Use of jwa polypeptide in the preparation of a preparation for repairing skin photo-damage

Abstract

The present application relates to the application of JWA polypeptide in the preparation of a preparation for repairing skin photo-damage, and the amino acid sequence of the polypeptide is shown as I or II: I: FPGSDRF-Z; II: X-FPGSDRF-Z; wherein, the amino acid S is modified by phosphorylation, and X and Z are respectively an amino acid or an amino acid sequence. Through the synergistic effect of the material microenvironment support and the biological regulation of the JWA polypeptide, the JWA polypeptide loaded with the gamma-PGA / HA hydrogel can inhibit the NOD2 / RIPK2 / NF-kappa B inflammatory axis, restore the integrin alpha v beta 3 related adhesion and the ZO-1 related barrier, effectively repair the UVB induced skin photo-damage, and no obvious systemic toxicity is found, thereby providing a new local repair technical means for the UVB induced skin photo-damage.

Description

Technical Field

[0001] This invention relates to the application of a JWA polypeptide in the preparation of agents for repairing photodamage to the skin, particularly suitable for repairing UVB-induced photodamage to the skin, and belongs to the field of biomedicine and biomaterials technology. Background Technology

[0002] The skin is the body's first line of defense against environmental damage. The ultraviolet radiation humans are exposed to mainly includes UVA (long-wave ultraviolet) and UVB (medium-wave ultraviolet). UVB has strong biological effects, inducing acute erythema, desquamation, crusting, epidermal hyperplasia, inflammatory responses, and barrier dysfunction. Excessive UVB exposure can also cause intracellular reactive oxygen species accumulation, DNA damage, mitochondrial dysfunction, and apoptosis, leading to decreased fibroblast migration, dermal collagen homeostasis imbalance, and delayed tissue repair.

[0003] Current interventions for UV-related skin damage mainly include sunscreens, antioxidants, anti-inflammatory drugs, retinoids, and physical therapy. However, most of these measures are primarily for pre-irradiation protection or target a single point of damage. For local repair after UVB irradiation, issues remain, including insufficient drug stability, short skin retention time, low local bioavailability, and difficulty in simultaneously addressing antioxidant, anti-inflammatory, and tissue remodeling effects. Therefore, it is necessary to develop topical formulations that combine highly efficient local delivery with multi-stage repair capabilities.

[0004] JWA (GenBank, AF070523, 1998) is a conserved, multifunctional gene from nematodes to humans, responsible for maintaining tissues and organs against oxidative stress, repairing DNA damage, and reducing apoptosis. JP1 (sequence: FPGSDRFGGGG-RGD, S-site phosphorylation) is a targeted integrin oligopeptide kinase. Its core sequence is derived from the functional structure of the JWA protein and was designed and screened by Zhou Jianwei's team, the discoverers and namers of the JWA gene, based on fundamental research evidence regarding the relationship between the gene's structure and function. Due to the conserved and multifunctional nature of the JWA gene, JP1 has been granted multiple patents for various indications due to its exhibiting numerous important biological functions. Research evidence shows that JP1 can directly penetrate the brain, skin, and eye, and performs various biological functions, including inhibiting melanoma growth and metastasis, repairing vascular structures, activating hair follicle stem cells, suppressing inflammatory responses, and enhancing tissue repair. The RGD structure contained in JP1 provides targeting for its interaction with integrin-related receptors (such as integrin αvβ3). Research evidence indicates that JP1 exerts its function by targeting and binding to integrin molecules on the cell membrane before entering the cytoplasm. These properties suggest that JP1 may be an effective candidate for repairing UVB-induced skin damage. However, JP1 is a water-soluble peptide, and although its biological function is closely related to changes in UVB-induced skin photodamage, inflammation, and adhesion-related signaling pathways, its skin penetration, local retention time in the microenvironment, and stability may be insufficient. To determine the repair efficacy of JP1 against UVB-induced skin damage, it is necessary to investigate whether using a loaded topical delivery platform can enhance JP1's skin penetration, retention, and local bioavailability, as well as its repair effect on UVB-damaged skin. Furthermore, it is essential to investigate whether the loaded delivery system of JP1 produces any side effects.

[0005] Biomimetic extracellular matrix (ECM) hydrogels offer promising strategies for improving local drug retention, modulating the microenvironment of injured skin, and promoting tissue regeneration. γ-Polyglutamic acid (γ-PGA) possesses good biocompatibility and biodegradability, making it a viable alternative to collagen-like ECMs. Hyaluronic acid (HA) is an important component of natural ECMs, exhibiting moisturizing, antioxidant, and immunomodulatory functions. Previous studies have shown that γ-PGA / HA-based hydrogels possess good biocompatibility and a porous three-dimensional network structure, providing a suitable microenvironment for cell adhesion, proliferation, and migration, and promoting collagen deposition and tissue repair. However, using γ-PGA / HA-based hydrogels alone as a skin photodamage repair agent lacks precise biological evidence for repair, resulting in limited skincare effects. Furthermore, for target molecules with well-defined functions (such as JP1), there is insufficient research evidence to support the selection of γ-PGA / HA hydrogels as carriers among numerous ECM hydrogels. Therefore, although γ-PGA / HA hydrogels may be promising carriers for local drug delivery and skin repair, further research is needed to confirm whether they can load target molecules with specific functions (such as JP1) for skin photodamage repair and whether they can exert synergistic effects. Summary of the Invention

[0006] The main objective of this invention is to overcome the problems of insufficient local retention, single action mechanism, and limited tissue repair effect of existing skin repair techniques after UVB irradiation, and to provide an application of JWA polypeptide in the preparation of skin photodamage repair agents, which can improve UVB-induced erythema, desquamation, crusting and epidermal hyperplasia, and no obvious systemic toxicity was observed.

[0007] The technical solution of this invention to solve its technical problem is as follows:

[0008] The use of a polypeptide, characterized in that the polypeptide is a JWA polypeptide; the use is for preparing a formulation for repairing photodamage to the skin caused by ultraviolet radiation;

[0009] The amino acid sequence of the JWA polypeptide is shown in I or II:

[0010] I: FPGSDRF-Z;

[0011] II: X-FPGSDRF-Z;

[0012] Among them, amino acid S is phosphorylated, and X and Z are amino acids or amino acid sequences, respectively;

[0013] X is selected from one of F, (R)9, (R)9-F, 6-aminohexanoic acid, 6-aminohexanoic acid-F, 6-aminohexanoic acid-(R)9, and 6-aminohexanoic acid-(R)9-F;

[0014] Z is selected from (G). n -RGD、A-(G) n -RGD is one of them, where n is an integer greater than or equal to 0, and the value of n ranges from 0 to 10.

[0015] Preferably, the preparation is a topical preparation for repairing UVB-induced skin photodamage; the skin photodamage includes at least one of erythema, desquamation, crusting, and epidermal hyperplasia.

[0016] Preferably, the formulation includes a carrier, wherein the carrier is a γ-PGA / HA hydrogel.

[0017] Preferably, the function of the formulation is to simultaneously reduce oxidative stress, DNA damage, mitochondrial dysfunction, apoptosis and inflammatory response, and promote cell migration, collagen reconstruction and epidermal barrier restoration.

