A recombinant humanized elastin with improved endogenous skin aging efficacy and application thereof

Abstract

The application provides a recombinant humanized elastin with improved endogenous skin aging efficacy and an application thereof, and belongs to the technical field of skin medicine and biological materials. The recombinant humanized elastin is based on a human elastin core functional sequence, and is recombined, optimized and efficiently expressed by using a synthetic biology strategy, and exhibits excellent stability and biological activity. The recombinant humanized elastin can be used to construct a skin repair dressing, and can be used for remodeling an extracellular matrix network structure, and can be used for synergistically improving skin elasticity decline, collagen loss and high oxidative stress and other endogenous aging characteristics. Animal experiments prove that a product based on the recombinant humanized elastin has good tissue repair capacity, biocompatibility and antioxidant activity, and can be widely applied to functional skin care products and medical cosmetic preparations.

Description

Technical Field

[0001] This invention relates to the interdisciplinary field of dermatology and biomaterials, specifically to an active ingredient based on recombinant elastin and its application in improving endogenous skin aging, falling within the scope of protein engineering and the development of anti-aging skin products. Background Technology

[0002] The skin, the largest organ in the human body, is mainly composed of the epidermis, dermis, and subcutaneous tissue, and plays a vital role in protecting the body from external aggressors. Skin aging is a complex biological process driven by genetic, environmental, and physiological factors, generally divided into extrinsic aging (such as ultraviolet radiation) and endogenous aging (i.e., the natural decline that occurs with age). Endogenous skin aging is characterized by a decrease in the content of collagen and elastin in the dermis, disordered fiber arrangement, accelerated degradation of the extracellular matrix (ECM), increased water loss, and decreased antioxidant capacity.

[0003] Elastin is one of the most important structural proteins in the skin, accounting for approximately 2% to 5% of the dermal dry weight. It plays a crucial role in maintaining skin elasticity, enabling the skin to resist repeated mechanical stretching and maintain structural stability. In the dermis, collagen primarily provides structural support, while elastin endows the skin with excellent elasticity and reversible deformation capacity. Elastin synthesis peaks in late embryonic development and almost ceases in adulthood, and once damaged, it is difficult to repair naturally. During aging, under the combined influence of endogenous and exogenous factors, the expression of matrix metalloproteinases (MMPs) in the skin is significantly upregulated, leading to accelerated elastin degradation, structural breakage, and the gradual disintegration of the elastic network. Simultaneously, the expression levels of key proteins essential for elastic fiber formation, such as lysyl oxidase (LOX) and fibrillin-1, decrease, further inhibiting the efficiency of new elastic fiber formation. The synergistic effect of these factors causes the skin to gradually lose elasticity, resulting in typical signs of aging such as increased wrinkles and sagging.

[0004] Current anti-aging products primarily use antioxidants, hyaluronic acid, recombinant collagen, or peptides as main ingredients, focusing on improving skin hydration, firmness, and radiance. However, these ingredients mostly target the collagen system and have limited ability to rebuild the elastin network, failing to fundamentally address the problem of declining skin elasticity. Although a small amount of elastin extracts are used in skincare products, they are mostly derived from animal tissues, presenting issues such as low biocompatibility, high immunogenicity, and poor batch stability, making it difficult to meet the high-quality raw material requirements of medical aesthetics and functional skincare products.

[0005] With the development of biosynthesis and protein engineering technologies, the preparation of human elastin using gene recombination technology has become a safe, stable, and highly controllable alternative. By precisely designing expression sequences and constructing recombinant expression systems, it is possible to obtain elastin with high purity, low immunogenicity, and natural functional structures. However, specific functional validation and mechanistic studies targeting endogenous skin aging are currently lacking, particularly systematic evidence regarding its role in promoting elastic fiber regeneration, alleviating oxidative stress, and rebuilding the ECM network. Summary of the Invention

[0006] To address the shortcomings of the existing technologies, this invention provides a recombinant humanized elastin with the effect of improving endogenous aging and its applications. The recombinant humanized elastin is designed and expressed through synthetic biology based on the core functional region sequence of human elastin. It exhibits good stability, biocompatibility, and bioactivity, and can achieve anti-aging through antioxidant activity and promotion of extracellular matrix remodeling, effectively delaying endogenous skin aging. It can be widely used in cosmetics or dermatological medicine.

[0007] To achieve the above objectives, the specific technical solution of the present invention is as follows:

[0008] In a first aspect, the present invention provides a recombinant humanized elastin with the effect of improving endogenous skin aging, wherein the amino acid sequence of the recombinant humanized elastin is as follows:

[0009] (a) The amino acid sequence shown in SEQ ID NO. 1; or

[0010] (b) An amino acid sequence obtained by substituting, deleting and / or adding one or more amino acids based on the sequence shown in SEQ ID NO. 1, and having the same function as the protein shown in SEQ ID NO. 1.

[0011] Furthermore, the recombinant humanized elastin is obtained by treating the precursor elastin with a protease.

[0012] Furthermore, the amino acid sequence of the precursor elastin is as follows;

[0013] (a) The amino acid sequence shown in SEQ ID NO. 2; or

[0014] (b) An amino acid sequence obtained by substituting, deleting and / or adding one or more amino acids based on the sequence shown in SEQ ID NO. 2, and having the same function as the protein shown in SEQ ID NO. 2.

[0015] Furthermore, the nucleotide sequence of the gene encoding the precursor elastin is as follows:

[0016] (a) The nucleotide sequence shown in SEQ ID NO. 3; or

[0017] (b) A nucleotide sequence of the nucleotide sequence shown in SEQ ID NO. 3 that has been substituted, deleted and / or added with one or more nucleotides, and that expresses the same protein as the nucleotide sequence shown in SEQ ID NO. 2.

