CN-122005750-A - Application of bombyx mori neuropeptide BommoOKA _type4 in preparation of anti-melanoma medicine

Abstract

The invention discloses an application of silkworm neuropeptide BommoOKA _Type4 in preparing a medicine for inhibiting growth of melanoma cells, and belongs to the technical field of medical biology. Aiming at the technical problems that melanoma has high malignancy degree, is easy to transfer and has limited treatment means, and the silkworm neuropeptide BommoOKA _type4 is mainly used for whitening skin before, and whether the silkworm neuropeptide can directly inhibit the growth of melanoma cells is not clear, the invention provides the application of the silkworm neuropeptide in preparing a medicament for inhibiting the melanoma cells. The bombyx mori neuropeptides can inhibit the gene expression and protein function of melanin synthesis key enzymes TYR, TRP1 and TRP2 by down regulating the expression of a core transcription factor MITF, so that the proliferation of melanoma cells is directly inhibited while the melanin synthesis is inhibited, and therefore, the bombyx mori neuropeptides can be used for preparing medicines for treating melanoma, and a novel peptide candidate substance is provided for the treatment of melanoma.

Inventors

• WANG PINGYANG
• HUANG XUHUA
• LU CHUNXIA
• LIU KAILI
• HUANG SHENHUI
• TANG MINGYAN
• CHEN XIAOQING
• Liang Xiangliu
• XU WENWEN
• CHEN JING
• MO BINGQIAO
• XIAO XIAO
• LI ANHUA
• LIU FENMEI
• LIANG GUIQIU
• WANG XIA
• CUI QIUYING
• ZHANG YULI
• TANG LIANG
• JIANG MANGUI

Assignees

• 广西壮族自治区蚕业技术推广站(广西壮族自治区蚕种质量检验检疫站、广西壮族自治区蚕业科学研究院)

Dates

Publication Date

20260512

Application Date

20260121

Claims (8)

1. 1. The application of the bombyx mori neuropeptide BommoOKA _type4 in preparing a medicine for inhibiting the growth of melanoma cells is provided, wherein the amino acid sequence of the bombyx mori neuropeptide BommoOKA _type4 is shown as SEQ ID NO. 1.
2. 2. A pharmaceutical composition for inhibiting the growth of melanoma cells, comprising the bombyx mori neuropeptide BommoOKA _type4 of claim 1 and a pharmaceutically acceptable carrier.
3. 3. A method of preparing a pharmaceutical composition according to claim 2, comprising the steps of: S1, dissolving silkworm neuropeptide BommoOKA _type4 powder in phosphate buffer solution to prepare polypeptide stock solution, wherein the concentration of the polypeptide stock solution is 10-20 mM; S2, preparing a carrier solution from a pharmaceutically acceptable carrier, wherein the mass volume fraction of the carrier in the carrier solution is 1-5 g/100mL; s3, mixing the polypeptide stock solution with the carrier solution, controlling the mixing volume ratio between 1:10 and 1:50, stirring 30-60 min on a magnetic stirrer at a rotating speed of 100-300 rpm, and filtering and sterilizing the mixed solution through a filter membrane with the pore diameter of 0.22 mu m; and S4, subpackaging the filtered and sterilized solution into sterile glass bottles or sterile plastic containers under the sterile condition, sealing the containers after subpackaging, and preserving at the temperature of 2-8 ℃ to obtain the pharmaceutical composition.
4. 4. A method of preparing a pharmaceutical composition according to claim 3, wherein the pharmaceutically acceptable carrier is a liposome.
5. 5. The method of preparing a pharmaceutical composition according to claim 4, wherein the carrier solution is a liposome dispersion, the method of preparing the liposome dispersion comprising the steps of: S2.1, mixing dipalmitoyl phosphatidylcholine, cholesterol and distearoyl phosphatidylethanolamine-polyethylene glycol 2000 according to a molar ratio of 50-70:30-40:5-10, dissolving in chloroform, and evaporating under reduced pressure at 35-45 ℃ on a rotary evaporator for 30-60 min to form a uniform lipid film; S2.2, placing the lipid film in a vacuum drying oven to be dried for 4-6 h so as to thoroughly remove organic solvent residues; S2.3, hydrating the dried lipid film with water for injection at 50-60 ℃ for 40-80 min to obtain liposome dispersion liquid.
6. 6. The method of preparing a pharmaceutical composition according to claim 5, wherein in step S3, the polypeptide stock solution and the liposome dispersion are mixed in a volume ratio of 1:15-1:25, and stirred on a magnetic stirrer at a rotation speed of 150-250 rpm, while the mixed solution is placed in a water bath at 25-37 ℃ for incubation of 50-70 min; After the incubation is finished, extruding the mixed solution through a polycarbonate membrane with the aperture of 80-120 nm for 7-12 times by using an extruder to obtain liposome suspension carrying polypeptide; the liposome suspension was filter sterilized by passing through a filter membrane having a pore size of 0.22 μm.
7. 7. The method for preparing the pharmaceutical composition according to claim 6, wherein the liposome suspension loaded with the polypeptide is subjected to stabilization and purification treatment, and the specific process is as follows: S3.1, placing the liposome suspension loaded with the polypeptide into a program cooling instrument, cooling from 45 ℃ to 4 ℃ at a rate of 0.5-1 ℃ per minute, and keeping at 6-10 h at 4 ℃; S3.2, after the maintenance is finished, recovering the suspension to room temperature at 20-25 ℃; S3.3, placing the suspension recovered to room temperature in an ultracentrifuge, centrifuging 45-60 min under the conditions of 4 ℃ and 150000-200000 g, and carefully removing the supernatant; s3.4, adding precooled sucrose-histidine buffer solution with the pH value of 6.8-7.4 into the centrifugally precipitated liposome precipitation block, wherein the concentration of sucrose in the sucrose-histidine buffer solution is 8-12 g/100mL, and the concentration of histidine is 5-20 mM; S3.5, gently suspending the liposome precipitation block on a vortex oscillator to obtain a concentrated and purified liposome suspension; S3.6 the concentrated and purified liposome suspension was filter sterilized by passing through a filter membrane having a pore size of 0.22. Mu.m.
8. 8. The method of preparing a pharmaceutical composition according to claim 7, wherein the step of bringing the suspension back to room temperature at 20-25 ℃ comprises: The suspension which is kept at the temperature of 4 ℃ and is 6-10 h is placed in a temperature control device with a program temperature control function, the temperature is slowly increased from 4 ℃ to 20-25 ℃ at the temperature increasing rate of 0.5-2 ℃ per minute, and 10-30 min is kept after the target temperature is reached, so that the process of recovering to the room temperature is completed.

