Application of hylarana guentheri multifunctional polypeptide Cak-HG in preparation of medicine for treating psoriasis

Abstract

The invention provides an application of Cath-HG in preparation of a medicine for preventing and / or treating psoriasis. Experiments prove that Cath-HG has a good curative effect on an imiquimod-induced mouse psoriasis model, can improve skin injury, reduce PASI score, reduce inflammatory cytokine mRNA level, reduce spleen coefficient, recover the number of platelets and neutrophils, inhibit platelet activation and platelet-neutrophil compound formation, reduce neutrophil infiltration and activation, and can be used for treating psoriasis. The release of extracellular traps of neutrophils is inhibited.

Description

Technical Field

[0001] The invention belongs to the technical field of biopharmaceuticals, and specifically relates to an application of a multifunctional polypeptide of Rana sutchuenensis Cath-HG in the preparation of a drug for treating psoriasis. Background Art

[0002] As a chronic and relapsing inflammatory skin disease, psoriasis has long been a key research topic in the medical field. The pathological mechanism of this disease is complex, involving the interaction of multiple factors such as immune cells, inflammatory mediators, and cell signal transduction pathways. Despite the continuous innovation of treatment methods, the treatment of psoriasis still faces multiple challenges, including repeated attacks of the disease and drug side effects. Therefore, actively exploring new therapeutic targets and strategies is of great significance to improving the quality of life of patients with psoriasis.

[0003] Abnormal increase in neutrophils is a significant pathological feature of psoriasis. Under the stimulation of multiple factors such as immune complexes, cytokines and chemokines, these cells will release neutrophil extracellular traps (NETs), thereby aggravating the local inflammatory response and promoting excessive proliferation of keratinocytes. In addition, the role of platelets in psoriasis cannot be ignored. Although the specific mechanism of action of platelets in psoriasis is not yet fully understood, studies have pointed out that platelet activation is one of the important mechanisms of psoriasis. In the early stages of inflammation or endothelial cell damage, platelets are activated, which in turn attracts neutrophils and forms platelet-neutrophil complexes at the lesion site, thereby aggravating the development and deterioration of psoriasis. Therefore, inhibiting platelet activation and blocking the formation of platelet-neutrophil complexes are expected to become a new strategy for the treatment of psoriasis.

[0004] It is worth mentioning that Cath-HG, as a multifunctional polypeptide secreted from the skin of the amphibian water frog, has multiple biological activities such as antibacterial, inhibition of serine protease activity, insulin release activity, LPS neutralization, and inhibition of macrophage activation. It has shown good application prospects in the treatment of diseases such as acne infection and sepsis. For example, the publication number of CN106432483A discloses the serine protease inhibitory peptide from the skin of a water frog and its gene and pharmaceutical application, which discloses that the serine protease inhibitory peptide from the skin of a water frog is used to prepare drugs for treating infectious diseases caused by pathogenic microorganisms, treating diseases related to elevated blood sugar or anti-tumor, and in the preparation of drugs for preventing or treating gastritis or pancreatitis. However, it only discloses the effects of the serine protease inhibitory peptide from the skin of a water frog in inhibiting bacteria and fungi, the activity of serine protease inhibitors and promoting insulin secretion. Another example is the application of the multifunctional polypeptide Cath-HG of a water frog in pharmaceuticals and cosmetics with the publication number of CN113413459B, which discloses that the multifunctional polypeptide Cath-HG of a water frog is used to prepare drugs against Propionibacterium acnes. However, it only discloses the bactericidal effect of the multifunctional polypeptide of a water frog on Propionibacterium acnes ATCC6919. In other words, neither discloses the application of the multifunctional polypeptide Cath-HG of a water frog in preparing drugs for preventing or treating psoriasis, and there is no report on the use of Cath-HG to prevent or treat psoriasis. Summary of the invention

[0005] In view of the deficiencies in the prior art, the object of the present invention is to provide a multifunctional polypeptide of Rana sutchuenensis Cath-HG, which is used for preparing a preparation and / or a medicine for preventing and / or treating psoriasis.

