Polypeptide with anti-aging and repairing effects, composition and application thereof

Abstract

The invention belongs to the technical field of anti-aging substances, and particularly relates to a polypeptide with anti-aging and repairing effects, a composition and application of the polypeptide, the amino acid sequence of the polypeptide is shown as SEQ ID NO: 1, and the SEQ ID NO: 1 is FFRKVLKLIRKIWR.The polypeptide can promote growth of fibroblasts and regulate the fibroblasts to synthesize collagen, and the anti-aging and repairing effects are achieved. And proliferation and migration of keratinocytes and proliferation of vascular endothelial cells are promoted. Therefore, the mask has the effects of improving the loss of skin collagen, enhancing the moisturizing effect, efficiently repairing the skin, resisting wrinkles, removing scars and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of anti-aging substances, and specifically relates to a polypeptide with anti-aging and repairing effects, a composition and uses thereof. Background Art

[0002] Skin aging is a multifactorial process that depends on intrinsic factors (genetic, hormonal and metabolic) and extrinsic factors (long-term exposure to UV rays, smoking, air pollution, chemicals and poor nutrition, etc.). Aging adversely affects the connective tissue in the skin and leads to a reduction in elastin and collagen fibers, resulting in fine lines and wrinkles.

[0003] The skin is the most important physical barrier for humans to resist mechanical damage, extreme temperatures, microbial infection, radiation and other environmental factors. It is mainly composed of a keratinized complex epidermis composed of keratinocytes and a thick collagen-rich inner layer mainly composed of nerve endings, blood vessels, lymphatic vessels and fibroblasts. The epidermis plays an important role in the water retention and repair functions of the skin. The main component of the dermis is collagen, which is composed of collagen fibers synthesized and secreted by fibroblasts. More than 90% of the dermal fibers are composed of interstitial collagen, mainly type I and type III, which can provide tensile strength and mechanical resistance to the skin. Therefore, collagen is essential for skin health, and both photoaging and intrinsic aging will reduce its presence in the body, resulting in a reduction in skin thickness and a loss of elasticity and flexibility.

[0004] Wound healing is a multi-step biological process that occurs spontaneously in the local wound surface of the body, which can be divided into hemostasis, inflammation, proliferation, epithelialization and remodeling. These processes require the participation and coordination of multiple cell types and factors. Among them, fibroblasts play a leading role in collagen synthesis, wound contraction and extracellular matrix remodeling. Vascular endothelial cells proliferate, migrate, and participate in the formation of granulation tissue. Keratinocytes proliferate and migrate to the edge of the wound surface, grow intensively to cover the wound surface, and drive re-epithelialization. Therefore, fibroblasts, keratinocytes and vascular endothelial cells all play a vital role in wound healing.

[0005] CN118557700A discloses an application of ELABELA polypeptide in anti-aging, and its polypeptide sequence is QRPVNLTMRRKLRKHNCLQRRCMPLHSRVPFP. It improves the proliferation ability of mesenchymal stem cells, improves the autophagy level of aged mesenchymal stem cells, promotes aged mesenchymal stem cells to enter the cell cycle, and reduces the aging degree of mesenchymal stem cells, thereby achieving the purpose of reducing or delaying individual aging. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a polypeptide, composition and use thereof with anti-aging and repair effects. The polypeptide can promote the growth of fibroblasts and stimulate fibroblasts to produce collagen, thereby improving skin collagen loss, enhancing moisturizing effect, efficiently repairing skin, anti-wrinkle and scar removal.

[0007] An embodiment of the present invention provides a polypeptide with anti-aging and repair effects, the amino acid sequence of the polypeptide is shown in SEQ ID NO: 1, and SEQ ID NO: 1 is FFRKVLKLIRKIWR.

[0008] Preferably, the N-terminus of the polypeptide is acetylated and the C-terminus is amidated, that is, Ac-FFRKVLKLIRKIWR-NH2, to improve its stability.

