An active polypeptide NP-VV-3 that promotes oral ulcer healing and its applications

Abstract

This invention discloses an active polypeptide NP-VV-3 that promotes oral ulcer healing and its applications, belonging to the field of biomedical technology. The amino acid sequence of the active polypeptide NP-VV-3 is: VVPAFVLLKKAIRIMFKRNC. In in vitro experiments, polypeptide NP-VV-3 significantly promoted scratch closure of human immortalized keratinocytes at concentrations as low as 1 nM, demonstrating a dose-dependent healing-promoting ability. Transwell migration assays further confirmed that polypeptide NP-VV-3 significantly enhanced the migration ability of HaCaT cells at concentrations of 1 nM and 10 nM, indicating that it can accelerate wound repair by promoting epithelial cell migration. The polypeptide NP-VV-3 provided by this invention provides an important candidate molecule for the development of novel, highly effective, and low-irritation drugs for the treatment of oral ulcers.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an active polypeptide NP-VV-3 that promotes the healing of oral ulcers and its applications. Background Technology

[0002] Oral ulcers are a common oral mucosal disease that causes pain and discomfort. Current treatments are mostly symptomatic, with limited effectiveness and high recurrence rates, impacting quality of life. According to a study based on the U.S. National Health and Nutrition Examination Survey, the occurrence of oral ulcers is significantly associated with age, sex, household income, hyperlipidemia, depression, smoking, and alcohol consumption. Oral ulcers can occur in people of all ages, with a prevalence of 5-20%. Clinically, the management of oral ulcers still faces many challenges. Although various treatment options are available, including topical medications, systemic drugs, and biologics, there is currently no universally accepted targeted therapy. Commonly used medications include topical treatments such as the antibiotic chlorhexidine, chlorhexidine-dexamethasone compound film, and dexamethasone patches. These drugs are effective, but their side effects include local irritation and fungal infections. Therefore, the development of new, more effective, safer, and less side-effect-promoting drugs for oral ulcer healing is urgently needed.

[0003] This invention aims to provide a method derived from the Yunnan frog ( Nidirana pleuraden NP-VV-3, a polypeptide for the skin, has excellent activity in promoting the healing of oral ulcers. Summary of the Invention

[0004] The first objective of this invention is to provide a polypeptide with activity that promotes the healing of oral ulcers, and the second objective of this invention is to provide applications of said polypeptide.

[0005] The first objective of this invention is achieved by providing an active polypeptide NP-VV-3 that promotes oral ulcer healing, having the amino acid sequence shown in SEQ ID NO:1: VVPAFVLLKKAIRIMFKRNC.

[0006] The second objective of this invention is achieved by using the polypeptide in the preparation of a medicament for treating oral ulcers.

[0007] The beneficial effects of this invention are as follows:

[0008] The polypeptide NP-VV-3 provided in this invention is derived from the skin of the Yunnan frog and has shown strong activity in promoting oral ulcer healing in both cellular and animal experiments. In vitro experiments showed that NP-VV-3 at concentrations as low as 1 nM significantly promoted scratch closure of human immortalized keratinocytes. Transwell cell migration assays further confirmed that both 1 nM and 10 nM of NP-VV-3 significantly enhanced the migration ability of HaCaT cells, indicating that NP-VV-3 can accelerate wound repair by promoting epithelial cell migration. In an SD rat oral ulcer model, local administration of 1 nM, 10 nM, and 100 nM of NP-VV-3 significantly improved the ulcer healing rate, showing a concentration-dependent trend. These results indicate that the polypeptide NP-VV-3 of this invention has low toxicity and high safety, providing an important candidate molecule for the development of novel, highly effective, and low-irritation drugs for the treatment of oral ulcers. Attached Figure Description

[0009] Figure 1 This is a quantitative diagram of the cytotoxicity of the peptide NP-VV-3 against HaCaT cells;

[0010] Figure 2 The following diagram illustrates the effect of the present invention's polypeptide NP-VV-3 on HaCaT cell scratch healing: the left diagram shows the cell scratch healing status, and the right diagram shows the quantitative analysis of cell scratch healing.

[0011] Figure 3 The following figures show the detection results of the ability of the polypeptide NP-VV-3 of the present invention to promote the migration of HaCaT cells: the left figure is a macroscopic diagram of cell migration, and the right figure is a quantitative diagram of cell migration.

[0012] Figure 4 The following figures illustrate the healing effect of the NP-VV-3 polypeptide of this invention on the oral ulcer model in SD rats on day 7 after drug administration: the left figure shows the healing of oral ulcers in rats, and the right figure shows the quantitative analysis of oral ulcer healing in rats. Detailed Implementation

[0013] 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.

