Application of chrysin in preparation of medicine for treating mammalian NFAT2 related diseases

Abstract

The invention relates to an application of chrysin in preparation of drugs for treating mammalian NFAT2 related diseases. It is confirmed for the first time that chrysin shows significant anti-inflammatory activity in a KD mouse model and a cell injury model. This effect may be achieved by modulating NFAT2. The discovery has important significance for understanding and potentially treating the vascular endothelial injury of the Kawasaki disease, and a new insight is provided for treating the Kawasaki disease by using traditional Chinese medicines.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to application of chrysin in preparing medicine for treating NFAT2-related diseases in mammals. Background Art

[0002] Kawasaki disease (KD), also known as mucocutaneous lymph node syndrome, is characterized by inflammatory changes in small and medium-sized immune blood vessels. KD has become the most common cause of acquired heart disease combined with coronary artery aneurysms in children. The incidence of coronary artery aneurysms in patients with Kawasaki disease who are not promptly and effectively treated is approximately 20-25%; even with timely and standardized treatment, 3%-5% of patients may still develop coronary artery disease. Vascular endothelial dysfunction is involved in the pathogenesis of coronary artery damage in KD. In the acute phase of the disease, circulating monocytes and neutrophils adhere to the surface of endothelial cells and release a large number of inflammatory mediators. The subsequent series of events ultimately leads to endothelial cell damage. The signals of multiple inflammatory mediators are significantly increased in children with acute KD. Therefore, inhibiting inflammation is an important clinical strategy for the management and treatment of KD, and exploring effective therapeutic strategies to reduce inflammation and damage to endothelial cells is the key to alleviating the prognosis of KD and reducing cardiovascular complications.

[0003] The etiology of KD is still unclear, and its inflammatory response is a complex biological process involving various signaling pathways and molecules. Among them, nuclear factor of activated T cells (NFAT) plays an important role in controlling cell activation and inflammatory responses. Our previous studies have also shown that regulatory pathways involving the interaction between NFAT and leukocytes can promote the release of inflammatory factors, chemokines, adhesion molecules, growth factors, and induced enzymes, and play a role in vascular damage in KD. SNPs in the Ca2+ / NFAT signaling pathway may play a crucial role in KD. In addition, studies have shown that serum from KD patients can increase the proliferation and angiogenesis of HCAECs, accompanied by increased levels of NFAT2, IL-8, and other cytokines, and the translocation of NFAT2 to the nucleus. Our research group also confirmed that among the NFAT gene family, NFAT2 showed the strongest transcriptional activity in peripheral blood mononuclear cells of KD patients (NCBI: GSE210094), which supports earlier research results and emphasizes that NFAT2 may be involved in the pathogenesis of KD. Although NFAT activation is induced by cytokines, the exact signaling pathways responsible for this process in the context of KD remain to be fully elucidated. Because dysregulation of NFAT2 signaling contributes to the development of KD-associated vascular damage, further investigation of the molecular mechanisms controlling NFAT2 activation and downstream signaling events in KD could provide valuable insights into the pathogenesis of the disease.

[0004] In recent years, an increasing number of studies have focused on the regulatory role of plant-derived compounds in inflammatory responses. Chrysin is a natural flavonoid compound that is widely found in honey, propolis, and fruits. It has a wide range of biological activities, including the prevention of oxidative stress, inflammation, and neurodegeneration. As part of the human diet, chrysin is considered a promising compound that can be used to prevent many diseases, including cancer, diabetes, and neurodegenerative diseases. Existing studies have shown that chrysin can inhibit inflammatory responses through various mechanisms, has a wide range of anti-inflammatory properties, and shows great therapeutic potential in various disease models. Summary of the invention

[0005] Problem that the invention aims to solve

[0006] The purpose of the present invention is to provide a new method for treating vascular endothelial damage caused by Kawasaki disease.

[0007] Solutions for solving problems

[0008] The present invention provides an application of chrysin in preparing a medicine for treating NFAT2-related diseases in mammals.

[0009] Preferably, the drug is a drug that inhibits the expression of NFAT2 protein.

[0010] Preferably, the NFAT2-related diseases include Kawasaki disease, chronic lymphocytic leukemia, systemic lupus erythematosus, cardiomyopathy, and myocarditis.