[0009] The embodiment of the present invention provides a composition, comprising the polypeptide and other auxiliary materials.

[0010] Preferably, the composition is a medicine or a cosmetic.

[0011] Preferably, the cosmetic is in the form of freeze-dried powder, essence, lotion, cream or facial mask.

[0012] The embodiment of the present invention provides a use of the polypeptide, wherein the polypeptide is used to prepare substances with anti-aging and repair effects.

[0013] Preferably, the polypeptide is used to promote the growth of fibroblasts and regulate the mixing of fibroblasts into collagen via the TGF-β / Smad signaling pathway.

[0014] Preferably, the polypeptide is used to promote keratinocyte proliferation and promote re-epithelialization during wound repair.

[0015] Preferably, the polypeptide is used to promote the proliferation and migration of vascular endothelial cells and promote wound repair.

[0016] The beneficial effect of the present invention is that the polypeptide of the present invention has new functions in anti-aging and repair. The polypeptide can promote the growth of fibroblasts and promote the synthesis of collagen in fibroblasts by activating the TGF-β / Smad signaling pathway. After a certain amount of collagen is synthesized, the signaling pathway can be inhibited, thereby preventing the formation of scars during skin repair. Therefore, the polypeptide of the present invention has the effects of improving the loss of skin collagen, enhancing the moisturizing effect, efficiently repairing the skin, anti-wrinkle and scar removal, and can be used to prepare anti-aging and skin repair drugs or cosmetics. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is the effect of peptides on fibroblast proliferation.

[0018] in Figure 1 (a) is the cell activity data after 1 day of treatment. Figure 1 (b) Cell viability data after 3 days of treatment.

[0019] Figure 2 The promoting effect of peptides on keratinocyte proliferation.

[0020] in Figure 2 (a) is the cell activity data after 1 day of treatment. Figure 2 (b) Cell viability data after 3 days of treatment.

[0021] Figure 3 The promoting effect of peptides on the proliferation of vascular endothelial cells.

[0022] in Figure 3 (a) is the cell activity data after 1 day of treatment. Figure 3 (b) Cell viability data after 3 days of treatment.

[0023] Figure 4 It is the effect of peptides on fibroblast migration.

[0024] Figure 5 It is the effect of peptides on the migration of vascular endothelial cells.

[0025] Figure 6 It is the effect of polypeptide on the expression of fibroblast-related functional proteins.

[0026] Figure 7 Repair effect of peptides on full-thickness skin defects on the back of mice. DETAILED DESCRIPTION

[0027] Example 1 A polypeptide with anti-aging and repairing effects, the amino acid sequence of the polypeptide is shown in SEQ ID NO: 1. SEQ ID NO: 1 is FFRKVLKLIRKIWR.

[0028] Example 2 The effects of the peptides on fibroblast proliferation were measured.

[0029] Human fibroblasts, keratinocytes and vascular endothelial cells were cultured in a culture system containing DMEM / F12 (1:1) medium, 10% fetal bovine serum (FBS), 10 ng / ml epidermal growth factor (EGF), 5 μg / ml insulin, 100 IU / ml penicillin and 100 μg / ml streptomycin.

[0030] When the cell density reached 80-90%, an appropriate number of cells were inoculated in a 96-well plate and cultured for 12 hours. 0, 1, 2, 4, 6, and 8 μg / ml of polypeptide (FFRKVLKLIRKIWR) and reference peptide (FFRKVLKLIRKIFR) were added to each well, and the amino acid sequence of the reference peptide was SEQ ID NO: 2). After continuous culture for 24 and 72 hours, 10 μL of CCK-8 reagent was added to the original culture medium, mixed, and cultured in a 37°C constant temperature incubator for 30 minutes. The OD value was measured at 450 nm using an enzyme-linked immunosorbent assay detector to evaluate cell activity.

