Application of polypeptide in preparation of medicine for treating psoriasis

Abstract

The invention discloses an application of a polypeptide Apelin-13 in a medicine for treating psoriasis, in particular to psoriasis-like skin lesion. In an imiquimod (IMQ) cream induced mouse psoriasis model, intraperitoneal injection of Apelin-13 can significantly reduce scale, erythema and skin thickness, and can inhibit proliferation of epithelial spine cells. HE staining and immunohistochemical staining are carried out on the skin tissue of an experimental mouse, and the result shows that Apelin-13 can reduce the expression of the psoriasis marker protein K17, the thickness of the surface layer of the back side skin of the experimental mouse and the downward extension of a spinous layer. Experimental mouse serum ELISA (enzyme-linked immunosorbent assay) detection results show that the Apelin-13 can be used for down-regulating IL-17A (interleukin-17A) which is highly expressed in psoriasis. The polypeptide Apelin-13 can be used as a medicine for treating psoriasis, especially a medicine for treating psoriasis-like skin lesion.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the use of a polypeptide in preparing a drug for treating psoriasis, especially the use of the polypeptide Apelin-13 targeting K17 and IL-17A in preparing a drug for treating psoriasis-like skin lesions. Background Art

[0002] Psoriasis, also known as psoriasis, is a common chronic inflammatory skin disease in humans. About 1% to 3% of people worldwide suffer from psoriasis, and about 0.5% of people in China suffer from psoriasis (K. Yan et al., Safety and efficacy of methotrexate for Chinese adults with psoriasis with and without psoriasis. Jama Dermatol 155, 327-334 (2019).). There are five types of psoriasis reported so far, namely: plaque psoriasis (also known as psoriasis vulgaris); guttate or eruptive psoriasis, also known as intertrigo psoriasis or tinea psoriasis; pustular psoriasis; erythrodermic and inverse psoriasis. Among them, chronic plaque psoriasis (psoriasis vulgaris) is the most common form of the disease, accounting for about 90% of cases. Psoriasis can affect any part of the skin, and the typical lesions are monomorphic, showing erythematous plaques covered with white scales. Psoriasis plaques can be not only few, but also expand to larger areas, or manifest as erythroderma, involving the entire body surface (W.-H. et al., Psoriasis. Lancet 386, 983-994 (2015)). In addition, psoriasis can also induce a variety of complications, such as psoriatic arthritis, metabolic syndrome, non-melanoma skin cancer, cardiovascular disease, etc., which have a significant negative impact on the patient's quality of life (W.Zhou et al., Luteolin attenuates imiquimod–induced psoriasis-like skin lesions in BALB / c mice via suppression of inflammationresponse. Biomed Pharmacother 131, (2020).), so psoriasis is no longer considered a disease that only affects the skin, but is regarded as a systemic inflammatory disease.