[0018] Preferably, the N-terminus of the polypeptide is acetylated and the C-terminus is amidated; the amino acid sequence of the polypeptide is one of SEQ ID No. 1 to SEQ ID No. 39; and the amino acid configuration of each amino acid in the FPGSDRF sequence of the polypeptide is L-type or D-type.

[0019] The present invention also proposes:

[0020] An formulation for repairing photodamage to the skin caused by ultraviolet radiation, characterized in that the formulation comprises JWA peptide-loaded γ-PGA / HA hydrogel;

[0021] The amino acid sequence of the JWA polypeptide is shown in I or II:

[0022] I: FPGSDRF-Z;

[0023] II: X-FPGSDRF-Z;

[0024] Among them, amino acid S is phosphorylated, and X and Z are amino acids or amino acid sequences, respectively;

[0025] X is selected from one of F, (R)9, (R)9-F, 6-aminohexanoic acid, 6-aminohexanoic acid-F, 6-aminohexanoic acid-(R)9, and 6-aminohexanoic acid-(R)9-F;

[0026] Z is selected from (G). n -RGD、A-(G) n -RGD is one of them, where n is an integer greater than or equal to 0, and the value of n ranges from 0 to 10.

[0027] Preferably, the N-terminus of the polypeptide is acetylated and the C-terminus is amidated, the amino acid sequence of the polypeptide is one of SEQ ID No. 1 to SEQ ID No. 39, and the conformation of each amino acid in the FPGSDRF sequence of the polypeptide is L-type or D-type; or, the formulation is a topical formulation for repairing UVB-induced skin photodamage; or, the preparation process of the JWA polypeptide-loaded γ-PGA / HA hydrogel is as follows: γ-PGA / HA hydrogel is prepared by crosslinking γ-PGA and HA with PEGDE, and the JWA polypeptide is uniformly loaded in the γ-PGA / HA hydrogel network to obtain the JWA polypeptide-loaded γ-PGA / HA hydrogel.

[0028] The present invention also proposes:

[0029] The formulation described above is used to prepare products for repairing skin photodamage caused by ultraviolet radiation.

[0030] Preferably, the product is a topical product for repairing skin photodamage caused by UVB.

[0031] Preferably, the product is a skin care product or cosmetic product.

[0032] The polypeptides involved in this invention are a subset of the series of polypeptides described in Chinese invention patents CN201310178099X and CN103239710B. Practical research has confirmed that the series of JWA polypeptides-loaded γ-PGA / HA hydrogels, represented by JP1, through the synergistic effect of material microenvironment support and JWA polypeptide bioregulation, inhibit the NOD2 / RIPK2 / NF-κB inflammatory axis, restore integrin αvβ3-related adhesion and ZO-1-related barrier, and effectively repair UVB-induced skin photodamage without significant systemic toxicity, providing a new local repair technique for UVB-induced skin photodamage. Attached Figure Description

[0033] Figure 1 This is a schematic diagram illustrating the mechanism of action of JP1-loaded γ-PGA / HA hydrogel in repairing UVB-induced acute skin photodamage in Example 1 of the present invention.

[0034] Figure 2This is a schematic diagram illustrating the characterization, release behavior, cytocompatibility, and cellular uptake of the JP1-loaded γ-PGA / HA hydrogel in Example 1 of this invention. (A) Schematic diagram of the design of JP1 and the preparation process of the JP1-loaded γ-PGA / HA hydrogel. (B) FTIR spectra of γ-PGA, HA, and γ-PGA / HA hydrogel. (C, D) Rheological analysis of γ-PGA / HA solution and γ-PGA / HA hydrogel, including strain scanning and frequency scanning. (E) Viscosity changes of γ-PGA / HA solution and γ-PGA / HA hydrogel at different shear rates. (F) Scanning electron microscope image showing the porous microstructure of the γ-PGA / HA hydrogel. Scale bar = 200 μm. (G) Time-dependent swelling curve of γ-PGA / HA hydrogel in PBS at 37 °C. (H) Enzymatic degradation curve of γ-PGA / HA hydrogel in PBS containing hyaluronidase and papain at 37 °C, expressed as percentage of remaining mass. (I) In vitro release curves of JP1 from γ-PGA / HA solution and γ-PGA / HA hydrogel at pH 7.0 and 37 °C. (J, K) Representative live / dead cell staining images of HDFs and HaCaT cells after 1 and 3 days of treatment with γ-PGA / HA hydrogel, JP1, or JP1-loaded γ-PGA / HA hydrogel. Green indicates live cells, and red indicates dead cells. Scale bar = 1000 μm. (L, M) Representative fluorescence images of FITC-JP1 uptake by HDFs and HaCaT cells after 4 h and 8 h of treatment. Green indicates FITC-JP1, and blue indicates DAPI. Scale bar = 50 μm. Data are expressed as mean ± standard deviation (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001; ns indicates no statistical significance.

[0035] Figure 3 This is a schematic diagram illustrating the injectability and shape retention capability of the γ-PGA / HA hydrogel in Example 1 of the present invention.

[0036] Figure 4 This diagram illustrates the effect of different concentrations of JP1 and γ-PGA / HA hydrogel on the viability of UVB-damaged cells in Example 1 of this invention. (A, B) Viability of UVB-damaged HDFs and HaCaT cells after treatment with different concentrations of JP1 was detected using CCK-8 assay. (C, D) Viability of UVB-damaged HDFs and HaCaT cells after treatment with different concentrations of γ-PGA / HA hydrogel was detected using CCK-8 assay. Data are expressed as mean ± standard deviation (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001; ns indicates no statistically significant difference.

[0037] Figure 5This is a schematic diagram illustrating how JP1-loaded γ-PGA / HA hydrogel alleviates UVB-induced oxidative damage and DNA damage in Example 1 of this invention. (A) CCK-8 analysis of the viability of UVB-damaged HDFs and HaCaT cells after different treatments. (B) Representative morphological images of UVB-damaged HDFs and HaCaT cells after different treatments. Scale bar = 1000 μm. (C) Representative fluorescence images of HDFs and HaCaT cells after DCFH-DA staining. Scale bar = 1000 μm. (D) Representative flow cytometry histograms of intracellular ROS levels in HDFs and HaCaT cells after DCFH-DA staining. (E, F) Representative immunofluorescence staining images and quantitative analysis of γ-H2AX in HDFs and HaCaT cells. Red represents γ-H2AX, and blue represents DAPI. Scale bar = 100 μm. (G, H) Representative scratch images and quantitative analysis of cell migration of HDFs at 0, 12, and 24 h after different treatments. Scale bar = 1000 μm. Representative Western blot bands and quantitative analysis of γ-H2AX expression in (I, J) HDFs and HaCaT cells. Data are expressed as mean ± standard deviation (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001; ns indicates no statistical significance.

[0038] Figure 6 This is a schematic diagram illustrating the quantitative analysis of ROS levels in HDFs and HaCaT cells in Example 1 of this invention. (A, B) Quantitative analysis of DCFH-DA fluorescence intensity in HDFs and HaCaT cells was performed based on fluorescence staining images. (C, D) Quantitative analysis of ROS levels in HDFs and HaCaT cells after DCFH-DA staining was performed using flow cytometry. Data are expressed as mean ± standard deviation (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001; ns indicates no statistically significant difference.

