阅读说明：本技术 一种活性十肽及其在制备保护听觉毛细胞产品中的应用 (Active decapeptide and application thereof in preparation of auditory hair cell protection product ) 是由 孙晨 高燕 张姗姗 刘可春 张云 巴帅康 于 2020-04-20 设计创作，主要内容包括：本发明涉及一种活性十肽及其在制备保护听觉毛细胞产品中的应用。一种活性十肽在保护听觉毛细胞方面的应用,所述十肽的氨基酸序列如SEQ ID NO.1所示。本发明通过将含10个氨基酸残基的活性肽用于庆大霉素抗生素所导致的听觉毛细胞损伤中；检测后首次发现,该多肽化合物可以通过减少机体对庆大霉素的吸收,减少毛细胞的凋亡,进而对听觉毛细胞起到保护作用,改善耳毒性药物对听觉毛细胞的损害,且毒副作用小,是一种很有开发前景的听力保护药物,具有广阔的市场前景。(The invention relates to an active decapeptide and application thereof in preparation of products for protecting auditory hair cells. The application of an active decapeptide in protecting auditory hair cells, wherein the amino acid sequence of the decapeptide is shown as SEQ ID NO. 1. The invention uses the active peptide containing 10 amino acid residues in auditory hair cell damage caused by gentamicin antibiotics; the polypeptide compound is found for the first time after detection to play a role in protecting auditory hair cells by reducing the absorption of organisms on gentamicin and reducing the apoptosis of hair cells, improve the damage of ototoxic drugs on the auditory hair cells, have small toxic and side effects, are hearing protection drugs with a good development prospect and have a wide market prospect.)

1. An active decapeptide, the amino acid sequence of which is shown in SEQ ID NO. 1.

2. A method for extracting the active decapeptide according to claim 1, comprising the steps of:

(1) removing shells of the rapana venosa, taking all soft tissue parts, grinding, adding an acid solution with the pH value of 1.0-4.0, adding pepsin with the weight of 5-20% of the weight of the soft tissue, performing oscillatory enzymolysis for 1-5 hours at 35-40 ℃, adjusting the pH value to 7.0-9.0, adding trypsin and chymotrypsin with the weight of 5-20% of the weight of the soft tissue, performing oscillatory enzymolysis for 1-5 hours at 35-40 ℃, centrifuging, taking supernatant, concentrating and freeze-drying to obtain the rapana venosa polypeptide extract;

(2) redissolving the rapana venosa polypeptide extract prepared in the step (1) by using a buffer salt solution, carrying out column separation by using Sephadex G25, eluting 5 column volumes by using a buffer salt solution with the pH value of 6.0-8.0 as an eluent, collecting fractions of column volumes of 3 rd-5 th, freeze-drying, dissolving dry powder saline, separating by using Sephadex L H-20, collecting a sample by using the saline as the eluent at the speed of 10m L/45 min, collecting one part every 45min, combining 14 th-20 th parts of active section eluent, and concentrating to prepare a polypeptide active section crude extract;

(3) dissolving the crude extract of the active polypeptide segment prepared in the step (2) by using ammonium acetate buffer solution with the concentration of 10mM and the pH value of 5.8-6.2, filtering the solution by using a 4.5 mu m microporous membrane, separating the solution by using a Welch HI L IC Amide column, wherein the binary mobile phase comprises acetonitrile and ammonium acetate buffer solution with the concentration of 10mM and the pH value of 5.8-6.2, the volume ratio of the acetonitrile to the ammonium acetate buffer solution is 85:15, and the flow rate is 0.8 ml/min^-1Collecting eluent with an absorption peak with in-vitro DPPH free radical scavenging activity at 210nm, identifying and determining amino acid composition, and freeze-drying to obtain the polypeptide with the function of resisting oxidative stress damage.

3. The method according to claim 2, wherein in the step (1), the pepsin enzymolysis pH value is 2.0-3.0, and the trypsin and chymotrypsin enzymolysis pH value is 7.2-8.0.

4. The method according to claim 3, wherein in the step (1), the pH regulator is hydrochloric acid and sodium hydroxide.

5. The method of claim 2, wherein in step (1), the ratio of the enzymatic activities of trypsin and chymotrypsin is 1: (0.2-5).

6. The method according to claim 2, wherein in the step (2), the buffer salt system is a phosphate buffer system, and the pH value is 6.8-7.2.

7. The method of claim 2, wherein in step (3), the amino acid composition is identified and determined by L C-MS protein identification technique.

