Recombinant extracellular matrix protein composition and application thereof in preparation of anti-skin photoaging product

Abstract

The invention belongs to the technical field of biological medicine and skin repair, and particularly relates to a recombinant extracellular matrix protein composition and application thereof in preparation of products for resisting skin photoaging. The recombinant extracellular matrix protein composition is obtained by mixing recombinant humanized III collagen, recombinant humanized chemical fibronectin and recombinant humanized elastin. Tests show that the recombinant extracellular matrix protein composition can improve the survival rate of cells damaged by UVB, reduce the generation of ROS, improve the activity of SOD, CAT and GSH-Px and the level of GSH, and reduce the content of lipid peroxide MDA. The recombinant extracellular matrix protein composition has an effective anti-oxidative damage effect and can resist skin cell damage caused by UVB.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and skin repair technology, and specifically relates to a recombinant extracellular matrix protein composition and its application in the preparation of anti-skin photoaging products. Technical Background

[0002] Ultraviolet (UV) radiation is one of the major environmental factors that cause skin damage. Its mechanisms of action include: 1) Oxidative stress: UVB (280-320 nm) penetrates the epidermis, inducing a burst of reactive oxygen species (ROS), leading to lipid peroxidation, protein carbonylation, and mitochondrial DNA damage. 2) Matrix degradation: VA (320-400 nm) activates matrix metalloproteinases (MMP-1, MMP-3, and MMP-9) in dermal fibroblasts, degrading type I and III collagen and elastic fibers, causing skin sagging and wrinkling. 3) Inflammatory response: UV radiation stimulates keratinocytes to release proinflammatory cytokines (IL-6, TNF-α, and IL-1β), leading to erythema, edema, and impaired barrier function. 4) DNA photodamage: UVB induces the formation of thymine dimers (CPDs) and 6-4 photoproducts (6-4PPs), interfering with DNA replication and repair and increasing the risk of mutations. The ultimate clinical consequences of long-term UV exposure are photoaging (wrinkles and hyperpigmentation), immunosuppression, and an increased risk of skin cancer.

[0003] Current treatments for UV-induced skin damage primarily include antioxidants, anti-inflammatory drugs, and physical protection, but these methods all have drawbacks. For example, there are limitations associated with single targets. Some antioxidants, such as vitamin C and vitamin E, only scavenge ROS and are unable to repair degraded ECM components or reverse DNA damage. Anti-inflammatory drugs, such as glucocorticoids, can provide short-term relief of redness and swelling, but long-term use can lead to skin atrophy and do not promote structural repair. Common sunscreens, such as zinc oxide, only prevent UV penetration and are ineffective against deep damage that has already occurred. Animal-derived proteins are also commonly used as cosmetic ingredients for UV protection, but these foreign proteins are highly immunogenic (allergy rates of 3-5%) and lack functional receptor binding domains, resulting in low repair efficiency. Transdermal efficiency is low, and natural collagen (molecular weight > 300 kDa) and elastin have difficulty penetrating the stratum corneum, with actual bioavailability of less than 1%. Furthermore, traditional emulsions or gels are unable to maintain protein activity and are prone to denaturation and inactivation during storage. It can be seen that the prominent problem at this stage in treating UV skin damage is the lack of a synergistic repair network for UV damage. Existing products mostly target a single pathological link (such as only inhibiting MMP or anti-oxidation) and are unable to repair collagen, elastic fibers and barrier structures at the same time, resulting in unsustainable therapeutic effects.

[0004] Therefore, it is very necessary to provide a product with multi-target synergistic anti-photoaging, especially skin damage caused by ultraviolet (UV) rays. Summary of the Invention

[0005] One of the objectives of the present invention is to provide a recombinant extracellular matrix protein composition, comprising recombinant humanized collagen III, recombinant humanized fibronectin, and recombinant humanized elastin mixed in a set ratio.

[0006] Preferably, the set ratio is 19:4.4:1 by mass.

[0007] A second object of the present invention is to provide the use of the recombinant extracellular matrix protein composition described above in the preparation of an antioxidant damage product.

[0008] A third object of the present invention is to provide a use of the recombinant extracellular matrix protein composition described above in the preparation of products for resisting skin photoaging.

[0009] A fourth object of the present invention is to provide a use of the recombinant extracellular matrix protein composition described above in the preparation of a product for resisting skin damage caused by ultraviolet rays.

[0010] Preferably, the product is at least one of medicine, cosmetics, health care products and food.

[0011] Preferably, the product also includes excipients acceptable to medicines, cosmetics, health products, and foods.

[0012] Preferably, the product is a recombinant extracellular matrix protein dressing, which is prepared from 36 parts by mass of a recombinant extracellular matrix protein composition, 6 parts by mass of mannitol, 0.6 parts by mass of a pH regulator, 0.4 parts by mass of a buffer and 57 parts by mass of water.