[0039] Figure 7This is a schematic diagram illustrating how JP1-loaded γ-PGA / HA hydrogel alleviates UVB-induced apoptosis in Example 1 of this invention. (A) Representative TUNEL staining images of HDFs irradiated with UVB after different treatments. Scale bar = 1000 μm. (B) Representative TUNEL staining images of HaCaT cells irradiated with UVB after different treatments. Scale bar = 200 μm. (C, D) Quantitative analysis of TUNEL-positive apoptotic cells in HDFs and HaCaT cells. (E) Representative immunofluorescence staining images of BAX-positive cells in HaCaT cells. Scale bar = 100 μm. (F) Representative immunofluorescence staining images of Bcl-2-positive cells in HaCaT cells. Scale bar = 100 μm. (G, H) Quantitative analysis of BAX and Bcl-2 fluorescence intensity in HaCaT cells. (I) Detection of HDF apoptosis using Annexin V-FITC / PI double staining combined with flow cytometry. (J) Quantitative analysis of the total apoptosis rate of HDFs. (K, L) Representative Western blot images of apoptosis-related protein expression in HDFs and HaCaT cells after different treatments. (MR) Quantitative analysis of BAX, Bcl-2, and Caspase-3 protein expression in HDFs and HaCaT cells. Data are expressed as mean ± standard deviation (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001; ns indicates no statistical significance.

[0040] Figure 8 This is a schematic diagram illustrating how JP1-loaded γ-PGA / HA hydrogel regulates the expression of apoptosis-related proteins and alleviates UVB-induced mitochondrial dysfunction in Example 1 of this invention. (A, B) Representative immunofluorescence staining images and quantitative analysis of BAX expression in HDFs after different treatments. Red represents BAX, and blue represents DAPI. Scale bar = 100 μm. (C, D) Representative immunofluorescence staining images and quantitative analysis of Bcl-2 expression in HDFs after different treatments. Red represents Bcl-2, and blue represents DAPI. Scale bar = 100 μm. (E, F) Representative JC-1 staining images of HaCaT cells after different treatments and quantitative analysis of the JC-1 aggregate / monomer fluorescence intensity ratio. Red fluorescence represents JC-1 aggregates, and green fluorescence represents JC-1 monomers. Scale bar = 1000 μm. Data are expressed as mean ± standard deviation (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001; ns indicates no statistical significance.

[0041] Figure 9This is a schematic diagram illustrating the inhibition of UVB-induced NF-κB inflammatory signals in HDFs and HaCaT cells by JP1-loaded γ-PGA / HA hydrogel in Example 1 of this invention. (A) Representative immunofluorescence staining images of NF-κB and p-NF-κB in HDFs after UVB irradiation with different treatments. Scale bar = 100 μm. (B) Quantitative analysis of the fluorescence intensity of NF-κB and p-NF-κB in HDFs. (C) Representative immunofluorescence staining images of NF-κB and p-NF-κB in HaCaT cells after UVB irradiation with different treatments. Scale bar = 100 μm. (D) Quantitative analysis of the fluorescence intensity of NF-κB and p-NF-κB in HaCaT cells. (E) Representative Western blot images of NF-κB pathway-related protein expression in HDFs. (F) Quantitative analysis of the p-NF-κB / NF-κB ratio and IκBα protein expression in HDFs. (G) Representative Western blot images of NF-κB pathway-related protein expression in HaCaT cells. (H) Quantitative analysis of p-NF-κB / NF-κB ratio and IκBα protein expression in HaCaT cells. (I) qRT-PCR analysis of IL-1β, IL-6, and TNF-α mRNA expression levels in HDFs. (J) qRT-PCR analysis of IL-1β, IL-6, and TNF-α mRNA expression levels in HaCaT cells. Data are expressed as mean ± standard deviation (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001; ns indicates no statistical significance.

[0042] Figure 10 This is a schematic diagram of RNA sequencing analysis of HDFs after UVB irradiation and JP1-loaded γ-PGA / HA hydrogel treatment in Example 1 of this invention. (A) Venn diagram showing the intersection of differentially expressed genes in the Control group, UVB group, and UVB+γ-PGA / HA+JP1 group of HDFs. (B) Heatmap and cluster analysis of differentially expressed genes, showing representative enriched pathways. (C) Expression trends of 6 gene clusters. (D) Enrichment analysis of GO biological processes in each gene cluster. (E) Enrichment analysis of KEGG pathways in different gene clusters. (F, G) The 12 KEGG pathways with the highest enrichment in gene clusters C4 and C5. (H) qRT-PCR was used to verify the mRNA expression levels of NOD1, NOD2, and RIPK2. Data are expressed as mean ± standard deviation (n=3). *P<0.05, **P<0.01, ***P<0.001; ns indicates no statistical significance.

[0043] Figure 11This is a schematic diagram illustrating the involvement of the NOD2 / RIPK2 / NF-κB signaling pathway in Example 1 of this invention, verified through NOD2 overexpression and inhibition experiments. (A) Western blot and quantitative analysis were used to verify the overexpression efficiency of NOD2 in HDFs. (B, C) In the NOD2 overexpression experiment, Western blot was used to detect the expression of NOD2 / RIPK2 and NF-κB pathway-related proteins in HDFs after different treatments. (DG) Quantitative analysis of NOD2 / GAPDH, p-RIPK2 / RIPK2, p-NF-κB / NF-κB, and IκBα / GAPDH protein levels. (HK) qRT-PCR analysis of RIPK2 and inflammatory cytokine mRNA expression in NOD2-overexpressing HDFs. (L, M) After treatment with GSK717 alone or in combination with JP1-loaded γ-PGA / HA hydrogels, Western blot was used to detect the expression of NOD2 / RIPK2 and NF-κB pathway-related proteins. (NQ) Quantitative analysis of corresponding protein levels in the GSK717 inhibition assay. (RV) qRT-PCR analysis of NOD2, RIPK2, and inflammatory cytokine mRNA expression after different treatments. Data are expressed as mean ± standard deviation (n=3). *P<0.05, **P<0.01, ***P<0.001; ns indicates no statistically significant difference.

[0044] Figure 12 This is a schematic diagram illustrating the effect of NOD2 inhibitor addition on key pathway indicators as detected by immunofluorescence in Example 1 of this invention. (A) Representative immunofluorescence staining images and quantitative analysis of NOD2 expression in HDFs. Green represents NOD2, and blue represents DAPI. Scale bar = 100 μm. (B) Representative immunofluorescence staining images and quantitative analysis of p-RIPK2 expression in HDFs. Green represents p-RIPK2, and blue represents DAPI. Scale bar = 100 μm. Data are expressed as mean ± standard deviation (n = 3). ***P < 0.001.