[0003] Psoriasis is a complex inflammatory skin disease mediated by multiple cells, including keratinocytes, T cells, endothelial cells, macrophages, and dendritic cells (J. Gao et al., Daphnetin inhibits proliferation andinflammatory response in humanHaCaT keratinocytes and ameliorates imiquimod-induced psoriasis-like skin lesion in mice. Biol Res 53, (2020).). Among them, keratinocytes are a kind of resident skin cells, which are both participants and victims. The balance of keratinocyte proliferation and apoptosis is crucial to maintain skin homeostasis. In psoriasis lesions, this balance is broken. Changes in keratinocyte differentiation in psoriasis are characterized by upregulation of the early differentiation marker keratin (M. Kastelan et al., Apoptosis inpsoriasis. ActaDermatovener Cr 17, 182-186 (2009).), and keratin 17 (K17) is overexpressed in the epidermis of psoriasis. K17 has pro-proliferative and pro-inflammatory effects on keratinocytes, thereby participating in the pathogenesis of psoriasis. In addition, immune imbalance of T lymphocyte subsets is a hallmark of psoriasis (R. Wu et al., MicroRNA-210 overexpression promotes psoriasis-like inflammation by inducing Th1 and Th17 cell differentiation. J Clin Invest 128, 2551-2568 (2018).). Among them, the IL-23 / Th17 cell axis plays a crucial role in the pathogenesis of psoriasis (A. Chiricozzi et al., Role of IL-23 in the pathogenesis of psoriasis: a novel potential therapeutic target Expert Opin Ther Tar 18, 513-525 (2014).).Th17 cells are the main source of IL-17 family cytokines, and IL-17 has been considered a key cytokine for the establishment and maintenance of the psoriasis phenotype (K. El Malki et al., An alternative pathway of imiquimod-induced psoriasis-like skin inflammation in the absence of interleukin-17 receptor a signaling. J Invest Dermatol 133, 441-451 (2013).). IL-17A binds to receptors expressed on keratinocytes, dendritic cells, dermal fibroblasts, and endothelial cells (MG Works et al., Inhibition of TYK2 and JAK1 ameliorates imiquimod-induced psoriasis-like dermatitis by inhibiting IL-22 and the IL-23 / IL-17 axis. J Immunol 193, 3278-3287 (2014)). When the IL-23 receptor (IL-23R) is activated, it promotes the development of Th17 cells, which are characterized by the production of inflammatory cytokines, including IL-17A, IL-17F, and IL-22 (A. Di Cesare et al., The IL-23 / Th17 Axis in the Immunopathogenesisof Psoriasis. J Invest Dermatol 129, 1339-1350 (2009).). Dermal γδ T cells, as the main and active effector T cells in imiquimod (IMQ)-induced psoriasis, rapidly produce IL-17A and IL-22, triggering and amplifying inflammation (S. Pantelyushin et al., Rorγt+ innate lymphocytes and γδ Tcells initiate psoriasiform plaque formation in mice. J Clin Invest 122, 2252-2256 (2012).). IL-17 works synergistically with interferon-γ (IFN-γ) and TNF-α to stimulate keratinocyte proliferation and secrete a large number of inflammatory cytokines, such as IL-6, TGF-β, IL-18, IL-17, IL-12, IL-15, TNF-α, GM-CSF, ICAM-1, etc.These inflammatory cytokines can actively recruit and activate neutrophils, accelerate the accumulation of T cells into the epidermis, and lead to epidermal hyperplasia, acanthosis, and hyperkeratosis (Z.-z. Huang et al., Artesunate alleviatesimiquimod-induced psoriasis-like dermatitisin BALB / c mice. IntImmunopharmacol 75, (2009).).

[0004] Psoriasis can cause systemic damage and seriously affect the quality of life of patients. Established systemic drugs for the treatment of psoriasis include methotrexate, cyclosporine, and avermectin. Although these biologics have achieved certain clinical efficacy, psoriasis cannot be completely cured, the relapse rate is high, and long-term use of existing treatment options has considerable side effects. In addition, several biologics have been developed and approved for the treatment of psoriasis in the past decade. Except for etanercept, which is a fusion protein, all approved biologics are monoclonal antibodies (Abs). TNF-α inhibitors / antagonists, Etanercept, Adalimumab, Infliximab, etc. are approved for the treatment of psoriasis and psoriatic arthritis. Although these monoclonal antibodies have greatly improved the efficacy of psoriasis patients, there are still some problems: single effect, high price, and uncertainty of long-term antagonism of only one cytokine. Ustekinumab interferes with the development of Th17 lymphocytes, which are important effector cells in psoriatic inflammation. Ustekinumab, a drug that blocks IL-12 and IL-23, is also approved for this indication. Secukinumab was approved as the first biologic to block IL-17A, a key factor in the production of TH17 and other cells. Biologics have a good safety profile with only a slight increase in opportunistic infections (JH Saurat al., Efficacy and safety results from the randomized controlled comparative study of adalimumab vs. methotrexate vs. placebo in patients with psoriasis (CHAMPION). Br J Dermatol 158, 558-566 (2008).). TNF-α inhibitors are often used after phototherapy when conventional systemic therapy has failed, is not tolerated, or is contraindicated. This second-line use is partly due to the high direct cost of the drug, which is approximately ten times that of conventional systemic drugs.

[0005] Methotrexate (MTX) is a folic acid antagonist that is listed as one of the first choice drugs in multiple treatment guidelines for moderate to severe psoriasis. It has been widely used for injection and oral routes for decades. Although MTX has significant anti-psoriatic effects and is more cost-effective than other treatments, its adverse reactions, such as gastrointestinal discomfort (such as nausea, vomiting, diarrhea, abdominal pain and anorexia), stomatitis, bone marrow suppression, hepatotoxicity, pulmonary fibrosis, discomfort and alopecia, limit its long-term clinical application (H. Duet al., Hyaluronic acid-based dissolving microneedle patch loaded with methotrexate for improved treatment of psoriasis. Acs Appl Mater Inter11, 43588-43598 (2019).).

