Application of amphibian active peptide Ha-CATH1 in preparation of inflammatory dermatosis relieving preparation and inflammatory dermatosis relieving preparation

Abstract

The invention discloses application of amphibian active peptide Ha-CATH1 in preparation of an inflammatory dermatosis relieving preparation and the inflammatory dermatosis relieving preparation, and relates to the field of biomedicine, and the amino acid sequence of the active peptide Ha-CATH1 is as shown in SEQ ID NO: 1. Experiments prove that the amphibian active peptide Ha-CATH1 can relieve skin erythema, scales and other symptoms caused by inflammatory dermatosis, relieve local and systemic inflammatory reactions, and can be used for improving the inflammatory dermatosis. The amphibian active peptide Ha-CATH1 sequence is low in concentration, can play a role, is efficient, has non-toxic metabolites, and can be applied to preparation of preparations for relieving inflammatory dermatosis.

Description

Technical Field

[0001] The present application relates to the field of biomedicine, in particular to the application of an amphibian active peptide Ha-CATH1 in the preparation of an inflammatory skin disease remission preparation and an inflammatory skin disease remission preparation. Background Art

[0002] Inflammatory skin diseases are a group of diseases caused by the destruction of skin tissues due to immune system disorders, including psoriasis, atopic dermatitis, eczema, contact dermatitis, urticaria, acne, rosacea, etc.

[0003] Among them, psoriasis is a chronic, recurrent, inflammatory, autoimmune skin disease. Clinically, psoriasis vulgaris is the main type, and the typical clinical manifestation is scaly erythema or plaques. Research shows that about 125 million people worldwide suffer from psoriasis, and the risk of metabolic syndrome, cardiovascular diseases, mental disorders and other diseases in moderate to severe psoriasis patients increases.

[0004] Although the exact cause of psoriasis has not been fully elucidated, genetic, immune and environmental factors play important roles in the occurrence and development of psoriasis. In the pathogenesis stage of psoriasis, the antimicrobial peptide LL-37 secreted by keratinocytes binds to its own nucleic acid to form a complex, which stimulates the activation of dendritic cells, and then secretes cytokines such as IL-23 to activate T cell subsets such as Th17, Th1, and Th22. The cytokines such as IL-17 and IL-22 secreted by the latter promote the proliferation of keratinocytes. The stimulated keratinocytes further secrete corresponding pro-inflammatory cytokines and chemokines, recruiting more T cells and neutrophils to infiltrate, thus forming a positive feedback loop between immune cells and keratinocytes to amplify the inflammatory response, and finally forming the pathological features of psoriasis such as epidermal thickening, abnormal keratinization, vascular tortuosity and dilation, and inflammatory cell infiltration.

[0005] Atopic dermatitis (AD) is mainly manifested as chronic recurrent eczematous dermatitis, accompanied by significant skin dryness and severe itching. The prevalence of atopic dermatitis in children in developed countries reaches 10-20%. In the past decade or so, the incidence of atopic dermatitis in China has been increasing continuously, affecting people of all ages.

[0006] The onset of atopic dermatitis is closely related to genetics and the environment. Immune abnormalities, skin barrier dysfunction, skin microbiota disorders, etc. are important links in the onset. Th2-type inflammation is a basic feature of atopic dermatitis, and IL-4 and IL-13 are important cytokines. Under the action of various pathogenic factors, Langerhans cells and dendritic cells present allergens and then initiate Th2-type inflammatory responses. Th2-type cytokines can inhibit keratinocytes from expressing skin barrier-related proteins, exacerbating skin barrier dysfunction. At the same time, scratching not only damages the skin barrier but also leads to the release of autoantigens, and the body produces IgE against autoantigens, making the body in an inflammatory and allergic state.

[0007] Current treatments for inflammatory skin diseases such as psoriasis and atopic dermatitis include: systemic treatment and topical treatment. Traditional drugs used in systemic treatment include: immunosuppressants, glucocorticoids, biological agents, etc.; topical drugs play a key role in the topical treatment of mild and moderate to severe patients. Currently, commonly used topical drugs in clinical practice include calcineurin inhibitors (such as tacrolimus, pimecrolimus), glucocorticoids (such as halometasone, mometasone furoate), etc. Since patients need to take drugs for a long time, and these drugs have many side effects, such as local itching and irritation, skin atrophy, telangiectasia, etc. These side effects can increase the risk of infection and skin malignancies. Moreover, the existing drugs in clinical practice have a slow onset of action, resulting in poor patient compliance and bringing a greater psychological and economic burden to patients. Therefore, new topical preparations need to be developed to solve current clinical problems.