[0045] Figure 13This is a schematic diagram illustrating the similarity between the inhibitory effect of JP1-loaded γ-PGA / HA hydrogel on UVB-induced inflammatory signaling and RIPK2 inhibition in Example 1 of this invention. (A) Western blot analysis of NOD2, RIPK2, and p-RIPK2 protein expression in HDFs after different treatments. (B, C) Quantitative analysis of NOD2 / GAPDH and p-RIPK2 / RIPK2 protein levels. (D) Western blot analysis of NF-κB, p-NF-κB, and IκBα protein expression. (E, F) Quantitative analysis of p-NF-κB / NF-κB and IκBα / GAPDH protein levels. (G, H) Representative immunofluorescence staining images of p-RIPK2 and p-NF-κB in HDFs. Green indicates the target protein, and blue indicates DAPI. Scale bar = 100 μm. (I, J) Quantitative analysis of the fluorescence intensity of p-NF-κB and p-RIPK2. (K, L) qRT-PCR analysis of NOD2 and RIPK2 mRNA expression. (MO) qRT-PCR analysis of inflammatory cytokines IL-1β, IL-6, and TNF-α mRNA expression. (P) Schematic diagram of the role of JP1-loaded γ-PGA / HA hydrogel and GSK583 in inhibiting the NOD2 / RIPK2 / NF-κB pathway in UVB-damaged HDFs. Data are expressed as mean ± standard deviation (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001; ns indicates no statistical significance.

[0046] Figure 14 This is a schematic diagram illustrating how JP1-loaded γ-PGA / HA hydrogel alleviates UVB-induced photodamage in mice in Example 1 of this invention. (A) Schematic diagram of the UVB-induced acute photodamage model and local treatment process. (B) Representative gross images of the back skin of mice in each group on day -3, day 0, and day 4. (C, E) Representative H&E staining images of skin tissue after different treatments and quantitative analysis of epidermal thickness. Scale bar = 200 μm. (D, F) Representative Masson staining images of back skin tissue and quantitative analysis of collagen area. Scale bar = 100 μm. (G) qRT-PCR analysis of IL-1β, IL-6, and TNF-α mRNA expression levels in skin tissue. (H) ELISA analysis of IL-1β, IL-6, and TNF-α levels in skin tissue. Data are expressed as mean ± standard deviation (n = 5). *P < 0.05, **P < 0.01, ***P < 0.001; ns indicates no statistical significance.

[0047] Figure 15This diagram illustrates how JP1-loaded γ-PGA / HA hydrogel alleviates epidermal damage and dermal inflammation in mice in Example 1 of this invention. (A, E) Representative immunofluorescence staining images and quantitative analysis of γ-H2AX in skin tissue. Red represents γ-H2AX, and blue represents DAPI. Scale bar = 100 μm. (B, F) Representative TUNEL staining images and quantitative analysis of apoptotic cells in skin tissue. Red represents TUNEL-positive cells, and blue represents DAPI. Scale bar = 100 μm. (C, G) Representative immunofluorescence staining images and quantitative analysis of p-NF-κB in skin tissue. Red represents p-NF-κB, and blue represents DAPI. Scale bar = 100 μm. (D, H) Representative immunofluorescence staining images and quantitative analysis of ZO-1 in skin tissue. Red represents ZO-1, and blue represents DAPI. Scale bar = 100 μm. Data are expressed as mean ± standard deviation (n = 5). *P<0.05, **P<0.01, ***P<0.001; ns indicates no statistical significance.

[0048] Figure 16 This is a schematic diagram illustrating how JP1 promotes integrin αvβ3-related repair in UVB-damaged skin and exhibits good in vivo biocompatibility in Example 1 of this invention. (A) Representative fluorescence images showing the skin penetration and local distribution of FITC-JP1 at 0.5 h and 4 h after local application of free JP1 or JP1-loaded γ-PGA / HA hydrogel to UVB-damaged skin. Green indicates FITC-JP1. Scale bar = 100 μm. (BD) qRT-PCR analysis of integrin αv, integrin β1, and integrin β3 mRNA expression in skin tissue after UVB irradiation and JP1 treatment. (E, F) Representative immunofluorescence staining images and quantitative analysis of integrin αv in skin tissue. Red indicates integrin αv, and blue indicates DAPI. Scale bar = 100 μm. (G, H) Representative immunofluorescence staining images and quantitative analysis of integrin β3 in skin tissue. Red indicates integrin β3, and blue indicates DAPI. Scale bar = 100 μm. (IM) qRT-PCR analysis of NOD2, RIPK2, IL-1β, IL-6, and TNF-α mRNA expression in UVB-damaged HDFs after JP1 treatment with or without Cyclo(-RGDfK)-mediated integrin αvβ3 inhibition. (N) Biochemical analysis of serum liver and kidney function-related indicators in different treatment groups, including ALT, AST, ALP, TP, ALB, GLOB, BUN, CREA, and UA. (O) Representative H&E staining images of major organs such as heart, liver, spleen, lung, and kidney. Scale bar = 100 μm. Data are expressed as mean ± standard deviation. *P < 0.05, **P < 0.01, ***P < 0.001; ns indicates no statistical significance. Detailed Implementation

[0049] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. However, the present invention is not limited to the examples given. Unless otherwise specified, all materials used are conventional materials (such as commercially available products), and all experimental methods used are conventional experimental methods.

[0050] Example 1

[0051] This embodiment is a systematic study on the effect of JP1-loaded γ-PGA / HA hydrogel on repairing UVB-induced skin photodamage.

[0052] I. Materials and Methods

[0053] 1. Experimental materials and JP1 peptide

[0054] The average molecular weight of γ-PGA is 700 kDa, and the average molecular weight of HA is 1000 kDa; the hydrogel crosslinking agent is PEGDE. JP1 was prepared by solid-phase synthesis with a purity greater than 98%, and the lyophilized powder was stored at −20 °C. The amino acid sequence of JP1 is SEQ ID No. 1: FPGSDRF-GGGG-RGD, with an RGD motif at the C-terminus. Amino acid S is phosphorylated, and its N-terminus and C-terminus are acetylated and amidated, respectively. The conformation of each amino acid in the FPGSDRF sequence is L-type or D-type. FITC-JP1 was used for cellular uptake and skin distribution experiments.

[0055] Cells included human dermal fibroblasts (HDFs) and HaCaT keratinocytes; experimental animals were 6-8 week old BALB / c mice weighing 18-20 g. The main detection reagents included CCK-8, Calcein AM / PI live / dead staining reagent, DCFH-DA, Annexin V-FITC / PI, JC-1, TUNEL reagent, and antibodies against γ-H2AX, BAX, Bcl-2, Caspase-3, NOD2, RIPK2, p-RIPK2, NF-κB, p-NF-κB, IκBα, ZO-1, integrin αv, and integrin β3.

[0056] 2. Preparation and characterization of JP1-loaded γ-PGA / HA hydrogel

[0057] γ-PGA / HA hydrogels were prepared by crosslinking γ-PGA and HA with PEGDE, and JP1 was uniformly loaded into the hydrogel network to obtain JP1-loaded γ-PGA / HA hydrogels. Figure 2(Figure A). The chemical structures of γ-PGA, HA and the composite hydrogel were determined by FTIR; strain scanning, frequency scanning and viscosity tests at different shear rates were performed using a rotational rheometer; the hydrogel was freeze-dried, the cross-section was sputter-coated with gold and then observed by scanning electron microscopy.

