CN111249440A - Application of small molecule short peptide in preparation of product for treating acne - Google Patents
Application of small molecule short peptide in preparation of product for treating acne Download PDFInfo
- Publication number
- CN111249440A CN111249440A CN202010151278.4A CN202010151278A CN111249440A CN 111249440 A CN111249440 A CN 111249440A CN 202010151278 A CN202010151278 A CN 202010151278A CN 111249440 A CN111249440 A CN 111249440A
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- short peptide
- small molecule
- acne
- product
- skin
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- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
Abstract
The invention discloses application of small molecule short peptide in preparation of a product for treating acne. The small molecule short peptide is a bFGF derived degradation short peptide consisting of 8 amino acid residues, and the amino acid sequence of the small molecule short peptide is as follows: LQLQAEER, the invention discovers that the small molecular short peptide can inhibit the hyperproliferation of sebaceous glands and reduce the grease secretion of skin, has high stability, small toxicity and side effect, is suitable for a wide range of people, has good transdermal property and can continuously act on a treatment part, so the small molecular short peptide can be used for resisting acne, and particularly can be used for treating acne caused by hormone level change in the adolescence and the pregnancy.
Description
Technical Field
The invention belongs to the field of biological medicines, and particularly relates to application of small-molecule short peptide in preparation of a product for treating acne.
Background
Acne is the most common chronic inflammatory disease of the pilosebaceous unit in adolescence. The pathogenesis of the disease is not completely understood because the pathogenesis of the disease is complicated. Currently, hyperseborrhoea secretion from the skin of a patient, abnormal keratinization in hair follicles, propionibacterium acnes colonized in pilo-sebaceous gland ducts and inflammatory factor mediators are considered as key factors in the development of acne lesions. The number of people afflicted with acne tends to increase continuously due to air pollution, stress caused by the accelerated pace of modern life, and imbalanced levels of sex hormones. In addition, certain drugs can also cause acne by stimulating the sebaceous glands, and in particular the formation of corticosteroids and anabolic steroids, such as facial "rosacea", is caused by the use of skin care products containing undesirable additives. Although acne does not affect normal activities, it has a great psychological and social impact on patients because it is frequently found on the face.
The secretion of hormones is relatively vigorous in both males and females in adolescence, and excessive androgen in the body can stimulate sebaceous glands near skin follicles, so that sebaceous gland cells are excessively proliferated, and a large amount of grease is secreted by enlarged sebaceous glands to block the follicles. At the same time, unsaturated fatty acids such as oleic acid and palmitoleic acid secreted from the skin induce abnormal keratinization and epidermal hyperplasia, increase calcium influx in keratinocytes, increase the cohesion of the proximal portion of the follicular infundibulum, and promote keratinocyte production. And at the infundibulum of the distal portion of the follicle, keratinocytes and sebum gradually accumulate to form comedones. Comedones are early symptoms of acne, and because swollen comedones damage the follicular wall, sebum and microorganisms readily penetrate the follicular wall to cause an inflammatory response by neutrophils, T cells.
To date, there are many methods that have been widely used in order to inhibit excessive secretion of sebaceous gland lipids and pathogenic bacteria that cause acne and inflammation of the pilo-sebaceous glands. In the aspect of using antibiotics, the local treatment of the colonization of propionibacterium acnes by clindamycin and erythromycin is commonly used to relieve the pro-inflammatory action of acne formation. In addition to its inhibitory effect on Propionibacterium acnes, erythromycin may act in the treatment Of acne by its inhibition Of P-450 enzyme activity, thereby delaying retinoid catabolism and attenuating retinoid-mediated FGFR2b signaling (AHMAD N, MUKHTAR H. Cytochrome P450: Atarget for drug depletion for skin diseases [ J ]. Journal Of investigative Dermatology,2004,123(3): 417-25.). However, several clinical trials have shown that chronic topical erythromycin use results in a gradual decrease in the effectiveness of acne treatment (DRENO B, FOURLC P, REYNAOD A, et al. Effect of zinc gluconate on propionibacterium acid resistance to erythromycins in antibiotic activity in the vitamin and in the vitamin study [ J ]. European J. of microbial chemistry: EJD,2005,15(3):152-5.), which may be associated with the development of antibiotic-resistant propionibacteria, and therefore topical antibiotic monotherapy is not recommended.
