Application of a composition containing sardine peptide in improving symptoms of atopic dermatitis

Abstract

The present invention provides the use of a composition containing sardine peptides in improving the symptoms of atopic dermatitis. The sardine peptides are a mixture of sardine dipeptides to pentapeptides. The oligopeptides contained in the sardine peptides have high biological activity. These oligopeptides constitute short anti-allergic active peptides that can regulate the function of T cells, especially Th2 cells. These cells play a key role in the allergic reaction of atopic dermatitis and can promote the production of IgE antibodies. Therefore, the short anti-allergic active peptides reduce the production of IgE by inhibiting the activation and proliferation of Th2 cells, thereby improving the symptoms of atopic dermatitis.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to the application of a composition containing sardine peptides in improving the symptoms of atopic dermatitis. Background Art

[0002] Atopic dermatitis (AD), also known as atopic eczema or hereditary allergic dermatitis, is a chronic, relapsing, inflammatory skin disease characterized by intense itching, dry skin, and an eczematous rash. Atopic dermatitis is often associated with skin barrier dysfunction, a long course, and frequent recurrences, placing significant physical and psychological burdens on patients and severely impacting their quality of life. The global prevalence of AD has steadily increased over the past 30 years, reaching 10% to 20% among children in developed countries.

[0003] The pathogenesis of AD is the result of a multifactorial interaction, including genetic factors, immune system abnormalities, skin barrier dysfunction, allergen exposure, and environmental factors. The immune response in AD is associated with abnormal T cell responses, particularly those of Th2 T cells. Overactivation of Th2 cells leads to the release of large amounts of cytokines such as IL-4, IL-5, and IL-13, which in turn promote the production of IgE antibodies by B cells, exacerbating allergic reactions. IgE binds to high-affinity receptors on the surfaces of mast cells and eosinophils, sensitizing these cells. Upon further exposure to allergens, these cells release inflammatory mediators such as histamine and leukotrienes, triggering skin inflammation and itching. Therefore, IgE plays a crucial role in the pathogenesis of AD, and developing an effective drug for the treatment of atopic dermatitis is of great practical significance. Summary of the Invention

[0004] Technical problem to be solved: In response to the above-mentioned problems, the purpose of the present invention is to propose the use of a composition containing sardine peptides in improving the symptoms of atopic dermatitis. Studies have confirmed that the composition containing sardine peptides has excellent efficacy and safety as a therapeutic agent for atopic dermatitis, can significantly improve the related symptoms of atopic dermatitis, and improve the quality of life of patients.

[0005] Sardines peptides are a mixture of oligopeptides obtained by processing sardines, with a molecular weight ranging from 200 to 900 Daltons. These oligopeptides possess high biological activity. The oligopeptides Ala-Leu, Gly-Trp, Ile-Phe, Met-Tyr, Phe-Leu, Val-Tyr, Arg-Val-Tyr, Ile-Val-Tyr, and Gly-Trp-Ala-Pro constitute short anti-allergic peptides, which play a significant role in improving the symptoms of atopic dermatitis. These peptides can modulate T cell function, particularly Th2 cells, which play a key role in allergic reactions in AD and promote IgE production. By inhibiting the activation and proliferation of Th2 cells, these peptides reduce IgE production and improve atopic dermatitis symptoms. The inventors conducted experiments in a mouse model of atopic dermatitis, demonstrating that a topical sardine peptide preparation effectively suppressed elevated IgE levels in mice, significantly reduced inflammatory cell infiltration, and decreased epidermal hyperplasia compared to a control group. Further trials in volunteers with atopic dermatitis demonstrated that applying a topical sardine peptide preparation effectively alleviated patients' itching symptoms and skin condition, significantly improving their quality of life.

[0006] Technical solution: Application of a composition containing sardine peptides in improving the symptoms of atopic dermatitis.

[0007] Furthermore, the sardine peptide is prepared from sardines by methods including microbial fermentation, acid method, alkali method, electrical method, artificial grafting method, gene expression method, and enzymatic hydrolysis method.

