阅读说明：本技术 一种活性十肽在保护听觉毛细胞方面的应用 (Application of active decapeptide in aspect of protecting auditory hair cells ) 是由 孙晨 张姗姗 刘可春 巴帅康 高燕 于 2020-05-14 设计创作，主要内容包括：本发明涉及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。检测后首次发现,该多肽化合物可以通过抑制庆大霉素在毛细胞内的过量积聚来对毛细胞起保护作用,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 application of active decapeptide in protecting auditory hair cells, wherein the amino acid sequence of the decapeptide is shown as SEQ ID NO. 1. The polypeptide compound is found for the first time after detection to play a role in protecting hair cells by inhibiting the excessive accumulation of gentamicin in the hair cells, improve the damage of ototoxic drugs to 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 application of active decapeptide as a drug effect component in preparing a drug for treating ear diseases, wherein the amino acid sequence of the active decapeptide is SEQ ID NO. 1.

2. The use of claim 1, wherein the otic disorder is an otic disorder caused by excessive absorption of aminoglycoside drugs by auditory hair cells.

3. An active decapeptide is used as an effective component and applied to the preparation of related health-care food for inhibiting excessive absorption of aminoglycoside drugs by auditory hair cells, wherein the amino acid sequence of the active decapeptide is SEQ ID NO. 1.

4. An aminoglycoside drug for alleviating ear injury, which is characterized by comprising active decapeptide, wherein the amino acid sequence of the active decapeptide is SEQ ID NO. 1.

Technical Field

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

Background

Aminoglycoside medicine is a kind of clinically common antibiotics, and has the characteristics of stable property, strong antibacterial ability, low medication cost and the like, so that the aminoglycoside medicine is widely applied clinically. However, these drugs have strong ototoxicity and can kill auditory hair cells in the inner ear of mammals, which in turn leads to sensorineural deafness. Gentamicin is one of the representatives of aminoglycoside drugs. Auditory hair cells are susceptible cells of aminoglycoside drugs. When the aminoglycoside acts on the body, it is very easy to accumulate in auditory hair cells, and further induces the apoptosis. In the mammalian auditory system, auditory hair cells are terminally differentiated cells that exit the cell cycle and, once damaged, fail to regenerate. Therefore, no effective medicine for preventing and treating sensorineural deafness caused by the action of aminoglycoside medicines exists up to now. The search for a novel compound can inhibit excessive absorption of auditory capillaries to aminoglycoside drugs, is helpful for preventing and treating sensorineural deafness and promoting the research and development of related therapeutic drugs, and has very important significance.

In the field of research on ototoxic control drugs, zebrafish is a new type of vertebrate model organism emerging in recent years, with typical inner ear auditory hair cells that are highly homologous to humans, both at the structural and molecular level. In addition, the lateral line of zebrafish has a large number of innervated lateral hair cells. The lateral line hair cells are highly homologous with auditory hair cells in human inner ear in the aspects of structure, function, gene regulation and the like, and are extremely sensitive to aminoglycoside drugs. When zebrafish developed to day 4, their hair cells had developed completely. At the moment, the whole body of the zebra fish is transparent, so the hair cells of the zebra fish can be observed in real time by means of technologies such as fluorescence labeling and the like. At present, a zebra fish model is used for searching a novel compound with a protective effect on auditory hair cells, and the novel compound becomes a key for treating sensorineural deafness.

The sachet (Neptunea arthritica cumingii) is a large predatory gastropod of the phylum Mollusca (Mollusca), class Gastropoda (Gastropoda), order Neogastropoda (Neovastropida), family Bombycidae (Buccidia). It is widely distributed in yellow sea and Bohai sea areas in China. At present, the research on the aspidium fragrans mainly focuses on genome, nutrition, reproductive characteristics and the like, and reports related to active ingredients of the aspidium fragrans are few. The inventor obtains a novel polypeptide from the pimpinella arguta earlier, and researches show that the polypeptide has the function of repairing oxidative damage. The polypeptide has already been applied for related patent (patent number: ZL201810915407.5), but the application of the polypeptide in the hearing protection field, in particular in the aspect of inhibiting excessive absorption of aminoglycoside drugs by auditory hair cells is not related.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides the application of decapeptide in preparing auditory hair cell protection products.

The technical scheme of the invention is as follows:

an active decapeptide, amino acid sequence SEQ ID No.1 is as follows:

Tyr-Ser-Gln-Leu-Glu-Asn-Glu-Phe-Asp-Arg(YSQLENEFDR)。

the active decapeptide is disclosed in patent No. ZL201810915407.5 (application No. 201810915407.5).

