Application of amphibian active peptide Ha-CATH1 in preparing inflammatory skin disease alleviating preparations, inflammatory skin disease alleviating preparations

Abstract

This application discloses the use of an amphibian active peptide, Ha-CATH1, in preparing a preparation for alleviating inflammatory skin diseases, and the preparation for alleviating inflammatory skin diseases. The application relates to the biomedical field, and the amino acid sequence of the active peptide, Ha-CATH1, is as shown in SEQ ID NO: 1. Experimental verification has shown that the amphibian active peptide, Ha-CATH1, can alleviate symptoms such as skin erythema and scaling caused by inflammatory skin diseases, reduce local and systemic inflammatory responses, and can be used to improve inflammatory skin diseases. The amphibian active peptide, Ha-CATH1, is effective at low concentrations, has high efficacy, and its metabolites are non-toxic. It can be used to prepare preparations for alleviating inflammatory skin diseases.

Description

Technical Field

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

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

[0003] Psoriasis is a chronic, relapsing, inflammatory, autoimmune skin disease, primarily characterized by psoriasis vulgaris, with typical clinical manifestations of scaly erythema or plaques. Studies show that approximately 125 million people worldwide suffer from psoriasis, and patients with moderate to severe psoriasis are at increased risk of developing metabolic syndrome, cardiovascular disease, and mental disorders.

[0004] Although the exact cause of psoriasis has not yet been fully elucidated, genetic, immune, and environmental factors play a significant role in the development and progression of psoriasis. During the pathogenesis of psoriasis, the antimicrobial peptide LL-37 secreted by keratinocytes binds to their own nucleic acids to form a complex, stimulating the activation of dendritic cells, which in turn secrete cytokines such as IL-23 to activate T cell subsets such as Th17, Th1, and Th22. The latter secrete cytokines such as IL-17 and IL-22, which promote the proliferation of keratinocytes. The stimulated keratinocytes further secrete corresponding proinflammatory cytokines and chemokines, recruiting more T cells and neutrophils to infiltrate, thus forming a positive feedback loop between immune cells and keratinocytes, amplifying the inflammatory response and ultimately leading to the pathological characteristics of psoriasis, such as epidermal thickening, abnormal keratinization, vascular tortuosity and dilation, and inflammatory cell infiltration.

[0005] Atopic dermatitis (AD) is characterized by chronic, recurrent eczematous dermatitis, accompanied by significant dry skin and severe itching. The prevalence of atopic dermatitis in children in developed countries is as high as 10-20%. In the past decade, the incidence of atopic dermatitis in my country has continued to increase, affecting people of all ages.

[0006] The onset of atopic dermatitis is closely related to genetics and the environment. Immune abnormalities, skin barrier dysfunction, and skin flora disorders are important links in the onset of the disease. Th2-type inflammation is the basic characteristic of atopic dermatitis, and IL-4 and IL-13 are important cytokines. Under the influence of various pathogenic factors, Langerhans cells and dendritic cells initiate a Th2-type inflammatory response after presenting allergens. 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. The body produces IgE against autoantigens, which puts the body in an inflammatory and allergic state.

[0007] Current treatments for inflammatory skin diseases such as psoriasis and atopic dermatitis include systemic and topical therapies. Traditional systemic medications include immunosuppressants, glucocorticoids, and biologics. Topical medications play a key role in the topical treatment of patients with mild to moderately severe disease. Commonly used topical medications include calcineurin inhibitors (such as tacrolimus and pimecrolimus) and glucocorticoids (such as halometasone and mometasone furoate). These medications require long-term use and are associated with numerous side effects, such as local pruritus, skin atrophy, and telangiectasia. These side effects increase the risk of infection and skin malignancies. Furthermore, existing medications are slow to take effect, leading to poor patient compliance and significant psychological and financial burdens. Therefore, the development of novel topical formulations is needed to address current clinical challenges.