[0013] Preferably, the pH regulator is any one or more combinations of citric acid, sodium citrate, lactic acid, sodium lactate, sodium hydroxide, arginine, and triethanolamine, and the buffer is any one or more combinations of phosphate, carbonate, and citrate.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] 1) The present invention discovers for the first time that a composition comprising recombinant humanized collagen III, recombinant humanized fibronectin, and recombinant humanized elastin, proteins derived from human skin extracellular matrix (ECM), can prevent and / or treat UV-induced cell damage at a low effective concentration and without toxic side effects. The present invention also provides an optimal ratio of recombinant humanized collagen III, recombinant humanized fibronectin, and recombinant humanized elastin of 19:4.4:1.

[0016] 2) Experiments have shown that the recombinant extracellular matrix protein composition provided by the present invention can improve the cell survival rate of HaCaT cells damaged by UVB, reduce the generation of ROS, increase the activity of SOD, CAT, GSH-Px and GSH levels, and reduce the content of lipid peroxide MDA, indicating that the recombinant extracellular matrix protein composition has an effective antioxidant effect and can resist UVB-induced skin cell damage.

[0017] 3) The present invention expands the new application field of recombinant extracellular matrix proteins and provides a new source and direction for the development of biological protein components for drugs to treat diseases related to ultraviolet-induced skin damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The invention relates to the protective effect of the recombinant extracellular matrix protein composition on UVB-induced HaCaT cell damage.

[0019] Figure 2 The effect of recombinant extracellular matrix protein composition on the intracellular ROS level induced by UVB in HaCaT cells.

[0020] Figure 3 The invention relates to the effect of the recombinant extracellular matrix protein composition on the activities of SOD, CAT, GSH-Px and the contents of GSH and MDA in HaCaT cells induced by UVB. DETAILED DESCRIPTION

[0021] The technical solution of this application is described in more detail below with reference to experiments and drawings.

[0022] Unless otherwise specified, the terms used herein have the meanings commonly understood by those skilled in the art. The ECM protein composition herein refers to a recombinant extracellular matrix protein composition.

[0023] Experimental Materials: Recombinant humanized collagen III, recombinant humanized fibronectin, and recombinant humanized elastin were obtained from Wuhu Interfil Biological Products Industry Research Institute Co., Ltd. (lyophilized powders) and prepared into an ECM protein composition at a ratio of 19:4.4:1 by weight. HaCaT cells were also obtained from Wuhu Interfil Biological Products Industry Research Institute Co., Ltd. SOD assay kits, CAT assay kits, ROS assay kits, MDA assay kits, GSH assay kits, and GSH-PX assay kits were purchased from Sigma-Aldrich.

[0024] Example 1

[0025] Protective effect of ECM protein composition on UVB-induced HaCaT cell damage

[0026] Cell treatment: 5000 HaCaT human immortalized keratinocytes in good growth condition were seeded into 96-well culture plates. After the cells adhered overnight, a normal control group, a UVB irradiation group, and an ECM protein composition group (total protein concentration 10EU) were set up, with 6 replicates for each concentration. After further culture for 12 hours, the cells were washed twice with PBS and then a thin layer of PBS was added. Except for the normal control group, the cells in the other groups were irradiated with UVB using an ultraviolet irradiation device at a dose of 142mJ / cm 2 , discard PBS, and wash twice with pre-cooled PBS. Set up a blank group without cells, add 100 μL fresh 1% DMEM and 10 μL CCK-8 solution to each well, place in the incubator and continue incubation for 1 hour, and then measure the absorbance of each well at 450 nm.

[0027] The cell viability was calculated according to the following formula:

[0028] Cell survival rate % = (OD value of sample group - OD value of blank group) / (OD value of control group - OD value of blank group) × 100%

[0029] The protective effect of the ECM protein composition of the present invention on UVB-induced HaCaT cell damage was detected by CCK-8 method. The results are as follows Figure 1 As shown in the results, the survival rate of HaCaT cells after irradiation with 142mJ / cm2 UVB was only 44% of that of the normal group, while the cell survival rate after the action of the ECM protein composition was significantly higher than that of the UVB group, and the survival rate of HaCaT cells reached 71% of the normal group, indicating that the ECM protein composition has a significant photoprotective effect on UVB-induced HaCaT cells.

[0030] Example 2

[0031] Antioxidative effect of ECM protein combination on UVB-induced HaCaT cell damage

[0032] Cell treatment: HaCaT human immortalized keratinocytes in good growth condition were seeded at 106 cells / well in a 6-well culture plate. After culturing for 12 hours to adhere to the wall, a normal control group, a UVB irradiation group, and an ECM protein composition group (total protein concentration 10EU) were set up, with 3 replicates in each group. After further culturing for 12 hours, the cells were washed twice with PBS, and then a thin layer of PBS was added. Except for the normal control group, the cells in the other groups were irradiated with UVB using an ultraviolet irradiation device at a dose of 142mJ / cm 2 , discard PBS and wash twice with pre-chilled PBS.

