Silk peptide SO1 for inhibiting skin photoaging and composition and application thereof

Abstract

The invention discloses a silk peptide SO1 for inhibiting skin photoaging as well as a composition and application thereof. The amino acid sequence of the polypeptide SO1 is EFDDIK. Experiments prove that SO1 can be combined with platelet-derived growth factor receptor beta (PDGFR beta) in a high affinity manner, NF [kappa] B and ERK signal channels at the downstream of the PDGFR beta can be stably regulated and controlled, expression of key senescence markers (TP53, P21 and P16) and senescence-related secretory phenotype factors (such as IL6 and MMPs) can be reduced in a concentration-dependent manner, reactive oxygen species (ROS) can be effectively eliminated, and senescence cell accumulation can be reduced. The discovery provides clear active ingredients and action targets for developing novel anti-skin photoaging drugs or cosmetics taking targeted cell aging as a core mechanism.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a silk peptide SO1 for inhibiting skin photoaging, and also to a composition containing the silk peptide SO1 and the application of the composition. Background Technology

[0002] Studies have clarified that ultraviolet (UV) radiation not only causes the acute reaction of "sunburn," but is also a major environmental factor contributing to chronic photoaging, photoimmunosuppression, and even skin cancer. UV radiation mainly includes UVA (315-400nm), UVB (280-315nm), and UVC (200-280nm). Among them, UVA accounts for up to 95% of the total UV radiation on the ground and has extremely strong penetrating power. It can damage the reticular layer of the dermis, producing a large amount of ROS and indirectly damaging DNA, and is considered to be the main environmental cause of photoaging. UVB is mainly absorbed by epidermal keratinocytes and basal cells, and can be directly absorbed by DNA, thereby producing cyclobutanepyrimidine dimers (CPD) and 6-4 photoproducts, which are initiating factors for mutation and carcinogenesis. However, UVC is almost completely filtered out by the ozone layer, and the general public's exposure is extremely low, except for acute burns caused by artificial light sources (sterilizing lamps, welding arcs). Clinical manifestations of UV-induced skin damage include epidermal necrosis, spongiosis, increased vascular permeability, reduced collagen, wrinkles, and age spots. Current treatments for photoaging primarily involve topical medications such as retinoids and antioxidants, which not only have limited effectiveness but also often come with side effects such as irritation and allergic reactions. Therefore, there is an urgent need for more effective treatments with fewer adverse reactions.

[0003] Silk fibroin and sericin, derived from silkworms, possess multiple benefits including anti-oxidation, anti-inflammation, moisturizing, and collagen induction, making them a hot ingredient in "natural-sustainable" cosmetics. Silk fibroin, a water-soluble globular protein encapsulating silk fibers, accounts for approximately 25%–30% of the total silk content. Its molecules contain about 50% hydrophilic amino acids (such as serine, aspartic acid, and glycine), endowing it with excellent water solubility, hygroscopicity, and biocompatibility. Studies have shown that silk fibroin possesses potential bioactivity in whitening and photoprotection. For example, sericin can intervene in melanin synthesis by inhibiting tyrosinase activity; enzymatic fragments with a molecular weight below 3kDa can reduce enzyme activity and Vmax through mixed-type inhibition, blocking L-DOPA oxidation. The tryptophan and tyrosine residues in sericin exhibit characteristic UV absorption in the 280-320nm wavelength range, suggesting that it can replace some organic sunscreens to reduce UV-induced skin damage. Furthermore, sericin can significantly downregulate COX-2 and IL-6 expression, reducing erythema and pigmentation, making it suitable for post-sun repair and sun protection products. While these studies have preliminarily revealed the multifunctional potential of sericin, existing hydrolyzed sericin products are mostly random enzymatic hydrolysis products with unclear active peptide sequences and ambiguous mechanisms of action. This leads to poor reproducibility of product efficacy, a lack of quality standards, and difficulty in passing cosmetic raw material safety assessments. Therefore, developing a sericin active peptide with a well-defined structure, clear mechanism of action, and controllable quality has become an important direction for addressing industry pain points. This research contributes to the understanding of peptide-based photoprotective mechanisms and the practical application of silk fibroin as a sustainable anti-photoaging resource. Summary of the Invention

[0004] In view of this, one objective of the present invention is to provide a silk peptide SO1 for inhibiting skin photoaging; a second objective of the present invention is to provide a composition containing the silk peptide SO1 for inhibiting skin photoaging; a third objective of the present invention is to provide the use of the silk peptide SO1 for inhibiting skin photoaging or the combination thereof in a medicament or cosmetic for preventing or treating skin photoaging; and a fourth objective of the present invention is to provide a method for inhibiting skin photoaging.