[0008] Furthermore, the sardine peptide is a mixture of sardine dipeptides to pentapeptides, including Ala-Leu, Ala-Trp, Arg-Tyr, Gly-Trp, Gly-Tyr, Ile-Phe, Ile-Trp, Ile-Tyr, Leu-Trp, Leu-Tyr, Lys-Trp, Met-Leu, Met-Phe, Met-Tyr, Phe-Leu, Thr-Tyr, Tyr-Leu, Va l-Phe, Val-Trp, Val-Tyr, Ala-Asn-Pro, Ala-Lys-Lys, Arg-Phe-His, Arg-Val-Tyr, Gly-Arg-Pro, Ile-Val-Tyr, Trp-Ile-Thr, Tyr-Val-Pro, Val-Val-Phe, Gly-Trp-Ala-Pro, Asn-Val-Pro-Ile-Tyr.

[0009] Furthermore, the composition comprises a sardine peptide composition and pharmaceutically or cosmetically acceptable excipients.

[0010] Furthermore, the mass concentration of the sardine peptide composition in the medicine is 0.01 to 50 wt.%.

[0011] Furthermore, the dosage forms of the composition include skin administration preparations, mucosal administration preparations, gastrointestinal administration preparations, respiratory tract administration preparations, cavity administration preparations, and injection administration preparations.

[0012] Furthermore, the uses of the composition include medicines, medical devices and cosmetics.

[0013] Furthermore, the frequency and duration of use of the composition vary depending on symptoms, age, dosage form, etc., but the standard is 2-3 times a day. The frequency of application can be increased depending on the severity of the symptoms, and the duration of use is 7-14 days.

[0014] Furthermore, the composition can be administered via a variety of routes, including dermal, mucosal, and gastrointestinal formulations, depending on the patient and symptoms. Specifically, it can be administered as a semisolid formulation such as a gel, ointment, or cream; a liquid formulation such as a paint, film, lotion, douche, suspension, emulsion, or solution; a gaseous formulation such as a spray, aerosol, or powder; or a topical formulation such as a patch, jellies, cataplasms, plasters, or powders. By selecting appropriate excipients, the composition can also be administered as a solid formulation, such as a tablet, pill, capsule, or granule. Any of these formulations can be prepared using known methods.

[0015] Furthermore, the preparation is preferably a preparation for skin administration, including gels, ointments, creams, paints, film coatings, lotions, rinses, suspensions, emulsions, solutions, sprays, aerosols, powder sprays, patches, jellies, papules, powders (dusts), etc.; particularly preferably, gels, sprays, lotions, and ointments.

[0016] The gel of the present invention may contain a matrix, a cross-linking agent, a solvent, a stabilizer, a accelerator, a humectant, and the like. Gelatin, pectin, carbomer, sodium carboxymethylcellulose (CMC-Na), polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), and the like may be used as the gel matrix; water, ethanol, and the like may be used as the solvent; and sodium alginate, chitosan, glycerol, propylene glycol, polyethylene glycol, beeswax, olive oil, and the like may be used as the humectant.

[0017] Liquid preparations are prepared by dissolving the active ingredient, a sardine peptide composition, in a matrix of ethanol, glycerol, propylene glycol, polyethylene glycol, water, or fatty oil. Preferably, the liquid preparation comprises an aqueous solution of the sardine peptide composition dissolved in physiological saline. In addition to physiological saline, the aqueous solution may also contain a small amount of an organic solvent such as ethanol, glycerol, propylene glycol, polyethylene glycol, or dimethyl sulfoxide.

[0018] Liquid preparations, in addition to the above-mentioned aqueous solution preparations, can also be in the form of aqueous or oily solutions, suspensions, emulsions, etc. In addition, they can also be provided as dry compositions that are reconstituted in water or an appropriate medium before use. Beneficial effects

[0019] The sardine peptides described in the present invention contain a variety of short peptides with high biological activity, including anti-allergic active short peptides Ala-Leu, Gly-Trp, Ile-Phe, Met-Tyr, Phe-Leu, Val-Tyr, Arg-Val-Tyr, Ile-Val-Tyr, and Gly-Trp-Ala-Pro. These anti-allergic active short peptides can regulate the function of T cells, particularly Th2 cells, which play a key role in AD allergic reactions. By inhibiting the activation and proliferation of Th2 cells, the anti-allergic active short peptides reduce the production of IgE and improve the symptoms of atopic dermatitis.