[0006] Apelin is an endogenous ligand of G protein-coupled receptor (APJ) and is widely distributed in the central nervous system and multiple organs of humans and animals. Apelin precursor protein is cleaved into a biologically active Apelin fragment, including Apelin-36, Apelin-17, Apelin-13 and Apelin-12 (K. Tatemoto et al., Isolation and characterization of a novel endogenous peptide ligand for the human APJ receptor. Biochem Bioph Res Co 251, 471-476 (1998).). Among them, Apelin-13 is the most biologically active polypeptide fragment of Apelin (D. Birmpili et al., Fluorinated apelin-13mediatesneuroprotective effects in multiple sclerosis models. Neurobiol Dis198, (2024).). The main form of apelin in plasma is pyroglutamine-Apelin-13, namely: Pyr-Apelin-13 (C. Mesmin et al., Liquid chromatography / tandem mass spectrometry assay for the absolute quantification of the expected circulating apelin peptides in human plasma. Rapid Commun Mass Sp 24, 2875-2884 (2010).). Apelin-13 has anti-inflammatory effects in various diseases by inhibiting the activity of pro-inflammatory factors TNF-α and IL-6 (X. Shen et al., Apelin-13 attenuates early brain injury through inhibiting inflammation and apoptosis inrats after experimental subarachnoid hemorrhage. Mol Biol Rep 49, 2107-2118 (2022).).There is increasing evidence that Apelin-13 regulates cell proliferation, differentiation and apoptosis and is a potential protective molecule against a variety of diseases (I. Falcão-Pires et al., Apelin: a novel neurohumoral modulator of the cardiovascular system. Pathophysiologic importance and potential use as atherapeutic target. Rev Port Cardiol 24,1263-1276 (2005).). Apelin-13 protects the brain, heart, kidneys and lungs from damage, and these effects are related to its anti-inflammatory, antioxidant and anti-apoptotic biological activities (H. Yin et al., Apelin-13 protects against cisplatin-induced ototoxicity by inhibiting apoptosis and regulating STAT1 and STAT3. Arch Toxicol 97, 2477-2493 (2023).). However, its application in the treatment of psoriasis-related diseases remains unknown.

[0007] In view of the problems of poor efficacy, serious side effects and easy recurrence in the existing drugs for treating psoriasis, the present invention provides a use of Apelin-13 in the preparation of drugs for treating psoriasis, which has known pharmacokinetic and toxicological effects, few side effects, low price and good development and application prospects. Summary of the invention

[0008] The purpose of the present invention is to overcome the defects of the prior art and provide an application of a polypeptide in the preparation of a drug for treating psoriasis-like skin lesions, especially the application of the polypeptide Apelin-13 targeting K17 and IL-17A in the preparation of a drug for treating psoriasis, which has the characteristics of less toxic and side effects, low price, good efficacy, etc.

[0009] To achieve the above object, the present invention adopts the following technical solution: A use of a polypeptide in preparing a drug for treating psoriasis, wherein the polypeptide is Apelin-13.

[0010] In the above application, the psoriasis includes various types of mild, moderate and severe psoriasis and psoriasis-related complications.

[0011] Furthermore, the present invention provides the use of the polypeptide in preparing a drug for treating psoriasis-like skin lesions, that is, discloses the use of the polypeptide Apelin-13 in preparing a drug for treating psoriasis-like skin lesions.

[0012] In the above application, the Apelin-13 is a fragment of Apelin, and its amino acid sequence is: Pyr-Arg-Pro-Arg-Leu-Ser-His-Lys-Gly-Pro-Met-Pro-Phe.

[0013] Furthermore, the polypeptide also includes modifications or alterations based on the amino acid sequence of the polypeptide Apelin-13.

[0014] The present invention also provides a pharmaceutical composition for treating psoriasis, wherein the pharmaceutical composition comprises the polypeptide Apelin-13.

[0015] Furthermore, in the above-mentioned pharmaceutical composition for treating psoriasis, the pharmaceutical composition can be administered orally, by injection or external use.

[0016] The present invention observes the effect of polypeptide Apelin-13 in an animal model of psoriasis, and the results show that polypeptide Apelin-13 can effectively inhibit the clinical symptoms and histopathological changes of psoriasis.