[0005] To achieve the above objectives, the present invention provides the following technical solution: A silk peptide SO1 for inhibiting photoaging of the skin, wherein the silk peptide SO1 can bind to the PDGFRβ receptor with a binding free energy of less than -10 kcal / mol.

[0006] Preferably, the amino acid sequence of the silk peptide SO1 is EFDDIK.

[0007] A composition containing the silk peptide SO1 for inhibiting skin photoaging.

[0008] The use of the silk peptide SO1 or the combination thereof for inhibiting skin photoaging in pharmaceuticals or cosmetics for the prevention or treatment of skin photoaging.

[0009] Preferably, the silk peptide SO1 of the present invention prevents or treats photoaging of the skin by targeting the PDGFRβ receptor and inhibiting the activation of the PDGFRβ / NF-κB / ERK signaling axis.

[0010] A method for inhibiting photoaging of the skin includes administering an effective amount of silk peptide SO1 to an individual in need, said silk peptide SO1 having the amino acid sequence EFDDIK.

[0011] Preferably, this invention reduces the generation of ultraviolet-induced reactive oxygen species (ROS) and / or decreases the number of β-galactosidase-positive cells.

[0012] The beneficial effects of this invention are as follows: This invention provides a peptide SO1 for inhibiting skin photoaging. This peptide has a high affinity for the PDGFRβ receptor on the cell membrane (binding free energy as low as -21.97 kcal / mol) and a stable binding mode. Molecular dynamics simulations confirm that its binding conformation is very stable, providing a solid basis for drug design based on this peptide. The core mechanism of this peptide in combating photoaging lies in inhibiting the "cellular senescence" signaling pathway, downregulating downstream key aging markers (such as TP53, P21, P16) and aging-related secretory phenotypes (SASPs, such as IL-6, MMPs), and inhibiting UV-induced ROS generation. Thus, it synergistically blocks the photoaging process at multiple levels—signaling pathways, gene expression, and protein function—resulting in a more thorough effect. It can be used as a highly effective active ingredient in the development of cosmetics or drugs; it has significant application value and broad prospects in the prevention and treatment of skin photoaging. Attached Figure Description

[0013] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the following figures are provided for illustration: Figure 1 To observe the microstructure of degummed silk fibroin using scanning electron microscopy; Figure 2 A statistical chart showing the concentration, extraction rate, and maximum molecular weight of silk protein; Figure 3 SDS-PAGE was used to determine the molecular weight of sericin. Figure 4 The inhibitory effects of different extraction methods on melanin; Figure 5 This describes the process of producing silk protein hydrolysates using the P-1 method. Figure 6 The effect of SEH on cell viability; Figure 7To evaluate the skincare efficacy of SEH; Figure 8 Changes in physiological parameters after continuous use of SEH; Figure 9 Results of the transcriptional response to UV-induced SEH treatment; Figure 10 Ridge map induced by UV irradiation after SEH processing; Figure 11 The results of the ternary graph analysis; Figure 12 The results of gene set enrichment analysis were performed on the "cellular senescence" pathway; Figure 13 Results of gene expression analysis for key pathways of "cellular senescence"; Figure 14 qPCR validation of MMP1, MMP9, IL6 and TNFα; Figure 15 Results of peptide identification and quantification in descending order of abundance; Figure 16 Clustering dendrogram analysis results; Figure 17 Find the intersection of KEGG enriched genes and draw a Venn diagram; Figure 18 To construct a protein-protein interaction network using STRING and Cytoscape; Figure 19 To analyze the expression levels of key receptors using RNA-seq expression profiling; Figure 20 Heatmap of binding energy between core peptide and highly expressed receptor; Figure 21 SO1 binds to specific amino acid residues of PDGFRβ through various stabilizing forces; Figure 22 The root mean square deviation (RMSD) of SO1 and PDGFRβ changes over time during molecular dynamics simulations. Figure 23 The root mean square wave (RMSF) is calculated based on the trajectory of molecular dynamics simulation. Figure 24 Let be the binding energy of each amino acid on SO1; Figure 25 The change in the number of hydrogen bonds in SO1 and PDGFRβ during molecular dynamics simulations; Figure 26 A diagram showing the conformational changes of SO1 and PDGFRβ during the simulation; Figure 27 SO1 inhibits UV-induced ROS expression; Figure 28SO1 inhibits UV-stimulated β-galactosidase; Figure 29 The results of Western blot analysis of the effect of SO1 on the NFκB / ERK signaling pathway; Figure 30 Results of SO1 inhibition of UV-induced aging-related gene mRNA levels; Figure 31 The results of ELISA detection of SO1 as an inflammatory target induced by UV inhibition. Detailed Implementation