[0018] Secondly, the present invention provides a method for preparing the recombinant humanized elastin, comprising the following steps:

[0019] (1) Synthesize the gene sequence SEQ ID NO.3 encoding the precursor elastin of the recombinant humanized elastin;

[0020] (2) The gene sequence is ligated to a vector, transformed into genetically engineered bacteria, and recombinant genetically engineered bacteria are constructed;

[0021] (3) The recombinant genetically engineered bacteria were expressed, the bacterial cells were collected, the supernatant was obtained by cleavage, and the precursor elastin was obtained by purification.

[0022] (4) Add the protease to the precursor elastin obtained by treatment, and purify it to obtain recombinant humanized elastin.

[0023] Furthermore, the protease includes thrombin, pepsin, trypsin, or bromelain.

[0024] Thirdly, the present invention provides a recombinant vector or recombinant genetically engineered bacteria carrying the precursor protein gene of the recombinant humanized elastin.

[0025] Furthermore, the recombinant vector includes pCold or pET; the recombinant engineered bacteria includes Escherichia coli.

[0026] Fourthly, the present invention provides the application of the recombinant humanized elastin in the preparation of products with the effect of improving endogenous aging.

[0027] Furthermore, the products for improving endogenous aging include, but are not limited to, cosmetics or medical aesthetic products.

[0028] Furthermore, the cosmetics include, but are not limited to, serums, lotions, essences, creams, masks, sprays, sunscreens, eye creams, foundations, and body lotions.

[0029] Furthermore, the medical aesthetic products include, but are not limited to, serums, gels, cold compresses, dressings, sponges, hyaluronic acid injection preparations, implants, and creams.

[0030] Fifthly, the present invention provides a skin repair dressing with the effect of improving endogenous aging, comprising the following components by weight percentage: 0.1%-1% recombinant humanized elastin, 5%-8% trehalose, 3%-5% glycerin, 0.2%-1% hyaluronic acid, 0.05%-0.5% arginine, 0.1%-0.5% carbomer, 0.1%-0.5% 1,2-hexanediol, 0.1%-0.5% p-hydroxyacetophenone, with the balance being water.

[0031] Furthermore, the skin repair dressing comprises the following components by weight percentage: 0.1% recombinant elastin, 5% trehalose, 3% glycerin, 1% hyaluronic acid, 0.5% arginine, 0.5% carbomer, 0.5% 1,2-hexanediol, 0.5% p-hydroxyacetophenone, with the balance being water.

[0032] Compared with the prior art, the advantages of the present invention are:

[0033] (1) The humanized elastin obtained by recombinant expression in this invention has a clear sequence conformation, avoiding the immune risks and batch inconsistencies caused by animal-derived extraction, and has good biosafety and industrial controllability.

[0034] (2) The skin repair dressing based on recombinant humanized elastin provided by the present invention has good stability and high bioactivity, and can effectively promote the proliferation, extension and migration of fibroblasts.

[0035] (3) The skin repair dressing based on recombinant humanized elastin provided by the present invention exhibits excellent post-sun exposure repair and reactive oxygen species scavenging capabilities in zebrafish models, and has reliable in vivo antioxidant protection.

[0036] (4) The skin repair dressing based on recombinant humanized elastin provided by the present invention can significantly alleviate the manifestations of endogenous aging skin, improve various typical aging characteristics such as dryness, wrinkles, barrier damage and decreased elasticity, and the repair effect is significantly better than existing commercially available products.

[0037] (5) The skin repair dressing based on recombinant humanized elastin provided by the present invention can balance the high oxidative stress microenvironment of aging skin, and effectively achieve dual regulation of skin anti-aging by enhancing the antioxidant system and inhibiting MMP activity.

[0038] (6) This invention provides an intervention method with a clear mechanism, clear components, and is safe and effective, filling the technical gap in the elastin reconstruction dimension of existing anti-aging products, and has good medical application prospects and industrial promotion value. Attached Figure Description

[0039] Figure 1This is an SDS-PAGE image of the recombinant humanized elastin RHE of this invention.

[0040] Figure 2 These are the results of the biocompatibility and bioactivity evaluation of the skin repair dressing of the present invention; wherein, Figure 2 a represents the results of the cytotoxicity experiment; Figure 2 b represents the results of the cell proliferation experiment; Figure 2 c represents the live cell count result; Figure 2 d represents the results of the live / dead cell staining experiment. Figure 2 e represents the result of the immunofluorescence staining experiment; Figure 2 f represents the results of the cell migration experiment; Figure 2 g represents the quantitative result of cell migration.

[0041] Figure 3 This is the evaluation result of the post-sun exposure repair efficacy of the skin repair dressing of the present invention; wherein, Figure 3 a is a schematic diagram of an experimental model for post-sun exposure repair; Figure 3 b represents the qualitative evaluation result of the post-sun repair efficacy; Figure 3 c represents the quantitative evaluation result of the post-sun repair efficacy;

[0042] Figure 4 This is the evaluation result of the antioxidant efficacy of the skin repair dressing of the present invention; wherein, Figure 4 a is a schematic diagram of the antioxidant experimental model; Figure 4 b represents the qualitative evaluation result of the antioxidant effect; Figure 4 c represents the quantitative evaluation result of antioxidant efficacy;

[0043] Figure 5 This is the evaluation result of the anti-aging efficacy of the skin repair dressing of the present invention; wherein, Figure 5 a is a schematic diagram of a D-galactose-induced endogenous aging model in mice; Figure 5 b. Dermoscopy findings; Figure 5 c represents the ultrasonic detection result; Figure 5 d represents the dermal density of the mouse; Figure 5 e represents the water content of the mouse's stratum corneum; Figure 5 f represents the TEWL test result; Figure 5 g represents the skin retraction time of the mouse;

[0044] Figure 6 The change in body weight of mice was used to evaluate the anti-aging efficacy of the skin repair dressing of the present invention.