[0058] The swelling properties of γ-PGA / HA hydrogels were determined using a gravimetric method. The weight of the freeze-dried hydrogel sample was recorded as W. d The sample was then immersed in 10 mmol / L, pH 7.4 PBS and incubated at 37 °C. Samples were removed at predetermined time points between 0 and 80 h, and after gently blotting away residual liquid with filter paper, they were weighed and recorded as W. r The swelling ratio of the hydrogel is calculated using the following formula:

[0059] Swelling rate (%) = (W r -W d ) / W d ×100%;

[0060] The in vitro degradation performance of γ-PGA / HA hydrogel was evaluated using an enzymatic degradation method. The initial wet mass of the hydrogel was weighed and recorded as W0, and placed in PBS containing 0.1 mg / mL hyaluronidase and 0.25 mg / mL papain, and incubated at 37 °C. The hydrogel was removed on days 1, 2, 3, 4, 5, and 6, and after gently blotting away residual liquid with filter paper, it was weighed and recorded as W0. t The remaining mass ratio of the hydrogel is calculated according to the following formula:

[0061] Remaining mass ratio (%) = W t / W0×100%;

[0062] Injectability test of γ-PGA / HA hydrogel: Methylene blue labeled γ-PGA / HA hydrogel was squeezed into the bottom of a plastic culture dish using a 20 mL syringe, and its injectability and shape retention were observed.

[0063] The in vitro release behavior of JP1 was detected by dialysis. Equal volumes of JP1-containing γ-PGA / HA solution and JP1-loaded γ-PGA / HA hydrogel were placed in dialysis bags and immersed in 40 mL of PBS (pH 7.0) release medium, and gently shaken at 37 °C. At 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12, 16, 20, and 24 h, appropriate amounts of the external release medium were collected, and an equal volume of fresh PBS was immediately added to maintain a constant total volume of the release system. The JP1 content in the release medium at different time points was determined using the BCA method. The JP1 release concentration was calculated based on the JP1 standard curve, and the release behavior of JP1 in γ-PGA / HA solution and γ-PGA / HA hydrogel was compared. All experiments were independently repeated three times.

[0064] Working concentrations of JP1 and γ-PGA / HA were screened in UVB-damaged HDFs and HaCaT cells, respectively. Subsequent cell experiments used 100 μM JP1 and 1.0 mg / mL γ-PGA / HA. Cell compatibility was assessed using live / dead staining. Cells were treated with FITC-JP1 for 4 h and 8 h to observe its time-dependent cellular uptake.

[0065] 3. UVB-induced cell photodamage model and grouping

[0066] HDFs and HaCaT cells were cultured in DMEM containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37 °C and 5% CO2. Cells were washed with PBS before irradiation, and HDFs were subjected to 60 mJ / cm² irradiation. 2 HaCaT cells were irradiated with UVB and treated with 20 mJ / cm². 2 UVB irradiation was performed with the lamp tube 15 cm away from the cells; the cells were then cultured in serum-free DMEM for 24 h after irradiation.

[0067] The experiment included four groups: Control group, UVB group, UVB+γ-PGA / HA group, UVB+JP1 group, and UVB+γ-PGA / HA+JP1 group. Each treatment was administered after UVB irradiation.

[0068] 4. Detection of cell viability, oxidative stress, DNA damage and migration

[0069] HDFs and HaCaT cell viability were detected using the CCK-8 assay. Intracellular ROS were detected by fluorescence microscopy and flow cytometry after incubation with 10 μM DCFH-DA for 30 min. DNA damage was evaluated using γ-H2AX immunofluorescence and Western blot. HDFs migration was evaluated using a scratch assay: HDFs migrated at a rate of 3 × 10⁻⁶ cells / year. 5Seeds were inoculated into 6-well plates, and after establishing a photodamage model, cross-shaped scratches were made. The scratch closure status was recorded at 0, 12 and 24 h.

[0070] 5. Detection of apoptosis and mitochondrial membrane potential

[0071] TUNEL staining was used to detect apoptosis in HDFs and HaCaT cells; Annexin V-FITC / PI double staining combined with flow cytometry was used to determine the proportion of early and late apoptosis in HDFs; immunofluorescence and Western blot were used to detect the expression of BAX, Bcl-2 and Caspase-3; mitochondrial membrane potential was evaluated by incubation with 10 μM JC-1 for 30 min and the red / green fluorescence ratio was used to evaluate the mitochondrial membrane potential.

[0072] 6. Detection of NF-κB inflammatory signals

[0073] Immunofluorescence was used to observe the expression of NF-κB and p-NF-κB and the nuclear translocation of p65; Western blot was used to detect the p-NF-κB / NF-κB ratio and IκBα expression; and qRT-PCR was used to detect the mRNA levels of IL-1β, IL-6 and TNF-α.

[0074] 7. RNA sequencing and enrichment analysis

[0075] Total RNA was extracted from HDFs in the Control, UVB, and UVB+γ-PGA / HA+JP1 groups. After quality testing, cDNA libraries were constructed and Illumina high-throughput sequencing was performed. The raw data underwent quality control and reference genome alignment. Differentially expressed genes were screened based on |log2fold change|≥1 and corrected P-value <0.05, followed by principal component analysis, cluster analysis, GO biological process analysis, and KEGG pathway enrichment analysis.

[0076] 8. Validation of the NOD2 / RIPK2 / NF-κB pathway

[0077] When HDFs fusion reached approximately 80%, a NOD2 overexpression plasmid was transfected with Lipofectamine 3000, and a UVB damage model was established 48 h after transfection. Pretreatment with either the NOD2 inhibitor GSK717 or the RIPK2 inhibitor GSK583 for 2 h was then performed before UVB irradiation and corresponding drug administration. Changes in NOD2, RIPK2, p-RIPK2, NF-κB, p-NF-κB, IκBα, and inflammatory factors were detected by Western blot, immunofluorescence, and qRT-PCR.

[0078] 9. Acute UVB photodamage model in BALB / c mice

[0079] BALB / c mice were randomly divided into four groups (n=5 per group) after hair removal on their backs: Control group, UVB group, UVB+γ-PGA / HA group, UVB+JP1 group, and UVB+γ-PGA / HA+JP1 group. Except for the Control group, the mice received 900 mJ / cm² radiation on their backs once daily. 2 UVB irradiation for 3 consecutive days. After modeling is completed, the corresponding preparation is applied topically once daily for 4 consecutive days. Figure 14 (See Figure A). The Control and UVB groups were treated with a vector composed of 30% saline, 20% anhydrous ethanol, and 50% propylene glycol. Mice were euthanized after treatment, and dorsal skin and serum were collected.

[0080] 10. Skin tissue repair and inflammation detection

[0081] Erythema, desquamation, and crusting of the mouse dorsal skin were recorded. Epidermal thickness and morphology were evaluated by H&E staining, and dermal collagen area and arrangement were evaluated by Masson staining. IL-1β, IL-6, and TNF-α in skin tissue were detected by qRT-PCR and ELISA. The expression and distribution of γ-H2AX, apoptosis, p-NF-κB, and ZO-1 were detected by immunofluorescence or TUNEL assay.