[0031] The experimental results are as follows Figure 1 As shown, it can be seen that compared with the control group, the peptide (FFRKVLKLIRKIWR) had no cytotoxicity to the cells after treating fibroblasts for 24 and 72 hours, and at a concentration of 2-6 μg / ml, the peptide (FFRKVLKLIRKIWR) had a good promoting effect on the proliferation activity of fibroblasts. However, the comparison peptide (FFRKVLKLIRKIFR), which has only one amino acid difference from the peptide (FFRKVLKLIRKIWR) and also has good antibacterial activity, did not significantly promote the proliferation of fibroblasts at the same concentration.

[0032] Depend on Figure 2 and 3 As shown, the polypeptide (FFRKVLKLIRKIWR) had no cytotoxicity to keratinocytes and vascular endothelial cells after treatment for 24 and 72 hours, and at a concentration of 1-6 μg / ml, the polypeptide (FFRKVLKLIRKIWR) had a good promoting effect on the proliferation activity of keratinocytes and vascular endothelial cells.

[0033] Example 3 The effects of the peptides on the migration of fibroblasts and endothelial cells were measured.

[0034] Fibroblasts were cultured to the logarithmic growth phase and inoculated in appropriate amounts in 6-well plates. When the cell density reached 100%, a 200 μL pipette tip was used to vertically scratch the middle of the well plate from top to bottom, and then the floating cells were washed with phosphate-buffered saline. Serum-free DMEM medium and 0, 2, and 4 μg / ml peptide (FFRKVLKLIRKIWR) medium were added to the well plate, respectively. Photos were taken under a microscope on days 0, 1, 3, and 5, and the scratch healing area was analyzed using Image J.

[0035] The experimental results are as follows Figure 4As shown. Fibroblast migration is a key process for effectively repairing skin damage. We studied the regulatory effect of different concentrations of peptide (FFRKVLKLIRKIWR) on fibroblast migration through scratch experiments. Compared with the control group, 2μg / ml and 4μg / ml of peptide (FFRKVLKLIRKIWR) can effectively induce fibroblast migration to the scratch area 48 hours and 72 hours after scratching ( Figure 4 ), indicating that the peptide (FFRKVLKLIRKIWR) can effectively promote the migration of fibroblasts, thereby promoting the repair of skin damage.

[0036] Depend on Figure 5 It can be seen that compared with the control group, 2 μg / m peptide (FFRKVLKLIRKIWR) can effectively induce vascular endothelial cells to migrate to the scratch area at 48 hours and 72 hours after scratching ( Figure 5 ), indicating that the peptide (FFRKVLKLIRKIWR) can effectively promote the migration of vascular endothelial cells, thereby promoting the repair of skin damage.

[0037] However, at all concentrations, the peptide (FFRKVLKLIRKIWR) had no significant effect on the migration ability of keratinocytes.

[0038] Example 4 Measuring the expression of functional proteins in fibroblasts Western blot was used to detect the expression of Smad2, Smad3, and Smad7, downstream proteins of the TGF-β classical signaling pathway, in fibroblasts after 3, 5, and 7 days of peptide (FFRKVLKLIRKIWR) intervention. The experimental results are shown in Figure 6 shown.

[0039] The results showed that after 2μg / ml peptide (FFRKVLKLIRKIWR) intervened in fibroblasts for 3 days, type I collagen and fibronectin increased. At the same time, the expression of Smad2, Smad3, and Smad7 in fibroblasts was downregulated, indicating that the TGF-β signaling pathway was activated. After 5 days of intervention, the expression levels of type I collagen, type III collagen, and fibronectin in fibroblasts continued to increase, but the expression of Smad7 was upregulated, indicating that the signaling pathway began to be inhibited. After 7 days of intervention, collagen I also increased compared with the control group (blank control), and collagen III expression decreased, indicating that the peptide (FFRKVLKLIRKIWR) can promote the conversion of collagen I to collagen III in fibroblasts. Previous studies have reported that excessive production of TGF-β may promote scar formation and fibrosis, while the peptide (FFRKVLKLIRKIWR) can promote cell collagen synthesis by activating the TGF-β / Smad signaling pathway in the early stage of skin repair, and inhibiting this signaling pathway in the late stage of repair can prevent scar formation during skin repair.