[0045] Figure 7 This is the evaluation result of the anti-aging efficacy of the skin repair dressing of the present invention; wherein, Figure 7 a shows the results of H&E, Masson, and Verhöeff staining; Figure 7b represents the epidermal thickness of an H&E image measured using ImageJ software. Figure 7 c represents the collagen fiber content in Masson images quantified using ImageJ software. Figure 7 d represents the elastic fiber content in Verhöeff images quantified using ImageJ software;

[0046] Figure 8 The evaluation results of the skin repair dressing of the present invention on balancing the endogenous aging and oxidative stress environment in vivo; wherein, Figure 8 a represents the malondialdehyde content; Figure 8 b represents superoxide dismutase activity; Figure 8 c represents catalase activity; Figure 8 d represents the glutathione content; Figure 8 e is a schematic diagram of a recombinant elastin skin repair dressing that balances endogenous aging and oxidative stress in the body;

[0047] Figure 9 The results of RT-qPCR detection of related gene expression; among them, Figure 9 'a' represents the LOX representation result; Figure 9 b represents the expression result of Fibrillin-1; Figure 9 c represents the expression result of MMP-2; Figure 9 d represents the expression result of MMP-12; Figure 9 e represents the expression result of Elastin; Figure 9 f represents the expression result of Col I; Figure 9 g represents the expression result of Col III; Figure 9 h is a schematic diagram illustrating how recombinant elastin skin repair dressings promote extracellular matrix remodeling. Detailed Implementation

[0048] The technical solution of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0049] This invention provides a recombinant humanized elastin with the effect of improving endogenous skin aging, wherein the amino acid sequence of the recombinant humanized elastin is as follows:

[0050] (a) The amino acid sequence shown in SEQ ID NO. 1; or

[0051] (b) An amino acid sequence obtained by substituting, deleting and / or adding one or more amino acids based on the sequence shown in SEQ ID NO. 1, and having the same function as the protein shown in SEQ ID NO. 1.

[0052] The method for preparing the recombinant humanized elastin includes the following steps:

[0053] (1) Synthesize the gene sequence SEQ ID NO.3 encoding the precursor elastin of the recombinant humanized elastin;

[0054] (2) The gene sequence is ligated to a vector, transformed into genetically engineered bacteria, and recombinant genetically engineered bacteria are constructed;

[0055] (3) The recombinant genetically engineered bacteria were expressed, the bacterial cells were collected, the supernatant was obtained by cleavage, and the precursor elastin was obtained by purification.

[0056] (4) Add the protease to the precursor elastin obtained by treatment, and purify it to obtain recombinant humanized elastin.

[0057] In some examples, the protease includes thrombin, pepsin, trypsin, or bromelain.

[0058] The present invention also provides a recombinant vector or recombinant genetically engineered bacteria carrying the precursor protein gene of the recombinant humanized elastin.

[0059] In some examples, the recombinant vector includes pCold or pET; the recombinant engineered bacteria includes Escherichia coli.

[0060] The present invention also provides the application of the recombinant humanized elastin in the preparation of products with the effect of improving endogenous aging.

[0061] In some examples, the products for improving endogenous aging include, but are not limited to, cosmetics or medical aesthetic products. The cosmetics include, but are not limited to, serums, lotions, essences, creams, masks, sprays, sunscreens, eye creams, foundations, and body lotions. The medical aesthetic products include, but are not limited to, serums, gels, cooling patches, dressings, sponges, mesotherapy preparations, implants, and creams.

[0062] The present invention also provides a skin repair dressing with the effect of improving endogenous aging, comprising the following components by weight percentage: 0.1%-1% recombinant humanized elastin, 5%-8% trehalose, 3%-5% glycerin, 0.2%-1% hyaluronic acid, 0.05%-0.5% arginine, 0.1%-0.5% carbomer, 0.1%-0.5% 1,2-hexanediol, 0.1%-0.5% p-hydroxyacetophenone, with the balance being water.

[0063] In the following specific embodiments, unless otherwise specified, the methods used are conventional methods and the reagents used are conventional reagents that can be obtained commercially.

[0064] Example 1: A recombinant humanized elastin with the effect of improving endogenous aging

[0065] The recombinant humanized elastin RHE provided in this embodiment has the amino acid sequence shown in SEQ ID NO. 1. This protein is obtained by enzymatic digestion of precursor elastin; the specific preparation steps are as follows:

[0066] (1) Design of precursor elastin sequence

[0067] First, the precursor protein sequence of recombinant humanized elastin RHE was determined, and the specific amino acid sequence is shown in SEQ ID NO. 2.

[0068] (2) Construction of expression strains

[0069] A gene encoding the precursor elastin was synthesized, with the nucleotide sequence shown in SEQ ID NO. 3. This sequence was cloned into an expression vector, and the vector construction was verified by DNA sequencing. The recombinant plasmid was then transformed into *E. coli* BL21(DE3) competent cells to obtain an engineered strain expressing the precursor protein. Successfully transformed strains were added to glycerol and stored at -80 °C for subsequent use.

[0070] (3) Expression and purification of recombinant proteins

[0071] 200 μL of glycerol-containing bacterial culture was inoculated into 200 mL of LB broth containing antibiotics and incubated overnight in a shaker at 37 °C. Subsequently, 2% of the inoculum was transferred to 1 L of LB broth containing antibiotics for further amplification. When the OD of the bacterial culture... 600 When the pH reached 0.8-1.0, the shaker temperature was lowered to 25 °C, and IPTG was added to a final concentration of 1 mM for induction of expression. The cells were cultured overnight. After harvesting the cells, they were centrifuged at 4 °C and 3200 rpm for 30 min, the supernatant was discarded, and the cell pellet was collected. The cells were resuspended in a lysis buffer (pH 7.4) containing 20 mM sodium phosphate, 0.5 M sodium chloride, and 20 mM imidazole. After sonication and centrifugation again, the supernatant was collected. The supernatant was purified using a nickel affinity chromatography column to obtain the precursor elastin. Thrombin was added to the purified protein solution to a final concentration of 2-4 U / mL, and the protein was digested under suitable conditions to remove the N-terminal tag. Further purification was then performed to remove enzymes and impurities. Finally, the protein was lyophilized to obtain high-purity recombinant humanized elastin (RHE).

