGC.
[0091] The upstream primer for amplifying TNFRSF14 is shown in SEQ ID NO.45, and the downstream primer is shown in SEQ ID NO.46.
[0092] SEQ ID NO. 45: GTGCAGTCCAGGTTATCGTGT.
[0093] SEQ ID NO. 46: CACTTGCTTAGGCCATTGAGG.
[0094] The upstream primer for amplifying TNFRSF21 is shown in SEQ ID NO.47, and the downstream primer is shown in SEQ ID NO.48.
[0095] SEQ ID NO. 47: ATTGGCACATACCGCCATGTT.
[0096] SEQ ID NO. 48: GGCTTGTGTTGGTACAATGCTC.
[0097] Compared with the prior art, the present invention has at least the following beneficial effects:
[0098] 1. This invention discovers that the prepared β-glucan has the effect of reducing inflammation. Through transcriptomics, a series of genes related to skin extracellular matrix inflammation were screened, and it was found that β-glucan can significantly reduce the expression level of the above genes, proving that this β-glucan can be used for the development and application of various cosmetic products with the effect of inhibiting skin extracellular matrix inflammation.
[0099] 2. Experimental verification of this invention shows that the expression levels of the screened inflammatory genes significantly increased in inflammatory damaged cells, indicating that they can also serve as a reference for future evaluation of the ability of other raw materials or cosmetics to reduce skin extracellular matrix inflammation. This invention has developed a series of primers for skin extracellular matrix inflammation-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
[0100] Figure 1 This is a heatmap for transcriptomics analysis. Detailed Implementation
[0101] 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.
[0102] Example 1
[0103] This embodiment describes the preparation of β-glucan.
[0104] *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.
[0105] 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.
[0106] Example 2
[0107] This embodiment uses transcriptomics sequencing to study the effects of recombinant collagen on the skin in Example 1.
[0108] 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 received 2% ultrapure water; the drug-treated group received 2% SPG solution; the inflammation group received lipopolysaccharide (LPS) at a final concentration of 10 μg / mL to induce an inflammation model; and the repair group received LPS at a final concentration of 10 μg / mL to induce an inflammation model, along with 2% SPG solution. All cells were cultured for 24 h and then sent to BGI Genomics for transcriptomics sequencing.
[0109] Transcriptomic 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 group of genes that could serve as inflammatory biomarkers. The selected genes are as follows: IL1A, IL1B, IL1R1, IL1RAP, IL6, NFKB2, FOSB, FOSL1, JUN, JUND, CCL2, PTGS2, MMP10, MMP12, MMP13, CXCL8, CD86, PLAU, GADD45A, TNFSF18, TNFRSF10B, TNFRSF12A, TNFRSF14, and TNFRSF21.
[0110] The selected genes are all key participants in the inflammatory response and can serve as core inflammatory biomarkers: IL1A, IL1B, IL6, and CXCL8 are classic pro-inflammatory cytokines that mediate inflammation initiation, fever and pain, acute phase response, and neutrophil recruitment, respectively. Their expression is positively correlated with the severity of inflammation in scenarios such as infection and autoimmune diseases. IL1R1 and IL1RAP, as receptors and accessory proteins of IL1-like factors, reflect the sensitivity of cells to IL1 signaling and indicate the state of local inflammatory activation. NFKB2, FOSB, FOSL1, JUN, and JUND belong to transcriptional regulatory factors (NF-κB or AP-1 family), which drive inflammatory responses by regulating the transcription of pro-inflammatory factors and chemokines. Increased activity of these genes is closely related to chronic inflammation and inflammation-related diseases. CCL2, as a chemokine, can recruit monocytes to inflammatory lesions, and CD86, as... Immune co-stimulatory molecules regulate T cell-mediated adaptive inflammation, reflecting the degree of infiltration of inflammatory immune cells and the activation status of antigen-presenting cells, respectively. PTGS2 (COX-2) is a key enzyme in prostaglandin synthesis, mediating inflammatory pain and vasodilation, and is induced to be highly expressed in inflammatory lesions. MMP10, MMP12, and MMP13 are matrix metalloproteinases that participate in the repair of inflammatory tissue damage, elastin degradation, and cartilage destruction, respectively, and their expression is correlated with the degree of inflammation-mediated tissue damage. PLAU participates in fibrinolysis and tissue remodeling, GADD45A is associated with inflammatory stress and cell cycle regulation, and TNFSF18, TNFRSF10B, TNFRSF12A, and TNFRSF21 regulate inflammation and apoptosis through the TNF / TNFR signaling pathway, playing a role in the occurrence and development of inflammation. Their expression changes can serve as important monitoring indicators of inflammatory status.
