An active peptide OA-RD17 that promotes skin wound repair and its application

Abstract

The present invention discloses a peptide active for promoting skin wound repair, OA-RD17, and its application. The amino acid sequence of the peptide OA-RD17 is RDYCTPEDCDYDFSFPI. The peptide OA-RD17 can be used in the preparation of a drug for treating and promoting skin wound repair. The present invention provides a novel peptide, OA-RD17, that promotes wound repair both in vivo and in vitro, exhibiting excellent skin tissue regeneration activity. OA-RD17, consisting of only 17 amino acids, is easy to synthesize at low cost, providing a new approach for the development of skin wound repair drugs and a new direction for the development of drugs for promoting skin wound treatment.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to an active polypeptide OA-RD17 that promotes skin wound repair and its application. Background Art

[0002] The skin, the largest organ in the human body, is not only the body's primary natural protective barrier but also plays a vital role in numerous physiological processes. Once the skin is damaged, the body immediately initiates a cascade of precisely regulated wound repair processes to quickly restore the skin's barrier function and maintain a stable internal environment. However, the regenerative process of wounded skin is susceptible to the effects of both the body's own disease and the external environment, leading to chronic non-healing wounds or excessive wound healing. Chronic non-healing wounds and excessive wound healing not only impair the normal physiological functions of the skin but also incur high clinical treatment costs. The number of affected patients is increasing year by year, placing tremendous psychological pressure and financial burden on patients and placing a heavy burden on healthcare systems worldwide. Therefore, rapid and efficient regeneration and repair of the skin after injury plays an important role in reestablishing the body's normal physiological functions, restoring internal homeostasis, and alleviating the psychological and financial burden on patients and society.

[0003] In recent years, stem cell therapy, tissue engineering techniques, and keratinocyte transplantation have provided novel therapeutic strategies for wound repair. However, these approaches are hindered by limitations such as high costs and long treatment cycles, hindering widespread adoption. Drug therapy still plays a crucial role in the treatment of skin wounds. Common clinical wound repair agents include small molecule compounds, growth factors (rh-bFGF, EGF), plant and animal-derived drugs, and low-adhesion dressings. While these agents demonstrate promising results, they also suffer from numerous limitations, including difficulty in synthesis, high costs, demanding storage conditions, instability of active ingredients, and the potential for scarring, making them insufficient for clinically effective treatments for skin wounds. Therefore, the discovery of novel and highly effective lead molecules that promote skin tissue regeneration and the exploration of their molecular mechanisms of wound repair are crucial and necessary.

[0004] Compared to small-molecule drugs, peptides exhibit greater activity, lower toxicity, improved safety, a high development success rate, and enhanced targeting. Furthermore, peptide research and development offers a broad commercial market, making them a promising area of drug development. Amphibian-derived bioactive peptides are emerging as promising candidates for tissue regeneration drug development and are expected to become a key source of lead molecules for novel regenerative drugs. However, these studies are limited and remain in their infancy.

[0005] The present invention aims to provide a wound repair promoting active peptide which exhibits good skin wound repair promoting activity both in vivo and in vitro. Summary of the Invention

[0006] The first object of the present invention is to provide an active polypeptide OA-RD17 that promotes skin wound repair. The second object of the present invention is to provide applications of the active polypeptide OA-RD17.

[0007] The first object of the present invention is achieved in this way. The amino acid sequence of the skin wound repair promoting active polypeptide OA-RD17 is RDYCTPEDCDYDFSFPI, as shown in SEQ ID NO.1.

[0008] The second object of the present invention is achieved in that the skin wound repair promoting active polypeptide OA-RD17 is used in the preparation of a drug for treating and promoting skin wound repair.

[0009] The present invention provides a novel peptide, OA-RD17, which promotes wound repair both in vivo and in vitro, exhibiting excellent skin tissue regeneration activity. OA-RD17, consisting of only 17 amino acids, is easily synthesized at low cost, providing a new avenue for the development of skin wound repair drugs and a new direction for the development of drugs that promote the treatment of skin wounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 is the primary structure diagram of the polypeptide OA-RD17 of the present invention;

[0011] Figure 2 This is a graph showing the activity of the polypeptide OA-RD17 of the present invention in promoting scratch repair in HaCaT cells;

[0012] Figure 3 This is a graph showing the HaCaT cell proliferation promoting activity of the polypeptide OA-RD17 of the present invention;

