Polypeptide compound and application thereof

Abstract

The invention provides a polypeptide compound and application thereof, and belongs to the technical field of medical or dressing preparations. According to the invention, three specific polypeptides are synergistically proportioned to obtain the polypeptide compound, and the polypeptide compound has the effect of repairing a skin basement membrane in a targeted manner. The polypeptide compound has the advantages of being small in molecular weight, high in safety, low in allergy risk and high in permeability, and has excellent repairing and anti-aging functions. Experiments prove that the polypeptide compound provided by the invention, as an active peptide raw material, can remarkably promote the production of endogenous collagen IV, VII and XVII of skin, is also beneficial to repairing skin extracellular matrix, resisting DNA damage, improving skin cycle protein, inhibiting release of inflammatory factors and the like, can effectively repair a basement membrane, and has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of medical or cosmetic preparations, and particularly to polypeptide complexes and their applications. Background Art

[0002] One of the core manifestations of skin aging is the structural collapse of the epidermal-dermal junction (DEJ), which is mainly manifested as the degradation of the collagen networks of type IV, type VII, and type XVII. As the dynamic interface between the epidermis and the dermis, the DEJ not only undertakes the physiological functions of mechanical anchoring and nutrient exchange, but is also the core hub for maintaining the integrity of the skin barrier and regulating cell signal transduction. The integrity of its three-dimensional network structure depends on the three-dimensional synergistic effects of the basement membrane skeleton composed of type IV collagen, the anchoring fiber system formed by type VII collagen, and the transmembrane signal transduction network mediated by type XVII collagen. With age, the DEJ layer significantly thins, and the loss of reticular ridges leads to a reduction in its surface area, presenting a flat appearance, which in turn affects the skin's resistance to shear forces and makes the skin more vulnerable to damage.

[0003] The anti-aging solutions in the prior art mainly focus on exogenous macromolecular collagen supplementation or single polypeptide targeting, but these methods have obvious limitations. For example, exogenous macromolecular collagen supplementation is difficult to effectively penetrate into the DEJ layer and reconstruct its complex three-dimensional network structure; while single polypeptide targeting has limited action targets and cannot comprehensively activate the key signal pathways of the DEJ layer. In addition, the complexity of the DEJ layer makes it a major challenge in the field of anti-aging research.

[0004] Chinese Patent (CN118873431A) discloses a polypeptide composition for promoting skin repair, including palmitoyl tripeptide-5, acetyl hexapeptide-37, snow lotus stem cell extract, etc., which can synergistically enhance the skin barrier function and is suitable for the repair of sensitive skin, but this solution does not disclose the repair effect on the skin DEJ. Summary of the Invention

[0005] The purpose of the present invention is to provide a polypeptide complex and its applications, which have the advantages of small molecular weight, high safety, low allergy risk, and strong permeability, and also have excellent repair and anti-aging functions.

[0006] In order to achieve the above-mentioned invention purpose, the present invention provides the following technical solutions: The present invention provides a polypeptide complex, which contains polypeptide Pep-IV with an amino acid sequence as shown in SEQ ID NO.1, polypeptide Pep-VII with an amino acid sequence as shown in SEQ ID NO.2, and polypeptide Pep-XVII with an amino acid sequence as shown in SEQ ID NO.3.

[0007] Preferably, the weight ratio of polypeptide Pep-IV, polypeptide Pep-VII and polypeptide Pep-XVII in the polypeptide complex is 1:0.8-1.2:0.8-1.2.

[0008] The present invention also provides the application of the above-mentioned polypeptide complex in the preparation of products for repairing the skin basement membrane.

[0009] The present invention also provides the application of the above-mentioned polypeptide complex in the preparation of anti-aging products.

[0010] The present invention also provides the application of the above-mentioned polypeptide complex in the preparation of a promoter for promoting collagen synthesis.