[0072] The protein was analyzed by SDS-PAGE electrophoresis, and the electrophoresis results are as follows: Figure 1 As shown in the figure, RHE appears as a single band, indicating that the prepared elastin has high purity and meets the quality requirements for subsequent biological function evaluation and product application.

[0073] Example 2: A skin repair dressing with the effect of improving endogenous aging

[0074] The skin repair dressing of this embodiment is composed of the following components by weight percentage: 0.1% recombinant humanized elastin RHE prepared in Example 1, 5% trehalose, 3% glycerol, 1% hyaluronic acid, 0.5% arginine, 0.5% carbomer 940, 0.5% 1,2-hexanediol, 0.5% p-hydroxyacetophenone, and the balance being water.

[0075] The preparation process of the skin repair dressing is as follows: Carbomer 940 is dispersed in deionized water, stirred thoroughly and evenly, and heated to 85 ℃ to form a gel matrix; trehalose, glycerin and hyaluronic acid are added in sequence, stirred until completely dissolved, homogenized for 3 min, and kept warm for 10-15 min; arginine, 1,2-hexanediol and p-hydroxyacetophenone are added in sequence, and stirred and dispersed evenly; the temperature is lowered to 30 ℃ and recombinant humanized elastin RHE prepared in Example 1 is added, stirred until completely dissolved and transparent, discharged and allowed to stand to obtain skin repair dressing RHE gel.

[0076] Performance evaluation of the skin repair dressing prepared in Example 2

[0077] 1. Stability evaluation of skin repair dressings

[0078] Phase separation of the skin repair dressing was assessed by accelerated centrifugation: 5 g of the skin repair dressing sample was added to a centrifuge tube and centrifuged three times at 3000 rpm for 30 min each time. After confirming no phase separation occurred, the skin repair dressing sample was stored in an oven at 40 ℃ and a refrigerator at 4 ℃, respectively. The stability of the skin repair dressing was assessed by odor, physical appearance, pH value, and viscosity. The analysis period was 3 months. The pH value was determined according to Chinese standards QB / T 2872-2017 and GB / T13531.1-2008, by diluting the skin repair dressing sample with distilled water at a ratio of 1:10. Viscosity was measured using an Anton Paar rheometer (MCR 302, Anton Paar GmbH, Germany) at 100 s⁻¹. -1 The measurements were performed at a fixed shear rate. The test results are shown in Table 1.

[0079] Table 1: Stability Evaluation Results of Skin Repair Dressings

[0080]

[0081] As shown in Table 1, the skin repair dressing prepared by this invention has good stability. After centrifugation, heat resistance, and cold resistance tests, the pH value of the skin repair dressing remained within the physiological range of the skin, approximately 6.4; at 100 s... -1At a shear rate of [specific value], the viscosity of the skin repair dressing remained essentially unchanged. Furthermore, after three months of centrifugation, heating, and cold resistance tests, the skin repair dressing was uniformly transparent, without delamination, and exhibited no noticeable odor. In summary, the recombinant elastin skin repair dressing provided by this invention possesses excellent stability and can be stored for a long period.

[0082] 2. Evaluation of the biocompatibility and bioactivity of skin repair dressings

[0083] 2.1 Cytotoxicity assay

[0084] Fibroblasts (L929) that have grown to 70%-80% of the culture dish area were harvested, digested with 0.25% trypsin, and reconstituted into a cell density of 1×10⁶ cells / year using complete culture medium (10% FBS, 1% Penicillin–Streptomycin, DMEM). 5 Cell suspension at 100 μL / mL was seeded into 96-well plates and incubated at 37 ℃ with 5% CO2 saturated humidity for 24 h. The complete culture medium was aspirated, and skin repair dressing diluted with high-glucose DMEM medium at different ratios (x 1, x 10, x 20, x 100, with 4 replicates per ratio) was added to the experimental groups. The control group contained cells cultured in DMEM medium (Blank), and the blank group contained cell-free DMEM medium. Both groups were incubated at 37 ℃ with 5% CO2 saturated humidity for another 24 h. 10 μL of CCK-8 reagent was added to each group, and the cells were incubated for 1.5 h. The absorbance (OD value) of each well was measured at 450 nm using an ELISA reader. Cell viability was calculated based on the mean absorbance of each group using the following formula:

[0085]

[0086] The experimental results are shown in Figure 2 a. As shown in the figure, the skin repair dressing RHE gel of the present invention at different concentrations showed no cytotoxicity and good safety.

[0087] 2.2 Cell proliferation experiment

[0088] One mL of skin repair dressing and a commercially available recombinant collagen gel were each immersed in 9 mL of DMEM high-glucose medium and named RHE gel and RHC gel, respectively, for subsequent cell experiments. L929 cells that had grown to 70%-80% confluence of the culture dish were digested with 0.25% trypsin and cultured in complete medium to a cell density of 1 x 10⁻⁶ cells / mL. 5Cell suspension at 100 μL / mL was seeded into 96-well plates and cultured at 37 °C with 5% CO2 saturated humidity. After 24 h of culture, the complete culture medium was aspirated. RHE gel and RHC gel were added to the experimental groups (4 replicates), while the blank control group contained cells cultured in DMEM medium, and the blank group contained cell-free DMEM medium. Cells were cultured at 37 °C with 5% CO2 saturated humidity for 1, 3, and 5 days, respectively. At the specified time points, 10 μL of CCK-8 reagent was added to each group, and the cells were incubated for 1.5 h. The absorbance (OD value) of each well was measured at 450 nm using an ELISA reader. The relative cell proliferation rate (RGR) was calculated based on the mean absorbance of each group using the following formula:

[0089]

[0090] The experimental results are shown in Figure 2 b. As shown in the figure, compared with the blank control group (100%), the relative cell proliferation rate of the skin repair dressing of the present invention was greater than 100% on days 1, 3, and 5, and showed an increasing trend with time, significantly higher than that of commercially available recombinant collagen gel. This indicates that the skin repair dressing based on recombinant humanized elastin provided by the present invention can promote fibroblast proliferation.