[0082] 11. Validation of JP1 skin distribution and integrin αvβ3-related mechanisms

[0083] FITC-JP1 solution or FITC-JP1-loaded γ-PGA / HA hydrogel was applied topically to the dorsal skin of mice damaged by UVB. Skin tissue sections were collected at 0.5 h and 4 h to observe the penetration depth and local distribution of FITC-JP1. Integrin αv, integrin β1, and integrin β3 were detected by qRT-PCR and immunofluorescence.

[0084] To verify whether integrin αvβ3-related recognition is involved in the anti-inflammatory effect of JP1, HDFs were pretreated with Cyclo(-RGDfK), a selective competitive inhibitor of integrin αvβ3, and then JP1 was administered. The mRNA expression of NOD2, RIPK2, IL-1β, IL-6 and TNF-α was then detected.

[0085] 12. In vivo safety and statistical analysis

[0086] Serum biochemical assays were used to detect liver and kidney function indicators such as ALT, AST, ALP, TP, ALB, GLOB, BUN, CREA, and UA. Hematoxylin and eosin (H&E) staining was performed on the heart, liver, spleen, lungs, and kidneys. Data were analyzed using GraphPad Prism 10.0. One-way ANOVA was used for comparisons between groups, followed by Tukey multiple comparisons, with at least three independent replicates. P < 0.05 was considered statistically significant.

[0087] II. Experimental Results

[0088] (1) such as Figure 2 , Figure 3 As shown, JP1 was designed from the functional fragment of the JWA protein and was used to construct a JP1-loaded γ-PGA / HA hydrogel together with γ-PGA, HA and PEGDE. Figure 2 (Figure A). FTIR results show that the γ-PGA / HA hydrogel retains the main characteristic absorption peaks of γ-PGA and HA, at approximately 3430 cm⁻¹. -1 A broad peak of O–H / N–H stretching vibration is visible at this location, while the peak shape and intensity of some absorption peaks change, indicating that there is an intermolecular interaction between γ-PGA and HA, and the hydrogel system has been successfully formed. Figure 2 Figure B). Rheological results show that, compared with the γ-PGA / HA solution, the storage modulus G′ and loss modulus G″ of the γ-PGA / HA hydrogel are generally increased; within the test range, G″ of the hydrogel is generally higher than G′, indicating that the system has a certain degree of viscoelasticity and is mainly viscous in response. Figure 2 (See Figures C and D). As the shear rate increases, the viscosity of the γ-PGA / HA hydrogel gradually decreases, exhibiting significant shear-thinning properties. Figure 2 (Figure E). Further injection tests showed that the γ-PGA / HA hydrogel could be continuously and uniformly extruded smoothly through the syringe needle, without obvious blockage or breakage during injection. After the hydrogel was deposited at the bottom of the plastic culture dish, it maintained a relatively clear shape outline without obvious diffusion, indicating that it has good injectability and a certain shape retention ability. This result is consistent with its shear-thinning characteristics. Figure 3 Scanning electron microscopy results showed that an interconnected porous three-dimensional network structure was formed inside the γ-PGA / HA hydrogel. Figure 2 (See F-plot). The swelling experiment results showed that the swelling ratio of the γ-PGA / HA hydrogel gradually increased with time and tended to stabilize after about 40 h, with an equilibrium swelling ratio of about 40%. Figure 2 (G diagram). In the enzymatic degradation experiment, the hydrogel initially showed a temporary increase in mass due to water absorption and swelling, followed by a gradual decrease in the remaining mass, and near-complete degradation on day 6, indicating its good degradability. Figure 2(H diagram). In vitro release experiments showed that JP1 in γ-PGA / HA solution was rapidly released in the early stage, reaching a high concentration at about 4-6 h, and then slightly decreased; in contrast, JP1 in JP1-loaded γ-PGA / HA hydrogel showed a continuous and gradual release, and tended to stabilize after about 16-20 h, indicating that the hydrogel can prolong the release process of JP1. Figure 2 Figure I). Live / dead cell staining results showed that after 1 and 3 days of treatment with γ-PGA / HA hydrogel, JP1, or JP1-loaded γ-PGA / HA hydrogel, HDFs and HaCaT cells were predominantly green live cells with few red dead cells, indicating that none of the treatments caused significant cytotoxicity and demonstrated good cell compatibility. Figure 2 J and K plots). Cell uptake results showed that FITC-JP1 fluorescence signal was weak in HDFs and HaCaT cells after 4 h of incubation, while the cytoplasmic green fluorescence was significantly enhanced after 8 h of incubation, indicating that JP1 can be effectively taken up by both cell types, and that its cellular uptake is time-dependent. Figure 2 L-diagram and M-diagram).

[0089] (2) such as Figure 4 , Figure 5 and Figure 6 As shown, UVB irradiation significantly reduced the viability of HDFs and HaCaT cells. Different concentrations of JP1 and γ-PGA / HA hydrogels exhibited varying degrees of repair effects on UVB-induced cell damage, with 100 μM JP1 and 1.0 mg / mL γ-PGA / HA hydrogel showing better cell viability recovery effects. Figure 4 Based on this, after treatment with JP1 loaded with γ-PGA / HA hydrogel, the viability of HDFs and HaCaT cells recovered to 75.61% and 76.06%, respectively, which was better than treatment with JP1 or γ-PGA / HA hydrogel alone. Figure 5 (Figures A-C). Meanwhile, this composite hydrogel reduced the relative ROS fluorescence intensity in HDFs and HaCaT cells to 1.25 and 1.11, respectively. Figure 5 The D diagram, Figure 6 ), and significantly reduced the fluorescence intensity and protein expression level of the DNA damage marker γ-H2AX. Figure 5 The E and F diagrams, I and J diagrams). Furthermore, the scratch test results show ( Figure 5 (G and H diagrams) JP1-loaded γ-PGA / HA hydrogel significantly improved the migration rate and scratch closure of HDFs at 12 h and 24 h, indicating that it can effectively alleviate UVB-induced oxidative stress and DNA damage, and promote the migration and repair of damaged cells.

[0090] (3) such as Figure 7 and Figure 8 As shown, UVB significantly increased the TUNEL-positive signal in HDFs and HaCaT cells. The total apoptosis rate of HDFs increased from 8.40±0.94% in the Control group to 36.20±4.22%; after treatment with γ-PGA / HA and JP1 alone, it decreased to 24.48±1.45% and 24.41±1.40%, respectively, and further decreased to approximately 17.54% with the composite hydrogel. Figure 7 Figures A-D, I, and J). The composite hydrogel reduced BAX and Caspase-3, restored Bcl-2, and increased the red / green fluorescence ratio of JC-1, indicating that it can maintain mitochondrial membrane potential and alleviate apoptosis. Figure 7 E-plot - H-plot, K-plot - R-plot; Figure 8 ).

[0091] (4) such as Figure 9 As shown, UVB enhances NF-κB and p-NF-κB signaling and promotes p65 nuclear translocation, while increasing the p-NF-κB / NF-κB ratio and decreasing IκBα expression. JP1-loaded γ-PGA / HA hydrogel significantly reversed these changes and reduced the expression of IL-1β, IL-6, and TNF-α in HDFs and HaCaT cells, with an overall inhibitory effect stronger than γ-PGA / HA or JP1 treatment alone.