[0011] Preferably, the collagen includes COL4A1, COL7A1, COL17A1, COL6A2, COL8A1, COL14A1 and COL16A1.

[0012] The present invention also provides the application of the above-mentioned polypeptide complex in the preparation of products for inhibiting skin inflammation.

[0013] The present invention also provides the application of the above-mentioned polypeptide complex in the preparation of products for promoting DNA damage repair or reagents for regulating skin cell cycle proteins.

[0014] The present invention also provides the application of the above-mentioned polypeptide complex in the preparation of a reagent for promoting LAMA2 gene expression or a reagent for inhibiting MMP19 gene expression.

[0015] The present invention also provides a skin care product containing the above-mentioned polypeptide complex.

[0016] Advantages of the present invention: In the present invention, three specific polypeptides are synergistically proportioned to obtain a polypeptide complex with the efficacy of targeting and repairing the skin basement membrane. This polypeptide complex has the advantages of small molecular weight, high safety, low allergy risk and strong permeability, and also has excellent repair and anti-aging functions. Experiments have proved that the polypeptide complex provided by the present invention can significantly promote the production of endogenous type IV, VII, and XVII collagen in the skin as an active peptide raw material, and is also beneficial to repairing the skin extracellular matrix, resisting DNA damage, improving skin cyclin, inhibiting the release of inflammatory factors, etc. It can effectively repair the basement membrane and has broad application prospects. Description of the drawings

[0017] Figure 1A Effect of different ratios of Pep-IV, Pep-VII, and Pep-XVII on the COL4A1 in the Figure 1B Effect of different ratios of Pep-IV, Pep-VII, and Pep-XVII on theCOL7A1 Effect; Figure 1C For the effects of different ratios of Pep-IV, Pep-VII, and Pep-XVII on COL17A1 in the DEJ; Figure 2A For the effects of the polypeptide complex and single collagen of the same sequence on COL4A1 in the epidermal-dermal junction layer; Figure 2B For the effects of the polypeptide complex and single collagen of the same sequence on COL7A1 in the epidermal-dermal junction layer; Figure 2C For the effects of the polypeptide complex and single collagen of the same sequence on COL17A1 in the epidermal-dermal junction layer; Figure 3 For the effects of the polypeptide complex on LAMA2 gene expression; Figure 4 For the effects of the polypeptide complex on IL-11 gene expression; Figure 5 For the results of the human Raman permeability test of the polypeptide complex. Detailed implementation manners

[0018] The present invention provides a polypeptide complex, which contains polypeptide Pep-IV with an amino acid sequence shown in SEQ ID NO.1, polypeptide Pep-VII with an amino acid sequence shown in SEQ ID NO.2, and polypeptide Pep-XVII with an amino acid sequence shown in SEQ ID NO.3. Preferably, the weight ratio of polypeptide Pep-IV, polypeptide Pep-VII, and polypeptide Pep-XVII in the polypeptide complex is 1:0.8-1.2:0.8-1.2.

[0019] The present invention also provides the application of the above polypeptide complex in the preparation of products for repairing the skin basement membrane. In the present invention, the "products for repairing the skin basement membrane" refer to skin care or medical products for repairing or strengthening the basement membrane structure (skin basement membrane) at the junction of the epidermis and the dermis. By helping to restore the function of the basement membrane, skin problems caused by damaged basement membrane can be improved. In the present invention, the dosage form of the product can be selected from essence, lotion, cream, gel, spray, ointment, patch, lyophilized powder, oil, microneedle, ball, ampoule, mud mask, milk cream, aerosol, or nano microemulsion, etc.

[0020] The present invention also provides the application of the above polypeptide complex in the preparation of anti-aging products. In the present invention, the "anti-aging products" refer to skin care preparations for delaying skin aging manifestations, and the products may also include active compounding ingredients, excipients, etc.

