Active polypeptide Tigerin-OL1 capable of promoting healing of oral ulcer and application of active polypeptide Tigerin-OL1

Abstract

The invention discloses an active polypeptide Tigerin-OL1 capable of promoting healing of oral ulcer and application of the active polypeptide Tigerin-OL1. The active polypeptide Tigerin-OL1 capable of promoting healing of the oral ulcer has an amino acid sequence ICTMIPISRCY as shown in SEQ ID No.1 (sequence identifier number 1). The application of the polypeptide Tigerin-OL1 is the application of the polypeptide Tigerin-OL1 in preparation of drugs for promoting healing of oral ulcer. The polypeptide Tigerin-OL1 provided by the invention has excellent oral ulcer healing promoting activity in vivo and in vitro, only contains 11 amino acids and is easy to synthesize, and a new way is provided for development of drugs for promoting oral ulcer healing.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to an oral ulcer healing promoting active polypeptide Tigerin-OL1 and an application thereof. Background Art

[0002] Oral ulcers are a very common oral disease that not only causes pain to patients, but also interferes with normal physiological activities, such as eating and speaking. Oral ulcers can occur in people of different ages, and according to surveys, the prevalence rate is 5% to 20%.

[0003] The current drugs for treating oral ulcers include: analgesic, anti-inflammatory and hormone drugs, such as lidocaine gel, compound chlorhexidine dexamethasone film and prednisone acetate tablets. However, these drugs have limited effects, and some require intravenous injection or frequent administration, which may accelerate tumor growth. Therefore, it is particularly important to develop and identify new oral ulcer healing drugs that are efficient, safe and have few side effects. Peptides have attracted much attention because of their low molecular weight, high specificity, strong activity, few side effects, and multiple functions such as hypoglycemic, antibacterial, antioxidant, and neuroprotective. However, there are currently few reports on animal-derived oral ulcer healing peptides.

[0004] The present invention aims to provide a safe and efficient active peptide Tigerin-OL1 derived from Rana maritima for promoting the healing of oral ulcers. Summary of the invention

[0005] The first purpose of the present invention is to provide an active polypeptide Tigerin-OL1 that promotes oral ulcer healing, and the second purpose of the present invention is to provide the application of the polypeptide.

[0006] The first object of the present invention is achieved in that the oral ulcer healing promoting active polypeptide Tigerin-OL1 has an amino acid sequence as shown in SEQ ID No. 1: ICTMIPISRCY.

[0007] The second object of the present invention is achieved in that the polypeptide Tigerin-OL1 is used in the preparation of a drug for promoting oral ulcer healing.

[0008] The beneficial effects of the present invention are as follows: the polypeptide Tigerin-OL1 provided by the present invention has excellent oral ulcer healing activity both in vivo and in vitro, and the polypeptide has only 11 amino acids and is easy to synthesize, thus providing a new approach for the development of drugs for promoting oral ulcer healing. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1The results of the test of the ability of peptide Tigerin-OL1 to promote the migration of rat oral keratinocytes; Figure A is the migration of rat oral keratinocytes under electron microscope, and Figure B is the migration number of rat oral keratinocytes; Figure 2 The results of the in vivo and in vitro safety test of the peptide Tigerin-OL1; Figure A is the HE staining of important organs of rats, and Figure B is the effect of the peptide Tigerin-OL1 on cell viability; Figure 3 The results of the test of the effect of peptide Tigerin-OL1 on HaCat cell scratch healing; Figure A is a diagram of cell scratch healing, and Figure B is a quantitative diagram of cell scratch healing; Figure 4 These are graphs showing the wound healing effects of oral ulcers in modeled SD rats. Graph A shows the healing effects of oral ulcers in rats treated with peptides, and Graph B shows the quantitative healing of oral ulcers in multiple rats. DETAILED DESCRIPTION

[0010] The present invention will be further described in detail below in conjunction with 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 protection scope of the present invention.

[0011] The invention provides an oral ulcer healing promoting active polypeptide Tigerin-OL1, whose amino acid sequence is: ICTMIPISRCY.

[0012] The polypeptide Tigerin-OL1 is screened from the cDNA library of Rana maritima and then prepared by conventional chemical synthesis or gene expression.

[0013] The polypeptide Tigerin-OL1 of the present invention is synthesized by Wuhan Baiyixin Biotechnology Co., Ltd.