[0006] In a first aspect of the embodiments of the present application, there is provided a use of a multifunctional polypeptide of Rana salinarum, Cath-HG, in the preparation of a preparation and / or a medicine for preventing and / or treating psoriasis.

[0007] In some embodiments of the present application, the ingredients of the preparation and / or medicine include: the multifunctional polypeptide Cath-HG of Rana malariae and a pharmaceutically acceptable carrier.

[0008] In some embodiments of the present application, the dosage form of the preparation and / or drug includes cream, ointment, tablet, granule, capsule, powder, oral liquid or injection.

[0009] In some embodiments of the present application, the pharmaceutically acceptable carrier includes a surfactant, an excipient, a stabilizer, a suspending agent, an isotonic agent, a preservative, a filler, a disintegrant, an antioxidant, an emulsifier, a coating agent, a binder, a lubricant and / or a flavoring agent.

[0010] In some embodiments of the present application, the multifunctional polypeptide Cath-HG of Rana sutchuenensis reduces the skin inflammation caused by psoriasis by inhibiting platelet activation, preventing the formation of platelet-neutrophil complexes and the activation of neutrophils.

[0011] In some embodiments of the present application, the unit dosage of the multifunctional polypeptide Cath-HG of Rana sutchuenensis is 0.001-1000 mg.

[0012] In some embodiments of the present application, the unit dosage of the multifunctional polypeptide Cath-HG of Rana sutchuenensis is 0.1-100 mg.

[0013] In some embodiments of the present application, the unit dosage of the multifunctional polypeptide Cath-HG of Rana sutchuenensis is 1-50 mg.

[0014] In some embodiments of the present application, the unit dosage of the multifunctional polypeptide Cath-HG of Rana sutchuenensis is 0.1-100 mg / kg.

[0015] In some embodiments of the present application, the unit dosage of the multifunctional polypeptide Cath-HG of Rana sutchuenensis is 1-30 mg / kg.

[0016] The beneficial effects of the present invention are:

[0017] The present invention innovatively proposes a new application of Cath-HG in the preparation of drugs for the prevention and / or treatment of psoriasis. After rigorous experimental verification, Cath-HG showed significant therapeutic effects in the imiquimod-induced mouse psoriasis model. The compound can effectively improve the degree of skin damage in the psoriasis mouse model, significantly reduce its PASI score, and significantly downregulate the mRNA levels of inflammatory cytokines (including IL-1β, IL-6, IL-17A, IL-22, IL-23 and TNF-α) in the lesions.

[0018] In addition, Cath-HG can effectively reduce the spleen coefficient of the psoriasis mouse model, promote the recovery of the number of platelets and neutrophils in the whole blood, inhibit the activation of platelets in the lesions, and inhibit the formation of platelet-neutrophil complexes. At the same time, it can also significantly inhibit the number of neutrophil infiltration in the lesions, as well as the activation of neutrophils and the release of neutrophil extracellular traps.

[0019] In summary, Cath-HG has broad application prospects in the preparation of drugs for preventing and / or treating psoriasis, and is expected to provide a new and effective means for the treatment of psoriasis. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Design of the experiment for the imiquimod-induced psoriasis mouse model;

[0021] Figure 2 The typical manifestations of the back skin of mice in different groups are shown;

[0022] Figure 3 is the spleen coefficient of mice in different groups;

[0023] Figure 4 PASI scores of mice in different groups;

[0024] Figure 5 The results of H&E staining of the back skin of mice in different groups;

[0025] Figure 6 is the mRNA level of inflammatory factors in the skin lesions of mice in different groups;

[0026] Figure 7 is the number of platelets in whole blood of mice in different groups;

[0027] Figure 8 is the number of whole blood neutrophils in different groups of mice;

[0028] Fig. 9 The platelet activation levels of mice in different groups;

[0029] Fig.10 Immunohistochemistry of neutrophils in skin lesions of mice in different groups;

[0030] Fig.11 Immunofluorescence staining of skin lesions in different groups of mice. DETAILED DESCRIPTION

[0031] The principles and features of the present invention are described below, and the examples are only used to explain the present invention and are not used to limit the scope of the present invention. The present invention is described in more detail by way of example in the following paragraphs. According to the following description and claims, the advantages and features of the present invention will become clearer.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0033] Example 1