[0021] The present invention also provides the use of the above polypeptide complex in the preparation of a promoter for promoting collagen synthesis. In the present invention, the "promoter for promoting collagen synthesis" refers to a preparation capable of increasing the amount of collagen produced in the skin, and its dosage form can be selected from gels, lyophilized powders, microneedles or rolling balls, etc.

[0022] The present invention also provides the use of the above polypeptide complex in the preparation of a product for inhibiting skin inflammation. In the present invention, the "product for inhibiting skin inflammation" refers to a nursing preparation for reducing skin inflammatory reactions, and its dosage form can be selected from creams, patches, sprays or mask preparations, etc.

[0023] The present invention also provides the use of the above polypeptide complex in the preparation of a product for promoting DNA damage repair or a skin cell cycle protein regulator. In the present invention, the "product for promoting DNA damage repair" refers to a preparation for repairing DNA damage in skin cells or cells cultured in vitro, and the "skin cell cycle protein regulator" refers to a preparation for regulating the expression of cell cycle-related proteins, and its dosage form can be selected from lyophilized powders, aerosols, nanoemulsions or oils, etc.

[0024] The present invention also provides the use of the above polypeptide complex in the preparation of LAMA2 a gene expression promoter or MMP19 a gene expression inhibitor. In the present invention, the LAMA2 "gene expression promoter" refers to a preparation for increasing LAMA2 the gene expression level, and the MMP19 "gene expression inhibitor" refers to a preparation for decreasing MMP19 the gene expression level, and can be used for regulating the gene expression level in related experiments.

[0025] The present invention also provides a skin care product containing the above polypeptide complex. In the present invention, the dosage form of the skin care product can be selected from essence, lotion, cream, gel or lyophilized powder, etc., and the compounding ingredients can be selected from moisturizers, antioxidants, emollients, soothers, film-forming agents, preservatives, emulsifiers, pH regulators, penetration enhancers, mineral components, amino acids, sugar derivatives or lipid components, etc.

[0026] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0027] Example 1 Mix the polypeptide Pep-IV with the amino acid sequence shown in SEQ ID NO.1, the polypeptide Pep-VII with the amino acid sequence shown in SEQ ID NO.2, and the polypeptide Pep-XVII with the amino acid sequence shown in SEQ ID NO.3 in a weight ratio of 1:1:1 to obtain a polypeptide complex.

[0028] Example 2 Mix the polypeptide Pep-IV with the amino acid sequence shown in SEQ ID NO.1, the polypeptide Pep-VII with the amino acid sequence shown in SEQ ID NO.2, and the polypeptide Pep-XVII with the amino acid sequence shown in SEQ ID NO.3 in a weight ratio of 1:0.8:1.2 to obtain a polypeptide complex.

[0029] Example 3 Mix the polypeptide Pep-IV with the amino acid sequence shown in SEQ ID NO.1, the polypeptide Pep-VII with the amino acid sequence shown in SEQ ID NO.2, and the polypeptide Pep-XVII with the amino acid sequence shown in SEQ ID NO.3 in a weight ratio of 1:1.2:0.8 to obtain a polypeptide complex.

[0030] Experimental Example Pep-IV, Pep-VII, and Pep-XVII were selected through the database, and the sequences are shown in Table 1 below: Table 1 Sequences of Pep-IV, Pep-VII, and Pep-XVII

[0031] Under the mass ratio (Pep-IV : Pep-VII : Pep-XVII) = (10% - 40%) : (10% - 70%) : (15% - 50%), explore the promoting effect of this composite polypeptide on type IV, type VII, and type XVII collagens. Finally, the only ratio with the best promoting effect on the three key collagens in the DEJ was screened out, which is (Pep-IV : Pep-VII : Pep-XVII) = 1 : 1 : 1, and the composite polypeptide obtained at this ratio was named the polypeptide complex.

