Active polypeptide for promoting healing of oral ulcer and application thereof

Abstract

The invention discloses an active polypeptide for promoting healing of oral ulcer and application thereof, and belongs to the technical field of biological medicines. The amino acid sequence of the active polypeptide NPTX3 capable of promoting healing of the oral ulcer is DLKPIAEDIPSLEK. An in-vitro experiment result shows that the polypeptide NPTX3 can remarkably promote the scratch closure of the human immortalized keratinocytes at the concentration as low as 500 pM. Transwell migration experiments further prove that the polypeptide NPTX3 can obviously enhance the migration ability of HaCaT cells at the concentrations of 1nM and 10nM, which prompts that the polypeptide NPTX3 may accelerate wound repair by promoting epithelial cell migration. In an SD (Sprague Dawley) rat oral ulcer model, the ulcer healing rate can be obviously improved by locally applying the polypeptide NPTX3 (1 nM, 10 nM). The polypeptide NPTX3 provided by the invention shows excellent activity of promoting cell migration and tissue repair at an extremely low dose, has the advantages of low effective dose, strong biological activity, high safety and the like, and provides an efficient and mild new choice for drug treatment of oral ulcer.

Description

An active polypeptide that promotes oral ulcer healing and its application Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an active polypeptide that promotes oral ulcer healing and its applications. Background Technology

[0002] Oral ulcers are a common clinical disease of the oral mucosa, occurring in people of all ages, and are characterized by high recurrence and self-limitation. Their etiology is complex, involving multiple factors such as genetic susceptibility, immune dysfunction, nutritional deficiencies (e.g., B vitamins, iron, zinc), psychological stress, local trauma, and poor dietary habits. Epidemiological surveys show that the prevalence of oral ulcers is approximately 5%–20%, and has been increasing in recent years. Especially among contemporary young people, the fast pace of life, increased psychological stress, and preference for spicy, greasy, and other irritating foods further exacerbate the risk of this disease.

[0003] Currently, clinical medications for oral ulcers mainly fall into two categories: topical and systemic drugs. Commonly used topical preparations include corticosteroids (such as triamcinolone acetonide oral ointment) and compound antibacterial-anti-inflammatory agents (such as compound chlorhexidine dexamethasone film). Although these drugs can relieve symptoms and promote healing to some extent, they generally have strong local irritation and long-term use may cause mucosal atrophy or dysbiosis, limiting their clinical application.

[0004] Therefore, developing a novel therapeutic drug that is highly effective, safe, has few side effects, and can significantly promote the healing of oral ulcers has significant clinical implications and application prospects. In recent years, toxic peptides have attracted widespread attention due to their small molecular weight, stable structure, strong biological activity, high targeting, and low toxicity. Studies have shown that certain animal-derived toxic peptides possess multiple biological functions, including antibacterial, anti-inflammatory, antioxidant, cell proliferation-promoting, and tissue repair effects, demonstrating good pharmaceutical potential in metabolic diseases, infectious diseases, and neurodegenerative diseases. However, there are currently few reports on the effects of toxic animal-derived peptides on promoting oral ulcer healing. This invention aims to provide a peptide molecule, NPTX3, identified from the venom glands of the spider *Nephilaclavata*, that has activity in promoting oral ulcer healing. Summary of the Invention

[0005] The first objective of this invention is to provide an active polypeptide NPTX3 that promotes the healing of oral ulcers, and the second objective of this invention is to provide the application of the polypeptide.

[0006] The first objective of this invention is achieved by providing an active polypeptide NPTX3 that promotes oral ulcer healing, having the amino acid sequence DLKPIAEDIPSLEK as shown in SEQ ID NO:1.

[0007] The second objective of this invention is achieved by using the polypeptide in the preparation of a drug that promotes the healing of oral ulcers.

[0008] A drug that promotes the healing of oral ulcers, wherein the drug has NPTX3 as its active ingredient.

[0009] The beneficial effects of this invention are as follows: This invention provides an active polypeptide NPTX3 derived from the venom glands of the spider *Nephila clavata*, which exhibits significant bioactivity in promoting oral ulcer healing. In vitro experiments show that NPTX3 significantly promotes scratch closure of human immortalized keratinocytes at concentrations as low as 500 pM. Transwell migration assays further confirm that NPTX3 at concentrations of 1 nM and 10 nM significantly enhances the migration ability of HaCaT cells, suggesting that it may accelerate wound repair by promoting epithelial cell migration. In an SD rat oral ulcer model, topical application of NPTX3 (1 nM, 10 nM) significantly improved the ulcer healing rate. In summary, the NPTX3 provided by this invention exhibits excellent cell migration and tissue repair activity at extremely low doses, possessing advantages such as low effective dose, strong bioactivity, and high safety, providing a highly effective and gentle new option for the drug treatment of oral ulcers. Attached Figure Description