[0017] Compared with the prior art, the present invention has the following beneficial effects: In the present invention, the classic animal model of psoriasis is to apply 5% imiquimod (IMQ) cream to the back skin of adult Balb / c mice (10 weeks old) for 5-7 consecutive days to induce skin lesions. This model is used to detect the therapeutic effect of Apelin-13 on psoriasis. The results show that the polypeptide Apelin-13 can effectively inhibit the skin symptoms and pathological changes induced by IMQ.

[0018] In addition, the present invention also performs HE staining and immunohistochemistry on the skin tissue of the mouse of the animal model, and performs ELISA analysis on the serum to detect the effect of Apelin-13 on the thickness of the surface layer of the psoriasis skin, and the expression of IL-17A and K17. The results show that IMQ induction can cause thickening of the mouse skin epidermis, high expression of IL-17A in serum, and expression of K17 in skin tissue, while Apelin-13 can significantly reduce the increase in skin epidermal thickness, the expression of IL-17A in serum, and the expression of K17 in skin tissue.

[0019] This study found that the peptide Apelin-13 can downregulate the expression of K17 in the skin tissue of the mouse psoriasis model and the expression of IL-17A in the serum. It is known that K17 is an autoantigen specifically expressed in psoriasis epidermal keratinocytes, and its high expression is positively correlated with the severity of skin lesions. IL-17A is highly expressed in psoriasis and lowly expressed in normal tissues. The application of IL-17A antagonists can treat psoriasis. We speculate that Apelin-13 reduces psoriasis-like lesions by downregulating the expression of K17 and the differentiation and activation of Th17 cells. Our research group found that the efficacy of Apelin-13 in the treatment of psoriasis is equivalent to MTX, providing another new and effective way to treat psoriasis.

[0020] Based on the above studies, it is found that the polypeptide Apelin-13 has a good therapeutic activity against psoriasis, which is closely related to Apelin-13 inhibiting the changes in the gold standard skin epidermal thickness of psoriasis, the highly expressed IL-17A in psoriasis, and the expression of the psoriasis marker molecule K17. Therefore, it can be used as an active ingredient to prepare drugs for the treatment of psoriasis. In order to further improve the efficacy, the polypeptide Apelin-13 can also be compounded with other active ingredients to prepare drugs for the treatment of psoriasis. When preparing these drugs, pharmaceutically acceptable carriers can also be added. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a gross morphological observation of the therapeutic effect of Apelin-13 on psoriasis model mice; Figure 2 Skin histological observation of psoriasis model mice treated with Apelin-13; Figure 3 PASI score (scaly) and statistical analysis of skin lesions in psoriasis model mice treated with Apelin-13; Figure 4 PASI score (erythema) and statistical analysis of skin lesions in psoriasis model mice treated with Apelin-13; Figure 5 PASI score (thickness) and statistical analysis of skin lesions in psoriasis model mice treated with Apelin-13; Figure 6 PASI score (comprehensive score) and statistical analysis of skin lesions in psoriasis model mice treated with Apelin-13; Figure 7 The expression of K17 in the skin of psoriasis model mice treated with Apelin-13 (immunohistochemistry); Figure 8 The content of IL-17A in the serum of psoriasis model mice treated with Apelin-13 (ELISA). DETAILED DESCRIPTION

[0022] In order to make the technical purpose, technical solution and beneficial effects of the present invention clearer, the technical solution of the present invention is further described below in conjunction with specific embodiments, but the implementation is intended to explain the present invention and cannot be understood as limiting the present invention.

[0023] In the following examples, if no specific techniques or conditions are specified, the techniques or conditions described in the literature in the art or the product instructions were used.

[0024] The polypeptide Apelin-13 of the present invention was synthesized by the manufacturer GL Biochem (Shanghai) Ltd. In addition, it can also be synthesized by referring to the method in the literature (EY Zhen, RE Higgs, JA Gutierrez, Pyroglutamyl apelin-13 identified as themajor apelin isoform in human plasma. Analytical Biochemistry 442, 1-9 (2013).).