[0014] The present invention also provides the use of the polypeptide Tigerin-OL1 in preparing a drug for promoting oral ulcer healing.

[0015] The present invention further provides a pharmaceutical composition, which comprises the polypeptide Tigerin-OL1 as an active agent and a pharmaceutically effective carrier.

[0016] Example 1 Identification of the ability of peptide Tigerin-OL1 to promote rat oral keratinocyte migration The Transwell migration assay was used to detect the ability of peptide Tigerin-OL1 to promote the migration of rat oral keratinocytes at a concentration of 100 pM~1 µM. The experiment was divided into two groups, a blank control group (sterile PBS) and peptide Tigerin-OL1 concentration groups (100 pM~1µM). The logarithmic phase rat oral keratinocytes were digested with trypsin and resuspended in fresh DMEM / F12 medium without FBS at a concentration of 1×10 6 / well was plated in the upper chamber of Transwell, and DMEM / F12 medium containing 10% FBS and peptide Tigerin-OL1 (100 pM~1 µM) were added to the lower chamber, and placed in a 37°C, 5% CO2 cell culture incubator for 24 hours. After 24 hours, the upper chamber was removed and fixed with 4% paraformaldehyde for 20 minutes, and then stained with 1 mg / ml crystal violet for 20 minutes. The upper chamber was washed 3 times with PBS, and the excess crystal violet was gently wiped with a cotton swab, and then photographed and recorded under an inverted microscope with a 10x field of view. After taking pictures, the crystals were dissolved with 33% acetic acid solution, and then 100 µL of the solution of each group of samples was aspirated and placed in a 96-well plate, and then placed in an enzyme-linked detector to detect the absorbance value of each reaction well at a wavelength of 572 nm and perform data statistics.

[0017] Results: Figure 1 As shown, the peptide Tigerin-OL1 can significantly promote keratinocyte migration.

[0018] Example 2 In vivo and in vitro safety testing of the peptide Tigerin-OL1 1. In vitro HaCaT cell MTS assay to evaluate the in vitro safety of active peptide Tigerin-OL1 HaCaT cells in the logarithmic growth phase were digested with trypsin, resuspended in fresh DMEM / F12 medium without FBS, and evenly plated in a 96-well plate at a density of 4000-8000 / well. After plating, they were placed in a 37-degree Celsius, 5% CO2 cell culture incubator for 4 hours until all cells were attached to the wall. They were divided into 6 groups, namely, blank control group, 100 pM peptide Tigerin-OL1 group, 1nM peptide Tigerin-OL1 group, 10nM peptide Tigerin-OL1 group, 100nM peptide Tigerin-OL1 group, and 1 µM peptide Tigerin-OL1 group. 10 µl PBS and peptide Tigerin-OL1 of each concentration were added to the corresponding wells, and the wells were returned to the cell culture incubator for continued culture for 24 hours. After 24 hours, 10µ Cell Titer96 was quickly added to each well under light-proof conditions. RAQucous One Solution reagent, gently mix and return to the cell culture incubator for 4 h to fully react. After the reaction is completed, place the 96-well plate in an enzyme-linked detector to detect the absorbance value of each reaction well at a wavelength of 490 nm. Figure 2 shown.

[0019] from Figure 2 As shown in Figure B, there was no statistical difference in the cell activity of each group, indicating that the peptide Tigerin-OL1 had no toxic effect on cells in vitro.

[0020] 2. HE staining of rat organs to evaluate the in vivo safety of active peptide Tigerin-OL1 The organs of SD rats in the normal group and the polypeptide group were fixed with 4% paraformaldehyde, and the organ tissues of the rats were analyzed by H&E staining. The organ tissues were first fixed in a fixative containing 4% paraformaldehyde (PFA) for 24 h. After fixation, the fixed tissues were dehydrated. Dehydration was performed with gradually increasing concentrations of ethanol (50%, 70%, 85%, 95% and 100%), with each step taking 1 h. Finally, xylene was used for transparent treatment and soaked in wax immersion solution to prepare for subsequent sectioning. After tissue embedding, the wax block was cut into slices with a thickness of about 5 μm using a microtome for HE staining. The slices were first dewaxed with xylene for 15 min each time, repeated twice. Then, 100%, 95%, 80%, and 70% ethanol were used for dehydration to rehydrate the tissue slices, followed by rinsing with deionized water. During the HE staining process, the slices are first stained with hematoxylin for 5-15 minutes, then rinsed to remove excess dye, and then immersed in differentiation solution for re-staining for 1-3 minutes, and then rinsed with deionized water until the excess dye is removed. After staining, dehydrate again with 70%, 80%, 95% and 100% ethanol, and finally transparent with xylene and resin sealing. Through the above steps, the prepared tissue sections can be used for optical microscopy observation to study its tissue structure and pathological changes from Figure 2 As shown in Figure A, the use of the peptide Tigerin-OL1 had no effect on the tissue structure of important organs and the cell arrangement morphology of rats in each group, indicating that the peptide has no toxic effect in the body.