[0010] Figure 1 shows the effect of the active peptide NPTX3 of the present invention on hemolytic activity, where Figure A shows the hemolysis situation, Figure B shows the quantitative hemolytic activity of peptide NPTX3; Figure C shows the quantitative effect of active peptide NPTX3 on HaCaT cell cytotoxicity; Figure 2 shows the effect of active peptide NPTX3 of the present invention on HaCaT cell scratch healing; the left figure shows the scratch healing situation of cells under peptide treatment, and the right figure shows the quantitative effect of scratch healing of cells under peptide treatment; Figure 3 shows the effect of active peptide NPTX3 of the present invention on HaCaT cell migration ability (Transwell experiment); the left figure shows the macroscopic display of cell migration, and the right figure shows the quantitative effect of peptide-induced cell migration; Figure 4 shows the effect of active peptide NPTX3 of the present invention on wound healing rate in SD rat oral ulcer model on day 7 after drug administration; the top figure shows the oral ulcer healing in rats treated with peptide, and the bottom figure shows the quantitative effect of oral ulcer healing in rats treated with peptide. Detailed Implementation

[0011] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments, but this does not limit the present invention in any way. Any modifications or improvements made based on the teachings of the present invention shall fall within the protection scope of the present invention.

[0012] Example 1 Synthesis of polypeptide NPTX3 The polypeptide NPTX3 was identified from the venom gland of *Gnaphalium affine*, and its amino acid sequence is DLKPIAEDIPSLEK, corresponding to SEQ ID No: 1. It was prepared by chemical solid-phase synthesis by Wuhan Baiyixin Biotechnology Co., Ltd.

[0013] Example 2: In vitro hemolytic activity and cytotoxicity detection of peptide NPTX3 I. Experimental Methods Male SD rats weighing 200±20g were selected. Based on the rat's weight, 0.4% chloral hydrate solution was injected intraperitoneally, inducing a deep coma. The eyeballs were quickly removed with forceps, and blood was rapidly dripped into an anticoagulant tube. 1ml of physiological saline was added to a 1.5ml centrifuge tube, followed by the blood. The tube was centrifuged at 3000rpm for 5 minutes at 4℃, and the supernatant was discarded. This process was repeated three times to obtain erythrocyte sediment. Each tube was resuspended in 1ml of physiological saline, and then 200µL of 0.1% Triton X-100 and different concentrations of peptide (100pM-100µM; all peptides used in this application were prepared with phosphate buffer) were added sequentially. Physiological saline was used as the negative control group, 0.1% Triton X-100 was used as the positive control group, and different concentrations of peptide NPTX3 groups (100pM-100µM) were incubated in a water bath at 37℃ for 30 min. 100µL of the supernatant was taken and the absorbance of each reaction well was measured at 540nm and the data were statistically analyzed.

[0014] HaCaT cells in logarithmic growth phase were digested with trypsin, centrifuged for 5 min at 1000 rpm, and then resuspended in fresh DMEM / F12 medium containing 10% fetal bovine serum (FBS). The cells were then cultured at a rate of 1 × 10⁻⁶ cells / mL. 4 Cells were seeded into 96-well plates with 100 µL of cell suspension per well. After seeding, the plates were gently shaken to mix thoroughly and incubated at 37°C with 5% CO2 for 4 hours until the cells adhered to the plate. The culture medium was then removed, and fresh DMEM / F12 medium (without fetal bovine serum) and NPTX3 (100 pM-1 µM) were added to each well. The plates were then incubated at 37°C with 5% CO2 for 24 hours. Fresh DMEM / F12 medium (without fetal bovine serum) and MTS reagent were mixed at a 9:1 ratio, and 100 µL of the prepared solution was added to each well. The plates were then incubated at 37°C with 5% CO2 for 2-4 hours in the dark. The absorbance of each well was then measured at 490 nm, and the data were statistically analyzed.

[0015] II. Results As shown in Figure 1A, at concentrations of 100 pM to 100 µM, there was no statistically significant difference compared to the negative control group, indicating that the active peptide NPTX3 had no hemolytic activity. As shown in Figure 1B, at concentrations of 100 pM to 1 µM, the cell activity of the active peptide NPTX3 was not statistically different from that of the PBS control group, indicating no cytotoxicity.

[0016] Example 3. Detection of the In Vitro Scratch Repair Activity of Peptide NPTX3 in HaCaT Cells I. Experimental Methods HaCaT cells in logarithmic growth phase were digested with trypsin and then suspended in fresh DMEM / F12 medium containing 10% fetal bovine serum (FBS). Cells were cultured at a rate of 2 × 10⁻⁶ cells / year. 5 Cells were seeded into 24-well plates at a ratio of 500 µL of cell suspension per well. After seeding, the plates were gently shaken to mix thoroughly and then incubated at 37°C with 5% CO2 until the cells reached monolayer confluence. Once the cells reached monolayer confluence, a clean 200 µL pipette tip was used to create a scratch on the bottom of each well from top to bottom. The liquid in the wells was then gently discarded, and the cells were washed with PBS buffer three times to remove detached cells and culture medium components. After washing, fresh DMEM / F12 medium without FBS was added to each well, resulting in four groups: blank control, positive control, 100 pM peptide group, 500 pM peptide group, and 1 nM peptide group. Sterile PBS, 100 ng / ml fibroblast growth factor (FGF), and different concentrations of peptide were added to the corresponding wells. After gentle mixing, the culture conditions were restored, and the wells were placed in a cell culture incubator for further incubation. After scratching, the changes in scratches in each well were recorded at 0h and 48h to assess the cell repair activity of the peptide. Microscopic photography was used during recording to obtain clear images of the scratches, enabling accurate comparison of cell migration and repair in subsequent analysis.