[0091] 2.3 Live and Dead Cell Staining Assay

[0092] Take L929 cells that have grown to 70%-80% of the culture dish area, digest them with 0.25% trypsin, and use complete culture medium to prepare a cell density of 1×10⁻⁶ cells. 6 Cell suspension was prepared at 1 mL / mL. 1 mL of cell suspension was seeded into a laser confocal microscopy dish and incubated at 37 °C with 5% CO2 saturated humidity. After 24 h of culture, the complete culture medium was aspirated. RHE gel and RHC gel were added to the experimental groups (3 replicates); the blank control group received an equal volume of high-glucose DMEM medium. Cells were incubated at 37 °C with 5% CO2 saturated humidity for 1, 3, and 5 days. At specified time points, live / dead cell staining was performed according to the Solarbio live / dead cell staining kit, with live cells indicated by green and dead cells by red. Images were taken under a 10x laser confocal inverted microscope, and the number of live cells was counted using ImageJ.

[0093] The experimental results are shown in Figure 2c, 2d. The figures show that the skin repair dressing of the present invention primarily observed viable cells on days 1, 3, and 5, with no signs of cell death. The viable cell counts were 802±93, 997±45, and 1101±87, respectively, significantly higher than the viable cell counts at the corresponding time points for commercially available recombinant collagen gel. This indicates that the skin repair dressing based on recombinant humanized elastin provided by the present invention can promote fibroblast proliferation.

[0094] 2.4 Immunofluorescence assay

[0095] Take L929 cells that have grown to 70%-80% of the culture dish area, digest them with 0.25% trypsin, and use complete culture medium to prepare a cell density of 1×10⁻⁶ cells. 6 Cell suspension was prepared at 1 mL / mL. 1 mL of cell suspension was seeded into a laser confocal microscopy culture dish and incubated at 37°C with 5% CO2 saturated humidity. After 24 h of cell culture, the complete culture medium was aspirated. In the experimental groups, RHE gel containing FBS and RHC gel containing FBS were added (three replicates were set up); the blank control group received an equal volume of high-glucose DMEM medium containing FBS. Cells were further incubated at 37°C with 5% CO2 saturated humidity for another 24 h. Actin filaments were labeled with Rhodamine-phalloidin (red), and cell nuclei were labeled with DAPI (blue), following the manufacturer's instructions. Images were taken under a 40x laser confocal inverted microscope.

[0096] The experimental results are shown in Figure 2 e. The figure shows that L929 cells in the blank control group exhibited typical physiological morphology. The experimental group, however, showed more cells exhibiting extended actin fibers, and the skin repair dressing of this invention showed more pronounced elongation and spindle-shaped fibroblasts compared to commercially available recombinant collagen gel. This indicates that the skin repair dressing based on recombinant humanized elastin provided by this invention has a positive effect on cell extension.

[0097] 2.5 Cell migration experiment

[0098] Take L929 cells that have grown to 70%-80% of the culture dish area, digest them with 0.25% trypsin, and use complete culture medium to prepare a cell density of 1×10⁻⁶ cells. 6Cell suspension was prepared at 1 / mL. Using a marker and a ruler, three horizontal lines were drawn at the bottom of each well of a 6-well plate. 1 mL of cell suspension was seeded into each well and incubated at 37 °C with 5% CO2 saturated humidity. After 24 h of culture, a 200 μL pipette tip was used to gently push downwards along the wells to create longitudinal scratches. The culture medium was then aspirated, and the cells were washed three times with PBS to remove any scraped cells. RHE gel and RHC gel were added to the experimental groups (three replicates); the control group received an equal volume of high-glucose DMEM medium. Cells were incubated at 37 °C with 5% CO2 saturated humidity for 0 h and 24 h, and photographed under a 10x microscope. Relative migration rate was calculated using ImageJ.

[0099] The experimental results are shown in Figure 2 f, 2g. The figure shows that the blank control group cells showed almost no migration, with a migration rate of 14.83±2.08%. The skin repair dressing of this invention, after 24 h of culture, showed a cell migration rate of 76.05±4.54%, significantly higher than the 39.35±2.29% of the commercially available recombinant collagen gel group. This indicates that the skin repair dressing based on recombinant humanized elastin provided by this invention can promote fibroblast migration.

[0100] 3. Evaluation of the post-sun exposure repair efficacy of skin repair dressings

[0101] Normally developed zebrafish juveniles (2 dpf) were randomly assigned to six-well cell culture plates, 10 fish per well. 3 mL of a 10-fold diluted skin repair dressing (RHE gel) was added, and the plates were incubated in a 28 ℃ water bath in the dark for 2 h. They were then irradiated three times with UVB, each time at a dose of 2.07 ± 0.18 J / cm². 2 The intervals were 30 minutes. Finally, the zebrafish were incubated in a 28 ℃ water bath in the dark for 22 h. Observation and photography were performed using a stereomicroscope, and quantitative analysis of the zebrafish tail fin area was conducted using ImageJ (experimental model see...). Figure 3 a) Calculate the caudal fin wrinkling rate (%) using the following formula:

[0102]

[0103] Zebrafish skin structure is similar to human skin structure, and both are easily sunburned under excessive ultraviolet radiation. The post-sunburn repair efficacy of skin repair dressings can be evaluated by analyzing the degree of wrinkling in the zebrafish tail fin. Experimental results are shown below. Figure 3b, 3c. The figures show that the degree of caudal fin wrinkling in zebrafish varied under different test substances. Specifically, the caudal fin wrinkling rate of zebrafish treated with the skin repair dressing diluted 10 times according to this invention was 94.92%, which was not significantly different from the Blank group, comparable to the PC (0.1 mg / mL tea polyphenol solution) group, and significantly higher than the RHC gel (commercially available recombinant collagen gel diluted 10 times) group (p < 0.05). These results indicate that the skin repair dressing RHE gel based on recombinant humanized elastin provided by this invention has a significant repairing effect on sunburn.