[0014] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0015] Example 1: Extraction and hydrolysis of silk protein Rinse the silkworm cocoons three times with pure water and cut them into pieces about 1 cm long. 2 After breaking down the silk into small pieces, different methods were used to extract the silk protein: the material-to-liquid ratio was 2% for both methods, and the silk protein was extracted using papain (800,000 U / L, 40℃) for 12 h and 8 h (P-1 and P-2, respectively), high temperature and high pressure (120℃) for 2 h, 1 h, and 0.5 h (HTHP-1, HTHP-2, and HTHP-3, respectively), and by neutral soap (0.5%), sodium carbonate (1%), and malic acid (1%) (NS, SC, and MA, respectively). After processing, the silk protein was filtered to obtain SEH, and finally freeze-dried for storage.

[0016] The microstructure of degummed silk fibroin was observed using a scanning electron microscope (Hitachi SU3500, Japan). The results showed that the sericin in silk fibroin could be effectively removed by methods such as papain hydrolysis and high temperature and pressure. Figure 1 Sericin concentration was determined using the BCA protein quantification kit (Beyotime P0012, China).

[0017] The sericin removal rate is calculated using the following formula: Degumming rate (%) = (Initial mass of dried cocoons − Mass of dried cocoons after degumming) / Initial mass of dried cocoons × 100% Statistical results are as follows Figure 2 As shown in the figure. The results indicate that the efficiency of papain hydrolysis for 12 hours and high-temperature and high-pressure extraction for 7 hours can reach over 25%.

[0018] The molecular weight of sericin was determined by SDS-PAGE: A sericin sample with an equal solid content was mixed with loading buffer, boiled for 5 min, and then electrophoresed in a 12% separating gel. After Coomassie brilliant blue staining, the sample was scanned and recorded using a Patch-clamp imaging system (Canon, Japan). SDS-PAGE results showed that papain hydrolysis of silk fibroin was the most effective in producing a low molecular weight peptide library, exhibiting the most thorough and consistent degradation, with the product components mainly concentrated below 25 kDa. Figure 3 ).

[0019] Example 2: Screening for the anti-melanin production activity of silk fibroin B16F10 melanocytes were treated with the sericin peptide libraries obtained by the eight methods in Example 1 for 12 h. The melanin content in the cells was expressed as "melanin per unit protein". The content was detected by dissolving half of the cells in 1 M NaOH at 80°C for 1 h and measuring the absorbance at 405 nm using a ELISA reader (Synergy H4, USA); the protein concentration of the other half of the cells was measured using the BCA method.

[0020] Semi-quantitative melanin content = melanin absorbance ÷ protein concentration.