[0014] This invention discloses a polypeptide NP-VV-3 with activity promoting oral ulcer healing. The polypeptide NP-VV-3 is derived from the skin of the Yunnan frog, and its amino acid sequence is: VVPAFVLLKKAIRIMFKRNC.

[0015] The present invention also provides the application of the polypeptide NP-VV-3 in the preparation of a drug for treating oral ulcers.

[0016] The drug uses polypeptide NP-VV-3 as its active ingredient.

[0017] Example 1 Synthesis of polypeptide NP-VV-3

[0018] The amino acid sequence of the polypeptide NP-VV-3 is VVPAFVLLKKAIRIMFKRNC. It was synthesized by Wuhan Baiyixin Biotechnology Co., Ltd. using chemical solid-phase synthesis.

[0019] Example 2: In vitro cytotoxicity assay of peptide NP-VV-3

[0020] I. Experimental Methods

[0021] 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 h until the cells adhered to the plate. The culture medium was then removed, and fresh DMEM / F12 medium (without fetal bovine serum) and different concentrations of NP-VV-3 (100 pM-1 µM) were added to each well. The plates were then incubated at 37°C with 5% CO2 for 24 h. Subsequently, 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 h in the dark. The absorbance of each well was then measured at 490 nm and the data were statistically analyzed.

[0022] II. Results

[0023] from Figure 1 It can be seen that at concentrations of 100 pM-1 µM, the NP-VV-3 peptide showed no statistically significant difference in cell activity compared to the PBS group, indicating no cytotoxicity.

[0024] Example 3: Detection of the in vitro scratch repair activity of peptide NP-VV-3 in HaCaT cells

[0025] I. Experimental Methods

[0026] 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 × 10⁶ cells / day. 5Cells 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 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 blank control, positive control, 1 nM peptide group, 10 nM peptide group, and 100 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 0 h and 48 h 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.

[0027] II. Results

[0028] from Figure 2 It can be seen that, compared with the control group PBS, the peptide NP-VV-3 at concentrations of 1 nM and 10 nM can significantly promote the healing of HaCaT cell scratches. The healing-promoting effect of peptide NP-VV-3 at a concentration of 1 nM is higher than that at 10 nM.

[0029] Example 4: Detection of the ability of peptide NP-VV-3 to promote HaCaT cell migration

[0030] The Transwell migration assay was used to detect the ability of peptides at concentrations ranging from 1 nM to 100 nM to promote the migration of human immortalized keratinocytes. The experiment consisted of three groups: a blank control group (sterile PBS), a positive control group (FGF 100 ng / ml), and peptide concentration groups (1 nM–100 nM). Log-phase human immortalized keratinocytes were digested with trypsin and resuspended in fresh DMEM / F12 medium without FBS at a concentration of 1 × 10⁻⁶. 6The 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 NP-VV-3 (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.

[0031] like Figure 3 As shown, the peptide NP-VV-3 at concentrations of 1 nM and 10 nM significantly promoted the migration of HaCaT cells compared to the blank control group, and the peptide NP-VV-3 had a similar migration-promoting efficiency to the positive drug FGF.

[0032] Example 5: Detection of the wound healing activity of peptide NP-VV-3 in an SD rat oral ulcer model.

[0033] I. Experimental Methods

[0034] The activity of peptide NP-VV-3 was assessed by evaluating the healing rate of an oral ulcer model in SD rats.

[0035] The oral ulcer-promoting activity of peptide NP-VV-3 in promoting oral ulcer healing was detected using an animal model induced by 50% acetic acid etching. Twenty-five male SD rats weighing 200±20 g were selected. Circular filter paper pieces (1 mm in 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 five groups of five: a blank control group (PBS group), a positive control group (Kangfuxin liquid group), and low, medium, and high concentration peptide groups (NP-VV-3: 1 nM, 10 nM, and 100 nM). The low, medium, and high concentration groups of the polypeptide were given 50 µL of NP-VV-3 (1 nM, 10 nM, and 100 nM), 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, and the oral ulcer area of ​​each group of model SD rats was recorded synchronously for 7 consecutive days.

[0036] II. Results

[0037] like Figure 4As shown, the peptide NP-VV-3, at concentrations of 1 nM, 10 nM, and 100 nM, significantly promoted the healing of oral ulcers in rats compared to the blank control group, with statistical significance (*p<0.01). Its healing-promoting efficiency was similar to that of the positive control drug, Kangfuxin Liquid.

Claims

1. A polypeptide NP-VV-3 with activity promoting oral ulcer healing, characterized in that, The amino acid sequence of the polypeptide NP-VV-3 is: VVPAFVLLKKAIRIMFKRNC.

2. The use of the polypeptide NP-VV-3 of claim 1 in the preparation of a drug for treating oral ulcers.

3. A medicine for treating oral ulcers, characterized in that, The drug uses the polypeptide NP-VV-3 described in claim 1 as its active ingredient.

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