[0008] A growing number of peptide drugs offer advantages such as high selectivity and non-toxic metabolites. Some have already been used clinically, and bioactive peptides have become a research hotspot in recent years. Amphibian-derived bioactive peptides are one such candidate. Due to their exposed skin and complex habitats, amphibians have evolved a natural defense system composed of a variety of bioactive peptides to protect against infections and various physical stimuli. These peptides, present in skin glands, are released in high concentrations in response to skin irritation and damage, exerting their effects. These peptides extracted from amphibian skin exhibit a variety of activities, including anti-infective, anti-inflammatory, immunomodulatory, wound healing, and antioxidant properties.

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

[0010] However, there are no reports on the use of this active peptide in the treatment of inflammatory skin diseases.

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

[0012] In response to the above technical problems, the present application provides an application of an amphibian active peptide Ha-CATH1 in the preparation of an inflammatory skin disease alleviating preparation and an inflammatory skin disease alleviating preparation.

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

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

[0017] Preferably, the preparation is used to reduce the extent of hyperkeratosis and parakeratosis and inflammatory cell infiltration after lesions of inflammatory skin diseases.

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

[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 of this application include:

[0022] 1) This application provides the use of the amphibian active peptide Ha-CATH1 in the preparation of a preparation for alleviating inflammatory skin diseases. Experimental verification has shown that this amphibian active peptide Ha-CATH1 can alleviate symptoms such as erythema, scaling, scratching, and scabs caused by inflammatory skin diseases, reduce local and systemic inflammatory responses, and can be used to improve inflammatory skin diseases. This amphibian active peptide Ha-CATH1 sequence is effective at low concentrations, is highly effective, and its metabolites are non-toxic. It can be used in the preparation of a preparation for alleviating inflammatory skin diseases.

[0023] 2) The application provides the use of the amphibian active peptide Ha-CATH1 in the preparation of an inflammatory skin disease alleviating preparation, and the inflammatory skin disease alleviating preparation. The active peptide Ha-CATH1 can reduce the range of hyperkeratosis and parakeratosis, improve inflammatory cell infiltration, and reduce the relative expression levels of the key inflammatory factors IL-23 and IL-1β genes in mouse plasma after treating imiquimod-induced psoriasis lesions. The 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 the active peptide can improve the symptoms of inflammatory skin diseases such as psoriasis and atopic dermatitis, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Figure 1 is a graph showing the experimental results of the active peptide Ha-CATH1 in Example 1 of the present application; A is a photograph of the skin lesions of mice in the blank control group, psoriasis model group, positive control group (calcipotriol ointment), and Ha-CATH1 group after treatment on days 1, 3, 5, and 7; B is a graph showing the PASI score results of experimental mice in the blank control group, psoriasis model group, positive control group (calcipotriol ointment), and Ha-CATH1 group ( C is the HE staining results of the skin tissue of the blank control group, psoriasis model group, positive control group (calcipotriol ointment), and Ha-CATH1 group after 7 days of treatment of experimental mice;

[0025] Figure 2 These are the experimental results of ELISA and qRT-PCR detection of the active peptide Ha-CATH1 in Example 1 of the present application; A is the result of 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 treated for 7 days, detected by ELISA method, *P<0.05, **P<0.01, ***P<0.001 compared with the model group; B is the relative expression of IL-1β in the skin lesion tissue of mice in the blank control group, psoriasis model group, positive control group (calcipotriol ointment), and Ha-CATH1 group treated for 7 days, detected by qRT-PCR, *P<0.05, **P<0.01, ***P<0.001 compared with the model group;

[0026] Figure 3 The experimental results of the active peptide Ha-CATH1 in Example 2 of the present application are shown in Figure A; wherein A is a photo of the skin lesions of the blank control group, atopic dermatitis model group, positive control group (halometasone ointment), and Ha-CATH1 group of experimental mice before treatment and on the 7th day of treatment; B is a photo of the skin lesion severity score results of the blank control group, atopic dermatitis model group, positive control group (halometasone ointment), and Ha-CATH1 group of experimental mice during treatment ( ), *P<0.05, **P<0.01, ***P<0.001 compared with the model group. DETAILED DESCRIPTION

[0027] 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 changes or improvements made based on the teachings of the present invention fall within the scope of protection of the present invention.