[0008] Polypeptide drugs emerging in an endless stream have advantages such as high selectivity and non-toxic metabolites. Some peptide drugs have been applied clinically, and bioactive peptides have also become a research hotspot in recent years. Bioactive peptides derived from amphibians are also one of them. Amphibians have naked skin and a complex living environment. To resist infections and various physical factor stimulations in nature, amphibians have evolved a natural defense system composed of various active peptides. These polypeptides present in skin glands are released at high concentrations when the skin is stimulated and damaged to play their roles. These polypeptides extracted from amphibian skin have various activities such as anti-infection, anti-inflammatory, immunomodulatory, promoting wound healing, and antioxidant.

[0009] The amphibian active peptide Ha-CATH1 is extracted from the skin of the Sichuan tree frog. For example, this active peptide Ha-CATH1 is disclosed in CN202410664870.2 and can be used to prepare drugs for promoting the repair of skin infections. This active peptide Ha-CATH1 can promote the healing of full-thickness skin infection wounds in mice caused by Pseudomonas aeruginosa.

[0010] However, there is no report in the existing literature on using this active peptide to treat inflammatory skin diseases.

[0011] The information disclosed in the background art section is only intended to enhance the overall understanding of the background of the present invention and should not be regarded as an admission or any form of suggestion that this information constitutes the prior art already known to those of ordinary skill in the art. Summary of the Invention

[0012] The present application provides an application of an amphibian active peptide Ha-CATH1 in the preparation of a preparation for relieving inflammatory skin diseases, and a preparation for relieving inflammatory skin diseases.

[0013] The present application provides an application of an amphibian active peptide Ha-CATH1 in the preparation of a preparation for relieving inflammatory skin diseases, and the amino acid sequence of the active peptide Ha-CATH1 is as shown in SEQ ID NO:1.

[0014] Preferably, the inflammatory skin disease is psoriasis or atopic dermatitis.

[0015] Another aspect of the present application further provides a preparation for relieving inflammatory skin diseases, including: the amino acid sequence of the active peptide Ha-CATH1 is as shown in SEQ ID NO:1.

[0016] Preferably, the inflammatory skin disease is psoriasis or atopic dermatitis.

[0017] Preferably, after the skin lesion of the inflammatory skin disease, the preparation is used to reduce the range of hyperkeratosis and parakeratosis and inflammatory cell infiltration.

[0018] Preferably, after the skin lesion of the inflammatory skin disease, the preparation is used to reduce the relative expression levels of the key inflammatory factors IL-23 and IL-1β genes in the plasma.

[0019] Preferably, the concentration of the active peptide Ha-CATH1 is 200 μg / ml.

[0020] Preferably, the dosage form is a solution or a spray.

[0021] The beneficial effects that the present application can produce include: 1) The application of the amphibian active peptide Ha-CATH1 provided by the present application in the preparation of a preparation for relieving inflammatory skin diseases and the preparation for relieving inflammatory skin diseases. Through experiments, it is verified that the amphibian active peptide Ha-CATH1 can relieve symptoms such as skin erythema, scales, scratches, and scabs caused by inflammatory skin diseases, reduce local and systemic inflammatory reactions, and can be used to improve inflammatory skin diseases. The amphibian active peptide Ha-CATH1 sequence can play a role at a low concentration, is efficient and its metabolites are non-toxic, and can be applied to the preparation of a preparation for relieving inflammatory skin diseases.