[0020] The sardine peptide of the present invention is a mixture of short peptides of 2-5 peptides. The short peptides have amphiphilicity and can easily penetrate the stratum corneum of the skin and be locally absorbed by the skin.

[0021] The sardine peptide composition of the present invention has a good effect in improving the symptoms of atopic dermatitis and can be prepared into a variety of dosage forms for administration, thereby improving symptoms such as itching and dry skin in patients, and no local side effects such as irritation and systemic side effects such as drowsiness are observed. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Photos of the right ears of mice in each group after the fourth stimulation (n=10-12).

[0023] Figure 2 The difference in thickness (swelling) of the right ear of mice in each group before and after DNCB stimulation (n=10-12, mean±SEM); compared with the blank group, ##p<0.01; compared with the model group, **p<0.01.

[0024] Figure 3 The figure shows the difference in ear weight of mice in each group (n=8-12, mean±SEM); compared with the blank group, ##p<0.01; compared with the model group, *p<0.05.

[0025] Figure 4 Figures show the thymus index and spleen index of mice in each group (n=10-12, mean±SEM); compared with the model group, **p<0.01, where a is the thymus index and b is the spleen index.

[0026] Figure 5Figure 3 Changes in serum IL-4, IFN-γ, and IgE levels in mice in each group (n=8, mean±SEM); compared with the blank group, #p<0.05, ##p<0.01; compared with the model group, *p<0.05, **p<0.01, where a is IL-4; b is IFN-γ; and c is IgE.

[0027] Figure 6 Figures are the HE staining results of the right ears of mice in each group (n=5). DETAILED DESCRIPTION

[0028] In the following examples, unless otherwise specified, the experimental instruments and raw materials involved are commercially available products.

[0029] Raw materials, see Table 1.

[0030] Table 1

[0031]

[0032] It has been determined that the sardine peptide composition Valtyron of Japan Xianwei Company is prepared by enzymatic hydrolysis and is a mixture of sardine dipeptides to pentapeptides; among them, the sardine dipeptides are Ala-Leu, Ala-Trp, Arg-Tyr, Gly-Trp, Gly-Tyr, Ile-Phe, Ile-Trp, Ile-Tyr, Leu-Trp, Leu-Tyr, Lys-Trp, Met-Leu, Met-Phe, Met-Tyr, Phe-Leu, Thr-Tyr, Tyr-Leu u, Val-Phe, Val-Trp, and Val-Tyr; sardine tripeptide is a combination of Ala-Asn-Pro, Ala-Lys-Lys, Arg-Phe-His, Arg-Val-Tyr, Gly-Arg-Pro, Ile-Val-Tyr, Trp-Ile-Thr, Tyr-Val-Pro, and Val-Val-Phe; sardine tetrapeptide is Gly-Trp-Ala-Pro; and sardine pentapeptide is Asn-Val-Pro-Ile-Tyr.

[0033] The present invention will be further described below with reference to the accompanying drawings and embodiments. The following embodiments are intended to explain the present invention, but the present invention is not limited to the following embodiments:

[0034] Example 1 Sardine Peptide Spray

[0035] The spray comprises a sardine peptide composition and related auxiliary materials. The content of each material in the spray is shown in Table 2.

[0036] Table 2

[0037]

[0038] The preparation method of the sardine peptide spray described in this embodiment comprises the following steps:

[0039] Accurately weigh each material according to the amount shown in Table 2, and dissolve each material in 0.9% physiological saline. After the material is completely dissolved, mix the components thoroughly and filter using a 0.22 μm microporous filter membrane to obtain a mixed aqueous solution; the mixed aqueous solution is canned in a 30 ml spray container to prepare the sardine peptide spray.

[0040] Example 2 Sardine Peptide Gel

[0041] This embodiment provides a method for preparing a sardine peptide gel, which comprises a sardine peptide composition and related auxiliary materials. The content of each material in the gel is shown in Table 3.