[0025] Example 1: Gross morphological observation of the therapeutic effect of Apelin-13 on psoriasis mouse model The experimental animals were 10-week-old female Balb / c mice purchased from Beijing Weitonglihua Biotechnology Co., Ltd. and kept in an SPF animal laboratory. After the mice were adapted for 1 week, the backs were shaved 1 day in advance and randomly divided into four groups: normal mouse blank control group (Control, n = 6), intraperitoneal injection of normal saline twice a day, 0.1 mL each time; white vaseline preparation was applied to the back lesion area once a day. Model control group (Model, normal saline / IMQ group, n = 6): normal saline was injected intraperitoneally twice a day, 0.1 mL each time; 62.5 mg, 5% IMQ cream was applied to the back lesion area of ​​each mouse once a day. Positive control drug group (MTX 1 mg / kg IMQ group, n = 6): MTX with a drug concentration of 1 mg / kg was injected intraperitoneally once a day, 0.1 mL each time, and 62.5 mg, 5% IMQ cream was applied to the back lesion area of ​​each mouse once a day. Experimental group: 0.2 mg / kg (n=6), 0.1 mg / kg (n=6) and 0.05 mg / kg (n=6) of Apelin-13 solution were intraperitoneally injected twice a day, 0.1 mL each time, and 62.5 mg, 5% IMQ cream was applied to the back lesion area of ​​each mouse once a day. After 7 consecutive days of treatment, the mice were killed, and the back skin and subcutaneous tissue were collected. Half of the back skin was fixed in a pre-prepared fixative (10% paraformaldehyde) for 24 hours for HE staining and immunohistochemistry experiments, and the other half of the back skin was placed in a cryovial and frozen in a liquid nitrogen tank for other experiments later.

[0026] The results are as follows Figure 1 As shown in the results, compared with the blank control group, the IMQ-induced skin lesions in the model control group showed typical psoriasis features including erythema, thickening and scaly skin lesions. The positive control drug MTX and the Apelin-13 experimental groups at different doses (0.05 mg / kg, 0.1 mg / kg, 0.2 mg / kg) were able to significantly inhibit these skin lesions, showing only slight calloused skin and a small amount of scales.

[0027] Example 2: Apelin-13 inhibits IMQ-induced mouse epithelial cell proliferation The fixed mouse skin tissue was dehydrated, waxed, paraffin-embedded, sliced, and attached to a slide, and baked at 60 °C for 3 h. Slice, and then put the slices into xylene I for 10 min, xylene II for 10 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, 95% ethanol for 5 min, 90% ethanol for 5 min, 80% ethanol for 5 min, 70% ethanol for 5 min, and distilled water for 5 min for dewaxing and hydration. The slices were stained with Harris hematoxylin for 3 min, washed with tap water, differentiated with 1% hydrochloric acid alcohol for a few seconds, rinsed with tap water, blued with 0.6% ammonia water, and rinsed with running water. The slices were stained with eosin stain for 3 min. The sections were placed in 95% ethanol I for 5 min, 95% ethanol II for 5 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, xylene I for 5 min, and xylene II for 5 min to dehydrate and make them transparent. The sections were taken out of xylene and dried slightly, and then sealed with neutral gum. Observation and image acquisition and analysis were performed under a pathological microscope.

[0028] The results are as follows Figure 2 Compared with the epidermis of mice in the blank control group, under the continuous stimulation of IMQ, the stratum corneum of the epithelium of mice in the model control group was significantly thickened and broken, and the prickle cells (keratinocytes) proliferated and formed large papillae extending to the subcutaneous tissue. The epithelial layer of mice in the positive control drug MTX group and the Apelin-13 experimental groups with different doses (0.05 mg / kg, 0.1 mg / kg and 0.2 mg / kg) was significantly thinned, close to the epithelial thickness of mice in the blank control group. This result shows that the peptide Apelin-13 can inhibit IMQ-induced mouse epithelial cell proliferation.

[0029] Example 3: Apelin-13 significantly reduces the PASI score of psoriasis mice The damaged areas of the back skin of each group of mice were scored using the website http: / / pasi.corti.li / , and the psoriasis score was calculated based on the proportion of psoriasis lesion area, redness (0-4), thickening (0-4), and severity of scaling symptoms (0-4) in the lesion area. Figures 3 to 6 .