[0011] Preferably, the NFAT2-related disease is Kawasaki disease.

[0012] Preferably, the administration of chrysin includes oral administration, transdermal administration, and injection.

[0013] Preferably, the administration of chrysin is oral.

[0014] Preferably, the mammal is a human.

[0015] Preferably, the dosage of chrysin is 2-15 mg / kg.

[0016] Preferably, the dosage of chrysin is 3-10 mg / kg.

[0017] Preferably, the dosage of chrysin is 4.4-8.8 mg / kg.

[0018] Effects of the Invention

[0019] 1. Chrysin can significantly reduce the expression of NFAT2 in the KD mouse model. Further molecular docking analysis showed that chrysin has a moderate binding affinity with NFAT2, indicating that chrysin may exert its anti-inflammatory effect through the NFAT2 signaling pathway, thereby alleviating endothelial damage.

[0020] 2. In the TNFα-stimulated HCAEC cell model, chrysin significantly reduced the secretion of proinflammatory cytokines such as IL-6, IL-8 and IL-23, and alleviated the overexpression of vascular adhesion molecules such as ICAM-1 and VCAM-1, indicating the anti-inflammatory effect of chrysin in vascular endothelial cells.

[0021] In summary, chrysin can significantly reduce coronary artery inflammation in the CAWS-induced KD mouse model, and alleviate vascular endothelial damage by regulating the expression of inflammatory cytokines (such as IL-6, IL-8, TNFα, etc.). Chrysin can improve endothelial cell function through the NFAT2 pathway, inhibit the secretion of proinflammatory factors, and regulate the expression of endothelial activation markers such as ICAM-1 and VCAM-1, ultimately improving vascular inflammation. The present invention confirms for the first time that chrysin exhibits significant anti-inflammatory activity in both the KD mouse model and the cell injury model. This effect may be achieved by regulating NFAT2. These findings are of great significance for the understanding and potential treatment of vascular endothelial damage in Kawasaki disease, and also provide new insights into the treatment of Kawasaki disease with traditional Chinese medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Chrysin was shown to improve cardiovascular inflammatory damage in KD model mice and reduce the expression level of peripheral inflammatory factors. (A) HE staining was used to evaluate cardiovascular inflammation in KD mice (magnification x50). In the control group, no obvious inflammatory infiltration was observed around the coronary arteries. The CAWS group showed obvious inflammatory infiltration. Low-dose and high-dose chrysin significantly reduced CAWS-induced inflammation, and the efficacy of the two doses was comparable. (B) EVG staining was used to evaluate the damage of vascular elastic fibers (magnification x200). Elastic fiber staining showed that the elastic fibers in the control group were arranged regularly and orderly, while the fibers in the CAWS model mice were arranged disorderly and loosely connected. Chrysin restored the regular arrangement of elastic fibers. (C) Heat map showing the expression trend of cytokines in different groups. (D) Relative expression levels of four key cytokines (IL-6, IL-17, TNFα, and MCP-1) in different groups.

[0023] Figure 2Chrysin inhibits the expression levels of inflammatory factors (A) IL-6, (B) IL-8, (C) IL-23, (D) ICAM, and (E) VCAM in endothelial cells of the injury model. Compared with the control group, TNFα significantly increased the levels of inflammatory cytokines IL-6, IL-23, and VCAM. Treatment with 25μmol / L or 50μmol / L chrysin significantly reduced the expression of IL-6, IL-8, IL-23, ICAM, and VCAM, reversing the pro-inflammatory effect of TNFα.

[0024] Figure 3 Molecular docking shows the affinity of chrysin to NFAT2, and immunohistochemistry shows that chrysin reduces NFAT2 expression in the inflammatory area of ​​the heart. (A) Molecular docking of chrysin to NFAT2, the grid score of chrysin binding to NFAT2 (1a66) is -50.83 kcal / mol, indicating a considerable binding affinity, and hydrophobic interactions exist between chrysin and the LEU6, PRO7, TRP4, ALA168, VAL167, and LEU165 residues of NFAT2, while hydrogen bonds are formed with ARG16, HIE9, and SER10 residues. (B) Immunohistochemistry shows the expression levels of NFAT2 protein in different groups (magnification × 200), and NFAT2 expression is increased in the CAWS-induced KD mouse model, and this trend is alleviated after chrysin treatment.