In addition, The symptoms of acne are exacerbated by androgens increasing sebum production by activation of The androgen receptor of sebaceous gland cells (TRIFU V, TIPLICA G S, NAUMESCU E, et al. cortex 17alpha-propionate 1% yield, a new potential antidandrogene for topical treatment of acne. A. cortex randomised, double-bed compatible residues V. plant and dtreotide 0.05% yield [ J ]. The skin joint of medicine, 2011,165(1): 177-83.). At The same time, The data show that androgen-insensitive or castrated males do not produce acne due to decreased sebum secretion, further highlighting The importance of androgen and androgen receptor interaction in The development of acne (IMPERATO-MCGINLEYJ, GAUTIER T, CAI L Q, et al. The androgen control of recipe production. students of subjects with a second hydrotestone specificity and complex androgen sensitivity [ J ]. The Journal of clinical endirinology and metabolism,1993,76(2): 524-8.). Clinically, the commonly used medicines include antiandrogen, antibiotics, glucocorticoids, retinoic acid and the like. Wherein the externally applied all-trans retinoic acid, adapalene and tazarotene are the first choice drugs for treating mild acne; oral retinoic acid Drugs are mainly used for severe acne, which can promote epidermal cell renewal, alter abnormal follicular keratinization and promote comedolytic dissolution, and treat acne by regulating epidermal cell proliferation and differentiation, stimulating new collagen formation and fibroblast proliferation and reducing inflammation (BALDWIN E, NIGHLAND M, KENDALL C, et al.40Years Of Topical cosmetic ingredients [ J ]. Journal Of Drugs In Dermatology,2013,12(6): 638-42), reversing abnormal keratinization Of the epithelium, resulting In a decrease In keratinocyte cohesion, but have teratogenic effects (marquench a L, vitamin In wounds, origin) and pathological changes Of skin, systemic properties Of acne [ J ] 210, 20; oral antiandrogens selectively promote degradation of the androgen receptor (YANG Z, CHANG Y J, YU IC, et al. ASC-J9 peptides and bulbar musculature renal degradation of androgen receptor [ J ]. Nature medicine,2007,13(3):348-53.), primarily in female patients; glucocorticoid drugs are commonly used for the treatment of fulminant acne or acne conglobata, but these drugs have major side effects. Phototherapy such as photodynamic therapy with 5-aminolevulinic acid is also developed in some hospitals, and the principle is that symbiotic bacteria can generate a large amount of photosensitive substances to emit singlet oxygen and other free radicals to cause the injury and apoptosis of sebaceous gland cells under the irradiation of light with a certain wavelength (MA L, XIANG L H, YU B, et al, Low-dose topical 5-aminoglutaric acid photodynamic therapy in the treatment of the pathological of pathological sensitivity of acne vulgaris [ J ]. Photodiotherapy and photodynamic therapy,2013,10(4):583-90.), so that the treatment of acne can be realized. In addition, the acne can be treated by methods of traditional Chinese medicine decoction, acupuncture and moxibustion and the like, but the treatment course is long. There is therefore an urgent need for new drugs that can target a variety of pathological processes of acne with fewer side effects.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide the application of the small molecular short peptide in preparing a product for treating acne, in particular the application in preparing a product for treating acne caused by the change of hormone level in puberty and gestation.
The purpose of the invention is realized by the following technical scheme: the application of a small molecule short peptide in preparing a product for treating acne; wherein, the amino acid sequence of the small molecule short peptide is shown as follows: LQLQAEER.
The acne is preferably acne caused by the change of hormone level in adolescence and gestation.
The effective concentration of the small molecule short peptide is 100 +/-10 mu g/ml.
The product is a medical product, a skin care product or a cosmetic; preferably medical products, including pharmaceuticals and the like.
The formulation types of the product include but are not limited to solution or dry powder; preferably obtained by dissolving small molecule short peptide in algal polysaccharide hydrogel.
The seaweed polysaccharide hydrogel is preferably obtained by the following method: adding the algal polysaccharide powder into water, and uniformly mixing to obtain the algal polysaccharide hydrogel.
The content of the algal polysaccharide in the algal polysaccharide hydrogel is preferably 10% (w/w).
The application of a small molecular short peptide in preparing a medicament for inhibiting the hyperproliferation of sebaceous glands, reducing the secretion of skin grease and/or reducing the content of triglyceride in sebaceous gland cells; wherein, the amino acid sequence of the small molecule short peptide is shown as follows: LQLQAEER.
The application of a small molecular short peptide in preparing a medicament for inhibiting the growth of human keratinocytes, human skin fibroblasts and/or human sebaceous gland cells; wherein, the amino acid sequence of the small molecule short peptide is shown as follows: LQLQAEER.
