Recombinant humanized elastin with effect of improving endogenous skin aging and application of recombinant humanized elastin

Abstract

The invention provides recombinant humanized elastin with an effect of improving endogenous skin aging and application of the recombinant humanized elastin, and belongs to the technical field of crossing of dermatology and biological materials. The recombinant humanized elastin is based on a human elastin core function sequence, recombinant optimization and efficient expression are carried out by adopting a synthetic biological strategy, and excellent stability and biological activity are shown. The recombinant humanized elastin can be used for constructing a skin repair dressing, and synergistically improves endogenous aging characteristics such as skin elasticity reduction, collagen loss and high oxidative stress by remodeling an extracellular matrix network structure. 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] The present invention relates to the technical field of dermatology and biomaterials, and specifically to an active ingredient based on recombinant elastin and its application in improving endogenous skin aging, belonging to the technical development scope of protein engineering and skin anti-aging products. Background Art

[0002] The skin is the largest organ in the human body, primarily composed of the epidermis, dermis, and subcutaneous tissue. It protects the body from external aggressors. Skin aging is a complex biological process driven by genetic, environmental, and physiological factors. It is generally divided into exogenous aging (such as ultraviolet radiation) and intrinsic aging (natural decline with age). Intrinsic skin aging is manifested by decreased collagen and elastin content in the dermis, fiber disorganization, increased 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, comprising approximately 2% to 5% of the dry weight of the dermis. It plays a key role in maintaining skin elasticity, enabling it to withstand repeated mechanical strain and maintain structural stability. In the dermis, collagen primarily provides structural support, while elastin confers the skin's excellent elasticity and reversible deformability. Elastin synthesis peaks in late embryonic development and virtually ceases in adulthood. Once damaged, it is difficult to repair naturally. During aging, due to a combination of endogenous and exogenous factors, the expression of matrix metalloproteinases (MMPs) in the skin increases significantly, leading to increased elastin degradation, structural fragmentation, and the gradual disintegration of the elastic network. Simultaneously, the expression of key proteins such as lysyl oxidase (LOX) and fibrillin-1, essential for elastic fiber formation, decreases, further inhibiting the efficient construction of new elastic fibers. These factors work together to cause a gradual loss of skin elasticity, resulting in the development of typical aging symptoms such as increased wrinkling and sagging.

[0004] Existing anti-aging products mostly use antioxidants, hyaluronic acid, recombinant collagen, or peptides as their primary ingredients, focusing on improving skin hydration, firmness, and radiance. However, these ingredients primarily target the collagen system, with limited ability to rebuild the elastin network and unable to fundamentally address the problem of declining skin elasticity. While a small number of elastin extracts have been used in skincare products, most are derived from animal tissue and present issues such as low biosafety, high immunogenicity, and poor batch stability, making them difficult to meet the high-quality raw material requirements of medical aesthetics and functional skincare products.

[0005] With the advancement of biosynthesis and protein engineering technologies, the production of human elastin using recombinant genetic 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 native functional structure. However, specific functional validation and mechanistic studies targeting intrinsic skin aging are currently lacking, particularly systematic evidence for its ability to promote elastic fiber regeneration, alleviate oxidative stress, and reconstruct ECM networks. Summary of the Invention

[0006] To address the shortcomings of the above-mentioned prior art, the present invention provides a recombinant humanized elastin protein with the efficacy of improving intrinsic aging, and its application. This recombinant humanized elastin protein, based on the core functional region sequence of human elastin, is designed and expressed through synthetic biology. It exhibits excellent stability, biocompatibility, and bioactivity. It can achieve anti-aging effects by providing antioxidant benefits and promoting extracellular matrix remodeling, effectively delaying intrinsic skin aging. It is widely applicable in the fields of cosmetics and dermatology.

[0007] To achieve the above object, the specific technical solutions of the present invention are as follows:

[0008] In a first aspect, the present invention provides a recombinant humanized elastin protein having the efficacy of improving endogenous skin aging, wherein the amino acid sequence of the recombinant humanized elastin protein is:

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

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

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

[0012] Furthermore, the amino acid sequence of the precursor elastin is:

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

[0014] (b) An amino acid sequence obtained by substitution, deletion and / or addition of one or more amino acids based on the sequence shown in SEQ ID NO. 2, and having substantially 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:

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

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

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

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

[0020] (2) Connecting the gene sequence to a vector, transforming genetically engineered bacteria, and constructing recombinant genetically engineered bacteria;

[0021] (3) Expressing the constructed recombinant genetically engineered bacteria, collecting the bacteria, crushing them to obtain the supernatant, and purifying the precursor elastin;

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

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

[0024] In a third aspect, the present invention provides a recombinant vector or a recombinant genetically engineered bacterium carrying the precursor protein gene of the recombinant humanized elastin.

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

[0026] In a fourth aspect, the present invention provides the use of the recombinant humanized elastin in the preparation of a product having the efficacy of improving endogenous aging.