[0033] Refer to the kit instructions to detect the levels of ROS, SOD, CAT, GSH-Px, GSH and MDA in each group of cells:

[0034] 1. Detection results of ROS levels in each group of cells

[0035] Intracellular ROS levels reflect the degree of cellular oxidation. Changes in intracellular ROS levels were monitored using the specific fluorescent probe 2',7'-dihydrodichlorofluorescein diacetate (DCFH-DA). DCFH-DA is lipid-soluble and can cross cell membranes and react with intracellular esterases to produce DCFH. Because DCFH cannot cross the cell membrane, the fluorescent probe can be loaded into cells and oxidized by intracellular ROS to fluorescent DCF. The DCF fluorescence intensity was measured using a fluorescence microplate reader at an excitation wavelength of 500 nm and an emission wavelength of 525 nm. The final intracellular ROS level was expressed as a fluorescence value.

[0036] The results are as follows Figure 2 As shown, 142mJ / cm 2 After UVB irradiation, the intracellular ROS level increased significantly, 3.6 times that of the normal group. After the ECM protein composition was applied, the cellular ROS level was significantly reduced, indicating that the ECM protein composition has a significant inhibitory effect on UVB-induced ROS generation in HaCaT cells.

[0037] 2. Test results of SOD, CAT, GSH-Px enzyme activities and GSH, MDA contents in each group of cells

[0038] Changes in the levels of intracellular antioxidant indicators such as SOD, CAT, GSH-Px, GSH, and MDA can indirectly reflect the degree of cell damage.

[0039] The results are as follows Figure 3 As shown in the results, compared with the normal group, the activities of SOD, CAT, GSH-Px and GSH content in the UVB-irradiated group were significantly reduced, and the MDA content was significantly increased, indicating that UVB irradiation disrupts the antioxidant balance in HaCaT cells and causes cell damage. The ECM protein composition has a significant improvement effect on the changes in SOD, CAT, GSH-Px, GSH and MDA levels in HaCaT cells induced by UVB.

[0040] Example 3

[0041] A recombinant extracellular matrix protein dressing is prepared from 36 parts by mass of a recombinant extracellular matrix protein composition, 6 parts by mass of mannitol, 0.6 parts by mass of a pH regulator, 0.4 parts by mass of a buffer and 57 parts by mass of water.

[0042] Among them, the recombinant extracellular matrix protein composition is obtained by mixing recombinant humanized collagen III, recombinant humanized fibronectin, and recombinant humanized elastin in a mass ratio of 19:4.4:1; the pH regulator is sodium citrate, the buffer is phosphate, and the pH value of the dressing is between 4.5-6.5.

[0043] Example 4

[0044] A recombinant extracellular matrix protein dressing is prepared from 36 parts by mass of a recombinant extracellular matrix protein composition, 6 parts by mass of mannitol, 0.6 parts by mass of a pH regulator, 0.4 parts by mass of a buffer and 57 parts by mass of water.

[0045] Among them, the recombinant extracellular matrix protein composition is obtained by mixing recombinant humanized collagen III, recombinant humanized fibronectin, and recombinant humanized elastin in a mass ratio of 19:4.4:1; the pH regulator is lactic acid and sodium lactate, the buffer is citrate, and the pH value of the dressing is between 4.5-6.5.

[0046] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A recombinant extracellular matrix protein composition, characterized in that: It includes recombinant humanized collagen III, recombinant humanized fibronectin, and recombinant humanized elastin mixed in a set ratio.

2. A recombinant extracellular matrix protein composition according to claim 1, characterized in that: The set ratio is 19:4.4:1 by mass.

3. Use of the recombinant extracellular matrix protein composition according to claim 1 in the preparation of an antioxidant damage product.

4. Use of the recombinant extracellular matrix protein composition according to claim 1 in the preparation of products for resisting skin photoaging.

5. Use of the recombinant extracellular matrix protein composition according to claim 1 in the preparation of a product for resisting skin damage caused by ultraviolet rays.

6. The use according to claim 3, 4 or 5, characterized in that The product is at least one of medicine, cosmetics, health care products and food.

7. The use according to claim 6, characterized in that The products also include excipients acceptable in medicines, cosmetics, health products and foods.

8. The use according to claim 3, 4 or 5, characterized in that The product is a recombinant extracellular matrix protein dressing, which is prepared from 36 parts by mass of a recombinant extracellular matrix protein composition, 6 parts by mass of mannitol, 0.6 parts by mass of a pH regulator, 0.4 parts by mass of a buffer and 57 parts by mass of water.

9. The use according to claim 8, characterized in that The pH regulator is any one or more combinations of citric acid, sodium citrate, lactic acid, sodium lactate, sodium hydroxide, arginine, and triethanolamine; and the buffer is any one or more combinations of phosphate, carbonate, and citrate.

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