[0042] Table 3

[0043]

[0044] The preparation method of the sardine peptide gel described in this embodiment comprises the following steps:

[0045] The sardine peptide composition and other ingredients were accurately weighed according to the amounts shown in Table 3. First, 4 grams of sardine peptide was dissolved in 40 grams of normal saline, and then 2 grams of chitosan was dissolved in the solution to obtain a chitosan sardine peptide solution. At the same time, 10 grams of sodium alginate was dissolved in the remaining normal saline, and then 2,4-dichlorobenzyl alcohol and menthol were dissolved in the sodium alginate solution in sequence. The chitosan sardine peptide solution was added to the sodium alginate solution and rapidly stirred to form a uniform transparent hydrogel.

[0046] After shaking until there is no sediment, the sardine peptide hydrogel can is placed into a 10 ml squeezable bottle.

[0047] Example 3 Sardine Peptide Skin Applicator

[0048] This embodiment provides a preparation of a sardine peptide skin application stick, which includes a sardine peptide composition and related substances as follows. The content of each material in the skin application stick is shown in Table 4.

[0049] Table 4

[0050]

[0051] The preparation method of the sardine peptide skin application stick described in this embodiment comprises the following steps:

[0052] Accurately weigh the materials according to the amounts shown in Table 4. Add peppermint oil, sweet orange oil, and lemon essential oil to refined olive oil in sequence. Add the ultrafine powder of the sardine peptide composition (fineness of less than 10 microns) to the above mixed oil under stirring. Emulsify under 80 Hz ultrasonic conditions and heat to 80°C to obtain an oil suspension solution. White beeswax is added to the oil suspension solution in batches. Pour the mixture into a mold at an appropriate temperature and cool it to produce 3.5 g sardine peptide skin application sticks.

[0053] Test Example 1

[0054] The sardine peptide spray, sardine peptide gel and sardine peptide skin application stick prepared in Examples 1, 2 and 3 were respectively given to patients with atopic dermatitis 2-3 times a day (4 times for those with severe symptoms). The patient's response was recorded after 7 consecutive days of use.

[0055] A total of 9 subjects participated and were randomly divided into 3 groups, with 3 subjects in each group.

[0056] Group 1: 3 subjects used the sardine peptide spray prepared in Example 1 to spray the affected area 3 times a day, morning, noon and evening.

[0057] Group 2: Three subjects applied the sardine peptide gel prepared in Example 2 to the affected area three times a day, morning, noon, and evening, with the application thickness being such that it was not noticeable to the naked eye.

[0058] Group 3: 3 subjects used the sardine peptide skin applicator prepared in Example 2 and applied it to the affected area twice a day, morning and evening. After 7 consecutive days of use, the effects are shown in Table 5.

[0059] Table 5

[0060]

[0061] Test Example 2: Pharmacodynamic Study of Sardines Peptide against Atopic Dermatitis

[0062] This test example uses 2,4-dinitrochlorobenzene (DNCB)-induced atopic dermatitis and eczema mice as a model to investigate the therapeutic effect of the sardine peptide gel described in Example 2 on DNCB-induced atopic dermatitis and eczema mice. The specific experimental method is as follows:

[0063] 1. Materials

[0064] 1.1 Experimental Animals

[0065] 100 SPF male ICR mice (17-22 g) were purchased from Liaoning Changsheng Biotechnology Co., Ltd., with a certificate number of 210726231101565653 and a license number of SCXK(Liao)2020-0001. They were housed in the SPF Animal Center of Shenyang Pharmaceutical University under a 12-h light cycle and an ambient temperature of 20-24°C with free access to food and water.

[0066] 1.2 Drug testing

[0067] Sardines peptide aqueous solution (homemade), sardine peptide gel from Example 2, sardine peptide refined sesame oil (homemade), clobetasol propionate ointment: Tangshan Red Star Pharmaceutical Co., Ltd., batch number: 46230202.

[0068] 1.3 Experimental Reagents

[0069] 2,4-Dinitrochlorobenzene (DNCB): Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number: C465514;

[0070] Sodium chloride injection: Jilin Province Cornell Co., Ltd., batch number: S17050311-2;

[0071] Iodine disinfectant: Shandong Lierkang Disinfection Technology Co., Ltd., batch number: 130811A;

[0072] Paraformaldehyde: Tianjin Bodi Chemical Co., Ltd., batch number: 20160402;

[0073] Disodium hydrogen phosphate: Tianjin Kaixin Chemical Industry Co., Ltd., batch number: 20170216;

[0074] Sodium dihydrogen phosphate: Shantou Xilong Chemical Plant Co., Ltd., batch number: 120201;

[0075] Potassium dihydrogen phosphate: Shantou Xilong Chemical Plant Co., Ltd., batch number: 120202;

[0076] Sodium chloride: Tianjin Hengxing Chemical Reagent Manufacturing Co., Ltd., batch number: 20170503;

[0077] Paraffin: Sinopharm Chemical Reagent Co., Ltd., batch number: 20160919;

[0078] Tissue embedding cassette: Jiangsu Shitai Experimental Equipment Co., Ltd.