[0034] Validation of Cath-HG as a drug for the prevention and / or treatment of psoriasis

[0035] 1. Materials and methods

[0036] 1.1. Modeling and treatment of psoriasis mouse model

[0037] Female BALB / c mice (weight range 20-25g, age 8 weeks) were purchased from the Experimental Animal Center of Southern Medical University. During the experiment, the mice were placed in constant environmental conditions, with the temperature maintained at 24±2℃, the humidity controlled at 50±5%, and followed a 12-hour light-dark cycle to ensure the normal functioning of their physiological rhythms. At the same time, the experimental environment provided sufficient bedding, food and clean water to meet the basic living needs of the mice.

[0038] All experimental operations related to mice were carried out in strict accordance with the established laboratory procedures to ensure the standardization and scientificity of the experimental process. In addition, all experimental protocols were strictly reviewed and approved by the Experimental Animal Ethics Committee of Southern Medical University to protect the rights and welfare of animals to the greatest extent.

[0039] After one week of adaptive feeding, the mice were numbered and weighed, and randomly divided into four experimental groups, each containing six mice. The groups were as follows: control group, model group, Cath-HG treatment group (dose of 1 mg / kg), and positive drug dexamethasone treatment group (dose of 2.5 mg / kg).

[0040] Before the experiment, use a razor to carefully remove the hair of the mouse back skin area of ​​about 3.0×5.0cm2, and evenly apply depilatory cream on the shaved area. After the depilatory cream works for 10 minutes, gently wipe off the depilatory cream with soaked absorbent paper, and then put the mouse back into the cage to adapt for three days.

[0041] Subsequently, imiquimod cream was used to establish a psoriasis model in mice. The specific operation was as follows: 62.5 mg of 5% imiquimod cream was weighed using a 1 mL syringe and evenly applied to the exposed skin on the back of mice in the model group and the treatment group with a cotton swab, while the control group mice were smeared with an equal amount of vaseline.

[0042] Four hours after application of the cream, the mice in the control and model groups received a subcutaneous injection of 100 μL of normal saline, while the mice in the Cath-HG group received a subcutaneous injection of 100 μL of Cath-HG (at a dose of 1 mg / kg), and the mice in the dexamethasone group received a subcutaneous injection of 100 μL of dexamethasone (at a dose of 2.5 mg / kg).

[0043] like Figure 1 As shown in the figure, during the whole experiment, the model construction and drug administration were carried out for seven days. On the eighth day of the experiment, the skin lesions of mice in each group were photographed and recorded. The results are shown in the figure. Figure 2Subsequently, the spleen tissue of the mouse was separated and taken out for photographing, and its weight was measured to calculate the spleen index (ratio of spleen weight to body weight). The results are shown in Figure 3 As shown, Figure 3 The data are expressed as mean ± SD, **P < 0.01, ***P < 0.001.

[0044] 1.2 PASI score

[0045] Before modeling each day, each group of mice was scored based on the severity of their skin lesions in three dimensions: erythema, scaling, and skin thickness. The scoring system uses a five-level system, from 0 to 4, where 0 represents no lesions, 1 represents mild lesions, 2 represents moderate lesions, 3 represents severe lesions, and 4 represents very severe lesions. The sum of the scores of the three dimensions is the PASI score, which is used to comprehensively reflect the degree of skin lesions in mice. The scoring results have been shown in detail in Figure 4 middle.

[0046] 1.3. H&E staining experimental steps

[0047] In this experiment, the skin of the lesion site of each group of mice was first carefully separated. Then, the samples were fixed with 4% paraformaldehyde solution to ensure the stability of their morphological structure. Next, the samples were embedded in paraffin to facilitate subsequent sectioning operations.

[0048] In order to obtain thin enough sections for easy observation, the wax blocks were carefully cut into slices with a thickness of 4 μm. Subsequently, the sections were stained using hematoxylin and eosin staining techniques to highlight the different structures and components in the tissue. After staining, neutral resin was used as a mounting medium to seal the sections to ensure their convenience in preservation and observation.