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

[0028] Furthermore, the cosmetics include but are not limited to liquid, lotion, essence, cream, facial mask, spray, sunscreen, eye cream, foundation, and body lotion.

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

[0030] In a fifth aspect, the present invention provides a skin repair dressing with the effect of improving endogenous aging, comprising the following components in mass percentage: 0.1%-1% of the recombinant humanized elastin, 5%-8% of trehalose, 3%-5% of glycerol, 0.2%-1% of hyaluronic acid, 0.05%-0.5% of arginine, 0.1%-0.5% of carbomer, 0.1%-0.5% of 1,2-hexanediol, 0.1%-0.5% of parahydroxyacetophenone, and the balance being water.

[0031] Furthermore, the skin repair dressing comprises the following components in percentage by mass: 0.1% of the recombinant elastin, 5% of trehalose, 3% of glycerol, 1% of hyaluronic acid, 0.5% of arginine, 0.5% of carbomer, 0.5% of 1,2-hexanediol, 0.5% of parahydroxyacetophenone, and the balance is water.

[0032] Compared with the prior art, the present invention is beneficial in that:

[0033] (1) The humanized elastin obtained by recombinant expression in the present invention has a clear sequence configuration, avoiding the immune risks and batch inconsistency problems 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 biological activity, 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 exhibited excellent post-sun repair and reactive oxygen species scavenging capabilities in a zebrafish model and possessed 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) The present invention provides an intervention method with a clear mechanism, clear ingredients, safety and effectiveness, which fills the technical gap of existing anti-aging products in the elastin reconstruction dimension and has good medical application prospects and industrial promotion value. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1This is an SDS-PAGE identification diagram of the recombinant humanized elastin RHE of the present invention;

[0040] Figure 2 The biocompatibility and bioactivity evaluation results of the skin repair dressing of the present invention are as follows; wherein, Figure 2 a is the result of cytotoxicity experiment; Figure 2 b shows the results of cell proliferation experiment; Figure 2 c is the result of viable cell counting; Figure 2 d is the result of live-dead cell staining experiment Figure 2 e is the result of immunofluorescence staining experiment; Figure 2 f is the result of cell migration experiment; Figure 2 g is the quantitative result of cell migration

[0041] Figure 3 The evaluation results of the after-sun repair efficacy of the skin repair dressing of the present invention are as follows; wherein, Figure 3 a is a schematic diagram of the after-sun repair experimental model; Figure 3 b is the qualitative evaluation result of after-sun repair efficacy; Figure 3 c is the quantitative evaluation result of after-sun repair efficacy;

[0042] Figure 4 is the evaluation result of the antioxidant effect 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 is the qualitative evaluation result of antioxidant efficacy; Figure 4 c is the quantitative evaluation result of antioxidant efficacy;

[0043] Figure 5 The results of the evaluation of the anti-aging efficacy of the skin repair dressing of the present invention are as follows: Figure 5 a is a schematic diagram of the D-galactose-induced endogenous aging model in mice; Figure 5 bDermatoscopy results; Figure 5 c is the ultrasonic detection result; Figure 5 d is the dermal density of mice; Figure 5 e is the water content of the mouse skin stratum corneum; Figure 5 f is the TEWL test result; Figure 5 g is the skin retraction time of mice;

[0044] Figure 6 The weight change of mice in the evaluation of the anti-aging efficacy of the skin repair dressing of the present invention;

[0045] Figure 7 The results of the evaluation of the anti-aging efficacy of the skin repair dressing of the present invention are as follows: Figure 7 a shows the results of H&E, Masson and Verhöeff staining; Figure 7b is the quantitative epidermal thickness of H&E images using Image J software; Figure 7 c is the quantitative collagen fiber content of Masson image using Image J software; Figure 7 d is the quantitative Verhöeff image elastic fiber content using Image J software;

[0046] Figure 8 This is the evaluation result of the skin repair dressing of the present invention in balancing the oxidative stress environment in the endogenous aging body; wherein, Figure 8 a is the malondialdehyde content; Figure 8 b is superoxide dismutase activity; Figure 8 c is catalase activity; Figure 8 d is the glutathione content; Figure 8 e is a schematic diagram of recombinant elastin skin repair dressing balancing endogenous oxidative stress in the body during aging;

[0047] Figure 9 The results of RT-qPCR detection of related gene expression; Figure 9 a is the expression result of LOX; Figure 9 b is the expression result of Fibrillin-1; Figure 9 c is the expression result of MMP-2; Figure 9 d shows the expression results of MMP-12; Figure 9 e is the expression result of Elastin; Figure 9 f is the expression result of Col I; Figure 9 g is the expression result of Col III; Figure 9 h is a schematic diagram of recombinant elastin skin repair dressing promoting extracellular matrix remodeling. DETAILED DESCRIPTION