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 excessive absorption of aminoglycoside drugs by auditory hair cells.

The active decapeptide is used as an active ingredient and applied to preparation of related health-care food for inhibiting excessive absorption of aminoglycoside drugs by auditory hair cells.

An aminoglycoside drug for alleviating ear injury, which is characterized by comprising active decapeptide, wherein the amino acid sequence of the active decapeptide is SEQ ID NO. 1.

Advantageous effects

The invention uses a bioactive peptide containing ten amino acid residues for inhibiting the excessive absorption of auditory capillary cells to aminoglycoside medicaments for the first time; the decapeptide is found for the first time after detection to be capable of effectively reducing absorption of the auditory capillary cells to gentamicin and further playing a role in protecting the auditory capillary cells, is small in toxic and side effects, is a hearing protection drug with a good development prospect, and has a wide market prospect.

Drawings

FIG. 1 is a photograph of hair cells after treatment of zebrafish with 70. mu.g/ml active decapeptide;

in the figure: a is a control group; b is a gentamicin making module; c is an active decapeptide treatment group; boxed are five groups (O1, MI1, MI2, O2, and IO4) of auditory hair cells around the ear.

FIG. 2 is a bar graph of hair cell count statistics after 70. mu.g/ml active decapeptide treatment of zebrafish, with the ordinate representing cell number in units of cells;

in the figure: denotes p < 0.001.

FIG. 3 is a photograph of hair cells after 90. mu.g/ml active decapeptide treatment of zebrafish;

in the figure: a is a control group; b is a gentamicin making module; c is an active decapeptide treatment group; the box is the five groups of auditory hair cells around the ear.

FIG. 4 is a histogram of hair cell count statistics after 90 μ g/ml active decapeptide treatment of zebrafish, with the ordinate representing cell number in units of cells;

in the figure: denotes p < 0.001.

FIG. 5 is a photograph of hair cells after treatment of zebrafish with 100. mu.g/ml active decapeptide;

in the figure: a is a control group; b is a gentamicin making module; c is an active decapeptide treatment group; the box is the five groups of auditory hair cells around the ear.

FIG. 6 is a bar graph of hair cell count statistics after treatment of zebrafish with 100 μ g/ml active decapeptide, with the ordinate representing cell number in units of cells;

in the figure: denotes p < 0.001.

FIG. 7 is a photograph showing the content of gentamicin in hair cells after treating zebrafish with 100. mu.g/ml active decapeptide.

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.

The contents of the examples, which are not specified in specific conditions, were carried out under conventional conditions; the reagents or instruments used are not indicated by the manufacturer, and are all common commercial products.

Preparing a reagent: the gentamicin standard was purchased from Qingdao Biotech, Inc., and was dissolved in physiological saline to prepare a 1000M stock solution. AAT is purchased from Texas Red dye (Texas Red, TR)The YO-PRO-1 dye was purchased from Saimer Feishell science Inc.

The water for embryo culture comprises the following components:

CaCl₂ 0.4mM，NaCl 5mM，MgSO₄0.16mM, KCl 0.17mM, deionized water.

Example 1

Protection of Zebra fish auditory hair cells by 70. mu.g/ml active decapeptide sample

Experimental methods

1. Pretreatment of zebra fish samples:

healthy sexually mature AB-line zebra fish were bred according to a male-female ratio of 1:1, placing the mixture into a mating jar, placing a partition plate in the middle, placing the mixture into a dark environment, drawing out the partition plate before lighting on the next day, stimulating the mixture by light to ovulate, fishing out adult fishes after ovulating for half an hour, and controlling the ovulating time within half an hour so as to reduce the difference of the development time between embryos. Collecting fertilized eggs, and disinfecting and cleaning the fertilized eggs; then the fertilized eggs are transferred into clean water for culturing zebra fish embryos, 0.2ppm methylene blue is added, light control culture is carried out at the temperature of 28 ℃ under the condition of 14h light/10 h dark cycle, 1/3 water for culturing the embryos is changed every 24h in the middle, and dead embryos are sucked out in time. Placing the zebra fish 6 days after birth under a dissecting mirror, selecting the zebra fish with normal development, placing the zebra fish into a 6-hole plate, wherein 20 fish in each hole, and adding a proper amount of water for embryo culture.

2. Active decapeptide treatment:

an active decapeptide with an amino acid sequence shown as SEQ ID NO.1 is synthesized by an artificial synthesis mode, and the decapeptide is synthesized by an Fmoc solid-phase synthesis method (Liu Zheng, Huangqiang. Fmoc solid-phase synthesis method [ J ]. proceedings of Guangxi national academy of sciences, 1999,5(2): 110-112.).