Compared with the prior art, the invention has the following advantages and effects:
1. compared with the existing common acne treatment medicines such as tretinoin, the short peptide of the invention can also inhibit the hyperproliferation of sebaceous glands and reduce the oil secretion of skin, has smaller toxicity and side effect, is wider in applicable population, can effectively inhibit the hyperproliferation of the sebaceous glands and reduce the oil secretion of the skin, has good transdermal property, can be used for resisting acne, and particularly can be used for treating acne caused by the change of hormone level in the adolescence and the gestation period.
2. No document reports that the sequence polypeptide can be used for treating acne, and the short peptide has only 8 amino acids, high stability, obvious use effect and advanced technology because the short peptide can continuously act on a treatment part.
Drawings
FIG. 1 is a graph showing the results of the colony formation assay for the inhibition of proliferation of short peptides on human keratinocytes (HACAT), Human Skin Fibroblasts (HSF) and human sebaceous gland cells (SZ 95).
FIG. 2 is a graph showing the results of the CCK-8 method for detecting the inhibitory effect of short peptides on human keratinocytes (HACAT), Human Skin Fibroblasts (HSF) and human sebaceous gland cells (SZ 95).
FIG. 3 is a graph showing the results of measurement of oil secretion of human sebaceous gland cells (SZ95) by the oil red O staining method; wherein, A is DHT control; b is the co-culture of DHT and short peptide.
FIG. 4 is a graph showing the results of triglyceride detection in human sebaceous gland cells by the triglyceride kit.
FIG. 5 is a schematic diagram of the establishment of a rabbit ear acne model; wherein A, B is a visual diagram of rabbit ear skin before and after modeling; C. d is a rabbit ear skin microscopic image before and after modeling; E. f is a staining graph of rabbit ear skin tissue HE before and after modeling.
FIG. 6 is a graph showing the effect of short peptides on recovery from acne in rabbit ears; wherein A is an intuitive picture of the skin recovery condition of the rabbit ears after the medicines in each group are smeared; b is a microscopic picture of the skin recovery condition of the rabbit ears after the medicines in each group are smeared; c is HE staining pattern of skin tissue after rabbit ears are smeared with each group of medicines.
FIG. 7 is a graph showing the results of a transdermal effect test of short peptides; wherein A is the hair follicle of rabbit ears observed under white light; and B is the condition of aggregation of FITC-P5 peptide on hair follicles observed under a fluorescence microscope.
Detailed Description
The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto. The experimental methods used in the following examples are all conventional methods unless otherwise specified, and the experimental materials, reagents and the like used in the examples are all commercially available unless otherwise specified. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
The small molecule short peptide related in the invention is a small molecule short peptide (short peptide for short) containing 8 amino acid residues, the peptide is derived from a peptide segment of basic fibroblast Growth Factor (bFGF, also called FGF2) which is naturally degraded in vivo, and the amino acid sequence of the peptide is as follows: LQLQAEER; the molecular weight is 986.09 Da; can be obtained by chemical synthesis or Chinese patent (patent number: 201410654935.1, name: a small anti-tumor molecular polypeptide and application).
The medium used in the present invention for culturing HACAT, HSF and SZ95 cells was DMEM medium from HyClone, USA.
Example 1
1. Clonogenic experiments the growth inhibitory effect of the short peptides on human keratinocytes (HACAT), Human Skin Fibroblasts (HSF) and human sebaceous gland cells (SZ95) was determined, wherein human sebaceous gland cells SZ95 were obtained from north na creative union, sozhou, human keratinocytes HACAT were obtained from shanghai famous biotechnology limited, and human fibroblasts HSF were obtained from shanghai continental biotechnology limited.
The cells of each group in the logarithmic growth phase were digested with trypsin and blown into single cells, diluted to a certain fold and seeded in 6-well plates (1.2X 10)6One/well) and gently rotated to disperse the cells evenly. The wells were incubated with 0. mu.M, 20. mu.M, 100. mu.M and 200. mu.M of the short peptide for 2 to 3 weeks, the medium was discarded, the cells were fixed, stained with crystal violet, visualized by photography and the number of cell colonies was counted.
The results are shown in FIG. 1: the experimental result shows that the short peptide can inhibit the growth of human skin keratinocyte (HACAT), fibroblast (HSF) and human sebaceous gland cell (SZ 95).
