Application of pilose antler polypeptide CNT14 in preparing medicine for promoting oral mucosa wound healing

Abstract

The present invention discloses the application of pilose antler polypeptide CNT14 in the preparation of a drug for promoting the healing of oral mucosal wounds. It is found through research that pilose antler polypeptide CNT14 can significantly promote the proliferation and migration of oral mucosal keratinocytes and oral mucosal fibroblasts in vitro, and can significantly promote the healing of rabbit oral mucosal wounds in vivo. The present invention not only broadens the application scope of pilose antler polypeptide CNT14, but also provides a new candidate drug for promoting the healing of oral mucosal wounds clinically.

Description

Technical Field

[0001] The present invention belongs to the technical field of the application of bioactive polypeptides, and particularly relates to the application of pilose antler polypeptide CNT14 in the pharmaceutical field. Background Art

[0002] Clinically, oral mucosal trauma may be caused by factors such as infection, trauma, periodontal disease, and autoimmune diseases, affecting people's appearance and functions such as swallowing and chewing. As a complex bacterium-containing environment, the oral cavity, if the wound cannot heal effectively after injury, will lead to the exposure of the wound, thereby increasing the risk of wound infection. Therefore, high-quality healing of oral mucosal trauma is a prerequisite for the success of various surgical techniques in the oral cavity. At present, for the soft tissue defects caused by oral mucosal trauma in clinical practice, the most commonly used method is mainly autologous soft tissue transplantation, but this will cause secondary trauma, increase the pain of patients, and cannot well restore the original anatomical shape. Therefore, finding effective technical means to promote the healing of oral mucosal trauma has become an urgent need in clinical practice.

[0003] Pilose antler is the unossified antler with velvet of male sika deer or red deer, and has been used in the field of traditional Chinese medicine in China since ancient times, with a usage time of more than two thousand years. Pilose antler polypeptides (PAP) are one of the main active components of pilose antler. In recent years, it has been found that they have pharmacological effects such as inhibiting tumor cells, improving memory disorders, promoting the regeneration of nervous system tissues, myocardial protection, and immune enhancement. A researcher isolated a monomer polypeptide CNT14 from sika deer antler, and its amino acid sequence is EPTVLDEVCLAHGP. The results of biological activity research show that this polypeptide can significantly promote the growth of mouse hippocampal nerve cells, indicating that it may have certain prospects in anti-aging drugs; at the same time, this polypeptide has a proliferative effect on epidermal cells, costal chondrocytes, and spleen cells, inferring that it may be a cell growth factor. Summary of the Invention

[0004] The purpose of the present invention is to investigate the effect of pilose antler polypeptide CNT14 on the healing of oral mucosal trauma, so as to develop its new use in the pharmaceutical field.

[0005] The results of biological activity research of the present invention show that pilose antler polypeptide CNT14 can significantly promote the proliferation and migration of oral mucosal keratinocytes and oral mucosal fibroblasts in vitro; in vivo, it can significantly promote the healing of rabbit oral mucosal trauma; indicating that pilose antler polypeptide CNT14 can be used to prepare drugs for promoting the healing of oral mucosal trauma.

[0006] For this reason, the present invention provides the following technical solution:

[0007] Use of pilose antler polypeptide CNT14 in the preparation of drugs for promoting the healing of oral mucosal trauma.

[0008] Furthermore, the drug for promoting oral mucosal wound healing is a drug that promotes the proliferation and migration of oral mucosal keratinocytes and oral mucosal fibroblasts.

[0009] The beneficial effects of the present invention are as follows: The present invention provides the application of pilose antler polypeptide CNT14 in the preparation of a drug for promoting oral mucosal wound healing, which not only broadens the application scope of pilose antler polypeptide CNT14, but also provides a new candidate drug for promoting oral mucosal wound healing clinically. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 It is a bar graph of cell proliferation after pilose antler polypeptide CNT14 stimulates normal human oral mucosal fibroblasts hOMF.

[0011] Figure 2 It is a bar graph of cell proliferation after pilose antler polypeptide CNT14 stimulates normal human oral mucosal keratinocytes hOK.

[0012] Figure 3 It is a result graph of the scratch test after 50 μg / ml pilose antler polypeptide CNT14 stimulates hOK cells.

[0013] Figure 4 It is a result graph of the scratch test after 50 μg / ml pilose antler polypeptide CNT14 stimulates hOMF cells.

[0014] Figure 5 It is a bar analysis graph of the scratch test result after pilose antler polypeptide CNT14 stimulates hOK cells.

[0015] Figure 6 It is a bar analysis graph of the scratch test result after pilose antler polypeptide CNT14 stimulates hOMF cells.

[0016] Figure 7 It is a result graph of pilose antler polypeptide CNT14 promoting oral mucosal wound healing in Japanese white rabbits in a parallel control experiment.

[0017] Figure 8 It is a result graph of pilose antler polypeptide CNT14 promoting oral mucosal wound healing in Japanese white rabbits in a self-control experiment.