[0048] The technical solutions of the present invention are described clearly and completely below. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. All other embodiments obtained by persons of ordinary skill in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0049] The present invention provides a recombinant humanized elastin protein having the efficacy of improving endogenous skin aging. The amino acid sequence of the recombinant humanized elastin protein is:

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

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

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

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

[0054] (2) Connecting the gene sequence to a vector, transforming genetically engineered bacteria, and constructing recombinant genetically engineered bacteria;

[0055] (3) Expressing the constructed recombinant genetically engineered bacteria, collecting the bacteria, crushing them to obtain the supernatant, and purifying the precursor elastin;

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

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

[0058] The present invention also provides a recombinant vector or a recombinant genetic engineering bacterium carrying the precursor protein gene of the recombinant humanized elastin.

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

[0060] The present invention also provides the use of the recombinant humanized elastin in preparing a product with the efficacy of improving endogenous aging.

[0061] In some examples, the products described for improving endogenous aging include, but are not limited to, cosmetics or medical cosmetic products. Cosmetics include, but are not limited to, concentrates, lotions, essences, creams, facial masks, sprays, sunscreens, eye creams, foundations, and body lotions. Medical cosmetic products include, but are not limited to, concentrates, gels, cold compresses, dressings, sponges, hydrating injection 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 in percentage by mass: 0.1%-1% of the recombinant humanized elastin, 5%-8% of trehalose, 3%-5% of glycerol, 0.2%-1% of hyaluronic acid, 0.05%-0.5% of arginine, 0.1%-0.5% of carbomer, 0.1%-0.5% of 1,2-hexanediol, 0.1%-0.5% of p-hydroxyacetophenone, and the balance being water.

[0063] In the following specific examples, unless otherwise specified, the methods used are conventional methods, and the reagents used are conventional reagents that can be obtained from commercial channels.

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

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

[0066] (1) Precursor elastin sequence design

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

[0068] (2) Construction of expression strain

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

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

[0071] Take 200 μL of glycerol culture and inoculate it into 200 mL of LB liquid medium containing antibiotics. Incubate it in a 37°C constant temperature shaker overnight. Then transfer 2% of the inoculum into 1 L of LB liquid medium containing antibiotics to continue the amplification culture. 600 When the value reaches 0.8-1.0, lower the shaker temperature to 25°C, add IPTG to a final concentration of 1 mM to induce expression, and continue culturing overnight. After harvesting, centrifuge at 4°C and 3200 rpm for 30 minutes, discard the supernatant, and collect the pellet. Resuspend the cells in lysis buffer (pH 7.4) containing 20 mM sodium phosphate, 0.5 M sodium chloride, and 20 mM imidazole, disrupt them by sonication, and centrifuge again. Collect the supernatant. Purify the supernatant using a nickel affinity chromatography column to obtain precursor elastin. Add thrombin to a final concentration of 2-4 U / mL to the purified protein solution. Enzymatic cleavage under appropriate conditions removes the N-terminal tag. Further purification is then performed to remove the enzyme and impurities, and finally, lyophilize to produce highly pure recombinant humanized elastin (RHE).

[0072] The protein was analyzed by SDS-PAGE electrophoresis. Figure 1 The figure shows that RHE appears as a single band, indicating that the prepared elastin has high purity and meets the quality requirements of 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 in percentage by mass: 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 is water.

[0075] The preparation process of the skin repair dressing is as follows: dispersing carbomer 940 in deionized water, stirring thoroughly to disperse evenly, and heating to 85°C to form a gel matrix; adding trehalose, glycerol, and hyaluronic acid in sequence, stirring until completely dissolved, homogenizing for 3 minutes, and keeping warm for 10-15 minutes; adding arginine, 1,2-hexanediol, and p-hydroxyacetophenone in sequence, stirring and dispersing evenly; cooling to 30°C, adding the recombinant humanized elastin RHE prepared in Example 1, stirring until completely dissolved and transparent, discharging and standing, and obtaining the skin repair dressing RHE gel.

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

[0077] 1. Stability evaluation of skin repair dressing

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

[0079] Table 1: Stability evaluation results of skin repair dressing

[0080]

[0081] As shown in Table 1, the skin repair dressing prepared by the present invention has good stability. After centrifugation, heat resistance and cold resistance tests, the pH value of the skin repair dressing remains within the physiological range of the skin, which is about 6.4. -1The skin repair dressing's viscosity remained essentially unchanged at a shear rate of 1000 nm. Furthermore, after three months of centrifugation, heating, and cold-resistance testing, the skin repair dressing remained uniform and transparent, exhibiting no delamination and no noticeable odor. In summary, the recombinant elastin skin repair dressing provided by the present invention exhibits excellent stability and is suitable for long-term storage.