8. Use of the active decapeptide according to claim 1 as a pharmaceutical effective ingredient in the manufacture of a medicament for the treatment of an otic disorder.

9. The use of claim 8, wherein the otic disorder is an otic disorder resulting from impaired auditory hair cells.

10. Use of the active decapeptide according to claim 1 as an active ingredient for the preparation of a health food for protecting auditory hair cells.

Technical Field

The invention relates to an active decapeptide and application thereof in preparation of products for protecting auditory hair cells, belonging to the technical field of biological medicines.

Background

Gentamicin (Gentamicin, Gen) is an aminoglycoside antibiotic commonly used for the treatment of bacterial infectious inflammation^[1]. However, the medicine has strong ototoxic effect and can kill hair cells of inner ears of mammals. The auditory hair cells in the inner ear are terminally differentiated cells that convert acoustic stimuli into electrical signals that produce hearing. Since mammalian auditory hair cells are highly differentiated cells that have exited the cell division phase and thus do not have the ability to regenerate. Thus, loss of mammalian auditory hair cells due to Gen action often results in permanent loss of hearing^[2]. Until now, there is no ideal therapeutic drug or method for sensorineural deafness due to auditory hair cell damage. How to protect auditory hair cells from Gen invasion has become the key to prevent and treat drug-induced deafness.

It has been shown that Gen ototoxicity is closely related to Reactive Oxygen Species (ROS)^[3]. With the prolonging of Gen medication time, the ROS content in auditory hair cells can be increased rapidly, excessive ROS can enable the hair cells to be in an oxidative stress state and damage DNA, protein, lipid and the like in the cells, and finally the cells are induced to be apoptotic. Therefore, the finding of an antioxidant substance capable of reducing the generation of ROS is very important, which is helpful for treating drug-induced deafness and promoting the research and development of related prevention and treatment drugs.

Zebra fish is newly emerging in recent years and can be used as a model organism for developing Gen drug-induced deafness prevention and treatment drugs. It is composed of a base, a cover and a coverHaving an inner ear structure similar to a human being^[4]Auditory hair cells in the inner ear are highly homologous to human beings in terms of development, structure, function, etc., and are easily damaged by ototoxic drugs. Zebrafish are rich in a number of innervated lateral auditory hair cells in addition to hair cells in the inner ear. The lateral line hair cells are very similar to human auditory hair cells in the aspects of structure, function, gene regulation, response to Gen and the like, and are convenient for living body operation. When zebrafish developed to day 4, their inner ear and lateral hair cells had developed completely. Since zebra fish is transparent in the whole body in the early development stage, the toxic effect of Gen on hair cells can be directly observed in real time by virtue of a living body imaging technology.

At present, by means of model organisms, a novel compound with obvious inhibition effect on drug ototoxicity damage is searched, and the novel compound becomes a key for protecting auditory hair cells and treating deafness diseases caused by the damage of the auditory hair cells.

U.S. patent No. US20120258921a1 (application No. US13440591) discloses peptides for the treatment of inflammation and therapeutic uses and methods of use thereof polypeptides containing transduction sequences inhibit cytokine activity and TNF- α secretion by interacting with Toll-like receptor signaling pathways.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides an active decapeptide and application thereof in preparing products for protecting auditory hair cells.

The technical scheme of the invention is as follows:

an active decapeptide, the amino acid sequence of which is shown in SEQ ID NO. 1.

A method for extracting the active decapeptide comprises the following steps:

(1) removing shells of the rapana venosa, taking all soft tissue parts, grinding, adding an acid solution with the pH value of 1.0-4.0, adding pepsin with the weight of 5-20% of the weight of the soft tissue, performing oscillatory enzymolysis for 1-5 hours at 35-40 ℃, adjusting the pH value to 7.0-9.0, adding trypsin and chymotrypsin with the weight of 5-20% of the weight of the soft tissue, performing oscillatory enzymolysis for 1-5 hours at 35-40 ℃, centrifuging, taking supernatant, concentrating and freeze-drying to obtain the rapana venosa polypeptide extract;

(2) redissolving the rapana venosa polypeptide extract prepared in the step (1) by using a buffer salt solution, carrying out column separation by using Sephadex G25, eluting 5 column volumes by using a buffer salt solution with the pH value of 6.0-8.0 as an eluent, collecting fractions of column volumes of 3 rd-5 th, freeze-drying, dissolving dry powder saline, separating by using Sephadex L H-20, collecting a sample by using the saline as the eluent at the speed of 10m L/45 min, collecting one part every 45min, combining 14 th-20 th parts of active section eluent, and concentrating to prepare a polypeptide active section crude extract;