[0018] Figure 9 It is a line graph of the effect of pilose antler polypeptide CNT14 on the oral mucosal wound healing rate in Japanese white rabbits in a parallel control experiment.

[0019] Figure 10 It is a line graph of the effect of pilose antler polypeptide CNT14 on the oral mucosal wound healing rate in Japanese white rabbits in a self-control experiment. DETAILED DESCRIPTION OF THE INVENTION

[0020] In order to make the objectives, technical solutions and beneficial effects of the present invention clearer, the preferred embodiments of the present invention will be described in detail below.

[0021] The pilose antler polypeptide CNT14 (amino acid sequence: EPTVLDEVCLAHGP, SEQ ID No. 1) used in the embodiments of the present invention was purchased from Nanjing Jie Peptide Biotechnology Co., Ltd.; normal human oral mucosa fibroblasts hOMF (Normal Human Oral Mucosa (P3) 500K fibroblast cells) and normal human oral mucosa keratinocytes hOK (Normal Human Oral Mucosa (P2) 500K Keratinocytes cells) were both purchased from Cell Research Corp in Singapore; the experimental animal Japanese white rabbits were purchased from the Experimental Animal Department of Kunming Medical University.

[0022] I. Effects of pilose antler polypeptide CNT14 on the proliferation of oral mucosa keratinocytes and oral mucosa fibroblasts

[0023] The CCK-8 method was used to detect the effects of pilose antler polypeptide CNT14 on the proliferation of hOMF and hOK cells. hOMF and hOK in the logarithmic growth phase were respectively inoculated into 96-well plates, pre-cultured in an incubator at 37°C and 5% CO2 for 24 h, and an aqueous solution of pilose antler polypeptide CNT14 at 100 μg / ml was added to make the final concentrations of CNT14 be 10, 20, 50, 80, and 100 μg / ml respectively. At the same time, control wells and blank wells were set. After treatment for different times (12, 24, 48 h), CCK-8 reagent was added, and the mixture was incubated at room temperature for 2 h. The absorbance value of the cells was detected with an enzyme-labeled instrument at 450 nm.

[0024] The column chart of cell proliferation after stimulation of hOMF by pilose antler polypeptide CNT14 is as Figure 1 shown. Treatment with 50 μg / ml pilose antler polypeptide CNT14 for 24 h could significantly promote the proliferation of hOMF cells; treatment with 10 μg / ml, 50 μg / ml, and 80 μg / ml pilose antler polypeptide CNT14 for 48 h all showed obvious proliferation of hOMF cells; it indicates that treatment with pilose antler polypeptide CNT14 can promote the proliferation of hOMF cells.

[0025] The column chart of cell proliferation after stimulation of hOK by pilose antler polypeptide CNT14 is as Figure 2As shown in the figure, treating with 20 μg / ml and 50 μg / ml of pilose antler polypeptide CNT14 for 24 h can significantly promote the proliferation of hOK cells; treating with 10 μg / ml, 20 μg / ml, 50 μg / ml, 80 μg / ml, and 100 μg / ml of pilose antler polypeptide CNT14 for 48 h can all significantly promote the proliferation of hOK cells, especially in the 50 μg / ml and 80 μg / ml groups, with extremely significant effects; indicating that pilose antler polypeptide CNT14 can promote the proliferation of hOK cells.

[0026] II. Effects of Pilose Antler Polypeptide CNT14 on the Migration of Oral Mucosal Keratinocytes and Oral Mucosal Fibroblasts

[0027] The scratch assay was used to detect the effects of pilose antler polypeptide CNT14 on the migration of hOMF and hOK cells. hOMF and hOK cells in the logarithmic growth phase were seeded into 6-well plates respectively. After the cells were confluent, they were scratched, and the scratches were made as perpendicular as possible to the horizontal lines on the back. The scratched cells were rinsed 3 times with PBS, and an aqueous solution of 100 μg / ml pilose antler polypeptide CNT14 was added to make the final concentrations of CNT14 10, 20, 50, 80, and 100 μg / ml respectively. At the same time, control wells were set up. The cells were cultured in an incubator at 37 °C and 5% CO2. Samples of hOK cells were taken at 0, 12, 24, and 48 h respectively, and samples of hOMF cells were taken at 0, 24, 48, and 72 h respectively, and then photographed.

[0028] The results of the scratch assay after stimulating hOK cells with 50 μg / ml of pilose antler polypeptide CNT14 are shown as Figure 3 in the figure. It can be seen that treating with 50 μg / ml of pilose antler polypeptide CNT14 can promote the migration of hOK cells. The column analysis chart of the results of the scratch assay after stimulating hOK cells with pilose antler polypeptide CNT14 is shown as Figure 5 in the figure. After treating with 10 μg / ml, 20 μg / ml, and 50 μg / ml of pilose antler polypeptide CNT14 for 24 h, the migration rate of hOK cells increased significantly; after treating with 10 μg / ml, 20 μg / ml, 50 μg / ml, 80 μg / ml, and 100 μg / ml of pilose antler polypeptide CNT14 for 48 h, the migration of hOK cells could be significantly promoted, especially in the 50 μg / ml, 80 μg / ml, and 100 μg / ml groups, with extremely significant effects; indicating that treating with pilose antler polypeptide CNT14 can promote the migration of hOK cells.