And (3) randomly dividing the zebra fish in the 6-hole plate in the step (1) into a control group, a gentamicin molding group and an active peptide treatment group, wherein each group comprises 3 parallel holes. The embryo culture water in the 6-well plate was removed, 2ml of embryo culture water was added to the control group, and the group was cultured in a constant temperature incubator at 28 ℃ for 1 hour; adding gentamicin with final concentration of 150 μ M into 2ml of embryo culture water added into the gentamicin molding group, and culturing in a constant temperature incubator at 28 ℃ for 1 h; active decapeptide treatment group 2ml of embryo culture water was added first with 70. mu.g/ml of active decapeptide, incubated in a 28 ℃ incubator for 1 hour, then simultaneously with 150. mu.M gentamicin and 70. mu.g/ml of active decapeptide, and further incubated in a 28 ℃ incubator for 1 hour.

3. Zebrafish auditory hair cell labeling:

and (3) after the treatment in the step 2 is finished, sucking liquid in the 6-hole plate, and adding embryo culture water to respectively clean each group of zebra fish. The YO-PRO-1 dye was then added to each of the groups of zebrafish at a final concentration of 2. mu.M, respectively, and incubated at 28 ℃ for 20min in the absence of light. After the incubation is finished, the YO-PRO-1 solution is sucked and removed, and embryo culture water is added to wash each group of zebra fish.

4. Counting the number of auditory hair cells:

and (3) placing the three groups of zebra fishes in 0.02% of tricaine solution by mass concentration respectively to make the zebra fishes anesthetized. Auditory hair cell images showing green fluorescence were collected under a fluorescence microscope. Five groups (O1, MI1, MI2, O2 and IO4) of auditory hair cells around the ears in each group of zebra fish were counted respectively according to the collected pictures.

The auditory hair cells of the five groups O1, MI1, MI2, O2 and IO4 surrounding the ear described above are disclosed in Rabble DW, Kruse GJ, Organization of the hierarchical line system in organizing zebraphish [ J ]. J Comp Neurol,2000,421: 189-.

The experimental results are shown in fig. 1 and fig. 2, and 70 μ g/ml active decapeptide can effectively alleviate gentamycin-induced auditory hair cell loss, and has statistical differences compared with gentamycin building blocks. The results prove that the active decapeptide at the concentration can effectively protect auditory hair cells in the body and improve the ototoxic damage induced by aminoglycoside antibiotics.

Example 2

Protective effect of 90 mu g/ml active decapeptide sample on zebra fish auditory hair cells

The pretreatment of zebra fish samples, labeling of zebra fish auditory hair cells and counting of the number of auditory hair cells are the same as in example 1.

Active decapeptide treatment:

an active decapeptide with an amino acid sequence shown as SEQ ID NO.1 is synthesized by adopting an artificial synthesis mode, and the decapeptide is synthesized by adopting an Fmoc solid-phase synthesis method (Liu Zheng, Huangqiang. Fmoc solid-phase synthesis method [ J ]. proceedings of Guangxi national academy of sciences, 1999,5(2): 110-.

The zebrafish in the 6-well plate were randomly divided into control, gentamicin molding and active peptide treatment groups, each group having 3 parallel wells. The embryo culture water in the 6-well plate was removed, 2ml of embryo culture water was added to the control group, and the group was cultured in a constant temperature incubator at 28 ℃ for 1 hour; adding gentamicin with final concentration of 150 μ M into 2ml of embryo culture water added into the gentamicin molding group, and culturing in a constant temperature incubator at 28 ℃ for 1 h; the active decapeptide treatment group was added to 2ml of embryo culture water, and 90. mu.g/ml of active decapeptide was first added, and cultured in a 28 ℃ incubator for 1 hour, and then gentamicin at a final concentration of 150. mu.M and 90. mu.g/ml of active decapeptide were simultaneously added, and the culture was continued in a 28 ℃ incubator for 1 hour.

The experimental results are shown in fig. 3 and 4, and the active decapeptide of 90 μ g/ml can effectively alleviate gentamicin-induced auditory hair cell loss, and has statistical difference compared with gentamicin modeling modules. The results prove that the active decapeptide at the concentration can effectively protect auditory hair cells in the body and improve the ototoxic damage induced by aminoglycoside antibiotics.

Example 3

Protection of Zebra fish auditory hair cells by 100. mu.g/ml active decapeptide sample

The pretreatment of zebra fish samples, labeling of zebra fish auditory hair cells and counting of the number of auditory hair cells are the same as in example 1.