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

[0083] 2.1 Cytotoxicity assay

[0084] Fibroblasts (L929) grown to 70%-80% of the culture dish area were obtained, digested with 0.25% trypsin, and prepared with complete culture medium (10% FBS, 1% Penicillin–Streptomycin, DMEM medium) to a cell density of 1×10 5 Cells were suspended at a concentration of 100 μL / mL. 100 μL of the cell suspension was transferred to a 96-well culture plate and incubated in a 37°C, 5% CO2, saturated humidity incubator for 24 h. The complete culture medium was aspirated, and skin repair dressings diluted with high-glucose DMEM medium at different times (x 1, x 10, x 20, x 100, with four replicates for each dilution) were added to the experimental group. The control group was cultured with cells in DMEM medium (Blank), and the blank group was cultured with DMEM medium without cells. Culture was continued in a 37°C, 5% CO2, saturated humidity incubator for 24 h. 10 μL of CCK-8 reagent was added to each group, and the cells were incubated in a cell culture incubator for 1.5 h. The absorbance (OD) of each well was measured at a wavelength of 450 nm using an enzyme-linked immunosorbent assay (ELISA). 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. The figure shows that the skin repair dressing RHE gel of the present invention at different concentrations has no cytotoxicity and good safety.

[0087] 2.2 Cell proliferation assay

[0088] 1 mL of skin repair dressing and commercially available recombinant collagen gel were soaked in 9 mL of DMEM high-glucose medium and named RHE gel and RHC gel respectively for subsequent cell experiments. L929 cells grown to 70%-80% of the culture dish area were obtained, digested with 0.25% trypsin, and cultured with complete medium to a cell density of 1x10 5Cell suspension of 100 μL / mL was inoculated into a 96-well culture plate and cultured in an incubator at 37 °C and 5% CO2 saturated humidity. After culturing the cells for 24 h, the complete culture medium was aspirated. RHE gel and RHC gel were added to the experimental groups (4 replicates were set up), the blank control group was cultured with DMEM medium, and the blank group was cultured with DMEM medium without cells. The cells were cultured in an incubator at 37 °C and 5% CO2 saturated humidity for 1 d, 3 d, and 5 d, respectively. At the designated time points, 10 μL of CCK-8 reagent was added to each group, incubated in a cell culture incubator for 1.5 h, and the absorbance (OD value) of each well was measured at a wavelength of 450 nm using an enzyme-linked immunosorbent assay. The relative proliferation rate (RGR) of the cells was calculated according to the mean absorbance of each group according to the following formula:

[0089]

[0090] The experimental results are shown in Figure 2 b. The figure shows that compared with the blank control group (100%), the skin repair dressing of the present invention achieved relative cell proliferation rates exceeding 100% on days 1, 3, and 5. This rate increased over time and was significantly higher than that of commercially available recombinant collagen gels. This demonstrates that the recombinant humanized elastin-based skin repair dressing provided by the present invention can promote fibroblast proliferation.

[0091] 2.3 Live and dead cell staining experiment

[0092] Take L929 cells grown to 70%-80% of the culture dish area, digest with 0.25% trypsin, and use complete culture medium to make the cell density of 1×10 6 Cells were suspended at a concentration of 100 cells / mL. 1 mL of the cell suspension was inoculated into a laser confocal microplate culture dish and cultured in a 37°C, 5% CO2, saturated humidity incubator. After 24 hours of culture, the complete culture medium was aspirated. RHE gel and RHC gel were added to the experimental groups (three replicates were set up); the blank control group received an equal volume of high-glucose DMEM medium. Cultures were continued in a 37°C, 5% CO2, saturated humidity incubator for 1, 3, and 5 days, respectively. At the designated time points, live / dead cell staining was performed according to the protocol provided with the Solebow Live / Dead Cell Staining Kit, with live cells stained green and dead cells stained red. Images were taken under a 10x laser confocal microscopy inverted microscope, and the number of live cells was counted using Image J.

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

[0094] 2.4 Immunofluorescence experiments

[0095] Take L929 cells grown to 70%-80% of the culture dish area, digest with 0.25% trypsin, and use complete culture medium to make the cell density of 1×10 6 Cells were suspended at a concentration of 1 cell / mL. 1 mL of the cell suspension was inoculated into a laser confocal microplate culture dish and cultured in a 37°C, 5% CO2, saturated humidity incubator. After 24 hours of cell culture, the complete culture medium was aspirated. RHE gel and RHC gel containing FBS were added to the experimental groups (three replicates were set up); the blank control group received an equal amount of high-glucose DMEM medium containing FBS. Culture was continued in a 37°C, 5% CO2, saturated humidity incubator for 24 hours. Actin fibers (red) were labeled with rhodamine-phalloidin, and nuclei (blue) were labeled with DAPI. Staining procedures were performed according to the instructions in the manufacturer's instructions. Images were taken under a 40x laser confocal microscopy inverted microscope.