[0021] In conclusion, the peptide Tigerin-OL1 is non-toxic both in vivo and in vitro.

[0022] Example 3 Detection of the activity of peptide Tigerin-OL1 in promoting scratch repair of HaCaT cells in vitro HaCaT cells in the logarithmic growth phase were digested by trypsin and then suspended in fresh DMEM / F12 medium containing 10% fetal bovine serum (FBS). The cells were seeded in a 24-well plate at a ratio of 2×10^5 cells / well, and 500 μL of cell suspension was added to each well. After seeding, they were gently shaken and placed in a cell culture incubator at 37 degrees Celsius and 5% CO2 for 10 h until the cells grew to a monolayer confluence state. When the cells reached monolayer confluence, a 200 μL clean pipette tip was used to evenly draw a straight line from top to bottom on the bottom of each well to create a scratch. Subsequently, the liquid in the well was gently discarded, and the cells in the well were washed with PBS buffer, and repeated 3 times to remove the detached cells and culture medium components. After washing, fresh DMEM / F12 medium without FBS was added to each well, which was divided into blank group, positive control group, 100 pM peptide Tigerin-OL1 group, 1 nM peptide Tigerin-OL1 group, 10 nM peptide Tigerin-OL1 group, 100 nM peptide Tigerin-OL1 group and 1 µM peptide Tigerin-OL1 group. Sterile PBS, fibroblast growth factor (FGF, 100 ng / ml) and different concentrations of peptide Tigerin-OL1 were added to the corresponding wells. After gently mixing, the culture conditions were restored and each well was placed in a cell culture incubator for continued culture. After scratching, the scratch changes of each well were recorded at 0 h and 24 h respectively to evaluate the cell repair activity of peptide Tigerin-OL1. During the recording, a microscope was used to take pictures to obtain clear scratch images so that subsequent analysis could accurately compare cell migration and repair.

[0023] Results: From Figure 3 It can be seen that the peptide Tigerin-OL1 at concentrations of 1 nM, 10 nM and 100 nM can promote the repair of HaCaT cell scratches, among which the repair effect of 10 nM peptide Tigerin-OL1 is the best.

[0024] Example 4 Detection of the activity of peptide Tigerin-OL1 in promoting oral ulcer healing in rats The oral ulcer animal model was induced by 50% acetic acid etching method to detect the activity of peptide Tigerin-OL1 in promoting oral ulcer healing. Male SD rats weighing 180-220 g were selected for the experiment and divided into 6 groups, namely Model group (model group PBS), Control group (positive control group), 100 pM peptide Tigerin-OL1 group, 1 nM peptide Tigerin-OL1 group, 10 nM peptide Tigerin-OL1 group, and 100 nM peptide Tigerin-OL1 group, with 6 rats in each group. The negative control group was given an equal amount of sterilized PBS, the positive control group was given an equal amount of FGF (100 ng / mL), and the peptide groups were given 20 µL of Tigerin-OL1 (100 pM, 1 nM, 10 nM, and 100 nM), respectively. The drug was administered topically with a pipette once in the morning and evening every day, and the oral ulcer area of ​​the modeled SD rats in each group was recorded synchronously for 7 consecutive days.

[0025] Results: From Figure 4 It can be seen that the peptide Tigerin-OL1 can promote the healing of oral ulcers in rats, and the peptide Tigerin-OL1 has a better effect at concentrations of 1 nM, 10 nM and 100 nM.

Claims

1. An active polypeptide Tigerin-OL1 that promotes oral ulcer healing, whose amino acid sequence is ICTMIPISRCY.

2. Use of the polypeptide Tigerin-OL1 according to claim 1 in the preparation of a drug for promoting oral ulcer healing.

3. A pharmaceutical composition comprising the polypeptide Tigerin-OL1 according to claim 1 as an active agent and a pharmaceutically effective carrier.

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