[0049] Finally, an optical microscope (brand: Olympus, origin: Japan) was used to collect images of the stained sections and conduct an in-depth analysis of the experimental results. Figure 5 As shown in the figure (scale bar = 200 μm), the figure clearly shows the microstructural characteristics of the skin in the lesion area of ​​each group of mice, providing strong data support for subsequent research.

[0050] 1.4. Reverse transcription real-time quantitative PCR (RT-qPCR) analysis

[0051] This experiment aims to accurately measure the expression levels of specific genes in the skin of the lesions of each group of mice by reverse transcription real-time quantitative PCR (RT-qPCR) technology. During the experiment, skin samples from the lesions of each group of mice were first obtained, and the total RNA was efficiently isolated using TRIzol reagent. Subsequently, the extracted total RNA was accurately quantified using Nanodrop 2000 (Thermo Scientific, USA) to ensure the accuracy of subsequent experiments. After obtaining sufficient RNA, it was reverse transcribed into cDNA using the PrimeScript RT kit (Takara, Japan) to provide a template for subsequent PCR reactions.

[0052] In the PCR reaction stage, SYBR master mix Ex Taq (Takara, Japan) was used as the fluorescent dye, and the experimental operation was performed on the LightCycler 480II (Roche, Switzerland), a high-performance real-time fluorescence quantitative PCR instrument. In order to eliminate the experimental errors between different samples, the relative expression level was standardized to the expression level of β-actin to obtain more reliable experimental results.

[0053] After the experiment, the data were sorted and analyzed, and the following Figure 6 The experimental results shown in the figure, Figure 6 Data are expressed as mean ± standard deviation, *P < 0.05, **P < 0.01, ***P < 0.001. At the same time, for the convenience of readers, the primer sequences used in this experiment are also organized into Table 1 for reference.

[0054] Table 1 qRT-PCR primers

[0055]

[0056]

[0057] 1.5. Platelet count analysis

[0058] Blood samples were collected from each group of mice using EDTA anticoagulant tubes. Subsequently, 100 μL of blood from each group of mice was accurately taken and the platelet count was accurately detected using a PE-6800 fully automatic blood analyzer (Pukang, China). The results have been recorded in detail in Figure 7 middle, Figure 7 The data are expressed as mean ± SD, *P < 0.05, **P < 0.01, for subsequent analysis and reference.

[0059] 1.6 Flow cytometry analysis

[0060] In this experiment, blood samples of mice in each group were collected through EDTA anticoagulant tubes. 50 μL of each sample was accurately taken and properly stored in EP tubes. Subsequently, according to the experimental requirements, 1 μL of PE-CD61 flow cytometry antibody, 1 μL of FITC-CD62P flow cytometry antibody, 1 μL of APC-Cy7-CD11b flow cytometry antibody, and 1 μL of PE-Cy7-Ly-6G flow cytometry antibody were accurately added to each tube of sample. To ensure that the antibody and the sample fully react, the sample was incubated in a constant 37°C environment in the dark for 30 minutes.

[0061] After the incubation process, to ensure complete lysis of red blood cells, 450 μL of red blood cell lysis solution was added to each tube of sample and mixed thoroughly, and then incubated again in the dark for 10 minutes. Then, the sample was centrifuged at 2000 rpm for 5 minutes to separate the cell components.

[0062] After centrifugation, the supernatant was carefully discarded, the precipitate was resuspended with 2 mL of PBS buffer to redisperse the cells in the liquid, and the resuspended liquid was transferred to a dedicated flow tube. Finally, the samples were tested using a high-performance flow cytometer (manufactured by Sysmex, Japan) to obtain accurate cell analysis data.

[0063] The experimental results are detailed in Figure 8 and Fig. 9 As shown, Figure 8 and Fig. 9 The data are expressed as mean ± SD, ***P < 0.001, and these results provide detailed information about specific cell types in mouse blood samples.

[0064] 1.7 Immunohistochemistry and immunofluorescence staining experiments

[0065] In this study, immunohistochemistry and immunofluorescence staining experiments were performed to deeply analyze the histological characteristics of the skin lesions of each group of mice. The specific steps are as follows:

[0066] First, the skin of the lesion site of each group of mice was separated and fixed with 4% paraformaldehyde. Then, paraffin embedding technology was used to make paraffin blocks from the fixed tissue blocks, which were cut into slices with a thickness of 4 μm for subsequent staining operations.