[0021] The results showed that, except for the NS library, all other libraries significantly inhibited melanin synthesis (P < 0.05). Among them, the P-1 library derived from papain hydrolysis showed the strongest inhibitory effect, even better than the positive control arbutin. Figure 4 Based on the above experimental results, the P-1 method was ultimately chosen to produce silk protein hydrolysate (SEH) for subsequent experiments. Figure 5 Further safety assessment of SEH in the NIH3T3 fibroblast model was conducted. Logarithmic-phase cells were seeded into 96-well plates until 80% confluence. After 24 h of sericin treatment, cells were incubated with CCK-8 for 30 min. Absorbance was measured at 450 nm, and cell viability was calculated. The results showed that SEH had no significant toxicity to NIH3T3 fibroblasts, and that SEH concentrations below 5.73 mg / mL had no significant effect on cell viability. Figure 6 This indicates that SEH has good biosafety.

[0022] Example 3: SEH inhibits melanin production during UV-induced photoaging. To evaluate the skincare efficacy of SEH, a small-scale, double-blind, half-face study was conducted. The participants were a 45-year-old male and a 25-year-old female. For 28 consecutive days, participants applied a cream containing 0.2% SEH to one side of their face (Table 1), while the other side of their face was treated with a completely SEH-free cream as a control. The results showed that, within the 4-week trial period, standardized imaging and time-series analysis revealed benign changes in skin pigmentation, UV spots, melasma, and redness, indicating that SEH effectively reduced pigmentation and erythema, and the tested area consistently showed better results than the control area. Figure 7 and Figure 8 These findings suggest that SEH can effectively inhibit early photoaging phenomena, such as pigmentation and erythema.

[0023] Table 1, Formula Table serial number Element Percentage (%) (Experimental Group) Percentage / % (control group) 1 glycerin 1.8 1.8 2 Xanthan Gum 0.2 0.2 3 Hydrogenated polydecene 0.4 0.4 4 Bis-PEG-15 methyl ether polydimethylsiloxane 1 1 5 Polydimethylsiloxane 1 1 6 SEH 0.2 0 7 pure water 95.4 95.6 Example 4: SEH inhibits melanocyte photoaging through cellular senescence signaling pathways To further investigate the molecular mechanism of SEH's inhibitory effect on pigmentation, human primary melanocytes were divided into three groups: a blank control group without any treatment, a UV irradiation group, and a group pretreated with 2 mg / mL SEH before UV irradiation. The UV irradiation conditions were UVA 75 mJ / cm². 2 +UVB 75 mJ / cm 2 After processing, transcriptome analysis was performed. The specific analysis methods are as follows: Three groups were set up: WT blank, 1 mg / mL SEH pretreatment + UV, and UV treatment alone (dose as above). Total RNA was extracted, and libraries were constructed and sequenced using the Illumina platform. Differentially expressed genes were screened using DESeq2, enriched with gseGO and gseKEGG using clusterProfiler (V3.5.1); box plots were drawn using ggplot2, and ternary plots were drawn using ggtern (V3.5.0). The calculations were performed on the Dawning high-performance computer.

[0024] Transcriptome sequencing revealed significant alterations in numerous inflammation- and aging-related pathways, with SEH exhibiting a remarkable protective effect. Notably, UV irradiation upregulated many inflammation-related signaling pathways, suggesting that melanin production-related pathways are not the core response to melanocyte photoaging, while inflammation and aging are more critical phenomena. To further understand the protective mechanism of SEH in skin photoaging, a comprehensive GSEA analysis was performed on three sample groups. Pathway enrichment analysis showed that SEH treatment effectively modulated UV-induced transcriptional responses. SEH primarily downregulated pro-inflammatory signaling pathways that were upregulated by UV irradiation, with the most significant inhibition observed in IL-17 signaling, TNF signaling, NFκB signaling, and chemokine-cytokine interactions. Figure 9Ridge mapping analysis showed that SEH could inhibit the upregulation of UV-induced inflammatory cascade responses, with the most significant protective effects manifested in the slowing of cellular senescence and the downregulation of various inflammatory responses, including the IL-17 signaling pathway and the cellular senescence pathway. Figure 10 Further ternary plot analysis revealed that the vast majority of pathways showing significant changes clustered in the lower left region, suggesting that SEH treatment primarily "offset" UV-induced changes rather than generating independent new effects. Notably, the cellular senescence pathway (marked by a red circle) had the highest enrichment score among all pathway categories, second only to the cancer pathway shown in purple. This pathway point is located in the lower left corner, adjacent to the SEH / UV vertex, strongly indicating that SEH primarily combats UV-induced photoaging by intervening in cellular senescence. Figure 11 Based on the above analysis, it can be inferred that cellular senescence is a key therapeutic target for SEH in combating UV-induced photoaging.