[0092] (5) such as Figure 10 As shown, RNA sequencing revealed good separation and reproducibility among the three sample groups. A total of 3050 overlapping differentially expressed genes were identified during the UVB damage and composite hydrogel repair process, and these genes could be clustered into six expression trends (…). Figure 10 Figures A-C). GO and KEGG analyses showed that differentially expressed genes mainly involved DNA replication and repair, cell cycle, extracellular matrix organization, apoptosis, and inflammatory immune pathways. Figure 10 Figures D and E); among them, cluster C4 is enriched in NF-κB, cytokine receptor interaction, necrosis and apoptosis pathway, and cluster C5 is enriched in NOD-like receptor, NF-κB, TNF, IL-17 and cytoplasmic DNA sensing pathway ( Figure 10 (F and G plots). qRT-PCR showed that UVB significantly increased NOD2 and RIPK2 expression, while the composite hydrogel reduced both, with no significant change in NOD1. Figure 10 (H diagram).

[0093] (6) For example Figure 11 and Figure 12 As shown, NOD2 overexpression enhances RIPK2 phosphorylation and NF-κB activation, decreases IκBα, and increases the expression of RIPK2, IL-1β, IL-6, and TNF-α, while partially weakening the inhibitory effect of the composite hydrogel. Figure 11Figure A-K). Both the NOD2 inhibitor GSK717 and the composite hydrogel inhibited NOD2 / RIPK2 / NF-κB signaling, and the combined treatment showed a stronger inhibitory trend overall, proving that NOD2 signaling is involved in the anti-inflammatory effect of the composite hydrogel. Figure 11 L-plot - V-plot; Figure 12 ).

[0094] (7) For example Figure 13 As shown, both the RIPK2 inhibitor GSK583 and JP1-loaded γ-PGA / HA hydrogel can reduce NOD2, p-RIPK2 / RIPK2, and p-NF-κB / NF-κB levels, restore IκBα, and reduce the transcriptional levels of NOD2, RIPK2, IL-1β, IL-6, and TNF-α, further supporting the view that the NOD2 / RIPK2 / NF-κB axis is an important pathway in JP1-loaded γ-PGA / HA hydrogel.

[0095] (8) such as Figure 14 As shown, erythema, desquamation, and crusting appeared on the backs of mice after UVB irradiation, with the JP1-loaded γ-PGA / HA hydrogel group showing the most significant improvement. Figure 14 Figure B). H&E showed that this group had the best inhibitory effect on UVB-induced epidermal hyperplasia (Figure B). Figure 14 Figures C and E); Masson staining showed that it increased the collagen-positive area and improved collagen arrangement (Figures C and E); Figure 14 (Figures D and F); the mRNA and protein levels of IL-1β, IL-6, and TNF-α in skin tissue were significantly decreased (Figures D and F). Figure 14 G-graph and H-graph).

[0096] (9) such as Figure 15 As shown, UVB increases the positive signals of γ-H2AX and TUNEL in skin tissue and significantly enhances p-NF-κB signal in the dermis, while disrupting the continuous distribution of ZO-1 in the epidermis. The composite hydrogel significantly reduces DNA damage, apoptosis, and inflammatory signals, and restores ZO-1 expression and epidermal barrier structure, showing superior effects compared to the single group.

[0097] (10) such as Figure 16 As shown, compared with free JP1, FITC-JP1 loaded with hydrogel exhibited earlier and stronger fluorescence signals in UVB-damaged epidermis, indicating that it improved local penetration and retention. Figure 16 Figure A). UVB reduces the expression of integrin αv and integrin β3, JP1 can partially restore both, while the change in integrin β1 is not significant ( Figure 16Figures B-H). Cyclo(-RGDfK) pretreatment attenuated the inhibitory effects of JP1 on NOD2, RIPK2, IL-1β, IL-6, and TNF-α, demonstrating that integrin αvβ3-related recognition is at least partially involved in JP1-mediated anti-inflammatory repair. Figure 16 (I-M diagrams). No significant abnormalities were observed in liver and kidney function indicators and histological examination of major organs in any treatment group. Figure 16 The N and O diagrams indicate that the topical formulation has good in vivo safety under the experimental conditions.

[0098] In summary, the overall mechanism of action of JP1-loaded γ-PGA / HA hydrogel is as follows: Figure 1 As shown: γ-PGA / HA hydrogel provides a mimicking extracellular matrix microenvironment and local delivery support, enabling JP1 to be continuously released and act on skin cells; JP1 enters cells by targeting integrin αvβ3-related recognition to regulate adhesion and repair signals, while inhibiting the NOD2 / RIPK2 / NF-κB inflammatory axis, thereby reducing oxidative stress, DNA damage, apoptosis and inflammation, and promoting collagen reconstruction and epidermal barrier recovery.

[0099] Example 2

[0100] In this embodiment, each JWA polypeptide shown in the table below (as shown in SEQ ID No. 2 to SEQ ID No. 39, note: the abbreviation for 6-aminocaproic acid is Acp) was tested according to the experimental methods in Example 1. The amino acid S of each JWA polypeptide was modified by phosphorylation.

[0101]

[0102] Due to space limitations, specific experimental data are not listed in this embodiment. The obtained experimental data show that the results of the detection of each JWA peptide according to the experimental methods in Example 1 are basically consistent with JP1.

[0103] Based on the research results represented by the above embodiments, the JWA peptide-loaded γ-PGA / HA hydrogel of the present invention, through the synergistic effect of material microenvironment support and JWA peptide bioregulation, inhibits the NOD2 / RIPK2 / NF-κB inflammatory axis, restores integrin αvβ3-related adhesion and ZO-1-related barrier, and can effectively repair UVB-induced skin photodamage.

[0104] In addition to the embodiments described above, the present invention may have other implementations. All technical solutions formed by equivalent substitution or equivalent transformation fall within the protection scope claimed by the present invention.

[0105] Summarize

[0106] This invention utilizes JWA peptide-loaded γ-PGA / HA hydrogel as the core to construct a local delivery system for repairing UVB-induced skin photodamage. First, the physicochemical properties, microstructure, JP1 release behavior, and biocompatibility of the hydrogel were evaluated using scanning electron microscopy, hydrogel swelling, degradation experiments, in vitro release experiments, and cell compatibility testing. Subsequently, UVB photodamage models were established in HDFs and HaCaT cells. The effects of the composite hydrogel on cell viability, oxidative stress, DNA damage, migration ability, mitochondrial function, and apoptosis were assessed using CCK-8 assays, ROS assays, γ-H2AX assays, scratch assays, TUNEL assays, flow cytometry, JC-1 staining, and apoptosis-related protein analysis.

[0107] Regarding the mechanism of action, changes in the NF-κB pathway and inflammatory factors were detected by immunofluorescence, Western blot, and qRT-PCR, and key signaling pathways related to repair were screened using RNA sequencing. Furthermore, NOD2 overexpression, the NOD2 inhibitor GSK717, and the RIPK2 inhibitor GSK583 were used to verify whether the JWA peptide-loaded γ-PGA / HA hydrogel functions by inhibiting the NOD2 / RIPK2 / NF-κB inflammatory axis; simultaneously, the integrin αvβ3 inhibitor Cyclo(-RGDfK) was used to verify the targeted binding and regulation of integrin αvβ3 signaling molecules by the JWA peptide.