[0032] The specific test process is as follows: Determine the COL4A1 、 COL7A1 and COL17A1 gene expression by RT-qPCR (real-time quantitative polymerase chain reaction): Seed HaCaT cells in the logarithmic growth phase at 1×10 6Cells were seeded at a density of [number] cells / well in a 6-well plate and cultured in an incubator at 37°C with 5% CO2 for 24 h. The culture medium was discarded, and the sample group was added with the sample (mixed at a set ratio, with the final concentration of the total peptide being 10 ppm, 10 mg / L, the same below), while the control group and the model group were added with serum-free medium. Then, they were cultured in an incubator at 37°C with 5% CO2 for 24 h. The culture medium was discarded, and the cells were treated with UVB (ultraviolet B band, 60 mJ / cm 2 ), and the cells in the blank control group did not need to be treated with UVB. After the treatment, serum-free medium was added, and they were cultured in an incubator at 37°C with 5% CO2 for 24 h. The supernatant was discarded, and the cells were collected after washing twice with PBS. RNA was extracted and reverse-transcribed into cDNA, followed by RT-qPCR.

[0033] The experimental results are as Figures 1A - 1C shown: After UVB irradiation, the relative expression levels of COL4A1, COL7A1, COL17A1 genes in the model group (NC) HaCaT cells were significantly decreased, indicating that the model was effective. When (Pep-IV : Pep-VII : Pep-XVII) = 1:1:1 (the final concentration of the total peptide was 10 ppm), the relative expression levels of COL4A1, COL7A1, COL17A1 genes were increased by 34.8%, 49.38%, and 75.77% respectively. This was the only ratio that significantly promoted the expression of the three collagen genes in the DEJ among the tested ratios. The composite polypeptide at this ratio was named polypeptide complex.

[0034] The comparative study results of the promoting effects of the polypeptide complex and the single collagen of the same sequence on the three collagens in the DEJ showed that the promoting effects of the polypeptide complex on type IV, type VII, and type XVII collagens were better than those of Pep-IV, Pep-VII, and Pep-XVII, with a synergistic effect.

[0035] The specific test process is as follows: The expression levels of COL4A1 , COL7A1 and COL17A1 genes were determined by RT-qPCR: HSF cells in the logarithmic growth phase were seeded at a density of 1×10 6 cells / well in a 6-well plate and cultured in an incubator at 37°C with 5% CO2 for 24 h. The culture medium was discarded, and the sample group was added with the sample (the final concentrations of the single sample and the mixed sample were 10 ppm respectively; the final concentration of the positive reference sample was 100 ppm VC + 7 ppm VE), while the control group and the model group were added with serum-free medium. Then, they were cultured in an incubator at 37°C with 5% CO2 for 24 h. The culture medium was discarded, and the cells were treated with UVB (60 mJ / cm 2)(Treat the cells (the blank control group does not need to be treated with UVB). After the treatment, add serum-free medium and culture in a 37°C, 5% CO2 incubator for 24 h; discard the supernatant, wash twice with PBS, and then collect the cells; extract RNA, reverse transcribe it into cDNA, and then perform RT-qPCR.)

[0036] The experimental results are as Figures 2A - 2C shown (1:1:1-DEJ in the figure is the polypeptide complex in the present invention, and 1:1:1-DEJ in the subsequent experimental results Figure 3 and Figure 4 is the same as above): After UVB irradiation, the relative expression levels of COL4A1 , COL7A1 , COL17A1 genes in the model group (NC) HSF cells COL4A1 , COL7A1 , COL17A1 were significantly decreased, indicating that the model was effective. The relative expression levels of

[0037] genes were increased by 27.01%, 49.48%, and 84.70% respectively by the polypeptide complex, and the promoting effects were significantly better than those of Pep-IV, Pep-VII, and Pep-XVII, indicating that the polypeptide complex was beneficial to targeting the strong and tough DEJ and promoting the expression of three collagens in DEJ.)