[0025] Furthermore, we performed gene set enrichment analysis (GSEA) targeting the "cellular senescence" pathway. The enrichment curve showed a significant negative enrichment score, indicating that SEH treatment significantly inhibited the activity of this pathway. Figure 12 Within this pathway, key genes—including cyclin-dependent kinase inhibitor 1A (CDKN1A), transcription factor p53 (TP53), NFκB subunit RELA, and SASP-related genes such as pro-inflammatory cytokines (IL6, IL8, IL18, TNFα) and matrix remodeling enzymes (MMP1, MMP9)—were downregulated to varying degrees. Figure 13 qPCR validation of MMP1, MMP9, IL6, and TNFα further confirmed the above results. Figure 14 These findings not only validate that "cellular senescence" may be one of the core mechanisms of photoaging, but also demonstrate that SEH can significantly delay cellular senescence. However, the molecular mechanisms by which SEH clears or inhibits senescent cells remain to be elucidated.

[0026] Example 5: Identification of bioactive peptides in SEH that target and highly express anti-aging receptors To investigate the correlation between the high efficacy of SEH in preventing UV-induced photoaging and its core active peptides, SEH was desalted and concentrated using C18 solid-phase extraction. Analysis was performed using Q Exactive high-resolution mass spectrometry (Thermo, Germany): precursor ions 350-1550 m / z, daughter ions from 110 m / z; DDA Top 20, dynamically excluded. Peptides were identified by comparing the spectra with the SilkDB database using SequestHT. A total of 105 peptides were identified in SEH, and their composition was determined by a bubble plot (…). Figure 15 The clustering dendrogram visually illustrates the origins and relative abundance of these peptides. Figure 16The peptides in SEH mainly originate from Sericin 1, Fib L, Seroin 1, Fib H and P25 proteins; the five most abundant peptides are DIPFFR, SITDLLR, DIPYHLR, YSSDSRDGSVSSSTG and EFDDIK, all of which are derived from Sericin 1, Fib L and Seroin.

[0027] To elucidate the anti-photoaging mechanism of SEH, OMIM and GeneCards were searched for "aging" and "senescence" to obtain the senescence gene set. The intersection of these genes with KEGG-enriched genes was calculated, and a Venn diagram was plotted. Hub genes were mapped to the STRING 9606 protein alias file (official naming conventions such as BioMart_HUGO). High-confidence interactions were extracted with a combined score ≥400 and visualized using Cytoscape 3.9.1. From the Venn diagram, 231 high-confidence overlapping genes were selected from 4206 "aging" entries and 674 KEGG pathway genes for subsequent analysis. Figure 17 Subsequently, a protein-protein interaction network was constructed using STRING and Cytoscape to identify a core functional module, whose key regulatory nodes include NFKBIA, STAT1, STAT3, TNF, and MAPK8. Figure 18 The network genes highly overlap with aging signaling pathways, with core genes including NFκB, STAT, TNF, TP53, and mTOR, further demonstrating that "cellular senescence" is the core mechanism by which SEH exerts its protective effect.

[0028] To delve deeper into potential peptide-receptor interactions, we focused on membrane proteins involved in cellular senescence pathways. RNA-seq expression profiling revealed varying expression levels of receptor proteins in cells, with TRPV4, IGF1R, PDGFRβ, and MC1R showing the highest expression abundance. Figure 19 These five characteristic peptides were molecularly docked with the aforementioned receptors. The protein crystal structure was downloaded from PDB, and the 3D structure of the peptides was constructed using PyMol V2.5.5. Energy minimization was performed using the MMFF94 force field. After dehydration, deionization, and ligand removal, the whole protein was used as the docking cassette. The structure was converted to PDBQT using ADFRsuite, and a global search was performed using AutoDock Vina (exhaustiveness=32) to select the optimal conformation. The optimal conformation was then displayed using PyMol and DS Visualizer. The resulting binding energy heatmap showed that the binding energies of multiple peptide-receptor pairs ranged from -11.644 to -6.557 kcal / mol. Figure 20Subsequently, complexes with binding energies below -10 kcal / mol—IGF1R-DIPFFR, PDGFRβ-EFDDIK, TRPV4-DIPFFR, TRPV4-DIPYHLR, TRPV4-EFDDIK, and TRPV4-SITDLLR—were selected for fine molecular docking to elucidate their microscopic mechanisms of action.