2. CCK-8 method for detecting growth inhibition effect of short peptide on human keratinocyte (HACAT), Human Skin Fibroblast (HSF) and human sebaceous gland cell (SZ95)
Respectively taking human keratinocyte (HACAT), Human Skin Fibroblast (HSF) and human sebaceous gland cell (SZ95), laying a 96-well plate according to 3000 cells per well, starving for 24 hours after adherence, taking short peptide, treating for 48 hours according to 5 multiplied concentration (0, 0.00256, 0.0128, 0.064, 0.32, 1.6, 8, 40 and 200 mu M), then treating by using CCK-8 reagent, and detecting OD value of each well by using a microplate reader.
As shown in FIG. 2, the short peptide inhibited the growth of human keratinocytes (HACAT), human dermal fibroblasts (HSF) and human sebaceous gland cells (SZ95), was less toxic, and its proliferation-inhibiting ability was only down-regulated by about 20% even at high concentrations. Thus, the short peptide has low cytotoxicity to skin sources.
3. Oil red O staining method for measuring oil secretion of SZ95 in human sebaceous gland cells
After a certain amount of human sebaceous gland cells SZ95 were plated in 6-well plates (1.2X 10)6One/well), 100nM DHT (dihydrotestosterone, available from Beijing Soilebao technologies, Inc.) was added for co-culture. The experimental group was treated with 100. mu.M of short peptide for 48 hours, the cells of the control group and the experimental group were fixed after removing the medium, and stained with oil red O for 10 minutes. The excess dye was then removed by rinsing with 60% (v/v) aqueous isopropanol. Finally, the cells were stained with hematoxylin for 20 seconds, rinsed with phosphate buffer (0.2M, pH7.2) and photographed under a microscope.
The results are shown in FIG. 3, and it can be seen from the experimental results that the human sebaceous gland cells SZ95 treated with the short peptide are stained with less red oil droplets, indicating that the short peptide can inhibit the secretion of oil from human sebaceous gland cells SZ 95.
4. Method for detecting triglyceride in human sebaceous gland cells
A certain amount of sebaceous gland cells SZ95 were seeded in 6-well plates (1.2X 10)6One/well), adding short peptides with different concentrations (20 μ M, 100 μ M and 200 μ M) into the wells the next day, taking no short peptide as a blank Control (Control), incubating for 48 hours, washing with PBS, collecting cells from each well into an EP tube, and separatingDiscarding the supernatant from the heart, and leaving the cell precipitate; then adding cell lysis solution into an EP tube, performing lysis on ice for 30 minutes, directly extracting a part of the lysis solution without centrifuging, mixing the part of the lysis solution with working solution, and determining the OD value; centrifuging the lysate, and determining the protein content of the cells by using a BCA kit; and finally, substituting the OD value and the protein content measured by the cells into a formula of a specification of a triglyceride kit, and calculating the relative concentration of triglyceride in the sebaceous gland cells SZ95 after different medicaments are treated. Wherein:
the results are shown in FIG. 4, and the experimental results show that the short peptide can reduce the content of triglyceride in the sebaceous gland cell SZ 95.
5. Establishment of rabbit ear acne model
The model was used to test the effect of the drug on the recovery of acne on rabbit ear skin.
In the experiment, a new zealand rabbit (12-week-old common-grade male new zealand rabbit with the weight of 2.0-2.5 kg and purchased from Xinhua laboratory animal farm in Huadu province in Guangzhou city) is selected, and an acne rabbit ear model is constructed by using Kligman method (American Academy of dermatological simplicity on social society [ J ]. Journal of the American Academy of Dermatology,1989,20(2Pt 1): 272-7), wherein the specific steps are as follows:
according to the Kligman method specified by a rabbit ear model, 1 time a day, the coal tar is dipped by a cotton swab and smeared on the inner side surface of a rabbit ear in a hairless range for 15 days continuously, and the thickness, hardness, redness and swelling of the rabbit ear, expansion of a hair follicle mouth, keratotic thrombosis and the like are observed during the period. After the model is made, photographing and HE staining are carried out, and the success of the model making is judged according to the character grading of the acne skin lesion and the histopathological examination established by the Chinese acne treatment guideline (2014 revision).
The results are shown in FIG. 5, which shows that the rabbit ear tissues are significantly thickened, hardened and roughened after molding. The follicular infundibulum is expanded, the follicular orifice is obviously seen with a plug, which is in an acne shape, and the follicular orifice is raised and has obvious desquamation, thus proving that the rabbit ear acne model is successfully molded.