[0028] Example

[0029] Unless otherwise specified, the materials and instruments used in the following examples were obtained from commercial channels; the detection methods used were all existing methods unless otherwise specified.

[0030] The active peptide Ha-CATH1 used in the following examples is a polypeptide encoded by the defense peptide gene of the Chinese amphibian Hyla cylindrica. It was prepared according to any of the methods disclosed in CN202410664870.2, "An Immunomodulatory Peptide Ha-CATH1 and Its Applications." Ha-CATH1 is composed of 31 amino acid residues, has a molecular weight of 3687.13 Daltons, an isoelectric point of 12, and its amino acid sequence is shown in SEQ ID NO: 1:

[0031] Phe Ser Arg Ser Arg Cys Gln Gly Arg Pro Cys Arg Leu Ala Gly Asp Arg

[0032] Ser Phe His Phe Gly Arg Leu Arg His Leu Asn Asn Glu Ile.

[0033] Example 1 Application of amphibian active peptide Ha-CATH1 in the preparation of psoriasis relief preparations

[0034] 1. Construction of psoriasis mouse model and treatment experiment

[0035] Twenty BALB / c female mice, 6-8 weeks old, weighing (20±2) g, were provided by the Department of Experimental Animal Science of Kunming Medical University and housed in an SPF-grade 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.

[0036] One day before the experiment, a 2 cm × 3 cm area on the back of the mice was depilated using a razor and depilatory cream. After modeling, 62.5 mg of 5% imiquimod ointment was applied to the back of all mice except the blank group every morning to induce psoriatic dermatitis. Six hours later, the backs of the mice in the Ha-CATH1 group were evenly coated with a polypeptide Ha-CATH1 solution (200 μg / ml, 200 μl / mouse). The psoriasis model group was treated with an equal amount of normal saline. The positive control group was treated with an appropriate amount of 0.005% calcipotriol ointment once a day for 7 consecutive days. The blank group received no treatment.

[0037] Photographs of mouse skin lesions were collected on days 1, 3, 5, and 7 before modeling. The Psoriasis Lesion Severity Index (PASI) score was assessed daily. The PASI, which measures erythema, scaling, and infiltration, is scored as 0, 1, 2, 3, and 4 for none, mild, moderate, severe, and extremely severe lesions, respectively. The sum of these scores constitutes the final score, with a higher total score indicating more severe lesions. Successful modeling was considered achieved when erythema, scaling, and infiltration were present on the back of the mouse, and pathological findings consistent with psoriasis were obtained. On day 8 of the experiment, plasma and lesional skin samples were collected and analyzed for HE staining, ELISA for plasma IL-23 levels, and qRT-PCR for IL-1β expression in lesional skin.

[0038] 2. Test results

[0039] The skin lesions of mice in each group on the 1st, 3rd, 5th and 7th days of the experiment were as follows Figure 1 As shown in A, the psoriasis model group had obvious erythema, scales and infiltration on the back skin. The scales and infiltration of the mice in the calcipotriol group were less severe than those in the model group, but some mice still had erythema. Compared with the model group, the scales on the back of the mice in the Ha-CATH1 group were significantly reduced, the erythema was lightened, and the skin infiltration was alleviated. The daily changes in PASI scores after the start of the experiment are shown in Figure 1 B, Daily PASI scores in the Ha-CATH1 group were lower than those in the model group and better than those in the calcipotriol-treated group, indicating that Ha-CATH1 can improve the skin lesions of imiquimod-induced psoriasis mice.

[0040] HE staining of the skin lesions of mice in each group after 7 days of treatment Figure 1 C, The model group showed extensive hyperkeratosis, minor parakeratosis, and residual cell nuclei. The dermis and epidermis showed uneven thickness, with a few elongated epidermal processes and abundant lymphocyte infiltration in the dermis, pathological findings consistent with psoriasis. Compared with the model group, the Ha-CATH1 group showed reduced hyperkeratosis and parakeratosis, as well as decreased inflammatory cell infiltration, suggesting that Ha-CATH1 can improve the pathological morphology of imiquimod-induced psoriasis in mice.