[0029] Example 6: SO1 peptide targets PDGFRβ receptor The protein crystal structure was downloaded using PDB, and the 3D peptide structure was constructed using PyMol V2.5.5. Energy minimization was performed using the MMFF94 force field. After dehydration, deionization, and ligand removal, the whole protein was used as the docking cassette. The structure was converted to PDBQT using ADFRsuite, and a global search was performed using AutoDock Vina (exhaustiveness=32) to select the optimal conformation. The optimal conformation was then displayed using PyMol and DS Visualizer. Molecular docking results revealed that SO1 (ESROin 1-derived EFDDIK) formed 30 interactions with PDGFRβ, exhibiting extremely strong peptide-receptor combinatorial interactions. These interactions included hydrogen bonds, charge attraction, and other non-covalent bonds (Table 2).

[0030] Table 2. Information on the interaction between PDGFRβ and EFDDIK Category Types From To 1 Electrostatic Attractive Charge X:ARG64 peptide:GLU1 2 Electrostatic Attractive Charge X:ARG115 peptide:ASP3 3 Electrostatic Attractive Charge X:ARG150 peptide:GLU1 4 Electrostatic Attractive Charge X:ARG150 peptide:ASP3 5 Electrostatic Attractive Charge X:ARG150 peptide:ASP4 6 Electrostatic Attractive Charge X:ARG177 peptide:ASP3 7 Electrostatic Attractive Charge peptide:GLU1 X:ASP131 8 Electrostatic Attractive Charge peptide:GLU1 X:GLU134 9 Electrostatic Attractive Charge peptide:LYS6 X:GLU134 10 Hydrogen Bond Conventional Hydrogen Bond X:ARG115 peptide:ASP3 11 Hydrogen Bond Conventional Hydrogen Bond X:ASN130 peptide:ASP4 12 Hydrogen Bond Conventional Hydrogen Bond X:ARG150 peptide:ASP4 13 Hydrogen Bond Conventional Hydrogen Bond X:ARG150 peptide:GLU1 14 Hydrogen Bond Conventional Hydrogen Bond X:ARG177 peptide:ASP3 15 Hydrogen Bond Conventional Hydrogen Bond peptide: GLU1 X: ASP131 16 Hydrogen Bond Conventional Hydrogen Bond peptide: ASP3 X: GLU97 17 Hydrogen Bond Conventional Hydrogen Bond peptide: LYS6 X: GLU134 18 Hydrogen Bond Conventional Hydrogen Bond peptide: LYS6 X: GLU134 19 Hydrogen Bond Conventional Hydrogen Bond peptide: LYS6 X: THR146 20 Hydrogen Bond Carbon Hydrogen Bond X: ILE145 peptide: LYS6 21 Hydrogen Bond Carbon Hydrogen Bond X: THR146 peptide: ILE5 22 Hydrogen Bond Carbon Hydrogen Bond X: PRO148 peptide: ILE5 23 Hydrogen Bond Carbon Hydrogen Bond X: ARG150 peptide: ASP4 24 Hydrogen Bond Carbon Hydrogen Bond peptide: LYS6 X: GLU133 25 Hydrogen Bond Carbon Hydrogen Bond peptide: LYS6 X: PHE136 26 Hydrogen Bond Pi-Donor Hydrogen Bond X: SER66 peptide: PHE2 27 Hydrophobic Pi-Pi T-shaped X: PHE99 peptide: PHE2 28 Hydrophobic Alkyl X: ARG177 [[ID=�3]]peptide: ILE5 29 Hydrophobic Alkyl peptide: LYS6 X: ILE137 30 Hydrophobic Pi-Alkyl peptide: PHE2 X: MET65 Subsequently, the high-affinity peptide SO1 (EFDDIK) derived from seroin-1 was finely characterized. Molecular modeling clearly demonstrated the binding mode of SO1 to PDGFRβ: both band-like and surface models showed that the peptide was stably embedded in the receptor's binding pocket. Interaction mapping further revealed that SO1 binds to specific amino acid residues of PDGFRβ through multiple stabilizing forces, including hydrogen bonds, C-H bonds, π-donor hydrogen bonds, and van der Waals forces. Figure 21 ).