[0025] Figure 4 Chrysin reduces NFAT2 expression in endothelial cell injury model. (A) NFAT2 protein level increases in endothelial cell injury model. (B) Chrysin reverses the increase in NFAT2 expression induced by TNFα. DETAILED DESCRIPTION

[0026] In order to make the technical solutions and beneficial effects of the present invention more clearly understandable, the following is a detailed description by listing specific embodiments. The drawings are not necessarily drawn to scale, and local features may be enlarged or reduced to more clearly show the details of the local features; unless otherwise defined, the technical and scientific terms used herein have the same meanings as those in the technical field to which this application belongs.

[0027] As used herein, the term "Kawasaki disease", "Kawasaki syndrome", "Kawasaki disease" or "KD" refers to mucocutaneous lymph node syndrome, an acute febrile rash disease with systemic vasculitis as the main pathological change.

[0028] As used herein, the term "NFAT" or "nuclear factor of activated T cells" is a family of transcription factors that are widely expressed in a variety of animal tissues and cells. It is a key regulatory point for multiple signal transduction pathways within cells, especially playing an important role in the immune system, nervous system development, axon growth and nervous system diseases.

[0029] As used herein, the term "chrysin" is also known as "cohen" and "5,7-dihydroxyflavonoid", and is a natural flavonoid compound widely found in nature, with a chemical formula of C 15 H 10 O4, molecular weight is 254.24, appearance is light yellow powder or light yellow prismatic crystals (crystallized from methanol), melting point is 285°C. Chrysin is insoluble in water, but soluble in alkali hydroxide solution, slightly soluble in ether, ethanol and chloroform.

[0030] As used herein, the term "CAWS" refers to Candida albicans aqueous solution, which is commonly used to induce the establishment of a Kawasaki disease systemic vasculitis model.

[0031] As used herein, the term "inflammatory cytokine" refers to cytokines that regulate various inflammatory responses, mainly including two types: pro-inflammatory cytokines and anti-inflammatory cytokines, such as TNFα, IL-6, IL-8 and IL-23.

[0032] As used herein, the term "endothelial activation marker" refers to a biomarker that is released or changes in expression when vascular endothelial cells are stimulated, such as ICAM-1, VCAM-1, etc. These markers can reflect the activation state of endothelial cells.

[0033] The present invention provides an application of chrysin in preparing a medicine for treating NFAT2-related diseases in mammals.

[0034] In certain embodiments, the drug is a drug that inhibits the expression of NFAT2 protein.

[0035] In certain embodiments, the NFAT2-related diseases include Kawasaki disease, chronic lymphocytic leukemia, systemic lupus erythematosus, rheumatoid arthritis, cardiomyopathy, and myocarditis.

[0036] In certain embodiments, the NFAT2-related disease is Kawasaki disease.

[0037] In certain embodiments, the administration of chrysin includes oral administration, transdermal administration, and injection.

[0038] In certain embodiments, the chrysin is administered orally.

[0039] In certain embodiments, the mammal is a mouse.

[0040] In certain embodiments, when the mammal is a mouse, the dosage of chrysin is 20 to 120 mg / kg.

[0041] In certain embodiments, when the mammal is a mouse, the amount of chrysin is 20 mg / kg, or 25 mg / kg, or 30 mg / kg, or 35 mg / kg, or 40 mg / kg, or 41 mg / kg, or 42 mg / kg, or 43 mg / kg, or 44 mg / kg, or 45 mg / kg, or 46 mg / kg, or 47 mg / kg, or 48 mg / kg, or 49 mg / kg, or 50 mg / kg, or 51 mg / kg, or 52 mg / kg, or 53 mg / kg, or 54 mg / kg, or 55 mg / kg, or 56 mg / kg, or 57 mg / kg, or 58 mg / kg, or 59 mg / kg, or 60 mg / kg, or 61 mg / kg. 1mg / kg, or 62mg / kg, or 63mg / kg, or 64mg / kg, or 65mg / kg, or 66mg / kg, or 67mg / kg, or 68mg / kg, or 69mg / kg, or 70mg / kg, or 71mg / kg, or 72mg / kg, or 73mg / kg, or 74mg / kg, or 75mg / kg, or 76mg / kg, or 77mg / kg, or 78mg / kg, or 79mg / kg, or 80mg / kg, or 85mg / kg, or 90mg / kg, or 95mg / kg, or 100mg / kg, or 105mg / kg, or 110mg / kg, or 115mg / kg, or 120mg / kg.