[0013] Figure 4 This is a graph showing the HaCaT cell migration promoting activity of the polypeptide OA-RD17 of the present invention;

[0014] Figure 5 This is a graph showing the activity of the polypeptide OA-RD17 of the present invention in promoting the repair of full-thickness cortical damage in mice in an animal model, wherein A is a graph showing the changes in full-thickness wound repair in mice, B is a graph showing the repair status of full-thickness wounds in mice, C is a HE staining graph of skin tissue in the full-thickness wound area of mice on days 4 and 8, D is a graph showing the epidermal regeneration thickness of skin tissue in the wound area of mice on days 4 and 8, and E is a graph showing the tissue regeneration thickness of skin tissue in the wound area of mice on days 4 and 8. DETAILED DESCRIPTION

[0015] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments, but the present invention is not limited in any way. Any changes or improvements made based on the teachings of the present invention fall within the scope of protection of the present invention.

[0016] The present invention provides an active polypeptide OA-RD17 derived from a frog with skin wound repair promoting effect. The amino acid sequence of the polypeptide OA-RD17 is RDYCTPEDCDYDFSFPI.

[0017] The present invention also provides the use of the polypeptide OA-RD17 in preparing a drug for treating and promoting skin wound repair.

[0018] The present invention further provides a pharmaceutical composition comprising the polypeptide OA-RD17 as an active agent and a pharmaceutically effective carrier.

[0019] Example 1 Screening of active peptide OA-RD17 to promote skin wound repair

[0020] Step 1: The sample was obtained from the skin of the Yunnan stink frog. Adult Yunnan stink frogs (from Yunnan Province, China) were housed in a 21 cm × 21 cm × 15 cm aquarium for 7 days to acclimate to the environment, with regular water changes and a diet of mealworms. The skin of the Yunnan stink frog was then rinsed with deionized water and quickly sacrificed by destroying the brain, and the skin was obtained.

[0021] Step 2: Construction of a cDNA library of the Yunnan stink frog: The skin tissue was obtained using a total RNA extraction kit (TIANGEN, China) to extract RNA from the skin tissue. After extraction, the RNA molecules were converted into cDNA molecules to establish a peptide cDNA library derived from the Yunnan stink frog skin tissue.

[0022] Step 3: To screen for cDNA encoding mature OA-RD17, polymerase chain reaction (PCR) templates were generated from cDNA synthesized using SMART technology and using 5' PCR primers (5'-CCAAA(G / C)ATGTTCACC(T / A)TGAAGAA-3') and 3' PCR primers (5'ATTCAGGCCGAGGCC GACA TG-3') synthesized by BGI (China). The resulting PCR products were cloned into competent Escherichia coli DH5a cells and sequenced using an Applied Biosystems DNA sequencer (ABI 3730XL, Foster City, CA, USA). The results are shown in Table 3. Figure 1The nucleotide sequence of the cDNA is shown in SEQ ID NO. 2. The cDNA sequence is 365 bp (containing 365 bases) and encodes 64 amino acid residues. The mature peptide sequence is "RDYCTPEDCDYDFSFPI" ( Figure 1 The base sequence is underlined in ( ). Through retrieval, the peptide was found to be a new peptide and was named OA-RD17.

[0023] The polypeptide OA-RD17 is then prepared by conventional chemical synthesis or gene expression.

[0024] The polypeptide OA-RD17 of the present invention was synthesized by Wuhan Baiyixin Biotechnology Co., Ltd.

[0025] Example 2 Detection of the activity of polypeptide OA-RD17 in promoting skin wound repair in vitro

[0026] 1. In vitro cell scratch repair activity detection

[0027] Keratinocytes were cultured in DMEM / F12 medium containing 10% fetal bovine serum and 1% antibiotics (100 U / mL penicillin, 100 U / mL streptomycin) at 37°C and 5% CO2. HaCaT cells were seeded in 24-well plates (2 × 10 5 Cells were plated at 400 μL per well (10 cells / well) until a confluent monolayer formed. A 200 μL sterile pipette tip was then used to scratch the cell monolayer to simulate a wound. After washing with PBS to remove cell debris, 500 μL of serum-free culture medium containing 50 μL of PBS (Vehicle), rh-bFGF (1 μg / mL), and OA-RD17 (10 nM) was added to each well. The cells were incubated at 37°C in a 5% CO2 atmosphere for 24 hours. Wound healing was recorded using an inverted microscope (Zeiss, Germany) at 0, 12, and 24 hours.