[0031] To verify the stability of this interaction, a 100 ns all-atom molecular dynamics simulation was performed. Peptide charge was calculated using antechamber+Gaussian 09 HF / 6-31G*, with a force field of GAFF2 (peptide) / ff14SB (protein). A 10 Å truncated octahedral water box with TIP3P was used, neutralized with Na⁺ / Cl⁻. Energy optimization was performed using a 2500-step steepest descent followed by a 2500-step conjugation gradient; equilibration was achieved by heating from 0 to 298.15 K for 200 ps, ​​followed by NVT for 500 ps and NPT for 500 ps; final 100 ns NPT production (298.15 K, 1 atm), truncated by 10 Å, with PME for long-range electrostatics, SHAKE for hydrogen bond confinement, and trajectory saved every 10 ps. The RMSD curves showed that the SO1 peptide (green) exhibited minimal fluctuation (≈1 Å), indicating extremely stable binding; the PDGFRβ (purple) backbone also remained intact throughout. Figure 22 RMSF residue flexibility assessment showed that fluctuations in most segments were ≤5 Å, suggesting that SO1 binding further "fixed" the core conformation of the receptor. Figure 23 The total binding energy of MM-GBSA was determined by decomposition of the binding free energy to be –21.97±1.49 kcal / mol, with the contributions from key residues being: GLU1 (–0.64±0.62), PHE2 (–2.72±0.35), ASP3 (–0.53±0.90), ASP4 (–1.56±0.94), ILE5 (–0.01±0.43), and LYS6 (–1.06±1.96 kcal / mol). Figure 24 Hydrogen bond monitoring showed that an average of 2–3 hydrogen bonds were maintained throughout the simulation, with a peak of up to 6, demonstrating the decisive role of hydrogen bonds in the stability of the complex. Figure 25 Snapshots at 10, 30, 50, 70, and 100 ns show that SO1 remained anchored within the binding cavity of PDGFRβ without significant displacement. Figure 26 ).

[0032] Example 7: SO1 peptide inhibits UV-induced ROS Reactive oxygen species (ROS) are a key factor inducing cellular senescence. ROS detection was performed using the following procedure: protein / peptide pretreatment → supernatant discarding → UV irradiation → incubation; 1 mL ROS probe was added, and the mixture was incubated at 37 °C with 5% CO2 for 30 min; the probe was discarded, the cells were washed three times with basal medium, and 1 mL of basal medium was added. The cells were then examined under a Leica DM1 microscope for imaging. Results showed that SO1 exhibited excellent anti-photoaging effects at multiple levels. SO1 effectively inhibited UV-induced ROS generation, even at a low concentration of 0.25 μM, demonstrating a significant inhibitory effect. Figure 27 ).

[0033] Example 8: SO1 peptide inhibits β-galactosidase expression after UV stimulation To verify the potent anti-aging activity of SO1 after UV irradiation, β-galactosidase staining was performed. The specific method was as follows: HaCaT was seeded in 6-well plates overnight, active protein / peptide pretreatment was performed → supernatant was discarded → UV irradiation was performed → incubation was performed; PBS was washed → 1 mL fixative was added at room temperature for 15 min → PBS was washed 3 times → staining solution was added, and the plates were incubated at 37 °C without CO2 for about 18 h; the staining solution was discarded, the plates were washed with PBS, and images were taken with a Leica DM18. SA-β-gal staining directly confirmed the potent anti-aging activity of SO1 after UV irradiation: starting from 0.25 μM, SO1 alleviated UV-induced aging in a concentration-dependent manner, and almost completely blocked the aging phenotype at 2.25 μM. Figure 28 ).