[0079] 95% ethanol: Liaohe Chemical Plant, Sujiatun District, Shenyang, batch number: 20160103;

[0080] Anhydrous ethanol: Tianjin Hengxing Chemical Reagent Manufacturing Co., Ltd., batch number: 20160226;

[0081] Xylene: Tianjin Fuyu Fine Chemical Co., Ltd., batch number: 20170103;

[0082] Mouse IFN-γ ELISA kit: Boster Biotechnology, catalog number: EK0375, 96T;

[0083] Mouse IL-4 ELISA kit: Wuhan Boster Biological, catalog number: EK0405, 96T;

[0084] Mouse IgE ELISA kit: Beijing Biosin Biotechnology, catalog number: bsk12030, 96T.

[0085] 1.4 Experimental Instruments

[0086] Vernier caliper: Menite Tools, MNT-150;

[0087] Multi-function microplate reader: Thermo Scientific, 5250030;

[0088] Paraffin slicer: Leica, Germany, RM2235;

[0089] Upright microscope: NIKON CORPORATION, DS-Ri2;

[0090] Microcentrifuge: Thermo Fisher Scientific, LEGEND MICRO 21R;

[0091] Pathological tissue bleaching and drying instrument: Changzhou Zhongwei Electronic Instrument Co., Ltd.

[0092] Precision constant temperature incubator: Shanghai Yiheng Scientific Instrument Co., Ltd.

[0093] YD-6D biological tissue embedding machine: Jinhua Yidi Medical Equipment Co., Ltd.

[0094] Transfer decolorization shaker: Haimen Qilin Bell Instrument Manufacturing Co., Ltd., TS-8;

[0095] -80 degrees low-temperature refrigerator: Qingdao Haier Special Electrical Appliance Co., Ltd., DW-86L386;

[0096] One ten-thousandth balance: Mettler Toledo (Shanghai) Co., Ltd.

[0097] 2. Experimental methods

[0098] 2.1 Experimental Grouping

[0099] There are 9 groups in total, as shown in Table 6 below.

[0100] Table 6

[0101]

[0102] 2.2 Model establishment

[0103] With the exception of the blank group, mice in all other groups had their abdomens shaved one day before the experiment, covering an area approximately 2.5 cm x 2.5 cm. On the first day of the experiment, 50 μL of a 1% 2,4-dinitrochlorobenzene (DNCB) solution (DNCB is prepared in acetone) was evenly applied to the shaved area for sensitization. A bolus application was performed on the second day. On the third day of the experiment, the hair on the back of the right ear was clipped and 10 μL of a 1% DNCB solution was applied for sensitization. Sensitization was repeated four times every three days (acetone was applied to the left ear). Successful modeling was considered successful if the mice showed signs of dry, rough, or thickened skin, or if they exhibited restlessness. Animals were sacrificed 24 hours after the final challenge.

[0104] 2.3 Dosage and administration time

[0105] Starting on day 3 of the experiment, mice were treated with 20 μL of the corresponding solvent three times daily on the dorsal surface of the right ear. The phosphate-buffered saline group was treated with PBS buffer, and the positive drug was applied once daily. Administration continued until the mice were sacrificed. If the challenge occurred on the day of the challenge, the drug was administered 1 hour before challenge. The thickness of the mid-section of the right ear was measured with a vernier caliper before induction and 24 hours after each challenge. The difference in right ear thickness before and after challenge was calculated for each mouse.