[0022] 2) The application of the amphibian active peptide Ha-CATH1 provided by this application in the preparation of a preparation for relieving inflammatory skin diseases, the preparation for relieving inflammatory skin diseases. After treating imiquimod-induced psoriasis lesions with the active peptide Ha-CATH1, the range of hyperkeratosis and parakeratosis can be reduced, and inflammatory cell infiltration can be improved; the relative expression levels of the key inflammatory factors IL-23 and IL-1β genes in the plasma of mice can be reduced; this active peptide can effectively reduce the erythema, edema, scratches, and scabs on the skin of atopic dermatitis model mice, and reduce the scratching behavior of mice, indicating that this active peptide can improve the symptoms of inflammatory skin diseases such as psoriasis and atopic dermatitis, and has good application prospects. Description of the Drawings

[0023] Figure 1 It is the experimental result diagram of the active peptide Ha-CATH1 in Example 1 of this application; among them, A is the photo of the skin lesion area of mice in the blank control group, psoriasis model group, positive control group (calcipotriol ointment), and Ha-CATH1 group after treatment for 1, 3, 5, and 7 days; B is the PASI score result diagram of the experimental mice in the blank control group, psoriasis model group, positive control group (calcipotriol ointment), and Ha-CATH1 group ( ) ; C is the HE staining result of the skin tissue of the experimental mice in the blank control group, psoriasis model group, positive control group (calcipotriol ointment), and Ha-CATH1 group after treatment for 7 days; Figure 2 It is the experimental result diagram of the ELISA detection and qRT-PCR detection of the active peptide Ha-CATH1 in Example 1 of this application; among them, A is the result diagram of the IL-23 content in the plasma of mice in the blank control group, psoriasis model group, positive control group (calcipotriol ointment), and Ha-CATH1 group after 7 days of experimental treatment. The detection was performed by ELISA method. Compared with the model group, *P<0.05, **P<0.01, ***P<0.001; B is the relative expression level of IL-1β in the skin lesion tissue of the experimental mice in the blank control group, psoriasis model group, positive control group (calcipotriol ointment), and Ha-CATH1 group after 7 days of experimental treatment. The detection was performed by qRT-PCR. Compared with the model group, *P<0.05, **P<0.01, ***P<0.001; Figure 3 It is the experimental result diagram of the active peptide Ha-CATH1 in Example 2 of this application; among them, A is the photo of the skin lesion performance of the experimental mice in the blank control group, atopic dermatitis model group, positive control group (halometasone ointment), and Ha-CATH1 group before treatment and on the 7th day of treatment; B is the result diagram of the skin lesion severity score of the experimental mice in the blank control group, atopic dermatitis model group, positive control group (halometasone ointment), and Ha-CATH1 group during the treatment period ( ), compared with the model group, *P<0.05, **P<0.01, ***P<0.001. Detailed implementation manners

[0024] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments, but the present invention is not limited in any way. Any transformation or improvement made based on the teachings of the present invention falls within the protection scope of the present invention.

[0025] Embodiment In the following embodiments, the materials and instruments used are obtained from commercial channels unless otherwise specified; the detection methods used are existing methods unless otherwise specified.

[0026] The active peptide Ha-CATH1 used in the following embodiments is a polypeptide encoded by the defense peptide gene of the Chinese amphibian Hyla annectans. It is prepared according to any method disclosed in CN202410664870.2 "An Immunomodulatory Peptide Ha-CATH1 and Its Application". Ha-CATH1 consists of 31 amino acid residues, with a molecular weight of 3687.13 daltons and an isoelectric point of 12. Its amino acid sequence is shown as SEQ ID NO:1: Phe Ser Arg Ser Arg Cys Gln Gly Arg Pro Cys Arg Leu Ala Gly Asp Arg Ser Phe His Phe Gly Arg Lea Arg His LeaAsnAsn Glu Ile.

[0027] Embodiment 1 Application of Amphibian Active Peptide Ha-CATH1 in the Preparation of Psoriasis Remission Preparation 1. Construction of Psoriasis Mouse Model and Treatment Experiment Twenty BALB / c female mice, 6-8 weeks old, with a body weight of (20±2) g, were provided by the Experimental Animal Department of Kunming Medical University and were housed in a SPF-level barrier environment. After one week of adaptive feeding, the mice were randomly divided into a blank control group, a psoriasis model group, a positive control group (calcipotriol ointment), and a Ha-CATH1 group, with 5 mice in each group.