[0067] In the immunohistochemistry experiment, the sections were incubated with the primary antibody of Ly-6G at 4°C overnight. After that, the sections were incubated with the secondary antibody at 37°C for 15 minutes for specific staining. Then, DAB was used for color development, so that the target antigen showed obvious color changes on the sections. Finally, the sections were imaged using an optical microscope (Olympus, Japan) and the results were analyzed.

[0068] In the immunofluorescence experiment, the sections were incubated with CD61 primary antibody and neutrophil single-line protease primary antibody at 4°C overnight. Subsequently, the sections were incubated with FITC-mouse IgG and Fluor647-rabbit IgG at 37°C for 15 minutes to achieve fluorescent staining. Finally, the sections were mounted with mounting medium containing DAPI, and images were collected and analyzed using a fluorescence microscope (Olympus, Japan).

[0069] Through immunohistochemistry and immunofluorescence staining experiments, the histological characteristic images of the skin lesions of each group of mice were successfully obtained, and the experimental results were deeply analyzed. Fig.10 (Scale bar = 200 μm) and Fig.11 (Scale bar = 100 μm), the experimental results show the specific staining of the skin in the lesion area of ​​mice in different groups, providing important experimental basis for subsequent research.

[0070] 1.8 Statistical analysis

[0071] All data were rigorously processed and presented in the form of mean ± SD to ensure the accuracy and reliability of the data. GraphPad Prism8 software was used for statistical analysis of the data, which is widely used and recognized in the field of scientific research. In the significance analysis, the one-way analysis of variance (ANOVA) method was used, which is a commonly used statistical method that can effectively evaluate the differences between different groups. When the P value is less than 0.05, the result is considered to be statistically significant, which helps to understand the connotation and rules of the data more accurately.

[0072] 2. Results

[0073] 2.1. Cath-HG improves the severity of psoriasis in mice

[0074] exist Figure 1 In the study, the imiquimod-induced psoriasis mouse model was used to study the therapeutic effect of Cath-HG, and dexamethasone was used as a positive control drug. Figure 2The comparison of the skin conditions of mice in each group after 7 days of modeling and drug administration is presented. The skin surface of mice in the blank control group was smooth and healthy, with no skin lesions or inflammation. In contrast, the back skin of mice with psoriasis induced by imiquimod showed typical characteristics of psoriasis lesions, including dark red plaques, skin bulges, and significant scale coverage. The skin condition of psoriasis mice treated with Cath-HG was significantly improved, as manifested by the fading of the color of skin erythema, the reduction of scales and skin wrinkles, and the significant reduction of skin bulges.

[0075] exist Figure 3 In the present study, it was observed that Cath-HG treatment significantly reduced spleen size and spleen coefficient in psoriasis mice. Figure 4 The effect of Cath-HG treatment on the skin scores of psoriasis mice was further demonstrated, and the results showed that the scores of erythema, scaling, and skin thickness, as well as the total PASI score, showed a significant downward trend. Figure 5 The H&E staining results in the study revealed that the skin sections of the model group mice showed typical psoriasis pathological features, such as significant thickening of the stratum corneum and stratum spinosum, disappearance or reduction of the granular layer, and abnormal keratinization process. After Cath-HG treatment, the pathological tissue morphology of the mice's skin was significantly restored.

[0076] In summary, the above experimental results show that Cath-HG treatment can effectively improve the disease severity of psoriasis mice and provide a new potential strategy for the treatment of psoriasis.

[0077] 2.2. After Cath-HG treatment, the inflammation level of psoriasis mice was significantly improved. Figure 6 As shown in the figure, the mRNA levels of IL-1β, IL-6, IL-17A, IL-22, IL-23 and TNF-α in the back skin of mice in the model group were significantly increased, indicating that the inflammatory response in the psoriasis lesions was aggravated. However, after receiving Cath-HG treatment, the mRNA levels of these pro-inflammatory factors were significantly reduced, which proved that Cath-HG has an effective inhibitory effect on the inflammatory response in psoriasis lesions.