[0037] Effect of the polypeptide complex on LAMA2 gene expression Using the co-culture system of keratinocytes incubated with the sample and fibroblasts stimulated by UVA (ultraviolet A band) + UVB, based on transcriptome sequencing analysis, the effect of the polypeptide complex on the genes related to ECM (extracellular matrix) proteins in skin cell signal transduction was studied. LAMA2 is a key gene of the laminin family and one of the main components of the extracellular matrix (ECM). It plays an important role in maintaining the basement membrane structure, cell adhesion and signal transduction, promoting cell migration and tissue repair, regulating immune response and inflammation, etc. The results showed that after the action of the polypeptide complex, the expression of LAMA2 gene could be significantly promoted, and the promotion rate was 102.26%, and the effect was better than that of the single collagen with the same sequence.)

[0038] The specific test process is as follows: (1) Cell seeding: After resuscitating the cells, when the plating rate reaches about 60%, seed keratinocytes (HaCaT) into a Transwell cell culture plate and seed fibroblasts into a 6-well plate, and incubate overnight in a CO2 incubator (37°C, 5% CO2). After the incubation, the fibroblasts continue to be incubated for 7.5 h, and then irradiated with UVA and UVB, and the irradiation dose is UVA (30 J / cm 2), and UVB (50 mJ / cm 2 ), with an irradiation time of 0.5 h. Keratinocytes (HaCaT) were subjected to subsequent drug administration operations; (2) Solution preparation: Prepare the working solutions of the test substances according to the test groups; (3) Drug administration: According to the test groups, when the cell seeding rate in the Transwell cell culture plate reached 40% - 60%, grouped drug administration was carried out, with 3 replicate wells set in each group. In the blank control group and the negative control group, 1 mL of culture medium was added to each well. In positive control group 1, 1 mL of culture medium containing EGF (1 ppb, 1 μg / L) was added to each well. In positive control group 2, 1 mL of culture medium containing retinol (10 μM) was added to each well. In the sample group, 1 mL of culture medium containing the test sample at the corresponding concentration (10 ppm) was added to each well. After drug administration, the Transwell cell culture plate was placed in a CO2 incubator (37 °C, 5% CO2) and cultured for 8 h; (4) Co - culture: The Transwell cell culture plate was placed on a 6 - well plate and cultured in a CO2 incubator (37 °C, 5% CO2) for 24 h; (5) Transcriptome sample collection: After incubation, the culture medium was discarded and the cells were collected; (6) After washing twice with PBS, RNA extraction and library construction were carried out; (7) Total RNA was extracted using TRIzol reagent according to the instructions. The purity and quantification of RNA were identified using a NanoDrop 2000 spectrophotometer (Thermo Scientific, USA), and the integrity of RNA was evaluated using an Agilent 2100 Bioanalyzer (Agilent Technologies, USA). A transcriptome library was constructed using the VAHTS Universal V5 RNA - seq Library Prep kit according to the instructions; (8) The library was sequenced and data processed using the Illumina Novaseq 6000 sequencing platform. The HISAT2 software was used for reference genome alignment, and the gene expression level (FPKM) was calculated; (9) Data screening criteria: Pair - wise comparisons were made between groups, and a p value < 0.05 was selected as candidate differential genes, and their gene functions were further investigated.

[0039] The experimental results are as Figure 3 shown: Compared with the blank group (BC), the ECM protein - related genes in the model group (NC) LAMA2Downregulation indicates the effectiveness of the model. Compared with the model group (NC), after the action of the polypeptide complex, the expression of the LAMA2 gene can be significantly promoted, with a promotion rate of 102.26%, and the effect is better than that of the single collagen with the same sequence.