[0029] The results of the scratch assay after stimulating hOMF cells with 50 μg / ml of pilose antler polypeptide CNT14 are shown as Figure 4 in the figure. It can be seen that treating with 50 μg / ml of pilose antler polypeptide CNT14 can promote the migration of hOMF cells. The column analysis chart of the results of the scratch assay after stimulating hOMF cells with pilose antler polypeptide CNT14 is shown as Figure 6As shown, when hOMF cells were treated with pilose antler polypeptide CNT14 at 20 μg / ml, 50 μg / ml, 80 μg / ml, and 100 μg / ml for 24 h, the migration rate of hOMF cells increased significantly; when treated with pilose antler polypeptide CNT14 at 20 μg / ml, 50 μg / ml, and 100 μg / ml for 48 h, the migration of hOMF cells was significantly promoted; when treated with pilose antler polypeptide CNT14 at 10 μg / ml, 20 μg / ml, 50 μg / ml, and 100 μg / ml for 72 h, the migration of hOK cells was also significantly promoted; especially in the 50 μg / ml group, when treated for 24, 48, and 72 h, the promotion of hOMF cell migration was extremely significant; it shows that the treatment with pilose antler polypeptide CNT14 can promote the migration of hOK cells.

[0030] III. Effects of Pilose Antler Polypeptide CNT14 on the Wound Healing of Rabbit Oral Mucosa

[0031] An oral mucosa wound model was constructed in Japanese white rabbits to observe the effects of pilose antler polypeptide CNT14 on the wound healing of rabbit oral mucosa. After the rabbits were anesthetized, a full-thickness flap of soft tissue with an area of about 0.5×0.5 cm was excised from the labial mucosa distal to the maxillary central incisors on the left (or right) side of each rabbit's oral cavity to construct an oral mucosa wound model; then 12 rabbits were randomly divided into two groups according to a parallel control experiment, with 6 as the control group and 6 as the experimental group; the 6 rabbits in the control group were used for a self-control experiment, with the left side as the control group and the right side as the experimental group; 0.5 ml of 100 μg / ml pilose antler polypeptide CNT14 aqueous solution was injected around the wound of the rabbits in the experimental group, and 0.5 ml of triple-distilled water was injected around the wound of the rabbits in the control group. After the experiment, the activities and feeding conditions of the rabbits were closely observed, and the healing of the wound was recorded with a camera. Photos were taken on the 3rd, 5th, 7th, 9th, and 11th days respectively, and the taken photos were processed with Image J software to calculate the wound healing rate and draw a line graph of the wound healing changes. 2 The results of the parallel control experiment on the promotion of rabbit oral mucosa wound healing by pilose antler polypeptide CNT14 are as shown in and. In the parallel control experiment, pilose antler polypeptide CNT14 could significantly promote the healing of rabbit oral mucosa wounds on the 3rd, 5th, 7th, 9th, and 11th days.

[0032] The results of the self-control experiment on the promotion of rabbit oral mucosa wound healing by pilose antler polypeptide CNT14 are as shown in and. In the self-control experiment, pilose antler polypeptide CNT14 could significantly promote the healing of rabbit oral mucosa wounds on the 3rd, 5th, 7th, 9th, and 11th days. Figure 7 and Figure 9 As shown, in the parallel control experiment, pilose antler polypeptide CNT14 could significantly promote the healing of rabbit oral mucosa wounds on the 3rd, 5th, 7th, 9th, and 11th days.

[0033] The results of the self-control experiment on the promotion of rabbit oral mucosa wound healing by pilose antler polypeptide CNT14 are as shown in and. Figure 8 and Figure 10 As shown, in the self-control experiment, pilose antler polypeptide CNT14 could significantly promote the healing of rabbit oral mucosa wounds on the 3rd, 5th, 7th, 9th, and 11th days.

[0034] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention rather than to limit them. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present invention. Sequence Listing <110> Kunming Stomatological Hospital Affiliated to Kunming Medical University <120> Application of Pilose Antler Polypeptide CNT14 in the Preparation of a Drug for Promoting Oral Mucosal Wound Healing <160> 1 <170> SIPOSequenceListing 1.0 <210> 1 <211> 14 <212> PRT <213> Sika deer (Cervus nippon Temminck) <400> 1 Glu Pro Thr Val Leu Asp Glu Val Cys Leu Ala His Gly Pro 1 5 10

Claims

1. Use of pilose antler polypeptide CNT14 in the preparation of a drug for promoting oral mucosa wound healing, wherein the amino acid sequence of the pilose antler polypeptide CNT14 is EPTVLDEVCLAHGP.

2. The application according to claim 1, wherein The drug for promoting oral mucosa wound healing is a drug for promoting the proliferation and migration of oral mucosa keratinocytes and oral mucosa fibroblasts.

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