[0096] The experimental results are shown in Figure 2 The figure shows that L929 cells in the blank control group exhibited typical physiological morphology. In the experimental group, more cells displayed extended actin fibers. Furthermore, the skin repair dressing of the present invention exhibited more pronounced spindle-shaped fibroblast elongation than commercially available recombinant collagen gels. This demonstrates that the skin repair dressing based on recombinant humanized elastin provided by the present invention has a positive effect on cell extension.

[0097] 2.5 Cell migration assay

[0098] Take L929 cells grown to 70%-80% of the culture dish area, digest with 0.25% trypsin, and use complete culture medium to make the cell density of 1×10 6Cell suspension was prepared at a concentration of 100 μg / mL. Three horizontal lines were drawn on the bottom of the well plate using a marker pen and a ruler. 1 mL of the cell suspension was inoculated into a 6-well culture plate and cultured in a 37°C, 5% CO2, saturated humidity incubator. After 24 hours of cell culture, a 200 μL pipette tip was aligned vertically with a ruler and gently pushed downward to create a scratch. The culture medium was aspirated, and the cells were rinsed three times with PBS to remove the scratched cells. RHE gel and RHC gel were added to the experimental groups (three replicates were set up); in the control group, an equal volume of high-glucose DMEM medium was added to the cells. The cells were cultured in a 37°C, 5% CO2, saturated humidity incubator for 0 and 24 hours, respectively. Images were taken under a 10x microscope, and relative migration rates were calculated using Image J.

[0099] The experimental results are shown in Figure 2 f, 2g. The figures show that in the blank control group, cells barely migrated, with a migration rate of 14.83±2.08%. After 24 hours of incubation, the cell migration rate in the skin repair dressing of the present invention was 76.05±4.54%, significantly higher than the 39.35±2.29% observed in the commercially available recombinant collagen gel. This demonstrates that the recombinant humanized elastin-based skin repair dressing provided by the present invention can promote fibroblast migration.

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

[0101] Normally developed zebrafish larvae (2 dpf) were randomly assigned to six-well cell culture plates, with 10 larvae per well. 3 mL of a 10-fold diluted skin repair dressing (RHE gel) was then added and incubated in a 28°C waterbath in the dark for 2 h. The cells were then irradiated three times with UVB at a dose of 2.07 ± 0.18 J / cm3. 2 The cells were then incubated for 22 h in a 28°C water bath in the dark. Images were taken using a stereo microscope, and the zebrafish tail fin area was quantitatively analyzed using Image J (see experimental model for details). Figure 3 a) Calculate the tail fin shrinkage rate (%) according to the following formula:

[0102]

[0103] The structure of zebrafish skin is similar to that of human skin and is easily sunburned under excessive ultraviolet radiation. By analyzing the degree of wrinkling of the zebrafish tail fin, the post-sun repair efficacy of skin repair dressings can be evaluated. Figure 3b, 3c. The figure shows that the degree of wrinkling of the zebrafish tail fin is different under the protection of different test substances. Among them, the wrinkling rate of the zebrafish tail fin treated with the skin repair dressing of the present invention diluted 10 times was 94.92%, which was not significantly different from the Blank group, was equivalent to the PC (0.1 mg / mL tea polyphenols solution) group, and was significantly higher than the RHC gel (commercially available recombinant collagen gel diluted 10 times) group (p < 0.05). The above results show that the skin repair dressing RHE gel based on recombinant humanized elastin provided by the present invention has a significant repair effect on sunburn.

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

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

[0106] CuSO4-induced oxidative stress in zebrafish is widely used to study whether the test substance has antioxidant effect. Figure 4 b, 4c. As shown in the figure, the fluorescence intensity of the Model group is the highest, proving that the model was successfully established. 0.1 mg / mL dipotassium glycyrrhizate solution was used as the positive control group (PC). The positive control group can effectively inhibit the formation of ROS. The fluorescence intensity of zebrafish treated with the skin repair dressing diluted 10 times was not significantly different from that of the PC group, and was significantly better than that of the RHC gel (recombinant collagen gel diluted 10 times) group. The above results show that the skin repair dressing RHE gel based on recombinant humanized elastin provided by the present invention exhibits good antioxidant efficacy.

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

[0108] 5.1 Experimental Procedure

[0109] After a 7-day adaptation period, 90 female Kunming mice were randomly divided into 5 groups, with 18 mice in each group: blank group (Blank), model group (Model), commercial VE milk group (VE), skin repair dressing group (RHE gel), and commercial recombinant collagen gel group (RHC gel). Then, the back hair was shaved short using an electric shaver, and then a depilatory cream was applied to remove the 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 / d) for 8 consecutive weeks. The Blank group was injected with 200 μL of 0.9 wt% saline every day. Two weeks after modeling, the mice in the treatment group were given 0.1 g of the corresponding sample smear treatment every day. The body weight of the mice was recorded weekly to monitor their health. Throughout the experiment, the mice had free access to food and water (see the animal experimental model). Figure 5 a).