Description

Application of bombyx mori neuropeptide BommoOKA _type4 in preparation of anti-melanoma medicine Technical Field The invention belongs to the technical field of medical biology, and particularly relates to application of bombyx mori neuropeptide BommoOKA _type4 in preparation of a medicine for inhibiting melanoma cells. Background Melanoma is a skin tumor with high malignancy and easy metastasis, and the treatment of the melanoma still needs to explore new effective means. In the prior art, some active ingredients derived from animals and plants have been confirmed to have an effect of inhibiting melanin synthesis, for example, some peptide substances have been used in the cosmetic field as whitening ingredients. However, the main research and development directions of these ingredients have focused on improving skin pigmentation, whether or not they have direct proliferation inhibitory activity on melanoma cells, and the related research reports have been insufficient. The bombyx mori neuropeptide BommoOKA _type4 has been found to inhibit tyrosinase activity and reduce pigmentation, and shows potential value in skin whitening, but whether the peptide can be applied to inhibit the growth of melanoma cells, so that a new candidate substance is provided for the treatment of melanoma, and no clear research conclusion and no published technical scheme exist before. Because the pathogenesis of melanoma is obviously different from that of common skin pigment metabolism, a peptide which is mainly used for regulating pigment metabolism is directly converted into an anti-tumor drug, and the technical difficulties of unclear action targets, unknown effective concentration, completely unknown biological activity and action mechanism in tumor cells and the like are faced. Disclosure of Invention It is an object of the present invention to solve at least the above problems and to provide at least the advantages to be described later. The invention also aims to provide an application of the bombyx mori neuropeptide BommoOKA _type4 in preparing a medicine for inhibiting melanoma cells, which can provide a novel peptide material selection for treating melanoma by inhibiting proliferation of the melanoma cells, reducing tyrosinase activity in cells and reducing melanin synthesis. To achieve these objects and other advantages and in accordance with the purpose of the invention, there is provided an application of bombyx mori neuropeptide BommoOKA _type4 in preparing a medicament for inhibiting growth of melanoma cells, wherein an amino acid sequence of bombyx mori neuropeptide BommoOKA _type4 is shown as SEQ ID No. 1. The bombyx mori neuropeptides BommoOKA _type4 can inhibit the gene expression and protein functions of a plurality of melanin synthesis key enzymes (TYR, TRP1 and TRP 2) comprehensively by down-regulating the expression of a core transcription factor MITF, and can inhibit the melanin synthesis and also directly inhibit the proliferation of melanoma cells. A pharmaceutical composition for inhibiting the growth of melanoma cells comprises the bombyx mori neuropeptide BommoOKA _type4 and a pharmaceutically acceptable carrier. A method of preparing a pharmaceutical composition for inhibiting melanoma cells comprising the steps of: S1, dissolving silkworm neuropeptide BommoOKA _type4 powder in phosphate buffer solution to prepare polypeptide stock solution, wherein the concentration of the polypeptide stock solution is 10-20 mM; S2, preparing a carrier solution from a pharmaceutically acceptable carrier, wherein the mass and volume fraction of the carrier in the carrier solution is 1% -5%; s3, mixing the polypeptide stock solution with the carrier solution, controlling the mixing volume ratio between 1:10 and 1:50, stirring 30-60 min on a magnetic stirrer at a rotating speed of 100-300 rpm, and filtering and sterilizing the mixed solution through a filter membrane with the pore diameter of 0.22 mu m; and S4, subpackaging the filtered and sterilized solution into sterile glass bottles or sterile plastic containers under the sterile condition, sealing the containers after subpackaging, and preserving at the temperature of 2-8 ℃ to obtain the pharmaceutical composition. The preparation method stably combines the raw material of the bombyx mori neuropeptide BommoOKA _type4 with biological activity with a pharmaceutically acceptable carrier through a set of standardized and controllable process flow, and finally prepares a sterile preparation form with uniform quality, which is suitable for use. The method comprises the steps of firstly dissolving polypeptide powder in a biocompatible phosphate buffer solution to prepare a stock solution with a specific concentration range (10-20 mM), wherein the stock solution aims at enabling the polypeptide to be fully dissolved and in a stable ion environment, preventing initial aggregation or degradation and providing a raw material solution with accurate concentration for the subse