[0034] Example 9: SO1 peptide inhibits photoaging through the NFκB / ERK signaling pathway Western blot analysis was conducted to detect the effects of SO1 on multiple aging-related signaling pathways. Specifically, peptide pretreatment was followed by UV irradiation, and after 6 h, the cells were washed twice with cold PBS. A lysis buffer containing inhibitor was added, and the cells were incubated on ice for 30 min. Cells were scraped off, incubated at 4°C and 12,000 rpm for 10 min, and the supernatant was collected for BCA quantification. An equal volume of protein was mixed with loading buffer, incubated at 95°C for 5 min, and then SDS-PAGE was performed. The cells were then transferred to a PVDF / NC membrane, blocked with 5% skim milk / BSA-TBST for 1 h, incubated overnight at 4°C with primary antibodies (CST 13586, 4695, 4370, 5170), washed with TBST, and incubated with secondary antibodies for 1 h (CST 5127). ECL chemiluminescence was performed, and the images were acquired using a gel imaging system and quantified using ImageJ. The results showed that SO1 can regulate multiple aging-related signaling pathways. UV irradiation significantly upregulated the expression of NFκB1 p105 precursor and its active subunit p50, suggesting activation of this pathway, while SO1 dose-dependently downregulated p105 and p50 levels. Similarly, UV radiation also enhances the phosphorylation of ERK1 / 2, a key upstream regulator of aging, while SO1 inhibits p-ERK1 / 2 activation in a concentration-dependent manner. Figure 29 ).

[0035] Based on the high affinity binding of SO1 to PDGFRβ and its regulatory role in the NFκB and ERK pathways, qRT-PCR further confirmed that SO1 has a significant "rescue" effect on genes in the cellular senescence pathway: UV irradiation can activate core senescence marker genes TP53, P21, and P16, while SO1 downregulates the mRNA expression of these genes in a concentration-dependent manner; simultaneously, it inhibits NF-κB1 transcription, thereby reducing the mRNA levels of the SASP-related inflammatory factor IL-1β and other senescence-related genes. Figure 30ELISA analysis also revealed a significant decrease in the protein levels of IL-6 and various MMPs in the culture supernatant. Figure 31 ).

[0036] Based on the analysis of the above experimental results, SO1 comprehensively improves UV-induced photoaging through the synergistic effect of the PDGFRβ / NFκB / ERK signaling axis: it inhibits core aging biomarkers and blocks SASP activation, providing an integrated therapeutic strategy for combating UV aging.

[0037] The above-described embodiments are merely preferred embodiments provided to fully illustrate the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are all within the scope of protection of the present invention. The scope of protection of the present invention is defined by the claims.

Claims

1. A silk peptide SO1 for inhibiting skin photoaging, characterized in that: The silk peptide SO1 can bind to the PDGFRβ receptor with a binding free energy of less than -10 kcal / mol.

2. The silk peptide SO1 for inhibiting skin photoaging according to claim 1, characterized in that: The amino acid sequence of the silk peptide SO1 is EFDDIK.

3. A composition containing the silk peptide SO1 of claim 1 for inhibiting skin photoaging.

4. The use of the silk peptide SO1 as described in claim 1 or 2 for inhibiting skin photoaging, or the combination as described in claim 3, in a medicament or cosmetic for preventing or treating skin photoaging.

5. The application according to claim 4, characterized in that, The silk peptide SO1 described above prevents or treats photoaging of the skin by targeting the PDGFRβ receptor and inhibiting the activation of the PDGFRβ / NF-κB / ERK signaling axis.

6. A method for inhibiting photoaging of the skin, characterized in that, This includes administering an effective amount of silk peptide SO1 to individuals in need, the amino acid sequence of which is EFDDIK.

7. The method according to claim 6, characterized in that: Reduce UV-induced reactive oxygen species (ROS) production and / or decrease the number of β-galactosidase-positive cells.

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