[0030] The results of Figures 3-6 show that IMQ can induce severe scaling, erythema and thickening symptoms in the mouse skin, and the PASI scores and comprehensive scores are significantly increased, indicating that the model is successful. The positive control drug MTX can alleviate various symptoms, and the peptide Apelin-13 has almost the same effect on alleviating various symptoms and PASI scores as MTX. The experimental results on the 7th day showed that compared with the blank control group, the scaling of the mice in the IMQ-induced model group ( Figure 3 ),erythema( Figure 4)、Thickening( Figure 5 ) and the total score ( Figure 6 ) and other indicators were significantly increased (all P values ​​< 0.01); compared with the IMQ model group, the scales of mice in the 0.05 mg / kg Apelin-13 experimental group, the 0.1 mg / kg Apelin-13 experimental group, and the 0.2 mg / kg Apelin-13 experimental group ( Figure 3 ),erythema( Figure 4 )、Thickening( Figure 5 ) and the total score ( Figure 6 ) and other indicators were significantly reduced (all P values ​​< 0.01).

[0031] Example 4: Analysis of immunohistochemistry results of psoriasis mouse skin tissue The following experimental process used a universal two-step kit from Zhongshan Jinqiao Company, which included reagent 1—endogenous peroxidase blocker; reagent 2—reaction enhancement solution; reagent 3—enhanced enzyme-labeled goat anti-mouse / rabbit IgG polymer, etc.

[0032] (1) Wash fresh skin tissue blocks with PBS and soak them in 10% formaldehyde solution overnight; (2) Soak the tissue blocks in 100%, 95%, 90%, 85% and 75% anhydrous ethanol for 1 h, respectively, and then soak the tissue blocks in xylene solution for 2 h; (3) Soak the tissue block in paraffin at 50-60 °C for 2 h, then pour it into the embedding frame together with the paraffin. After cooling, place it in a paraffin slicer and slice it to a thickness of 5 μm. (4) Place the prepared paraffin sections in a 60 °C oven for 3 h; (5) Dewaxing and rehydration: First, dewax the paraffin sections by soaking them in xylene I and xylene II for 10 min, and then hydrate them in anhydrous ethanol I, anhydrous ethanol II, 95% ethanol, 90% ethanol, 85% ethanol, 80% ethanol and 70% ethanol, respectively, for 5 min each. (6) Place the slices on a shaker and wash with distilled water for 5 min (to remove residual ethanol). (7) Add reagent 1 (endogenous peroxidase blocker) to completely cover the tissue, place in a humidified chamber, and incubate in the dark for 10 min. (8) Place the slices in a box containing 1 × PBS and wash on a shaker for 5 min. Repeat 3 times. (9) Antigen repair: There are two types of repair solutions: acidic and alkaline. Choose the appropriate repair solution according to the experimental requirements. Boil the repair solution at high heat in a microwave oven, then put the paraffin sections in, and boil them at medium-high heat for 20 seconds every 2 minutes for 15 minutes, then let the repair solution cool naturally at room temperature; (10) Wash with 1 × PBS on a shaker for 5 min, repeat 3 times (to wash away the residual repair solution); (11) Blocking: Drop 10% goat serum (prepared in PBS) on the tissue, place it in a wet box, and block it at room temperature for 1 hour; (12) Blocking with primary antibody: dilute the primary antibody [Keratin 17 (D12E5) XP (R) Rabbit mAb] with 1% BSA (prepared in PBS), add it dropwise to the tissue, and incubate it in a humidified box at 4 °C overnight. (13) After the slices return to room temperature, wash them with 1 × PBS on a shaker for 5 min. Repeat three times. (14) Add an appropriate amount of reagent 2 (reaction enhancement solution) and incubate in a wet box at room temperature in the dark for 20 min. (15) Wash with 1 × PBS on a shaker for 5 min, repeat three times; (16) Add reagent 3 (enhanced enzyme-labeled goat anti-mouse / rabbit IgG polymer) and incubate at room temperature in the dark for 30 min. (17) After incubation, wash three times with PBS, 5 min each time; (18) DAB color development: Add the prepared DAB color development solution to the tissue, color for 3-10 min in the dark, and rinse with tap water to terminate the reaction; (19) Stain with hematoxylin for 20-30 s and wash with distilled water for 5 min; (20) Dehydration and transparency: Soak the paraffin sections in 70% ethanol, 80% ethanol, 90% ethanol, anhydrous ethanol II and anhydrous ethanol I for 3 min respectively, and soak them in xylene II and xylene I for 10 min respectively; (21) Sealing with neutral gum: After the gel solidifies, observe the tissue structure and take photos under an Olympus pathology microscope.