[0041] ELISA test results 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 mouse plasma, and the difference was statistically significant.

[0042] qRT-PCR test results are as follows Figure 2 As shown in B, compared with the model group, the amphibian active peptide Ha-CATH1 reduced the relative expression of the inflammatory factor IL-1β gene in the skin lesion tissue of mice, and the effect was better than that of the calcipotriol group, and the difference was statistically significant.

[0043] Example 2 Application of amphibian active peptide Ha-CATH1 in the preparation of atopic dermatitis (AD) alleviating preparations

[0044] 1. Construction of an atopic dermatitis mouse model (AD model) and drug intervention

[0045] Twenty BALB / c female mice were randomly divided into blank control group, AD model group, positive control group (halometasone ointment), and Ha-CATH1 group, with 5 mice in each group.

[0046] One day in advance, a 2 cm × 3 cm area on the back of the mice was depilated using a razor and depilatory cream. On day 1, 200 μL of a 1% DNCB solution was applied to the back of the mice for three consecutive days. On days 6 and 9, 200 μL of a 0.5% DNCB solution was applied. No treatment was performed from days 10 to 19. On day 20, new hair on the back of the mice was removed again using a razor and depilatory cream. From days 20 to 22, 200 μL of a 1% DNCB solution was applied daily, and on days 25 and 28, 200 μL of a 0.5% DNCB solution was applied. A control group underwent hair removal alone.

[0047] The model was considered successful when erythema, edema, epidermal exfoliation, scratches, and scabs appeared on the back of the mouse.

[0048] Drug treatment began on day 29. The model group received 200 μL of normal saline, the positive control group received an appropriate amount of halometasone ointment, and the Ha-CATH1 group received Ha-CATH1 (200 μg / ml, 200 μL) once daily for 7 consecutive days. The blank group received no treatment.

[0049] On days 0, 1, 2, 3, 4, 5, 6, and 7 of treatment, mice were scored for skin lesion severity based on four criteria: erythema, edema / papules, exfoliation / scratching, and dryness / scabs. Scores range from 0 (no lesions), 1 (mild lesions, lesions present and require careful identification), 2 (moderate lesions, lesions immediately recognizable), and 3 (severe lesions, lesions very obvious). The sum of these scores is the total score, with higher scores indicating more severe lesions.

[0050] 2. Test results

[0051] At the end of the treatment trial, the skin lesions of mice were Figure 3 A. Compared with the pre-treatment group, mice repeatedly stimulated with DNCB showed obvious atopic dermatitis symptoms on their backs, such as erythema, scaling, edema, scratches, and scabs. Compared with the model group, 7 days of treatment with the peptide Ha-CATH1 reduced the erythema, edema, scratches, and scabs on the mice's skin, and reduced the scratching behavior of the mice. Compared with the blank group, the severity score of the skin lesions in mice after repeated DNCB stimulation was significantly increased, as shown in Figure 2. Figure 3 B. Starting from the fourth day of treatment, it can be seen that the severity score of skin lesions in mice treated with polypeptide Ha-CATH1 and halometasone ointment decreased and was significantly lower than that in the model group, thereby improving the atopic dermatitis lesions in mice.

[0052] 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.

[0053] Example 3 Preparation of active peptide preparation

[0054] Dissolve the amphibian active peptide Ha-CATH1 in physiological saline to prepare a 200 μg / ml solution. Apply the resulting solution evenly to the lesion area using a cotton swab or spray.

[0055] Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. An application of an amphibian active peptide Ha-CATH1 in the preparation of a preparation for alleviating inflammatory skin diseases, characterized in that: The amino acid sequence of the active peptide Ha-CATH1 is as shown in SEQ ID NO: 1; Inflammatory skin diseases are psoriasis or atopic dermatitis.

2. The use according to claim 1, characterized in that The concentration of the active peptide Ha-CATH1 in the preparation was 200 μg / ml.

3. The use according to claim 1, characterized in that The preparation is a solution or a spray.

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