[0111] 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.
[0112] Table 1
[0113]
[0114] The sequencing results showed that the expression levels of the aforementioned genes significantly increased after LPS induction, demonstrating that these genes can be considered inflammation-related genes. Furthermore, by adding the β-glucan prepared in this invention to the inflammation model, it was found that the expression levels of the aforementioned genes were significantly reduced compared to the inflammation group, proving that β-glucan can significantly inhibit the expression of the aforementioned inflammatory genes.
[0115] The expression change rate (Log2 Fold Change) of the above genes was calculated, as shown in Table 2. In the LPS-induced inflammation model compared to the control group (inflammation group / control group), a large number of inflammation-related genes were upregulated. The repair group formed after adding β-glucan, compared to the inflammation group (repair group / inflammation group), significantly inhibited the expression of these genes (showing a negative value). Comparing the repair group with the control group, the expression levels of related inflammatory genes remained essentially unchanged. This demonstrates the inhibitory effect of LPS on inflammation.
[0116] Table 2
[0117]
[0118] Example 3
[0119] Effects of different β-glucans on inflammatory genes
[0120] This embodiment investigates the effects of β-glucan prepared in Example 1, commercially available β-glucan 1, and commercially available β-glucan 2 on inflammatory genes.
[0121] 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 IL1A, IL1B, IL1R1, IL1RAP, IL6, NFKB2, FOSB, FOSL1, JUN, JUND, CCL2, PTGS2, MMP10, MMP12, MMP13, CXCL8, CD86, PLAU, GADD45A, TNFSF18, TNFRSF10B, TNFRSF12A, TNFRSF14, and TNFRSF21. The primers used for qPCR detection were SEQ ID NO.1-SEQ ID NO.48. After detection, the inhibition rates of β-glucan, commercially available β-glucan 1, and commercially available β-glucan 2 on inflammatory genes were obtained. The inhibition rate (Log2FC) = Log2(gene expression level in the β-glucan repair group / gene expression level in the inflammatory model group). The results are shown in Table 3.
[0122] Table 3
[0123]
[0124] The results above show that the β-glucan prepared in Example 1 has a significantly higher inhibitory effect on inflammation-related genes than other commercially available β-glucans.
[0125] Example 4
[0126] This embodiment performs ROC curve verification.
[0127] In this embodiment, ROC curve analysis was performed on all 24 inflammation-related genes screened in Example 2 to verify their diagnostic efficacy (sensitivity and specificity) as biomarkers in distinguishing inflammation models from normal cells.
[0128] In this embodiment, human fibroblasts (BJ cells) were used to prepare samples. In the inflammation group, lipopolysaccharide (LPS) was used to achieve a final concentration of 10 μg / mL to induce an inflammation model. Thirty replicate biological samples were prepared for both the control and untreated groups. The expression levels of all 24 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.
[0129] The ROC curve analysis results are shown in Table 4. The AUC values of all 24 genes were greater than 0.7, and the AUC values of 20 genes were greater than 0.8, indicating that this group of biomarkers has extremely high reliability in distinguishing inflammatory states.
[0130] The results above show that the 24 genes screened in this invention have excellent diagnostic performance and can be used as reliable biomarkers to evaluate the anti-inflammatory efficacy of cosmetic raw materials or products.
[0131] Table 4
[0132]
[0133] Example 5
[0134] This embodiment undergoes biological verification.
[0135] 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.
[0136] This embodiment uses immortalized human keratinocytes (HaCaT cells) and mouse macrophages (RAW264.7 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 24 h of culture, total RNA was extracted from the cells, and the expression changes of all 24 inflammatory 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.
[0137] The inhibitory effects of SPG on inflammatory gene expression in different cell models are shown in Table 5. In both HaCaT and RAW264.7 cell lines, SPG significantly inhibited LPS-induced high expression of inflammatory genes.
[0138] The SPG prepared in this invention significantly inhibited the high expression of LPS-induced inflammation-related genes in both human keratinocytes (HaCaT) and mouse macrophages (RAW264.7), and its inhibitory trend was highly consistent with the effect in BJ cells. This result fully demonstrates that the anti-inflammatory efficacy 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.
[0139] Table 5
[0140]
[0141] In summary, this invention, through transcriptomics, screened a series of genes related to skin extracellular matrix inflammation and demonstrated that the β-glucan in this invention can reduce the expression of these genes. Therefore, it can be used in the development and application of various cosmetic products with anti-inflammatory effects on the skin extracellular matrix. Furthermore, the aforementioned gene series can also serve as a reference for future evaluation of the ability of other raw materials or cosmetics to reduce skin extracellular matrix inflammation.