[0040] Effect of polypeptide complex on the expression of multiple collagen genes Using the co-culture system of keratinocytes after incubation with samples and fibroblasts after combined UVA+UVB stimulation, based on transcriptome sequencing analysis, the effect of the polypeptide complex on the expression of multiple collagen genes was studied. COL6A2, COL7A1, COL8A1, COL14A1, COL16A1 The proteins encoded by the genes are all collagens, which are one of the main components of the skin and endow the skin with strength, elasticity and firmness. MMP19 The protein encoded by the gene is an enzyme belonging to the matrix metalloproteinase (MMP) family. The catalytic domain of MMP-19 can hydrolyze basement membrane components type IV collagen, laminin and collagen, as well as large tenascin C isoforms, fibronectin and type I gelatin, indicating that MMP-19 is an effective basement membrane degrading enzyme that can hydrolyze multiple extracellular matrix components. The specific test process is the same as above.

[0041] The experimental results are shown in Table 2: Compared with the BC group, the NC group COL6A2, COL8A1, COL14A1, COL16A1 The relative gene expression levels decreased by 16.28%, 162.84%, 171.19%, 42.64% respectively, MMP19 The relative gene expression level increased by 34.78%, indicating the effectiveness of the model. Compared with the NC group, the polypeptide complex significantly promoted COL6A2, COL8A1, COL14A1, COL16A1 The relative gene expression levels, increased by 18.86%, 64.71%, 96.29%, 27.33% respectively, MMP19 The relative gene expression level decreased by 29.08%. The polypeptide complex is superior to the single collagen with the same sequence in promoting collagen expression and inhibiting collagen degradation.

[0042] Table 2 Effect of polypeptide complex on multiple collagen-related genes

[0043] *The expression ratio is the ratio of the expression levels between the two treatments. An expression ratio > 1 indicates an increase in expression level, and an expression ratio < 1 indicates a decrease in expression level; COL4, COL7, COL17 refer to Pep-IV, Pep-VII, Pep-XVII screened in Example 1 respectively.

[0044] Effect of polypeptide complex on DNA damage repair genes Using a co - culture system of keratinocytes after sample incubation and fibroblasts after UVA + UVB combined stimulation, based on transcriptome sequencing analysis, to study the effect of the polypeptide complex on DNA damage repair genes. NEIL3 The gene encodes a DNA glycosylase. This type of glycosylase initiates the first step of base excision repair by cleaving bases damaged by reactive oxygen species and introducing DNA strand breaks through related lyase reactions. LIG1 The gene encodes a member of the ATP - dependent DNA ligase protein family. The encoded protein plays a role in DNA replication, recombination, and base excision repair. And NEIL3 The gene and LIG1 The gene both belong to the base excision repair signaling pathway. MLH1 The gene, MSH6 The gene, MSH2 The gene, RFC1 The gene, RFC2 The gene, RFC3 The gene, RFC4 The gene, RFC5 The gene, EXO1 The gene, RPA2 The gene all belong to the mismatch repair signaling pathway. The specific test process is the same as above.

[0045] The experimental results are shown in Table 3: Compared with the BC group, the relative expression levels of NEIL3 and LIG1 genes in the NC group decreased by 133.10% and 98.42% respectively, indicating that the model is effective. Compared with the NC group, the polypeptide complex increased the relative expression levels of NEIL3 and LIG1 genes by 62.90% and 72.38% respectively. Compared with the BC group, the relative expression levels of MLH1 gene, MSH6 gene, MSH2 gene, RFC1 gene, RFC2 gene, RFC3 gene, RFC4 gene, RFC5 gene, EXO1 gene and RPA2 gene in the NC group decreased by 23.98%, 71.86%, 96.97%, 34.07%, 86.70%, 123.66% and 104.43%, 82.89%, 95.78% and 70.89% respectively, indicating that the model is effective. Compared with the NC group, the polypeptide complex increased the relative expression levels of MLH1 gene, MSH6 gene, MSH2 gene, RFC1 gene, RFC2 gene, RFC3 gene,RFC4 gene, RFC5 gene, EXO1 gene, and RPA2 the relative expression levels of the genes were increased by 14.24%, 52.29%, 52.69%, 22.20%, 56.29%, 66.44%, 51.82%, 47.68%, 67.08% and 40.58%, respectively. The polypeptide complex had a better promoting effect on DNA damage repair genes than the single collagen of the same sequence.