[0042] In certain embodiments, when the mammal is a mouse, the dosage of chrysin is 30-100 mg / kg.

[0043] In certain embodiments, when the mammal is a mouse, the dosage of chrysin is 40-80 mg / kg.

[0044] In certain embodiments, the mammal is a human.

[0045] In certain embodiments, when the mammal is a human, the amount of chrysin is 2-15 mg / kg.

[0046] In certain embodiments, when the mammal is a human, the amount of chrysin is 2 mg / kg, or 2.5 mg / kg, or 3 mg / kg, or 3.5 mg / kg, or 4 mg / kg, or 4.1 mg / kg, or 4.2 mg / kg, or 4.3 mg / kg, or 4.4 mg / kg, or 4.5 mg / kg, or 4.6 mg / kg, or 4.7 mg / kg, or 4.8 mg / kg, or 4.9 mg / kg, or 5 mg / kg, or 5.1mg / kg, or 5.2mg / kg, or 5.3mg / kg, or 5.4mg / kg, or 5.5mg / kg, or 5.6mg / kg, or 5.7mg / kg, or 5.8mg / kg, or 5.9mg / kg, or 6mg / kg, or 6.1mg / kg, or 6.2mg / kg, or 6.3mg / kg, or 6.4mg / kg, or 6.5mg / kg, or 6.6mg / kg, or 6.7mg / kg, or 6. 8mg / kg, or 6.9mg / kg, or 7mg / kg, or 7.1mg / kg, or 7.2mg / kg, or 7.3mg / kg, or 7.4mg / kg, or 7.5mg / kg, or 7.6mg / kg, or 7.7mg / kg, or 7.8mg / kg, or 7.9mg / kg, or 8mg / kg, or 8.1mg / kg, or 8.2mg / kg, or 8.3mg / kg, or 8.4mg / kg, or 8.5mg / kg, or 8.6mg / kg, or 8.7mg / kg, or 8.8mg / kg, or 8.9mg / kg, or 9mg / kg, or 9.5mg / kg, or 10mg / kg, or 10.5mg / kg, or 11mg / kg, or 11.5mg / kg, or 12mg / kg, or 12.5mg / kg, or 13mg / kg, or 13.5mg / kg, or 14mg / kg, or 14.5mg / kg, or 15mg / kg.

[0047] In certain embodiments, when the mammal is a human, the amount of chrysin is 3-10 mg / kg.

[0048] In certain embodiments, when the mammal is a human, the amount of chrysin is 4.4-8.8 mg / kg.

[0049] In certain embodiments, the administration of chrysin is transdermal administration.

[0050] In certain embodiments, the administration of chrysin is by injection.

[0051] Example 1: Establishment of mouse model

[0052] C57BL / 6J mice (4 weeks old) were used to replicate the Kawasaki disease mouse model, in which CAWS was a water-soluble substance prepared from Candida albicans NBRC1385 (China Industrial Microbiological Culture Collection Management Center), and chrysin was purchased from Sigma. The Kawasaki disease mouse model was simulated by intraperitoneal injection of CAWS (4 mg / mouse, for 5 days). The mice were randomly divided into four groups: control group (intraperitoneal injection of normal saline); KD group (intraperitoneal injection of CAWS); chrysin group 1 (intraperitoneal injection of CAWS, oral administration of 40 mg / kg of chrysin); chrysin group 2 (intraperitoneal injection of CAWS, oral administration of 80 mg / kg of chrysin). The mice in each group were killed on the 14th day and subjected to histological analysis. The results are shown in the following table. Figure 1 , 3 As shown in . Low-dose and high-dose chrysin significantly reduced CAWS-induced inflammation, restored the regular arrangement of elastic fibers, and effectively alleviated the increased expression of NFAT2, and the therapeutic effects of the two doses were comparable.