[0028] One day before the experiment, the back of the mice was depilated in a 2 cm × 3 cm area using a razor and depilatory cream. After the model was established, except for the blank group, the mice were smeared with 62.5 mg of 5% imiquimod ointment on the back every morning to induce psoriasiform dermatitis. Six hours later, the polypeptide Ha-CATH1 solution (200 μg / ml, 200 μl / mouse) was evenly smeared on the back of the mice in the Ha-CATH1 group, the psoriasis model group was smeared with an equal amount of normal saline, and the positive control group was smeared with an appropriate amount of 0.005% calcipotriol ointment once a day for 7 consecutive days. The blank group was not treated.

[0029] Before modeling on the 1st, 3rd, 5th, and 7th days, skin lesion photos of the mice were collected, and the psoriasis skin lesion severity index (PASI) was scored daily: mainly including three aspects of erythema, scale, and infiltration degree. According to the absence, mild, moderate, severe, and extremely severe skin lesions, they were recorded as 0 points, 1 point, 2 points, 3 points, and 4 points respectively. The sum of the scores of the three aspects was the final score, and the higher the total score, the more severe the skin lesions. When erythema, scale, infiltration symptoms appeared on the back of the mice and the skin lesion pathological results were consistent with the characteristics of psoriasis, it was considered that the modeling was successful. On the 8th day of the experiment, the plasma and skin lesions of the mice were taken, and HE staining, ELISA was used to measure the content of IL-23 in the plasma, and qRT-PCR was used to detect the relative expression level of IL-1β in the skin lesions.

[0030] 2. Test results On the 1st, 3rd, 5th, and 7th days of the experiment, the skin lesion manifestations of each group of mice were as Figure 1 shown in A. In the psoriasis model group, obvious erythema, scales, and infiltration appeared on the back skin. In the calcipotriol group, the scale and infiltration conditions of the mice were alleviated compared with the model group, but some erythema still remained. Compared with the model group, in the Ha-CATH1 group, the scales on the back of the mice were significantly reduced, the erythema was lightened, and the skin infiltration was alleviated. The daily change results of the PASI score after the start of the experiment are shown in Figure 1 B. The daily PASI score of the Ha-CATH1 group was lower than that of the model group and better than that of the calcipotriol treatment group. It shows that Ha-CATH1 can improve the skin lesion manifestations of imiquimod-induced psoriasis mice.

[0031] The HE staining of the skin lesions of each group of mice after 7 days of treatment is shown in Figure 1 C. In the model group, extensive hyperkeratosis and small areas of parakeratosis were visible, with residual cell nuclei, uneven thickness of the dermis and epidermis, a small amount of epidermal papilla elongation, and a large number of lymphocyte infiltrations in the dermis layer. The pathological manifestations were consistent with the characteristics of psoriasis. Compared with the model group, the ranges of hyperkeratosis and parakeratosis in the Ha-CATH1 group were reduced, and the inflammatory cell infiltration was decreased, suggesting that Ha-CATH1 can improve the histopathological morphology of the skin lesions of imiquimod-induced psoriasis mice.

[0032] The detection results by ELISA method are as Figure 2As shown in A, compared with the model group, the amphibian active peptide Ha-CATH1 reduced the level of the key inflammatory factor IL-23 in the plasma of mice, and the difference was statistically significant.

[0033] The results of qRT-PCR are as Figure 2 shown in B. Compared with the model group, the amphibian active peptide Ha-CATH1 reduced the relative expression level of the inflammatory factor IL-1β gene in the skin lesions of mice, and the effect was better than that of the calcipotriol group, and the difference was statistically significant.

[0034] Example 2 Application of amphibian active peptide Ha-CATH1 in the preparation of atopic dermatitis (AD) remission preparation 1. Construction of atopic dermatitis mouse model (AD model) and drug intervention Twenty BALB / c female mice were randomly divided into a blank control group, an AD model group, a positive control group (halometasone ointment), and a Ha-CATH1 group, with 5 mice in each group.