[0106] 2.4 Collection of materials

[0107] ① Blood Collection and Ear Pieces: 24 hours after the last challenge, blood was collected from the orbital cavity, serum was separated, and stored at -80°C until needed. Experimental animals were sacrificed, and a 0.9 cm diameter metal punch was used to punch a hole in the middle of the ear. Each ear piece was weighed using a 1 / 10,000 scale. The difference in ear weight was calculated as "right ear piece (experimental ear) weight - left ear piece (control ear) weight" for each mouse. Ear pieces were fixed in 4% paraformaldehyde for at least 24 hours and then embedded in paraffin for later use.

[0108] ② Remove the thymus and spleen: weigh them, calculate the thymus index and spleen index (= thymus or spleen weight / body weight × 100%), and store them in a -80℃ refrigerator until use.

[0109] 2.5 HE staining

[0110] 2.5.1 Dewaxing and hydration of paraffin sections

[0111] Place the slides in a constant temperature oven at 58-60°C for 3 h, and dewax in the following order: immerse in xylene I and II for 15 min each, immerse in anhydrous ethanol I and II for 5 min each, immerse in 95%, 85% and 75% ethanol for 5 min each, and rinse with PBS three times, 3 min each time.

[0112] 2.5.2 Hematoxylin counterstaining

[0113] The sections were lightly counterstained with hematoxylin solution for 3 min and immediately rinsed with tap water for 5 min.

[0114] 2.5.3 Hydrochloric acid-ethanol differentiation

[0115] The slices were placed in 1% hydrochloric acid ethanol and extracted 2-3 times, and then rinsed under tap water for 8 minutes.

[0116] 2.5.4 Dehydration and sealing

[0117] Dehydrate with 75%, 85% and 95% ethanol for 3 min, soak in anhydrous ethanol I and II for 5 min each, soak in xylene I and II for 10-15 min each, seal with neutral gum and observe under a microscope.

[0118] 2.6 Enzyme-linked immunosorbent assay (ELISA)

[0119] The operation was performed according to the kit instructions, and the standard curve and the content of each factor were calculated using CurveExpert software.

[0120] The operation of the mouse IFN-γ and IL-4 kit is briefly described as follows: 1) Add 100 μL of sample or standard and react at 37°C for 90 minutes; 2) Add biotin-labeled antibody and react at 37°C for 60 minutes, then wash three times; 3) Add ABC and react at 37°C for 30 minutes, then wash five times; 4) Add TMB and react at 37°C for 20 minutes; 5) Add stop solution and read the result at 450 nm on a microplate reader.

[0121] The Mouse IgE Kit procedure is briefly described as follows: 1) Add 100 μL of sample or standard, mix with the biotinylated antibody working solution, shake at room temperature for 120 minutes, and wash four times; 2) Add the enzyme conjugate working solution, shake at room temperature for 30 minutes, and wash four times; 3) Add the chromogenic substrate and react at room temperature for 20 minutes; 4) Add the stop solution and read the result at 450 nm using a microplate reader.

[0122] 2.7 Statistical Methods

[0123] Data were analyzed using SPSS 21.0. One-way analysis of variance was used for statistical significance. Intergroup comparisons were performed using the LSD method when variances were equal, and Dunnett's T3 method when variances were unequal. Experimental data are expressed as "mean ± SEM," and p < 0.05 was considered statistically significant.

[0124] 3. Experimental results

[0125] 3.1 Appearance of the right ear after the fourth DNCB challenge

[0126] After four DNCB stimulations, the right ear skin of mice in the blank group was normal and without damage; the right ear skin of mice in the model group showed dermatitis-like changes such as infiltrative erythema, erosion, and scab; compared with the model group, the erosion and exudation of the right ear skin of mice in the sardine peptide group and the positive drug group were significantly reduced ( Figure 1 ).

[0127] 3.2 Swelling of the right ear before and after DNCB stimulation

[0128] The thickness of the middle part of the right ear (experimental ear) was measured with a vernier caliper before induction and 24 hours after each challenge. The thickness of the right ear after challenge was subtracted from the thickness before induction to obtain the difference (i.e., swelling degree) for statistical analysis. The experimental results showed that compared with the blank group, the thickness of the right ear of the model group mice at each time point was significantly increased; compared with the model group, starting from the second challenge, the thickness of the right ear of the positive drug group mice was significantly reduced. The thickness of the right ear of the mice in the sardine peptide aqueous solution group and the gel group mice showed a downward trend, but no statistical difference was observed ( Figure 2 ).