[0046] Table 3 Effects of the polypeptide complex on DNA damage repair genes

[0047] * The expression ratio is the ratio of the expression levels between the two groups of treatments. An expression ratio > 1 indicates an increase in the expression level, and an expression ratio < 1 indicates a decrease in the expression level; COL4, COL7, and COL17 refer to Pep-IV, Pep-VII, and Pep-XVII screened in Example 1, respectively.

[0048] Effects of the polypeptide complex on cyclin-related genes Using the co-culture system of keratinocytes after incubation with the sample and fibroblasts after UVA+UVB combined stimulation, the effects of the polypeptide complex on cyclin-related genes were studied based on transcriptome sequencing analysis. CDK4 and CDK2 both genes encode a kind of cyclin-dependent kinase and belong to CDK the family, which is a key factor in cell cycle regulation, especially playing a core role in the G1 / S phase transition. The protein encoded by the CCNE2 gene belongs to the highly conserved cyclin family, and the members of this family are characterized by significant periodicity in protein abundance throughout the cell cycle. CCNA2 The gene encodes cyclin A2 of type A and is a member of the cyclin family. The specific test process is the same as above.

[0049] The experimental results are shown in Table 4: Compared with the BC group, the CDK4, CCNE2, CDK2, CCNA2 relative expression levels of the genes in the NC group were decreased by 30.56%, 158.97%, 124.72%, and 142.98%, respectively, indicating that the model was effective. Compared with the NC group, the polypeptide complex increased the CDK4, CCNE2, CDK2, CCNA2 relative expression levels of the genes by 26.30%, 115.53%, 71.81%, and 54.38%, respectively.

[0050] Table 4 Effects of the polypeptide complex on cyclin-related genes

[0051] *The expression ratio is the ratio of the expression levels between two groups of treatments. An expression ratio > 1 indicates an increase in expression level, while an expression ratio < 1 indicates a decrease in expression level; COL4, COL7, and COL17 refer to Pep-IV, Pep-VII, and Pep-XVII screened in Example 1, respectively.

[0052] Effect of the polypeptide complex on the IL-11 gene Using a co-culture system of keratinocytes after incubation with the sample and fibroblasts after combined UVA+UVB stimulation, based on transcriptome sequencing analysis, to study the effect of the polypeptide complex on IL-11 genes. IL-11 is a pro-inflammatory cytokine belonging to the IL-6 family. Inhibiting the signal of the pro-inflammatory protein IL-11 can significantly delay the aging of mammals. In certain inflammatory environments, IL-11 can stimulate fibroblasts to secrete inflammatory mediators such as IL-8 and VEGF, promoting angiogenesis and the recruitment of inflammatory cells, thereby exacerbating the inflammatory response. The specific test process is the same as above.

[0053] The experimental results are as Figure 4 shown: Compared with the BC group, the relative gene expression level in the NC group IL-11 significantly increased by 74.83%, indicating that the model is effective. Compared with the NC group, the relative gene expression level in the polypeptide complex group IL-11 significantly decreased by 30.05%, indicating that the polypeptide complex is beneficial for inhibiting skin inflammation and has a better effect than the single glue of the same sequence.

[0054] Test results of the Raman permeability of the polypeptide complex in human body Select the front area of the human forearm for Raman testing in the human body: The sample used for testing is DEJ Booter, and the application dosage is 5 mg / cm 2 , and a test skin area of 1×1 cm 2 is selected. Use a LabRAM Odyssey high-speed high-resolution confocal Raman spectrometer (HORIBA) to conduct tests at time points of 0 h, 0.5 h, 2 h, 4 h, and 6 h; the number of test subjects is 1 person, and the test area is 3 pieces of 1×1 cm 2 test skin area, and 5 points are selected in each test area for parallel testing 3 times. Calculate the relative permeability of the polypeptide complex.