Active decapeptide treatment:

an active decapeptide with an amino acid sequence shown as SEQ ID NO.1 is synthesized by an artificial synthesis mode, and the decapeptide is synthesized by an Fmoc solid-phase synthesis method (Liu Zheng, Huangqiang. Fmoc solid-phase synthesis method [ J ]. proceedings of Guangxi national academy of sciences, 1999,5(2): 110-112.).

The zebrafish in the 6-well plate were randomly divided into control, gentamicin molding and active peptide treatment groups, each group having 3 parallel wells. The embryo culture water in the 6-well plate was removed, 2ml of embryo culture water was added to the control group, and the group was cultured in a constant temperature incubator at 28 ℃ for 1 hour; adding gentamicin with final concentration of 150 μ M into 2ml of embryo culture water added into the gentamicin molding group, and culturing in a constant temperature incubator at 28 ℃ for 1 h; the active decapeptide treatment group was added to 2ml of embryo culture water, and 100. mu.g/ml of active decapeptide was first added, and cultured in a 28 ℃ incubator for 1 hour, and then gentamicin at a final concentration of 150. mu.M and 100. mu.g/ml of active decapeptide were simultaneously added, and the culture was continued in a 28 ℃ incubator for 1 hour.

The experimental results are shown in fig. 5 and 6, and the active decapeptide of 100 μ g/ml can effectively alleviate gentamicin-induced auditory hair cell loss, and has statistical differences compared with gentamicin building blocks. The results prove that the active decapeptide at the concentration can effectively protect auditory hair cells in the body and improve the ototoxic damage induced by aminoglycoside antibiotics.

Example 4

100 ug/ml active decapeptide sample can inhibit the accumulation of gentamicin in auditory hair cells

Zebrafish sample pretreatment was the same as in example 1.

Active decapeptide treatment:

an active decapeptide with an amino acid sequence shown as SEQ ID NO.1 is synthesized by an artificial synthesis mode, and the decapeptide is synthesized by an Fmoc solid-phase synthesis method (Liu Zheng, Huangqiang. Fmoc solid-phase synthesis method [ J ]. proceedings of Guangxi national academy of sciences, 1999,5(2): 110-112.).

TR, after being formulated as a 2mg/ml solution, was mixed with a 50mg/ml gentamicin solution at 22: 3 (volume ratio) and then kept out of the sun overnight to prepare the texas red-Gentamicin (GTTR) conjugate.

Zebrafish in 6-well plates were randomly divided into GTTR building blocks and active decapeptide treatment blocks. Removing embryo culture water in a 6-well plate, adding GTTR conjugate containing gentamicin with final concentration of 150 μ M into 2ml of embryo culture water added by a GTTR manufacturing module, and incubating for 1h in a constant temperature incubator at 28 ℃ in the dark; the active decapeptide treatment group was added to 2ml of embryo culture water, 100. mu.g/ml of active decapeptide was added, incubated for 1 hour in a 28 ℃ incubator, then simultaneously added with GTTR conjugate containing gentamicin at a final concentration of 150. mu.M and 100. mu.g/ml of active decapeptide, and further incubated for 1 hour in a 28 ℃ incubator in the absence of light. After the completion, the zebra fish is cleaned by embryo culture water, and a hair cell image showing red fluorescence is collected by using a laser confocal microscope.

The experimental results are shown in fig. 7, and a large amount of red fluorescence labeled gentamicin can be detected in the remaining hair cells of zebra fish in the GTTR model set. However, in the active decapeptide treated group, red fluorescence labeled gentamicin in hair cells was significantly reduced compared to the GTTR model group. The results indicate that the active decapeptide can inhibit the massive accumulation of gentamicin in hair cells, further confirming the auditory hair cell protection effect of the active decapeptide.

Through examples 1-4, the invention firstly discovers that the active decapeptide with the amino acid sequence shown as SEQ ID No.1 can inhibit excessive absorption of gentamicin by auditory capillary cells, and further plays a role in protecting gentamicin-induced drug-induced auditory capillary cell damage on the basis of the excessive absorption. The experimental result shows that the active decapeptide has the application prospect, the active decapeptide is used as a drug effect component to prepare the drugs for treating the ear diseases, and the further ear diseases are the ear diseases caused by excessive absorption of aminoglycoside drugs by auditory hair cells; the active decapeptide is used as an active ingredient and applied to preparation of related health-care food for inhibiting excessive absorption of aminoglycoside drugs by auditory hair cells.

SEQUENCE LISTING

<110> institute of biological research of academy of sciences of Shandong province

Qilu University of Technology

<120> application of active decapeptide in protecting auditory hair cells

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 10

<212> PRT

<213> Artificial sequence

<400> 1

Tyr Ser Gln Leu Glu Asn Glu Phe Asp Arg

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