[0104] 4. Evaluation of the antioxidant efficacy of skin repair dressings

[0105] Normally developed zebrafish juveniles at 3 dpf were randomly assigned to six-well cell culture plates, 10 fish per well. They were then treated with 3 mL of a 10-fold diluted skin repair dressing for 1 h. Oxidative stress was then induced in the zebrafish using 10 μM CuSO4 for 20 min. After washing the culture medium twice, the zebrafish were incubated with 20 μg / mL DCFH-DA solution for 40 min. After washing the culture medium three times, images were taken under a stereofluorescence microscope, and the average fluorescence intensity was quantified using ImageJ (experimental model see...). Figure 4 a).

[0106] CuSO4-induced oxidative stress in zebrafish is widely used to study whether test substances have antioxidant effects. Experimental results are shown below. Figure 4 b, 4c. The figure shows that the Model group had the highest fluorescence intensity, proving the successful establishment of the model. 0.1 mg / mL dipotassium glycyrrhizate solution served as the positive control group (PC). The positive control group effectively inhibited ROS formation. The fluorescence intensity of zebrafish treated with the skin repair dressing diluted 10 times was not significantly different from the PC group, but was significantly better than the RHC gel (recombinant collagen gel diluted 10 times) group. These results indicate that the skin repair dressing RHE gel based on recombinant humanized elastin provided by this invention exhibits good antioxidant effects.

[0107] 5. Evaluation of the anti-aging efficacy of skin repair dressings

[0108] 5.1 Experimental Procedure

[0109] Ninety female Kunming mice were randomly divided into five groups (n=18 per group) after a 7-day acclimatization period: Blank group, Model group, commercially available Vitamin E emulsion group (VE), Recombinant Human Skin Repair Dressing group (RHE gel), and commercially available Recombinant Human Collagen Gel group (RHC gel). The back hair was then shaved short with electric clippers, followed by application of depilatory cream to remove any remaining hair, exposing a 4×4 cm area of ​​skin. A mouse skin aging model was established by subcutaneous injection of 200 μL of D-galactose solution (500 mg / kg / day) for 8 consecutive weeks. The Blank group received daily injections of 200 μL of 0.9 wt% saline. Two weeks after model establishment, the treatment group mice were treated daily with 0.1 g of the corresponding sample. Mouse weight was recorded weekly to monitor their health. Throughout the experiment, mice had free access to food and water (see animal experimental models for details). Figure 5 a).

[0110] To observe the differences in skin aging before and after treatment in mice under physiological skin conditions, DermaLab was used at 2, 4, and 8 weeks of the experiment. ® The Combo instrument was used to perform dermoscopy, high-frequency ultrasound, dermal density, transepidermal water loss (TEWL), and stratum corneum moisture content measurements on the hair-removed areas of mice. Skin elasticity was measured according to the method of Tsukahara et al., by lifting the mouse skin from the midline as far as possible with the thumb and forefinger for 1 second, and then releasing it, and immediately recording the time required for the skin to return to its original state.

[0111] To assess the differences in the degree of skin aging relief in mice after treatment, histological staining analysis was performed on skin samples from different groups of mice at week 8. H&E staining was performed on skin sections to observe epidermal morphology and dermal density; Masson staining was performed on skin sections to observe collagen fiber content; and Verhöeff staining was performed on skin sections to observe elastic fiber content.

[0112] H&E staining: After dewaxing and dehydration of paraffin sections, HE staining is performed. After immersion in water, hematoxylin staining is performed for 5-8 min, followed by rinsing with running water for 1 min; differentiation in differentiation solution for 1-3 s, followed by rinsing for 1 min; blueing in blue solution for 30-60 s; alcoholization in 95% ethanol for 1 min; staining with alcohol-soluble eosin for 5-8 s; immersion in 95% ethanol I and II for 2-3 s each; dehydration in 100% ethanol I and II for 1 min each; clearing with xylene for 5-15 min; mounting with neutral resin, air drying at room temperature, and photographing.

[0113] Masson staining: After dewaxing and dehydration of paraffin sections, Masson trichrome staining is performed. Weigert iron hematoxylin staining is applied for 5-10 min, followed by rinsing; differentiation solution is applied for 5-15 s, followed by rinsing with water; blueing solution is applied for 1-5 min, followed by rinsing with water; Ponceau S and fuchsin staining is applied for 5-10 min; weak acid washing is performed for 1 min; phosphomolybdic acid solution washing is performed for 1-2 min; weak acid washing is performed for 1 min; aniline blue staining is performed for 2 min; rapid dehydration is performed with 95% ethanol; anhydrous ethanol I, II, and III are applied for 5-10 s each; xylene I, II, and III are applied for 1-2 min each; and neutral resin is used for mounting.

[0114] Verhöeff staining: After dewaxing and dehydration of paraffin sections, Verhöeff staining was performed. The prepared Verhöeff staining solution was used for 8-10 min, followed by rinsing; differentiation solution was used for 1 min, followed by rinsing with water; rapid deiodination with 95% ethanol; staining with alcohol-soluble eosin for 1 min; rapid dehydration with anhydrous ethanol; dewaxing with xylene until clear; and mounting with neutral resin.

[0115] To further verify the antioxidant efficacy of the recombinant elastin skin repair dressing in vivo, the differences in antioxidant activity in mice after treatment were observed. Following the instructions provided by Solarbio, malondialdehyde (MDA) content was measured using the micro-method, and superoxide dismutase (SOD) activity, catalase (CAT) activity, and reduced glutathione (GSH) content were measured spectrophotometrically. In short, skin tissue was first weighed, and the extract was added at a ratio of 0.1 g / mL. The tissue was homogenized using a tissue homogenizer. After centrifugation at 8000 g for 10 min at 4 ℃, the supernatant was collected. The MDA content, SOD activity, CAT activity, and GSH content were measured at the corresponding wavelengths using a microplate reader. Each sample was analyzed three times.