[0110] In order to observe the difference in the degree of skin aging before and after treatment in the physiological skin state of mice, DermaLab ® The -Combo instrument was used to examine the hair removal sites of mice using dermoscopy, high-frequency ultrasound, dermal density, transepidermal water loss (TEWL), and stratum corneum water content. Skin elasticity was measured according to the method of Tsukahara et al. The mouse skin was lifted as far as possible from the midline between the thumb and index finger for one second, then released. The time required for the skin to return to its original state was immediately recorded.

[0111] To assess the extent of skin aging alleviation after treatment, histological staining was performed on skin samples from the different groups at week 8. Skin sections were stained with H&E to observe epidermal morphology and dermal density, Masson's staining to observe collagen fiber content, and Verhöeff staining to observe elastic fiber content.

[0112] H&E staining: paraffin sections were dewaxed and dehydrated before HE staining. After immersion in water, the sections were stained with hematoxylin for 5-8 min and rinsed with running water for 1 min. Differentiation was performed with differentiation solution for 1-3 s and rinsed for 1 min. Blueing was performed with bluing solution for 30-60 s. The sections were immersed in 95% ethanol for 1 min. Alcoholization was performed with alcohol-soluble eosin for 5-8 s. The sections were immersed in 95% ethanol I and II for 2-3 s each. Dehydration was performed in 100% ethanol I and II for 1 min each. The sections were transparentized with xylene for 5-15 min. The sections were mounted with neutral gum, dried at room temperature, and photographed.

[0113] Masson staining: After dewaxing and dehydration, paraffin sections were stained with Masson trichrome, stained with Weigert iron hematoxylin for 5-10 min, rinsed, differentiated with differentiation solution for 5-15 s, washed with water, bluing solution for 1-5 min, washed with water, stained with Ponceau fuchsin for 5-10 min, washed with weak acid for 1 min, washed with phosphomolybdic acid solution for 1-2 min, washed with weak acid for 1 min, stained with aniline blue for 2 min, rapidly dehydrated with 95% ethanol, dehydrated with anhydrous ethanol I, II, and III for 5-10 s each, and sealed with xylene I, II, and III for 1-2 min each, and sealed with neutral gum.

[0114] Verhöeff staining: Paraffin sections were dewaxed and dehydrated before Verhöeff staining. The prepared Verhöeff stain was incubated for 8-10 min, followed by rinsing, differentiation in differentiation solution for 1 min, and water washing. The sections were rapidly deiodinated with 95% ethanol, stained with alcohol-soluble eosin for 1 min, rapidly dehydrated with anhydrous ethanol, dewaxed with xylene until transparent, and sealed with neutral gum.

[0115] To further validate the antioxidant efficacy of the recombinant elastin skin repair dressing in vivo, the differences in antioxidant activity after treatment were observed in mice. Following the instructions for the kit provided by Solebo, malondialdehyde (MDA) content was measured using a microassay, and superoxide dismutase (SOD) activity, catalase (CAT) activity, and reduced glutathione (GSH) content were measured spectrophotometrically. Briefly, skin tissue was 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 minutes at 4°C, the supernatant was collected for later use. MDA content, SOD activity, CAT activity, and GSH content were measured using a microplate reader at the corresponding wavelengths. Each sample was analyzed in triplicate.

[0116] To detect differences in the expression of genes related to extracellular matrix remodeling in mice after treatment, the expression levels of seven genes, including LOX, Fibrillin-1, MMP-2, MMP-12, Elastin, Col Ⅰ, and Col Ⅲ, were analyzed by 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 then gene expression levels were detected using TB Green Premix Ex Taq II. The primer sequences used for RT-qPCR are shown in Table 2. GAPDH gene was used as an internal reference gene for normalization, and 2 （-ΔΔCT） The relative expression levels of genes were calculated using the PCR method. Each sample was analyzed in triplicate.

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

[0118]

[0119] 5.2 Experimental Results

[0120] (1) Mouse health and epigenetic indicators

[0121] Aging can lead to a decline in physiological functions and ultimately to weight loss. During the experiment, the weight of mice Figure 6 The figure shows that at the beginning of the experiment, there was no significant difference in the average weight of mice in each group. By the eighth week, the average weight of mice in the Model group was lower than that in the Blank group, and there was no significant difference in the weight of mice in the recombinant elastin skin repair dressing group compared with the Blank group. This result indicates that the recombinant elastin skin repair dressing provided by the present invention does not affect the health of mice.