[0033] The results in Figure 7 show that compared with the blank control group, the expression of K17 in the model control group was significantly enhanced (positive: brown-yellow color was observed when the target antigen of the target tissue cells was detected, and DAB was used for color development; negative: brown-yellow color was not observed when the target antigen of the target tissue cells was detected, and DAB was used for color development). After administration of Apelin-13 at different doses (0.05 mg / kg, 0.1 mg / kg, and 0.2 mg / kg), the expression of K17 was significantly reduced. It can be seen that the application of Apelin-13 can reduce the expression of psoriasis marker proteins, thereby inhibiting the progression of psoriasis.

[0034] Example 5: Analysis of ELISA results of serum tissues of psoriasis mice The following experimental process used an ELISA kit from Elabscience, product number: E-EL-M0047. The reagents contained in the kit include standard / sample diluent, washing solution, stop solution, etc.

[0035] (1) 10-fold dilution: Take 12 μL of serum sample and add it to 108 μL of standard / sample diluent to make a 10-fold dilution; (2) Set up standard wells, blank wells, and sample wells respectively. Add 100 μL of the diluted standard to the standard wells. Add 100 μL of the standard / sample dilution to the blank wells, and add 100 μL of the sample to be tested to the remaining wells. Coat the ELISA plate and incubate at 37 °C for 90 min. (3) Shake off the liquid in the wells without washing. Add 100 μL of biotinylated antibody working solution to each well, cover the ELISA plate with a film, and incubate at 37 °C for 1 hour; (4) Shake off all the liquid in the wells, pat dry on clean absorbent paper, add 350 μL of washing solution to each well, soak for 1 min, absorb or shake off the liquid in the ELISA plate, pat dry, and repeat this washing step 3 times. After washing, proceed to the next step immediately, and do not let the microplate dry; (5) Add 100 μL of HRP conjugate working solution to each well, cover the plate with a film, and incubate at 37 °C for 30 min. (6) Shake off all liquid in the wells and wash the plate 5 times using the same method as step (4); (7) Add 90 μL of substrate solution (TMB) to each well, cover the plate with a film, and incubate at 37°C in the dark for about 15 min. Turn on the microplate reader 15 min in advance to preheat; (8) Add 50 μL of stop solution to each well to terminate the reaction; (9) Immediately measure the optical density (OD value) of each well at a wavelength of 450 nm using a microplate reader.

[0036] Result judgment 1. Calculate the average OD value of the standard and sample duplicate wells and subtract the OD value of the blank well as the correction value. With concentration as the horizontal axis and OD value as the vertical axis, fit the standard curve of the four-parameter logistic function on the double logarithmic coordinate axis.

[0037] 2. If the OD value of the sample is higher than the upper limit of the standard curve, it should be appropriately diluted and re-measured and multiplied by the corresponding dilution factor when calculating the sample concentration.

[0038] Results Figure 8As shown in Figure 8, the results of mouse serum ELISA showed that compared with the blank control group, the expression of IL-17A in the model control group was significantly enhanced (P<0.01); compared with the model control group, after treatment with 0.2 mg / kg Apelin-13 and MTX, the expression level of IL-17A was reduced (P<0.05, P<0.05). This result suggests that Apelin-13 may reverse psoriasis-like lesions by inhibiting the expression of IL-17A.

[0039] The above results show that the peptide Apelin-13 has a good effect in inhibiting IMQ-induced keratinocyte proliferation and lipid metabolism disorder, and shows good application prospects in the treatment of psoriasis.

Claims

1. Use of a polypeptide in the preparation of a drug for treating psoriasis, characterized in that: The polypeptide is Apelin-13.

2. The use according to claim 1, characterized in that The psoriasis includes various types of mild, moderate and severe psoriasis and psoriasis-related complications.

3. The use according to claim 1, characterized in that The polypeptide is used in preparing a drug for treating psoriasis-like skin lesions.

4. The use according to claim 1, characterized in that The Apelin-13 is a fragment of Apelin, and its amino acid sequence is: Pyr-Arg-Pro-Arg-Leu-Ser-His-Lys-Gly-Pro-Met-Pro-Phe.

5. The use according to claim 1, characterized in that The polypeptide includes modifications or alterations based on the amino acid sequence of the polypeptide Apelin-13.

6. A pharmaceutical composition for treating psoriasis, characterized in that: The pharmaceutical composition comprises the polypeptide Apelin-13.

7. The pharmaceutical composition for treating psoriasis according to claim 6, characterized in that: The pharmaceutical composition is administered orally, by injection or externally.

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