[0053] Example 2: Chrysin treatment and experimental design

[0054] Whole blood was collected from the mice in Example 1 for cytokine chip analysis of cytokine levels, and HE staining and scanning electron microscopy were used to observe the morphological changes of heart vessels. The results are as follows: Figure 1 shown. Figure 1 A HE staining showed that the vascular endothelial cells of the mice in the control group were intact and smooth, without inflammatory cell infiltration; the vascular endothelial cells of the mice in the model group were rough, with incomplete structure and disordered cell arrangement, and extensive infiltration of inflammatory cells was observed in the blood vessels and surrounding myocardial tissues; the vascular endothelial structure of the heart in the chrysin group was relatively complete, with less inflammatory cell infiltration. Figure 1 B EVG staining showed that the elastic fibers in the vascular walls of mice in the control group were arranged regularly and curled in an orderly manner; the elastic fibers in the vascular walls of mice in the model group were arranged disorderly, loose and broken; the elastic fibers in the chrysin group were relatively regular, with no obvious breaks. Figure 1 C cluster heat map shows the overall expression trend of cytokines in the control group, model group and chrysin group. Figure 1 D shows that compared with the control group, the expression of four key cytokines IL-6, IL-17, TNFα and MCP-1 in the model group was significantly increased, and compared with the model group, chrysin significantly reduced the expression levels of the four factors.

[0055] Example 3: Effects of Chrysin on Cytokine Levels and Changes in Vascular Adhesion Molecules

[0056] Human coronary artery endothelial cells (HCAEC) were treated with TNFα to simulate an endothelial cell injury model and evaluate the anti-inflammatory properties of chrysin. HCAECs were pretreated with TNFα for 0.5 h and then treated with chrysin. IL-6, IL-8, IL-23, ICAM, and VCAM- levels were evaluated 6 h after stimulation. Figure 2 As shown in Figure 3, TNFα treatment significantly increased the levels of inflammatory cytokines IL-6, IL-23, and adhesion molecule VCAM- compared with the control group. Treatment with 25 μmol / L or 50 μmol / L chrysin significantly reduced the expression of these markers and reversed the proinflammatory effects of TNFα.

[0057] Example 4: Regulatory effect of chrysin on NFAT2 signaling pathway

[0058] Western Blot was used to analyze the changes in NFAT2 expression under TNFα stimulation, and the regulatory effect of chrysin on the NFAT2 signaling pathway was studied. The molecular docking software DOCK 6.9 was used to analyze the binding affinity between NFAT2 and chrysin. The results are as follows Figure 3 As shown in Figure 2, the grid score of chrysin binding to NFAT2 (1a66) was -50.83 kcal / mol, indicating a comparable binding affinity. NFAT2 expression increased in the CAWS-induced KD mouse model, and this trend was alleviated in the chrysin group. The results of the regulatory effect of chrysin on the NFAT2 signaling pathway are shown in Figure 2. Figure 4 As shown, NFAT2 protein levels increased in the endothelial cell injury model, and chrysin reversed the increase in NFAT2 expression induced by TNFα.

[0059] It should be understood that the above embodiments are exemplary and are not intended to include all possible implementations included in the claims. Various modifications and changes may be made on the basis of the above embodiments without departing from the scope of the present disclosure. Similarly, the various technical features of the above embodiments may be arbitrarily combined to form other embodiments of the present invention that may not be explicitly described. Therefore, the above embodiments only express several implementations of the present invention and do not limit the scope of protection of the patent of the present invention.

Claims

1. A use of chrysin in the preparation of a drug for treating NFAT2-related diseases in mammals.

2. The use according to claim 1, characterized in that: The drug is a drug for inhibiting the expression of NFAT2 protein.

3. The use according to claim 1, characterized in that: The NFAT2-related diseases include Kawasaki disease, chronic lymphocytic leukemia, systemic lupus erythematosus, cardiomyopathy, and myocarditis.

4. The use according to claim 3, characterized in that: The NFAT2-related disease is Kawasaki disease.

5. The use according to claim 1, characterized in that: The administration methods of chrysin include oral administration, transdermal administration and injection.

6. The use according to claim 5, characterized in that: The administration method of chrysin is oral administration.

7. The use according to claim 1, characterized in that: The mammal is a human.

8. The use according to claim 7, characterized in that: The dosage of chrysin is 2-15 mg / kg.

9. The use according to claim 8, characterized in that: The dosage of chrysin is 3-10 mg / kg.

10. The use according to claim 9, characterized in that: The dosage of chrysin is 4.4-8.8 mg / kg.

Images