[0108] Finally, a UVB-induced acute skin photodamage model was established in BALB / c mice. The in vivo repair effect was evaluated by observing the appearance of skin lesions, H&E staining, Masson staining, immunofluorescence, detection of inflammatory factors, and skin barrier-related indicators. The effect of hydrogel on skin penetration and local retention of JWA peptide was analyzed by FITC-JP1 tracing. At the same time, serum biochemical indicators and histological changes of major organs were detected to evaluate its in vivo safety.

[0109] The overall research plan forms a complete research system of "material construction and characterization - in vitro functional evaluation - mechanism verification - animal efficacy and safety evaluation".

[0110] The main research results of this invention are as follows:

[0111] (1) This invention is the first to load JWA peptides containing RGD motifs onto γ-PGA / HA biomimetic hydrogels and use them for skin repair after UVB irradiation, thereby achieving the synergistic effect of JWA oligopeptide kinase regulation and hydrogel microenvironment support.

[0112] (2) The γ-PGA / HA biomimetic hydrogel has a shear-thinning and interconnected porous structure, which can realize the local delivery and sustained release of JWA peptides, improve their distribution and local retention in skin tissue, and has good cell compatibility, injectability and shape retention, making it easy to apply locally.

[0113] (3) JWA peptide-loaded γ-PGA / HA hydrogel, as a topical preparation, can simultaneously reduce oxidative stress, DNA damage, mitochondrial dysfunction, apoptosis and inflammatory response, and promote cell migration, collagen reconstruction and epidermal barrier recovery.

[0114] (4) This invention confirms that the NOD2 / RIPK2 / NF-κB inflammatory axis is an important regulatory pathway for the above-mentioned topical preparations, and confirms that integrin αvβ3-related recognition is involved in JWA peptide-mediated anti-inflammatory repair, providing a basis for the mechanism of action and subsequent targeted optimization.

[0115] (5) Animal experiments showed that the above-mentioned topical preparation could improve UVB-induced visible skin lesions, epidermal hyperplasia, collagen disorder and local inflammation; no obvious abnormalities were found in serum liver and kidney function indicators and histology of major organs, indicating that the preparation has good potential for local application.

[0116] In summary, this invention provides scientific evidence of a causal relationship between topical application of JWA peptides in repairing UVB-induced photodamage and the enhanced effectiveness of γ-PGA / HA-loaded hydrogels. The JWA peptide-loaded γ-PGA / HA hydrogel, formed by cross-linking γ-PGA and HA with polyethylene glycol diglycidyl ether and loading JWA peptides, exhibits shear thinning, interconnected porous structure, sustained release, and good cell compatibility. In vitro and in vivo experiments demonstrate that both the JWA peptides and the γ-PGA / HA-loaded hydrogel can target and exert their effects on cells, reducing reactive oxygen species and γ-H2AX levels generated by UVB, alleviating mitochondrial dysfunction and apoptosis, inhibiting the NOD2 / RIPK2 / NF-κB inflammatory axis and the expression of IL-1β, IL-6, and TNF-α, and restoring integrin αvβ3-related adhesion, dermal collagen structure, and ZO-1-related epidermal barrier in damaged skin cells. JWA peptide-loaded γ-PGA / HA hydrogel, as a topical formulation, can improve UVB-induced erythema, desquamation, crusting, and epidermal hyperplasia, without significant systemic toxicity, providing a new local repair technique for UVB-induced skin photodamage.

Claims

1. The use of a polypeptide, characterized in that, The polypeptide is JWA polypeptide; the intended use is for preparing a formulation to repair skin photodamage caused by ultraviolet radiation; The amino acid sequence of the JWA polypeptide is shown in I or II: I: FPGSDRF-Z; II: X-FPGSDRF-Z; Among them, amino acid S is phosphorylated, and X and Z are amino acids or amino acid sequences, respectively; X is selected from one of F, (R)9, (R)9-F, 6-aminohexanoic acid, 6-aminohexanoic acid-F, 6-aminohexanoic acid-(R)9, and 6-aminohexanoic acid-(R)9-F; Z is selected from (G). n -RGD、A-(G) n -RGD is one of them, where n is an integer greater than or equal to 0, and the value of n ranges from 0 to 10.

2. The use of the polypeptide according to claim 1, characterized in that, The formulation is a topical preparation for repairing UVB-induced skin photodamage; the skin photodamage includes at least one of erythema, desquamation, crusting, and epidermal hyperplasia.

3. The use of the polypeptide according to claim 1, characterized in that, The formulation includes a carrier, wherein the carrier is a γ-PGA / HA hydrogel.

4. The use of the polypeptide according to claim 3, characterized in that, The function of the formulation is to simultaneously reduce oxidative stress, DNA damage, mitochondrial dysfunction, apoptosis and inflammatory response, and promote cell migration, collagen reconstruction and epidermal barrier restoration.

5. The use of a polypeptide according to any one of claims 1 to 4, characterized in that, The N-terminus of the polypeptide is acetylated and the C-terminus is amidated; the amino acid sequence of the polypeptide is one of SEQ ID No. 1 to SEQ ID No. 39; in the polypeptide, the configuration of each amino acid in the sequence FPGSDRF is L-type or D-type.

6. A preparation for repairing photodamage to the skin caused by ultraviolet radiation, characterized in that, The formulation comprises JWA peptide-loaded γ-PGA / HA hydrogel; The amino acid sequence of the JWA polypeptide is shown in I or II: I: FPGSDRF-Z; II: X-FPGSDRF-Z; Among them, amino acid S is phosphorylated, and X and Z are amino acids or amino acid sequences, respectively; X is selected from one of F, (R)9, (R)9-F, 6-aminohexanoic acid, 6-aminohexanoic acid-F, 6-aminohexanoic acid-(R)9, and 6-aminohexanoic acid-(R)9-F; Z is selected from (G). n -RGD、A-(G) n -RGD is one of them, where n is an integer greater than or equal to 0, and the value of n ranges from 0 to 10.

7. The preparation for repairing skin photodamage caused by ultraviolet radiation according to claim 6, characterized in that, The peptide is acetylated at its N-terminus and amidated at its C-terminus. The amino acid sequence of the peptide is one of SEQ ID No. 1 to SEQ ID No.

39. The conformation of each amino acid in the FPGSDRF sequence of the peptide is L-type or D-type. Alternatively, the formulation is a topical formulation for repairing UVB-induced photodamage to the skin; Alternatively, the preparation process of the JWA peptide-loaded γ-PGA / HA hydrogel is as follows: γ-PGA / HA hydrogel is prepared by crosslinking γ-PGA and HA with PEGDE, and JWA peptide is uniformly loaded into the γ-PGA / HA hydrogel network to obtain JWA peptide-loaded γ-PGA / HA hydrogel.

8. Use of the formulation of claim 6 or 7 in the preparation of a product for repairing skin photodamage caused by ultraviolet radiation.

9. The use according to claim 8, characterized in that, The product is a topical product for repairing skin photodamage caused by UVB.

10. The use according to claim 8, characterized in that, The product in question is a skincare or cosmetic product.

Images