[0122] DermaLab ® -Combo dermatoscope probe was used to evaluate the macroscopic condition of the mouse back skin at different time points. The skin condition of the mice during the experiment was shown in Figure 5 b. The figure shows that at week 2, except for the Blank group, all groups showed aging symptoms such as dry skin, sagging, and fine wrinkles, and there was no significant difference. At week 4, the skin aging of the mice in the Model group deepened, the skin became rough, lost elasticity, and wrinkles increased. After 2 weeks of application, the skin condition of each treatment group improved. Compared with the VE group and the RHC gel group, the skin elasticity of the skin repair dressing group was restored, wrinkles were reduced, and the repair effect was the best. At week 8, the skin of the skin repair dressing group was smooth, elastic, and the epidermis was firm, with no significant difference from the Blank group. A small amount of wrinkles and slight dryness could still be observed on the backs of mice in the VE and RHC gel groups.

[0123] DermaLab ® -Combo high-frequency ultrasound probe, moisture probe and TEWL probe respectively detect the skin's dermal density, stratum corneum water content and skin barrier function. The ultrasound detection results are shown in Figure 5 c, Dermal density of mice Figure 5 d, Water content of mouse skin stratum corneum Figure 5 e, TEWL test results are shown in 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 between the treatment groups, but there were significant differences compared to the Blank group (P < 0.001). At week 4, compared to the Model group, dermal density and stratum corneum water content were significantly increased, and TEWL was significantly decreased in all treatment groups (P < 0.001). Compared to the VE and RHC gel groups, 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 state of the skin. 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 these indicators in the VE and RHC gel groups had not returned to normal levels. As shown above, the recombinant humanized elastin-based skin repair dressing provided by the present invention is the most effective in restoring dermal density, stratum corneum water content, and skin barrier function in aging mice. The skin retraction time of mice is shown in Figure 5 g. Skin elasticity depends on the function of elastic fibers. Decreased elasticity can lead to skin sagging 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 the present invention is significantly effective in increasing skin elasticity. In summary, the recombinant humanized elastin-based skin repair dressing RHE gel provided by the present invention is superior to commercially available VE emulsion and recombinant collagen gel in improving endogenous aging wrinkles, dryness, and sagging.

[0124] (2) Tissue staining

[0125] Aging causes the epidermis to become thinner and damages the structural integrity of the dermis. Figure 7 ) It was observed that the skin tissue of the mice in the Blank group was intact, the dermis and epidermis were tightly connected and wavy, the dermal fibers were evenly distributed, especially the collagen fiber and elastic fiber content were at normal levels. The epidermis of the mice in the Model group was thinner, and the dermal density was reduced, among which the collagen fibers were reduced and fragmented in some areas, and the elastic fibers were reduced and shortened. There was no significant difference in epidermal thickness between the skin repair dressing group and the Blank group. Its dermal fibers were tightly arranged, and the collagen content and elastic fiber content were not significantly different from those of the Blank group. Compared with the Model group, the epidermal thickness, collagen content and elastic fiber number of the VE and RHC gel groups were significantly increased (P < 0.001), but they still did not return to normal levels. In summary, the skin repair dressing RHE gel based on recombinant humanized elastin provided by the present invention showed significantly better efficacy than commercially available VE emulsion and recombinant collagen gel in repairing endogenous aging tissue damage, promoting collagen expression and increasing elastic fiber content.

[0126] (3) Oxidative stress

[0127] Skin aging can lead to ROS accumulation, thus inducing oxidative stress. MDA (malondialdehyde) is the main product of lipid peroxidation. Endogenous enzymes such as SOD (superoxide dismutase), CAT (catalase) and antioxidant small molecules such as GSH (glutathione) can inhibit the accumulation of ROS caused by aging. The content of MDA, SOD, CAT, and GSH can be found in the test results. Figure 8 The figure shows that the Model group has the highest MDA content and the lowest SOD, CAT and GSH levels, proving that the aging model was successfully established. There was no significant difference in the MDA, SOD, CAT and GSH levels between the skin repair dressing group and the Blank group. A preliminary study of its mechanism showed that the skin repair dressing of the present invention acts on the surface of aging skin, promoting the secretion of SOD, CAT and GSH by the skin. SOD makes the large amount of O2 produced by aging - It is rapidly converted into H2O2, which is then rapidly decomposed under the catalysis of CAT or the action of GSH, balancing the high oxidative stress. In addition, the reduction of reactive oxygen species also reduces the degree of cell membrane peroxidation and reduces MDA levels. Compared with the Model group, the VE group and the 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. The above results show that the skin repair dressing based on recombinant humanized elastin provided by the present invention, RHE gel, has better ability to improve endogenous aging oxidative stress than commercially available VE emulsion and recombinant collagen gel.