[0017] II. Results As shown in Figure 2, the active peptide NPTX3 at both 500 pM and 1 nM concentrations significantly promoted the healing of HaCaT cell scratches compared to the blank control group (PBS), with statistically significant differences (*p<0.01). The healing-promoting effect of NPTX3 at 1 nM concentration was higher than that at 500 pM, and at the 1 nM concentration, it had a similar cell healing rate to the positive control drug FGF.

[0018] Example 4. Detection of the ability of NPTX3 to promote the migration of HaCaT cells (Transwell assay) I. Experimental Methods The Transwell migration assay was used to detect the ability of the peptide to promote the migration of human immortalized keratinocytes at concentrations of 1nM-100nM. The experiment was divided into three groups: a blank control group (sterile PBS), a positive control group (FGF 100 ng / ml), and various peptide concentration groups (1nM-100nM). Logarithmic-phase human immortalized keratinocytes were digested with trypsin and resuspended in fresh DMEM / F12 medium without FBS at a concentration of 1×10⁻⁶. 6 The cells were seeded in the upper chamber of a Transwell plate, while the lower chamber was filled with DMEM / F12 medium containing 10% FBS and NPTX3 (1 nM-100 nM). The plate was then incubated at 37°C with 5% CO2 for 48 hours. After 48 hours, the upper chamber was removed and fixed with 4% paraformaldehyde for 20 minutes, followed by staining with 1 mg / ml crystal violet for 20 minutes. The upper chamber was washed three times with PBS, and excess crystal violet was gently wiped away with a cotton swab. The cells were then photographed under a 10x magnification microscope. After photographing, the crystals were dissolved in 33% acetic acid solution. 100 µL of each sample was then transferred to a 96-well plate and the absorbance at 572 nm was measured using an ELISA reader. Data were then statistically analyzed.

[0019] II. Results Figure 3 shows that the active peptide NPTX3 significantly promoted HaCaT cell migration at both 1 nM and 10 nM concentrations compared to the blank control group (PBS), with statistical significance (*p<0.01). At a concentration of 1 nM, NPTX3 showed a similar cell migration-promoting efficiency to the positive control drug FGF.

[0020] Example 5: Detection of the Activity of Active Peptide NPTX3 in Promoting Oral Ulcer Healing in Rats I. Experimental Methods An animal model of oral ulcers was induced using the 50% acetic acid etching method to detect the pro-oral ulcer healing activity of peptide NPTX3. Twenty-five male SD rats weighing 200±20g were selected. Circular filter paper pieces of 1mm size were cut and soaked in 50% glacial acetic acid. The filter paper was then placed on the lower lip mucosa of the rats for 30 seconds to establish an oral ulcer model. After modeling, the rats were divided into 5 groups of 5 rats each: blank control (PBS group), positive control (Kangfuxin liquid group), and low, medium, and high concentration peptide groups (NPTX3: 100pM, 1nM, 10nM). The low, medium, and high concentration groups of peptides were given 50µL of NPTX3 (100pM, 1nM, and 10nM), respectively. The blank control group was given an equal volume of sterile PBS, and the positive control group was given an equal volume of Kangfuxin original solution (KFX, manufacturer: Sichuan Good Doctor Panxi Pharmaceutical Co., Ltd.). The drugs were administered topically once in the morning and once in the evening for 7 consecutive days. The oral ulcer area of ​​the model SD rats in each group was recorded on the 3rd, 5th, and 7th days after administration.

[0021] II. Results (Figure 4) show that the active peptide NPTX3, at concentrations of 1 nM and 10 nM, significantly promoted the healing of oral ulcers in rats compared to the blank control (PBS) group, with statistical significance (*p<0.01). At concentrations of 1 nM and 10 nM, it showed similar healing-promoting efficiency to the positive control drug Kangfuxin liquid.

Claims

1. NPTX3, an active polypeptide that promotes oral ulcer healing, characterized in that... The amino acid sequence of the polypeptide NPTX3 is: DLKPIAEDIPSLEK.

2. The use of the polypeptide NPTX3 of claim 1 in the preparation of a drug to promote the healing of oral ulcers.

3. A drug for promoting the healing of oral ulcers, characterized in that, The drug uses the polypeptide NPTX3 described in claim 1 as its active ingredient.

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