[0035] One day in advance, the back of the mice in a 2 cm × 3 cm area was depilated with a razor and depilatory cream. On the first day of the experiment, 200 μL of 1% DNCB solution was applied to the back of the mice for 3 consecutive days; on the 6th and 9th days, 200 μL of 0.5% DNCB solution was applied; no treatment was performed on the 10th to 19th days; on the 20th day, the newly grown hair on the back of the mice was removed again with a razor and depilatory cream, and 200 μL of 1% DNCB solution was applied every day from the 20th to 22nd days, and 200 μL of 0.5% DNCB solution was applied on the 25th and 28th days. The blank group was only depilated.

[0036] When erythema, edema, epidermal exfoliation, scratches, crusts, etc. appeared on the back of the mice, it was considered that the model was successfully established.

[0037] On the 29th day, drug treatment was started. The model group was smeared with 200 μL of normal saline, the positive control group was smeared with an appropriate amount of halometasone ointment, and the Ha-CATH1 group was smeared with Ha-CATH1 (200 μg / ml, 200 μL), once a day for 7 consecutive days. The blank group was not treated.

[0038] On the 0th, 1st, 2nd, 3rd, 4th, 5th, 6th, and 7th days of treatment, the severity of skin lesions was scored according to four aspects of skin erythema, edema / papules, epidermal exfoliation / scratches, and dryness / crusts of the mice. Among them, 0 points (no skin lesions), 1 point (mild skin lesions, skin lesions exist and need to be carefully identified), 2 points (moderate skin lesions, skin lesions can be immediately identified), 3 points (severe skin lesions, skin lesions are very obvious). The sum of the scores in each aspect is the total score. The higher the score, the more severe the skin lesions.

[0039] 2. Test results At the end of the treatment test, the skin lesions of the mice were asFigure 3 A. It can be seen that compared with before treatment, obvious atopic dermatitis symptoms such as erythema, scales, edema, scratch marks, and crusts appeared on the backs of mice repeatedly stimulated with DNCB. Compared with the model group, after treatment with the polypeptide Ha-CATH1 for 7 days, the erythema, edema, scratch marks, and crusts on the skin of mice were alleviated, and the scratching behavior of mice was reduced. Compared with the blank group, the severity score of skin lesions in mice after repeated DNCB stimulation increased significantly, as shown in Figure 3 B. From the 4th day of treatment, it can be seen that the severity score of skin lesions in mice treated with the polypeptide Ha-CATH1 and halometasone ointment decreased, significantly lower than that of the model group, improving the skin lesions of atopic dermatitis in mice.

[0040] The above results indicate that the amphibian active peptide Ha-CATH1 can improve the symptoms of inflammatory skin diseases such as psoriasis and atopic dermatitis, and has good application prospects.

[0041] Example 3 Preparation of the Active Peptide Preparation The amphibian active peptide Ha-CATH1 was dissolved in physiological saline to prepare a solution with a concentration of 200 μg / ml. The obtained solution was evenly covered on the surface of the skin lesion area by means of cotton swab application or spray spraying.

[0042] Although the present invention has been described in detail with reference to the foregoing embodiments, for those skilled in the art, they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some of the technical features. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. Use of an amphibian active peptide Ha-CATH1 in the preparation of a preparation for relieving inflammatory skin diseases, characterized in that, The amino acid sequence of the active peptide Ha-CATH1 is as shown in SEQ ID NO:

1.

2. The application according to claim 1, wherein The inflammatory skin disease is psoriasis or atopic dermatitis.

3. A preparation for relieving inflammatory skin diseases, characterized in that, It includes: The amino acid sequence of the active peptide Ha-CATH1 is as shown in SEQ ID NO:

1.

4. The preparation according to claim 3, characterized in that, The inflammatory skin disease is psoriasis or atopic dermatitis.

5. The preparation according to claim 3, characterized in that, After the preparation is used for the skin lesions of inflammatory skin diseases, it can reduce the ranges of hyperkeratosis and parakeratosis, and inflammatory cell infiltration.

6. The preparation according to claim 3, characterized in that, After the preparation is used for the skin lesions of inflammatory skin diseases, it can reduce the relative expression levels of the key inflammatory factors IL-23 and IL-1β genes in plasma.

7. The preparation according to claim 3, characterized in that, The concentration of the active peptide Ha-CATH1 is 200 μg / ml.

8. The preparation according to claim 3, characterized in that, The dosage form is solution or spray.

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