6. Effect of short peptides on recovery of acne in Rabbit ears
After the rabbit ear acne model is successfully molded, 10% (w/w) algal polysaccharide hydrogel (algal polysaccharide powder is dissolved by ultrapure water and uniformly mixed to form gel) is used as a solvent to prepare short peptide with the concentration of 100 mu g/ml, the short peptide is smeared on rabbit ears every day, only 10% (w/w) algal polysaccharide hydrogel is smeared on a control group, nothing is smeared on a blank group, the recovery condition of rabbit ear acne of each group is observed after 20 days, and photographing and HE dyeing are carried out.
The results are shown in fig. 6, and show that the short peptide can obviously improve the symptoms of rabbit ear acne, the swelling of rabbit ears, the disappearance of epidermal keratinization and acne, the degree of sebaceous gland hyperplasia is reduced, pores are gradually restored, and the short peptide has a remarkable repairing effect on the rabbit ear acne.
7. Transdermal effect testing of short peptides
A short peptide with fluorescence (the short peptide is synthesized by the Beijing national center for nanometer science; the fluorescent substance is FITC label connected with the peptide) is used as the short peptide and is marked by a fluorescent dye, namely the fluorescent dye is connected to the short peptide through a chemical bond, wherein the weight ratio of the short peptide to the fluorescent substance is 50:1, the short peptide is synthesized by the Beijing national center for nanometer science in the experiment), the short peptide is smeared on rabbit (rabbit ear acne model; the construction method is the same as the above) ear skin for 6 hours, then the rabbit ear skin is sampled and frozen and sliced, and the fluorescence condition of the skin is observed under a fluorescence microscope.
The results are shown in fig. 7, which shows that the short peptide can penetrate the keratinized substance in the mouth and funnel of the hair follicle and extend to the sebaceous gland, thus demonstrating that the short peptide can directly reach the sebaceous gland tissue of the skin to act, and the transdermal effect is good.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Sequence listing
<110> river-south university
Application of <120> small molecule short peptide in preparation of product for treating acne
<160>1
<170>SIPOSequenceListing 1.0
<210>1
<211>8
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>1
Leu Gln Leu Gln Ala Glu Glu Arg
1 5
Claims (7)
1. The application of the small molecule short peptide in preparing the product for treating the acne is characterized in that the amino acid sequence of the small molecule short peptide is as follows: LQLQAEER.
2. Use according to claim 1, characterized in that: the effective concentration of the small molecule short peptide is 100 +/-10 mu g/ml.
3. Use according to claim 1, characterized in that: the product is a medical product, a skin care product or a cosmetic.
4. Use according to claim 1, characterized in that: the preparation type of the product is solution or dry powder.
5. Use according to claim 4, characterized in that: the product is obtained by dissolving micromolecular short peptide into algal polysaccharide hydrogel.
6. The application of the small molecule short peptide in preparing the medicine for inhibiting the hyperproliferation of sebaceous glands, reducing the secretion of skin grease and/or reducing the content of triglyceride in sebaceous gland cells is characterized in that the amino acid sequence of the small molecule short peptide is as follows: LQLQAEER.
7. The application of the small molecule short peptide in preparing the medicine for inhibiting the growth of human keratinocytes, human skin fibroblasts and/or human sebaceous gland cells is characterized in that the amino acid sequence of the small molecule short peptide is as follows: LQLQAEER.
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| WO2021175186A1 (en) * | 2020-03-06 | 2021-09-10 | 暨南大学 | Use of small molecule short peptide in preparation of product for treating acne |
| CN114681588A (en) * | 2022-02-17 | 2022-07-01 | 中山大学 | Application of polypeptide EN-9 in preparation of product for treating acne |
| CN116425836B (en) * | 2022-12-16 | 2024-04-16 | 中国医学科学院皮肤病医院(中国医学科学院皮肤病研究所) | A+ polypeptides/proteins and uses thereof |
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| CZ309857B6 (en) | 2022-03-31 | 2023-12-20 | Contipro A.S. | Hexapeptide, compositions comprising this hexapeptide, and topical use thereof |
| CN114573661B (en) * | 2022-04-27 | 2023-10-27 | 冯来坤 | Small molecular peptide and application thereof in promoting transdermal absorption of biomacromolecules |
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| CN114681588A (en) * | 2022-02-17 | 2022-07-01 | 中山大学 | Application of polypeptide EN-9 in preparation of product for treating acne |
| CN114681588B (en) * | 2022-02-17 | 2023-06-06 | 中山大学 | Application of polypeptide EN-9 in preparation of product for treating acne |
| CN116425836B (en) * | 2022-12-16 | 2024-04-16 | 中国医学科学院皮肤病医院(中国医学科学院皮肤病研究所) | A+ polypeptides/proteins and uses thereof |
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Application publication date: 20200609 |