[0078] 2.3. Cath-HG treatment helps restore the number of neutrophils and platelets in psoriasis mice to normal levels. Figure 7 As shown in Figure 2, the number of platelets in the whole blood of mice in the model group was significantly reduced, but after Cath-HG treatment, the number of platelets was restored. Figure 8 As shown, the ELISA test results showed that the number of neutrophils in the whole blood of the model group mice increased significantly, while Cath-HG treatment significantly decreased this number, indicating that Cath-HG has a significant regulatory effect on the number of neutrophils in psoriasis mice.

[0079] 2.4. Cath-HG can effectively reduce the platelet activation level in psoriasis mice. Fig. 9 As shown, the ELISA test results showed that the proportion of activated platelets in the whole blood of mice in the model group increased significantly, while after Cath-HG treatment, this proportion decreased significantly, proving that Cath-HG has an inhibitory effect on platelet activation in psoriasis mice.

[0080] 2.5. Cath-HG can inhibit the formation of platelet-neutrophil complexes in psoriasis mice. Fig.10 As shown in Figure 2, Ly-6G immunohistochemistry results showed that a large number of neutrophils infiltrated the dermis of the skin sections of the model group mice, while after Cath-HG treatment, the number of infiltrating neutrophils was significantly reduced. Fig.11 As shown in the figure, the immunofluorescence staining results of CD61 and neutrophil elastase showed that there was a large amount of platelet infiltration and neutrophil activation in the skin lesions of mice in the model group, and platelet-neutrophil complexes were formed. However, after Cath-HG treatment, the fluorescence quantity of CD61 and neutrophil elastase was significantly reduced, indicating that Cath-HG effectively inhibited the aggregation, activation and binding of platelets to neutrophils, thereby inhibiting the formation of platelet-neutrophil complexes in the skin lesions of psoriasis mice.

[0081] The above embodiments of the present invention are not intended to limit the protection scope of the present invention, and the implementation modes of the present invention are not limited thereto. All other modifications, replacements or changes made to the above structures of the present invention based on the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, should fall within the protection scope of the present invention.

Claims

1. Application of the multifunctional polypeptide Cath-HG of Rana sutchuenensis in the preparation of preparations and / or drugs for preventing and / or treating psoriasis.

2. The use according to claim 1, characterized in that: The ingredients of the preparation and / or medicine include: the multifunctional polypeptide Cath-HG of Rana sutchuenensis and a pharmaceutically acceptable carrier.

3. The use according to claim 2, characterized in that: The dosage forms of the preparation and / or drug include cream, ointment, tablet, granule, capsule, powder, oral liquid or injection.

4. The use according to claim 2, characterized in that: The pharmaceutically acceptable carrier includes a surfactant, an excipient, a stabilizer, a suspending agent, an isotonic agent, a preservative, a filler, a disintegrant, an antioxidant, an emulsifier, a coating agent, a binder, a lubricant and / or a flavoring agent.

5. The use according to any one of claims 1 to 4, characterized in that: The multifunctional polypeptide Cath-HG from the frog Cath-HG reduces skin inflammation caused by psoriasis by inhibiting platelet activation, preventing the formation of platelet-neutrophil complexes and the activation of neutrophils.

6. The use according to claim 2, characterized in that: The unit dosage of the multifunctional polypeptide Cath-HG of Rana sutchuenensis is 0.001-1000 mg.

7. The use according to claim 6, characterized in that: The unit dosage of the multifunctional polypeptide Cath-HG of Rana sutchuenensis is 0.1-100 mg.

8. The use according to claim 7, characterized in that: The unit dosage of the multifunctional polypeptide Cath-HG of Rana sutchuenensis is 1-50 mg.

9. The use according to claim 1, characterized in that: The unit dosage of the multifunctional polypeptide Cath-HG of Rana sutchuenensis is 0.1-100 mg / kg.

10. The use according to claim 9, characterized in that: The unit dosage of the multifunctional polypeptide Cath-HG of the frog is 1-30 mg / kg.

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