[0116] To detect the differential expression of extracellular matrix remodeling-related genes in mice after treatment, the expression levels of seven genes (LOX, Fibrillin-1, MMP-2, MMP-12, Elastin, Col I, and Col III) were analyzed using RT-qPCR. Total RNA was extracted from tissues using an RNA isolation kit, and the concentration and purity of the RNA samples were determined using a NanoDrop spectrophotometer. The extracted RNA was reverse transcribed to obtain cDNA templates for RT-qPCR amplification, and gene expression levels were then detected using TB GreenPremix Ex Taq II. The primer sequences used for RT-qPCR are shown in Table 2. The GAPDH gene was used as an internal control gene for normalization, and the expression levels were analyzed by 2... （-ΔΔCT） The relative expression level of genes was calculated using a method. Each sample was analyzed three times.

[0117] Table 2: Primer sequences used for RT-qPCR

[0118]

[0119] 5.2 Experimental Results

[0120] (1) Mouse health and epigenetic indicators

[0121] Aging leads to a decline in physiological function, ultimately resulting in weight loss. The weight of the mice during the experiment was observed... Figure 6 The figure shows that at the beginning of the experiment, there was no significant difference in the average body weight of the mice in each group; by week 8, the average body weight of the Model group mice was lower than that of the Blank group, while the body weight of the mice in the recombinant elastin skin repair dressing group was not significantly different from that of the Blank group. This result indicates that the recombinant elastin skin repair dressing provided by this invention does not affect the health of mice.

[0122] DermaLab ® The Combo dermoscopy probe was used to assess the macroscopic condition of the skin on the back of mice at various time points. The skin condition of the mice during the experiment is shown in [reference needed]. Figure 5 b. The figure shows that in week 2, all groups except the Blank group exhibited signs of aging such as dry skin, sagging, and fine wrinkles, with no significant differences. In week 4, the Model group mice showed increased skin aging, with rough skin, loss of elasticity, and increased wrinkles. After two weeks of topical treatment, the skin condition of all treatment groups improved. Compared with the VE group and the RHC gel group, the skin repair dressing group showed restored skin elasticity and reduced wrinkles, demonstrating the best repair effect. In week 8, the skin repair dressing group had smooth, elastic, and firm skin, with no significant difference from the Blank group. A small number of wrinkles and slight dryness were still observed on the backs of mice in the VE group and the RHC gel group.

[0123] DermaLab ® - The Combo high-frequency ultrasound probe, moisture probe, and TEWL probe were used to detect dermal density, stratum corneum hydration, and skin barrier function, respectively. The ultrasound results are shown in [link to results]. Figure 5 c, Dermal density of mice (see...) Figure 5 d. The water content of the stratum corneum of mouse skin is shown in [reference needed]. Figure 5 e. TEWL test results are shown in [link / reference]. Figure 5f. Aging leads to a decrease in dermal density and stratum corneum water content, and an increase in TEWL. The figure shows that at week 2, there were no significant differences in dermal density, stratum corneum water content, and TEWL among the treatment groups, but there were significant differences compared to the Blank group (P < 0.001). At week 4, compared to the Model group, each treatment group showed a significant increase in dermal density and stratum corneum water content, and a significant decrease in TEWL (P < 0.001). Compared to the VE group and RHC gel group, the skin repair dressing group showed the greatest increase in dermal density and stratum corneum water content, and the greatest decrease in TEWL, corresponding to the macroscopic skin condition. At week 8, there were no significant differences in dermal density, stratum corneum water content, and TEWL between the skin repair dressing group and the Blank group, and the indicators in the VE group and RHC gel group did not return to normal levels. Therefore, the skin repair dressing based on recombinant humanized elastin provided by this invention shows the best performance in restoring dermal density, stratum corneum water content, and skin barrier function in aging mice. The skin retraction time of mice is shown in the figure. Figure 5 g. Skin elasticity depends on the function of elastic fibers; decreased elasticity leads to skin laxity and prolonged skin retraction time. At week 4, there was no significant difference in skin retraction time between the skin repair dressing group and the Blank group, demonstrating that the skin repair dressing of this invention significantly increases skin elasticity. In summary, the skin repair dressing RHE gel based on recombinant humanized elastin provided by this invention is superior to commercially available VE lotion and recombinant collagen gel in improving endogenous aging wrinkles, dryness, and laxity.

[0124] (2) Tissue staining

[0125] Aging causes the epidermis to thin and disrupts the structural integrity of the dermis. Results were obtained using H&E, Masson, and Verhöeff staining (see staining results). Figure 7 The Blank group mice exhibited intact skin tissue with a tight, wavy connection between the dermis and epidermis, and uniform dermal fiber distribution, particularly with normal levels of collagen and elastin fibers. The Model group mice showed thinner epidermis and decreased dermal density, with reduced collagen fibers and fragmentation in some areas, as well as reduced and shortened elastin fibers. The skin repair dressing group showed no significant difference in epidermal thickness compared to the Blank group, with tightly packed dermal fibers and no significant difference in collagen and elastin content. Compared to the Model group, the VE and RHC gel groups showed significantly increased epidermal thickness, collagen content, and elastin fiber count (P < 0.001), but still did not return to normal levels. In conclusion, the RHE gel skin repair dressing based on recombinant humanized elastin provided by this invention demonstrates significantly superior efficacy compared to commercially available VE emulsion and recombinant collagen gel in repairing endogenous aging tissue damage, promoting collagen expression, and increasing elastin fiber content.