[0128] (4) RT-qPCR

[0129] Aging can lead to ECM degradation, especially a decrease in collagen and elastin content. LOX and Fibrillin-1 promote the cross-linking of tropoelastin to form mature elastic fibers. The upregulation of MMP-2 and MMP-12 can degrade elastic fibers. The results of RT-qPCR detection of related gene expression are shown in Figure 9 . The figure shows that the LOX and Fibrillin-1 genes in the skin repair dressing group were significantly upregulated compared with the Model group, VE group and RHC gel group; this proves that they promote the synthesis of elastin fibers and are beneficial to the recovery of skin elasticity. The expression of inactive MMP-2 and MMP-12 genes proves that they hinder the degradation of elastic fibers caused by aging. In addition, the upregulation of Elastin, Col I, and Col III genes in the skin repair dressing group corresponds to the results of increased elastic fiber and collagen volume fractions in tissue staining. The above results indicate that the skin repair dressing RHE gel based on recombinant humanized elastin provided by the present invention helps promote ECM remodeling.

[0130] In summary, the present invention provides a recombinant humanized elastin with the efficacy 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 biosafety. The skin repair dressing is non-toxic to fibroblasts in in vitro experiments and can significantly promote their proliferation, extension and migration; in the zebrafish UV sunburn and chemical oxidative stress models, RHE gel exhibits excellent after-sun repair and antioxidant activity; in the D-galactose-induced endogenous skin aging mouse model, RHE gel can significantly reduce wrinkle formation, increase dermal thickness and skin water content, reduce transepidermal water loss and shorten skin rebound time, and histological analysis further confirms that it can improve epidermal thinning and dermal collapse, significantly increase collagen and elastic fiber content and promote extracellular matrix network reconstruction; at the molecular mechanism level, RHE gel can upregulate LOX and Fibrillin-1 expression, inhibit MMP-2 and MMP-12 activity and significantly increase Col I / III expression, synergistically achieve skin tissue repair, and optimize the antioxidant microenvironment by reducing MDA levels and increasing SOD, CAT and GSH activities; compared with commercially available vitamin E emulsion and recombinant collagen gel, RHE Gel has significant advantages in restoring skin elasticity, reducing wrinkles and improving tissue structural integrity, showing a clear mechanism of action and broad industrial prospects.

[0131] The above specific embodiments describe the implementation of the present invention in detail, but the present invention is not limited to the specific details of 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 scope of protection of the present invention.

Claims

1. A recombinant humanized elastin having the effect of improving endogenous skin aging, characterized in that: The amino acid sequence of the recombinant humanized elastin is: (a) the amino acid sequence shown in SEQ ID NO. 1; or (b) An amino acid sequence obtained by substitution, deletion and / or addition of one or more amino acids based on the sequence shown in SEQ ID NO. 1, and having substantially the same function as the protein shown in SEQ ID NO.

1.

2. The recombinant humanized elastin having the efficacy of improving endogenous skin aging according to claim 1, characterized in that: The recombinant humanized elastin is obtained by treating precursor elastin with protease, and the amino acid sequence of the precursor elastin is: (a) the amino acid sequence shown in SEQ ID NO. 2; or (b) An amino acid sequence obtained by substitution, deletion and / or addition of one or more amino acids based on the sequence shown in SEQ ID NO. 2, and having substantially the same function as the protein shown in SEQ ID NO.

2.

3. The recombinant humanized elastin having the efficacy of improving endogenous skin aging according to claim 2, characterized in that: The gene sequence encoding the precursor elastin is: (a) the nucleotide sequence shown in SEQ ID NO. 3; or (b) A nucleotide sequence in which one or more nucleotides are substituted, deleted, and / or added to the nucleotide sequence shown in SEQ ID NO. 3 and which expresses the same protein as the nucleotide sequence shown in SEQ ID NO.

2.

4. The method for preparing the recombinant humanized elastin according to any one of claims 1 to 3, comprising the following steps: (1) Synthesizing the gene sequence SEQ ID NO. 3 encoding the precursor of the recombinant humanized elastin; (2) Connecting the gene sequence to a vector, transforming genetically engineered bacteria, and constructing recombinant genetically engineered bacteria; (3) Expressing the constructed recombinant genetically engineered bacteria, collecting the bacteria, crushing them to obtain the supernatant, and purifying the precursor elastin; (4) Add the precursor elastin obtained by protease 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 proteases include thrombin, pepsin, trypsin or bromelain.

6. A recombinant vector or recombinant genetically engineered bacterium carrying the precursor protein gene of the recombinant humanized elastin according to any one of claims 1 to 3.

7. Use of the recombinant humanized elastin according to any one of claims 1 to 3 in the preparation of a product for improving endogenous skin aging.

8. Use of the recombinant humanized elastin according to claim 7 in preparing a product for improving endogenous skin aging, characterized in that: The products include but are not limited to cosmetics or medical cosmetic products.

9. Use of the recombinant humanized elastin according to claim 8 in preparing a product for improving endogenous skin aging, characterized in that: The cosmetics include but are not limited to: stock solutions, lotions, essences, creams, facial masks, sprays, sunscreens, eye creams, foundations, and body lotions; the medical beauty products include but are not limited to: stock solutions, gels, cold compresses, dressings, sponges, hyaluronic acid injection preparations, implants, and creams.

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

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