(3) dissolving the crude extract of the active polypeptide segment prepared in the step (2) by using ammonium acetate buffer solution with the concentration of 10mM and the pH value of 5.8-6.2, filtering the solution by using a 4.5 mu m microporous membrane, and separating the solution by using a Welch HI L IC Amide column, wherein the binary mobile phase comprises Acetonitrile (ACN) and ammonium acetate buffer solution with the concentration of 10mM and the pH value of 5.8-6.2, the volume ratio of the Acetonitrile (ACN) to the ammonium acetate buffer solution is 85:15, and the flow rate is 0.8 ml/min^-1Collecting eluent with an absorption peak with in-vitro DPPH free radical scavenging activity at 210nm, identifying and determining amino acid composition, and freeze-drying to obtain the polypeptide with the function of resisting oxidative stress damage.

Preferably, in the step (1), the enzymolysis pH value of pepsin is 2.0-3.0, and the enzymolysis pH value of trypsin and chymotrypsin is 7.2-8.0; further preferably, in the step (1), the pH regulator is hydrochloric acid and sodium hydroxide.

Preferably, in the step (1), the enzyme activity ratio of trypsin to chymotrypsin is 1: (0.2-5).

According to the present invention, in the step (2), the buffer salt system is a phosphate buffer system, and the pH value is 6.8 to 7.2.

Preferably, in the step (3), L C-MS protein identification technology is adopted for identifying and determining amino acid composition.

The active decapeptide is used as a medicinal component for preparing a medicament for treating ear diseases.

Preferably, according to the present invention, the otic disorder is an otic disorder caused by damaged auditory hair cells.

The active decapeptide is used as an effective component in preparing health-care food for protecting auditory hair cells.

Advantageous effects

The invention uses active peptide containing 10 amino acid residues in auditory hair cell injury caused by gentamicin (Gen); the polypeptide compound is found for the first time after detection to reduce the absorption of gentamicin (Gen) by organisms and reduce the apoptosis of hair cells, so that the polypeptide compound has a protection effect on auditory hair cells, improves the damage of ototoxic medicaments to the auditory hair cells, has small toxic and side effects, is a hearing protection medicament with a good development prospect, and has a wide market prospect.

Drawings

FIG. 1 is a photograph of hair cells after 10. mu.g/ml active decapeptide treatment of zebrafish;

in the figure: the square frame is provided with five groups of auditory hair cells around the ear;

FIG. 2 is a histogram of hair cell counts after 10. mu.g/ml active decapeptide treatment of zebrafish;

in the figure: denotes p < 0.001;

FIG. 3 is a photograph of hair cells after 30. mu.g/ml active decapeptide treatment of zebrafish;

in the figure: the square frame is provided with five groups of auditory hair cells around the ear;

FIG. 4 is a histogram of hair cell counts after 30 μ g/ml active decapeptide treatment of zebrafish;

in the figure: denotes p < 0.001;

FIG. 5 is a photograph of hair cells after 50. mu.g/ml active decapeptide treatment of zebrafish;

in the figure: the square frame is provided with five groups of auditory hair cells around the ear;

FIG. 6 is a histogram of hair cell counts after 50. mu.g/ml active decapeptide treatment of zebrafish;

in the figure: denotes p < 0.001;

FIG. 7 is a photograph of Gen content in hair cells after treating zebrafish with active decapeptide at various concentrations;

FIG. 8 is a photograph showing the apoptosis of hair cells of zebra fish treated with active decapeptide at different concentrations;

in the figure: a is a control group; b is Gen make module; c is 10 mug/ml active decapeptide treatment group; d is 30 mug/ml active decapeptide treatment group; e is 50. mu.g/ml active decapeptide treatment group;

FIG. 9 is a map showing the results of the identification of L C-MS protein on the active polypeptide prepared in example 1.

Detailed Description

The technical solutions of the present invention are further described below with reference to the following embodiments and the drawings of the specification, but the scope of the present invention is not limited thereto.

Preparing a reagent: the gentamicin (Gen) standard was purchased from Qingdao Biotech, Inc. and was dissolved in physiological saline to prepare a 1000M stock solution. Purchase of AAT from Texas Red dyeThe TUNE L kit is purchased from Nanjing Novozam Biotech, Inc.;

the YO-PRO-1 dye was purchased from seimer feishell science ltd;