[0126] (3) Oxidative stress

[0127] Skin aging leads to the accumulation of reactive oxygen species (ROS), triggering oxidative stress. MDA (malondialdehyde) is a major product of lipid peroxidation. Endogenous enzymes such as SOD (superoxide dismutase) and CAT (catalase) and antioxidant small molecules such as GSH (glutathione) can inhibit the accumulation of ROS caused by aging. The results of MDA, SOD, CAT, and GSH content detection are shown below. Figure 8 The figure shows that the Model group had the highest MDA content and the lowest levels of SOD, CAT, and GSH, proving that the aging model was successfully established. The MDA, SOD, CAT, and GSH contents in the skin repair dressing group were not significantly different from those in the Blank group. Preliminary investigation of the mechanism reveals that the skin repair dressing of this invention acts on the surface of aging skin, promoting the secretion of SOD, CAT, and GSH. SOD reduces the large amount of ·O2 produced during aging. - The substance is rapidly converted to H2O2, which is then rapidly decomposed under the catalysis of CAT or the action of GSH, thus balancing the high oxidative stress. Furthermore, the reduction of reactive oxygen species also decreases the degree of cell membrane peroxidation and lowers MDA levels. Compared with the Model group, the VE group and RHC gel group significantly reduced MDA content and increased SOD, CAT, and GSH content (P < 0.001), but still did not return to normal levels. These results indicate that the skin repair dressing RHE gel based on recombinant humanized elastin provided by this invention has a better ability to improve endogenous aging oxidative stress than commercially available VE emulsion and recombinant collagen gel.

[0128] (4) RT-qPCR

[0129] Aging leads to ECM degradation, particularly a decrease in collagen and elastin content. LOX and fibrillin-1 promote the cross-linking of elastin to form mature elastic fibers. Upregulation of MMP-2 and MMP-12 can degrade elastic fibers. RT-qPCR results of related gene expression are shown below. Figure 9 The figure shows that the LOX and Fibrillin-1 genes in the skin repair dressing group were significantly upregulated compared to the Model, VE, and RHC gel groups, demonstrating their role in promoting elastin fiber synthesis and thus contributing to skin elasticity recovery. The inactive expression of MMP-2 and MMP-12 genes indicates that they inhibit age-related degradation of elastic fibers. Furthermore, the upregulation of Elastin, Col I, and Col III genes in the skin repair dressing group corresponds to the increased volume fraction of elastic fibers and collagen in tissue staining. These results indicate that the skin repair dressing RHE gel based on recombinant humanized elastin provided by this invention helps promote ECM remodeling.

[0130] In summary, this invention provides a recombinant humanized elastin with the effect of improving endogenous skin aging and a skin repair dressing RHE gel with the recombinant humanized elastin as the core active ingredient, which has excellent stability and biocompatibility. The skin repair dressing exhibited no toxicity to fibroblasts in in vitro experiments, significantly promoting their proliferation, extension, and migration. In zebrafish models of UV sunburn and chemical oxidative stress, RHE gel demonstrated excellent post-sunburn repair and antioxidant activity. In a mouse model of D-galactose-induced endogenous skin aging, RHE gel significantly reduced wrinkle formation, increased dermal thickness and skin hydration, decreased transepidermal water loss, and shortened skin rebound time. Histological analysis further confirmed its ability to improve epidermal thinning and dermal collapse, significantly increase collagen and elastin content, and promote extracellular matrix network reconstruction. At the molecular level, RHE gel upregulated LOX and Fibrillin-1 expression, inhibited MMP-2 and MMP-12 activity, and significantly increased Col I / III expression, synergistically achieving skin tissue repair. Simultaneously, it optimized the antioxidant microenvironment by reducing MDA levels and increasing SOD, CAT, and GSH activity. Compared to commercially available vitamin E emulsions and recombinant collagen gels, RHE gel... Gel has significant advantages in restoring skin elasticity, reducing wrinkles, and improving tissue structure integrity, demonstrating a clear mechanism of action and broad industrialization prospects.

[0131] The above detailed embodiments describe the implementation of the present invention; however, the present invention is not limited to the specific details described in the above embodiments. Within the scope of the claims and technical concept of the present invention, various simple modifications and changes can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

Claims

1. A recombinant humanized elastin with the effect of improving endogenous skin aging, characterized in that, The amino acid sequence of the recombinant humanized elastin is shown in SEQ ID NO.

1.

2. A recombinant humanized elastin precursor elastin with the effect of improving endogenous skin aging, characterized in that, The amino acid sequence of the precursor elastin is shown in SEQ ID NO.

2.

3. A gene encoding the precursor elastin of claim 2, characterized in that, The gene sequence is shown in SEQ ID NO.

3.

4. The method for preparing recombinant humanized elastin according to claim 1, characterized in that, Includes the following steps: (1) The gene sequence SEQ ID NO. 3 encoding the precursor elastin of the recombinant humanized elastin was synthesized; (2) The gene sequence is ligated to a vector, transformed into genetically engineered bacteria, and recombinant genetically engineered bacteria are constructed; (3) The recombinant genetically engineered bacteria were expressed, the bacterial cells were collected, the supernatant was obtained by cleavage, and the precursor elastin was obtained by purification. (4) Add the protease to the precursor elastin obtained by treatment, and purify it to obtain recombinant humanized elastin.

5. The method for preparing recombinant humanized elastin according to claim 4, characterized in that, The protease is thrombin.

6. A recombinant vector or recombinant genetically engineered bacterium carrying the gene for the precursor elastin of claim 3.

7. The use of the recombinant humanized elastin of claim 1 in the preparation of products that improve endogenous skin aging.

8. The application of the recombinant humanized elastin according to claim 7 in the preparation of products that improve endogenous skin aging, characterized in that, The products mentioned include cosmetics or medical aesthetic products.

9. The application of the recombinant humanized elastin according to claim 8 in the preparation of products that improve endogenous skin aging, characterized in that, The cosmetics include: serums, lotions, essences, creams, masks, sprays, and foundations; the medical aesthetic products include: serums, gels, dressings, sponges, hyaluronic acid injection preparations, implants, and creams.

10. A skin repair dressing with the effect of improving endogenous aging, characterized in that, Includes the following components by mass percentage: The recombinant humanized elastin of claim 1 comprises 0.1%-1%, trehalose 5%-8%, glycerol 3%-5%, hyaluronic acid 0.2%-1%, arginine 0.05%-0.5%, carbomer 0.1%-0.5%, 1,2-hexanediol 0.1%-0.5%, p-hydroxyacetophenone 0.1%-0.5%, with the balance being water.

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