[0040] Example 5 To detect the repair of full-thickness skin defects on the back of mice.

[0041] Sixty rats were randomly divided into a control group (full-thickness skin defect on the back) and a peptide (FFRKVLKLIRKIWR) group (200 μl of 2 μg / ml concentration was topically applied to the control group), with 30 rats in each group. The mice were anesthetized with 2% sodium pentobarbital. A π×1.5 cm area was formed in the middle of the back of the rat's head line using a scalpel and eye scissors. 2 Full-thickness skin defect. Apply the drug to the wound surface on the 1st, 2nd and 3rd day, twice a day. The control group was not given any drug. Gross observation was performed on the 1st, 3rd, 7th and 14th day. Ten mice in each group were killed on the 3rd, 7th and 14th day, and immunostaining was performed. The others were killed after the wound surface healed.

[0042] The experimental results are as follows Figure 7 As shown in the figure, compared with the control group, the peptide-treated group can significantly promote wound healing in mice. On the 7th, 10th and 14th days, the wound area was reduced to 54%, 84% and 98% of the original area, respectively, while the wound area of ​​the control group was reduced to 36%, 74% and 93%, respectively. The mice in each group were euthanized on the 3rd, 7th and 14th days after injury and histological evaluation was performed. Compared with the control group, the epidermis and dermis regeneration of peptide-treated mice increased, and the epidermal thickness decreased after 14 days. It shows that the peptide (FFRKVLKLIRKIWR) can effectively promote the repair of skin damage and prevent scar formation caused by excessive increase in skin thickness in the later stage.

[0043] A person skilled in the art should understand that the discussion of any of the above embodiments is merely illustrative and is not intended to imply that the scope of protection of the present application is limited to these examples. In line with the concept of the present application, the technical features in the above embodiments or different embodiments may be combined, the steps may be implemented in any order, and there are many other variations of different aspects of one or more embodiments of the present application as described above, which are not provided in detail for the sake of simplicity.

[0044] One or more embodiments of the present application are intended to cover all such substitutions, modifications and variations that fall within the broad scope of the present application. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of one or more embodiments of the present application should be included in the protection scope of the present application.

Claims

1. A polypeptide with anti-aging and repairing effects, characterized in that: The amino acid sequence of the polypeptide is shown in SEQ ID NO: 1, and SEQ ID NO: 1 is FFRKVLKLIRKIWR.

2. The polypeptide with anti-aging and repairing effects as claimed in claim 1, characterized in that: The N-terminus of the polypeptide is acetylated, and the C-terminus is amidated.

3. A composition, characterized in that: The method comprises the polypeptide as claimed in claim 1 or 2 and other auxiliary materials.

4. The composition according to claim 3, characterized in that The composition is a medicine or a cosmetic.

5. The composition according to claim 4, characterized in that The dosage form of the cosmetic is freeze-dried powder, essence, emulsion, cream or facial mask.

6. A use of the polypeptide according to claim 1 or 2, characterized in that: The polypeptide is used for preparing substances with anti-aging and repairing effects.

7. The use according to claim 6, characterized in that: The polypeptide is used for promoting the growth of fibroblasts and regulating the mixing of fibroblasts into collagen through the TGF-β / Smad signaling pathway.

8. The use according to claim 6, characterized in that: The polypeptide is used to promote the proliferation of keratinocytes and promote epithelialization during wound repair.

9. The use according to claim 6, characterized in that: The polypeptide is used for promoting the proliferation and migration of vascular endothelial cells and promoting wound repair.

Images