[0028] The results are as follows Figure 2 As shown in AB: Peptide OA-RD17 can significantly promote the scratch repair of keratinocytes, and the scratch repair effect is close to 100% after 24 hours of incubation with OA-RD17.

[0029] 2. In vitro cell proliferation activity assay

[0030] Keratinocytes were seeded in 96-well plates (5×10 3 After the cells adhered to the wall, different concentrations of peptide OA-RD17 (100 pM, 1 nM, 10 nM, 100 nM, 1 μM) were added. After continuous culture for 24 h, the changes in cell proliferation were detected using MTS reagent.

[0031] The results are as follows Figure 3 As shown: The peptide OA-RD17 promoted keratinocyte proliferation in a concentration gradient-dependent manner, and OA-RD17 exhibited good cell proliferation promoting activity at a concentration of 1 nM.

[0032] 3. In vitro cell migration activity assay

[0033] A trans-well plate with a filter membrane pore size of 8 μm was used as the upper chamber and inserted into a 24-well plate in the lower chamber. Keratinocytes and macrophages were then cultured at a cell density of 1×10 4 Cells were seeded into the upper chamber at 100 μg / well and OA-RD17 was added to a final concentration of 1 nM. Culture medium containing 10% FBS was then added to the lower chamber of the 24-well plate. The cells were cultured at 37°C for 24 hours. After washing with PBS, the cells were fixed with 4% paraformaldehyde and stained with 1 mg / mL crystal violet for 20 minutes. The upper chamber was washed with PBS to remove non-migrating cells, and the cells were photographed under an inverted microscope. After photographing, the crystal violet was completely eluted with 33% acetic acid. The absorbance (OD) was measured at 570 nm using a microplate reader to calculate the cell migration rate.

[0034] The results are as follows Figure 4 As shown in AB: Peptide OA-RD17 significantly promoted the migration of keratinocytes, and the number of migrated cells reached 2.34 times that of the blank control.

[0035] In summary, OA-RD17 has good in vitro wound repair activity.

[0036] Example 3 Detection of the activity of polypeptide OA-RD17 in promoting skin wound repair in vivo

[0037] Experimental method: Full-thickness cortical wound experiment in mice

[0038] 1. Kunming mice were anesthetized with 1% sodium pentobarbital (60 mg / kg). After removing the dorsal fur, two full-thickness wounds (10 mm in diameter) were created on both sides of the back using a biopsy needle. The mice were randomly divided into four groups: a blank control (PBS), a positive control (rh-bFGF, 100 ng / ml), a peptide group (OA-RD17, 1 nM), and a scrambled peptide group (1 nM). 20 µL of the drug was administered twice daily. Wound changes were recorded every other day until day 8 after surgery. Histopathological analysis of the dorsal wound tissue was performed on days 4 and 8 after surgery.

[0039] 2. To evaluate the regeneration of wound tissue in mice after treatment in different groups, hematoxylin andeosin (HE) staining was performed. Skin tissue was cut into 5 μm thick slices, dewaxed, graded hydrated, and then stained with HE. The regeneration of wound tissue was recorded using an optical microscope at the same magnification, as shown in Figure 2. Figure 5 As shown, the changes in the epidermis, new tissue, and epithelialization of skin with different pathological tissues were calculated using Image J software, and the data were statistically analyzed using Prism software.

[0040] result:

[0041] like Figure 5 As shown in AB: OA-RD17 can significantly promote the repair of full-thickness cortical wounds on the back of mice. After 8 days of treatment, the wound repair activity of OA-RD17 is similar to that of the positive control rh-bFGF, and the wound repair rate is close to 100%.

[0042] like Figure 5 CE shows: HE staining results showed that OA-RD17 significantly promoted the proliferation and migration of the wound epidermis of mice and significantly accelerated re-epithelialization. In addition, it also promoted the regeneration of granulation tissue in the wound area.

[0043] Summary: The wound repair peptide OA-RD17 provided by the present invention has strong skin tissue regeneration activity both in vivo and in vitro, and has certain clinical application value.

Claims

1. An active peptide OA-RD17 that promotes skin wound repair, characterized in that: The amino acid sequence of the polypeptide OA-RD17 is RDYCTPEDCDYDFSFPI.

2. Use of the polypeptide OA-RD17 according to claim 1 in the preparation of a drug for treating and promoting skin wound repair.

3. A pharmaceutical composition comprising the polypeptide OA-RD17 according to claim 1 as an active agent and a pharmaceutically effective carrier.

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