[0142] 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 reduce the expression levels of inflammation-related genes in the extracellular matrix of 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 reduce the expression levels of inflammation-related genes in the extracellular matrix of skin cells, characterized in that, The skin extracellular matrix inflammation-related genes include any one or a combination of at least two of the following: IL1A, IL1B, IL1R1, IL1RAP, IL6, NFKB2, FOSB, FOSL1, JUN, JUND, CCL2, PTGS2, MMP10, MMP12, MMP13, CXCL8, CD86, PLAU, GADD45A, TNFSF18, TNFRSF10B, TNFRSF12A, TNFRSF14, or TNFRSF21.
3. The use of β-glucan according to any one of claims 1 or 2 in the preparation of formulations that reduce the expression levels of inflammation-related genes in the extracellular matrix of 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 reduce the expression levels of inflammation-related genes in the extracellular matrix of 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 or 6, 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 maintain the anti-inflammatory capacity of the skin extracellular matrix, characterized in that, The biomarker is an extracellular matrix inflammation-related gene in the skin, which includes any one or a combination of at least two of the following: IL1A, IL1B, IL1R1, IL1RAP, IL6, NFKB2, FOSB, FOSL1, JUN, JUND, CCL2, PTGS2, MMP10, MMP12, MMP13, CXCL8, CD86, PLAU, GADD45A, TNFSF18, TNFRSF10B, TNFRSF12A, TNFRSF14, or TNFRSF21.
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 IL1A is shown in SEQ ID NO.1, and the downstream primer is shown in SEQ ID NO.2; The upstream primer for amplifying IL1B is shown in SEQ ID NO.3, and the downstream primer is shown in SEQ ID NO.4; The upstream primer for amplifying IL1R1 is shown in SEQ ID NO.5, and the downstream primer is shown in SEQ ID NO.6; The upstream primer for amplifying IL1RAP is shown in SEQ ID NO.7, and the downstream primer is shown in SEQ ID NO.8; The upstream primer for amplifying IL6 is shown in SEQ ID NO.9, and the downstream primer is shown in SEQ ID NO.10; The upstream primer for amplifying NFKB2 is shown in SEQ ID NO.11, and the downstream primer is shown in SEQ ID NO.12; The upstream primer for amplifying FOSB is shown in SEQ ID NO.12, and the downstream primer is shown in SEQ ID NO.14; The upstream primer for amplifying FOSL1 is shown in SEQ ID NO.15, and the downstream primer is shown in SEQ ID NO.16; The upstream primer for amplifying JUN is shown in SEQ ID NO.17, and the downstream primer is shown in SEQ ID NO.18; The upstream primer for amplifying JUND is shown in SEQ ID NO.19, and the downstream primer is shown in SEQ ID NO.20; The upstream primer for amplifying CCL2 is shown in SEQ ID NO.21, and the downstream primer is shown in SEQ ID NO.22; The upstream primer for amplifying PTGS2 is shown in SEQ ID NO.23, and the downstream primer is shown in SEQ ID NO.24; The upstream primer for amplifying MMP10 is shown in SEQ ID NO.25, and the downstream primer is shown in SEQ ID NO.26; The upstream primer for amplifying MMP12 is shown in SEQ ID NO.27, and the downstream primer is shown in SEQ ID NO.28; The upstream primer for amplifying MMP13 is shown in SEQ ID NO.29, and the downstream primer is shown in SEQ ID NO.30; The upstream primer for amplifying CXCL8 is shown in SEQ ID NO.31, and the downstream primer is shown in SEQ ID NO.32; The upstream primer for amplifying CD86 is shown in SEQ ID NO.33, and the downstream primer is shown in SEQ ID NO.34; The upstream primer for amplifying PLAU is shown in SEQ ID NO.35, and the downstream primer is shown in SEQ ID NO.36; The upstream primer for amplifying GADD45A is shown in SEQ ID NO.37, and the downstream primer is shown in SEQ ID NO.38; The upstream primer for amplifying TNFSF18 is shown in SEQ ID NO.39, and the downstream primer is shown in SEQ ID NO.40; The upstream primer for amplifying TNFRSF10B is shown in SEQ ID NO.41, and the downstream primer is shown in SEQ ID NO.42; The upstream primer for amplifying TNFRSF12A is shown in SEQ ID NO.43, and the downstream primer is shown in SEQ ID NO.44; The upstream primer for amplifying TNFRSF14 is shown in SEQ ID NO.45, and the downstream primer is shown in SEQ ID NO.46; The upstream primer for amplifying TNFRSF21 is shown in SEQ ID NO.47, and the downstream primer is shown in SEQ ID NO.48.