[0055] Raman spectral imaging data processing includes spectral preprocessing and data analysis. The spectral preprocessing process includes removing cosmic rays, spectral smoothing, removing background noise, baseline calibration, and spectral normalization, etc. Univariate data analysis is mainly for the biochemical substances corresponding to a specific peak position, and Raman spectral data analysis is performed to show the distribution of this substance in human skin.

[0056] For data analysis, the Labspec software was used to perform baseline calibration on the Raman spectrogram and confirm the positions of characteristic peaks, and calculations were carried out on the obtained Raman spectra, including peak intensity, peak shift, peak area, full width at half maximum, etc.; at the same time, the Labspec software was used for numerical analysis of the peak intensities corresponding to different depths and the drawing of their spatial distributions.

[0057] For the obtained Raman spectrogram, after spectral preprocessing, the initial appearance position of the Raman signal was used as the 0 μm position of the skin surface, and after feature selection, the changes in the keratin / lipid-related characteristic information and the changes in the water content in the skin with the depth in the skin were used to determine the junction between the stratum corneum and the viable epidermis and the junction between the viable epidermis and the dermis, so as to confirm the information related to the skin depth. Then, the characteristic Raman signal of this product, which is different from the skin intrinsic signal, was used to confirm its distribution in different skin depth spaces, so as to depict the penetration behavior of the product. The software was used to perform a normal distribution test of the data (Shapiro-Wilk Test). If it was normally distributed, a paired t test was used, otherwise a Wilcoxon signed-rank test for two related samples was used.

[0058] Relative permeability (%) = Raman spectral intensity of the active substance entering the skin / Raman spectral intensity of the active substance applied on the skin * 100% The experimental results are as Figure 5 shown: After using the polypeptide complex on the in-vivo skin of humans, the relative permeabilities of the polypeptide complex at 0.5 h, 2 h, 4 h, and 6 h were 13.57%, 30.88%, 52.35%, and 60.45% respectively. It can reach the DEJ layer in 2 h, effectively strengthening and toughening the skin basement membrane.

[0059] The above is only the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A polypeptide complex, characterized in that: The polypeptide complex contains a polypeptide Pep-IV with an amino acid sequence as shown in SEQ ID NO.1, a polypeptide Pep-VII with an amino acid sequence as shown in SEQ ID NO.2, and a polypeptide Pep-XVII with an amino acid sequence as shown in SEQ ID NO.

3.

2. The polypeptide complex according to claim 1, characterized in that The weight ratio of polypeptide Pep-IV, polypeptide Pep-VII and polypeptide Pep-XVII in the polypeptide complex is 1:0.8~1.2:0.8~1.

2.

3. Use of the polypeptide complex according to claim 1 or 2 in the preparation of a product for repairing skin basement membrane.

4. Use of the polypeptide complex according to claim 1 or 2 in the preparation of anti-aging products.

5. Use of the polypeptide complex according to claim 1 or 2 in the preparation of a collagen synthesis promoter.

6. The use according to claim 5, characterized in that: The collagens include COL4A1, COL7A1, COL17A1, COL6A2, COL8A1, COL14A1 and COL16A1.

7. Use of the polypeptide complex according to claim 1 or 2 in the preparation of a product for inhibiting skin inflammation.

8. Use of the polypeptide complex according to claim 1 or 2 in the preparation of a product for promoting DNA damage repair or a skin cell cycle protein regulating agent.

9. Use of the polypeptide complex according to claim 1 or 2 in the preparation of a LAMA2 gene expression promoting agent or a MMP19 gene expression inhibiting agent.

10. A skin care product, characterized in that: Containing the polypeptide complex according to claim 1 or 2.

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