[0129] 3.3 Difference in ear weight of mice in each group

[0130] A 0.9 cm diameter metal punch was used to punch a hole in the middle of the ear. The weight of each ear piece was measured using a 1 / 10,000 scale. The weight of the right ear piece (experimental ear) of each mouse was subtracted from the weight of the left ear piece (control ear) to obtain the ear weight difference, which was then statistically analyzed. The experimental results showed that compared with the blank group, the ear weight difference of the model group mice was significantly increased; compared with the model group, the ear weight difference of the high-dose sardine peptide aqueous solution group mice was significantly reduced ( Figure 3 ), indicating that sardine peptide aqueous solution can reduce the degree of ear swelling caused by DNCB.

[0131] 3.4 Thymus index and spleen index of mice in each group

[0132] Twenty-four hours after the last challenge, mice were killed, and the thymus and spleen were quickly isolated. The thymus and spleen indices were calculated by dividing the thymus or spleen weight by body weight × 100% for statistical analysis. The experimental results showed that compared with the model group, the thymus and spleen indices of mice in the positive drug clobetasol propionate group were significantly reduced. This is because clobetasol propionate is a glucocorticoid and has significant immunosuppressive effects. The spleen indices of mice in the high-dose sardine peptide aqueous solution group and the gel matrix group also decreased slightly. This result needs to be verified by subsequent repeated experiments ( Figure 4 ).

[0133] 3.5 Serum inflammatory factors and immunoglobulin E levels in mice in each group

[0134] ELISA kits were used to detect the levels of IL-4, IFNγ, and IgE in the serum of each group of mice. The experimental results showed that there was no significant difference in the levels of IL-4 and IFN-γ in the serum of each group of mice ( Figure 5 a, b); Compared with the blank group, the serum IgE levels of mice in the model group and PBS group were significantly increased; compared with the model group, the serum IgE levels of mice in the high-dose sardine peptide aqueous solution group, gel group, oil suspension group, and positive drug group were significantly decreased ( Figure 5 c). This suggests that sardine peptides may inhibit the overactivation of the immune system caused by DNCB.

[0135] 3.6 HE staining results of the right ears of mice in each group

[0136] No edema or inflammatory cell infiltration was observed in the epidermis, dermis, and subcutaneous tissue of mice in the blank group. Epidermal hyperplasia was observed in mice in the model group, PBS group, and gel matrix group, with obvious edema between and within epidermal cells and very significant inflammatory cell infiltration. Edema was still present in mice in the sardine peptide aqueous solution (especially high-dose) and gel groups, but inflammatory cell infiltration was significantly reduced and epidermal hyperplasia was reduced. Some epidermal hyperplasia and inflammatory cell infiltration were observed in mice in the sardine peptide oil solution group. Compared with the sardine peptide group, the pathological state of mice in the positive drug group was further alleviated ( Figure 6 ).

Claims

1. Application of a sardine peptide composition in the preparation of a drug for improving atopic dermatitis, characterized in that: The sardine peptide composition is a mixture of sardine dipeptides to pentapeptides, which are Ala-Leu, Ala-Trp, Arg-Tyr, Gly-Trp, Gly-Tyr, Ile-Phe, Ile-Trp, Ile-Tyr, Leu-Trp, Leu-Tyr, Lys-Trp, Met-Leu, Met-Phe, Met-Tyr, Phe-Leu, Thr-Tyr, Tyr-Leu, Val-P He, Val-Trp, Val-Tyr, Ala-Asn-Pro, Ala-Lys-Lys, Arg-Phe-His, Arg-Val-Tyr, Gly-Arg-Pro, Ile-Val-Tyr, Trp-Ile-Thr, Tyr-Val-Pro, Val-Val-Phe, Gly-Trp-Ala-Pro and Asn-Val-Pro-Ile-Tyr.

2. The use according to claim 1, characterized in that: The sardine peptide is prepared from sardines through methods including microbial fermentation, acid method, alkali method, electrical method, artificial grafting method, gene expression method and enzymolysis method.

3. The use according to claim 1, characterized in that: The drug further comprises pharmaceutically acceptable excipients.

4. The use according to claim 1, characterized in that: The mass concentration of the sardine peptide composition is 0.01 to 50 wt.%.

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