Application of IL-17A inhibitor in preparation of medicine for treating inflammatory diseases

Abstract

The invention relates to the technical field of biological medicines, in particular to application of an IL-17A inhibitor in preparation of medicines for treating inflammatory diseases. The invention provides application of an IL-17A inhibitor in preparation of a medicine for treating inflammatory diseases. Researches show that hesperetin has remarkable anti-inflammatory activity in vivo and in vitro, inflammatory reaction can be relieved by inhibiting generation of proinflammatory factors (TNF-alpha and IL-6) and regulating IL-23 / STAT3 / IL-17A signal channels, hesperetin serves as a natural medicine, generation of IL-17A can be more safely and stably inhibited, and hesperetin is a good IL-17A inhibitor.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the use of IL-17A inhibitors in the preparation of drugs for treating inflammatory diseases. Background Art

[0002] The interleukin 17 (IL-17) family is a cytokine subgroup composed of IL-17 (AF), which is produced by a variety of cells, including macrophages, Th17 cells, neutrophils, and NK cells. Since the discovery of IL-17A (also known as IL-17 or CTLA8) in 1993, the other five members of the family, IL-17B, IL-17C, IL-17D, IL-17E (also known as IL-25), and IL-17F, have been identified based on amino acid sequence homology. IL-17A, the most widely studied cytokine in this family, is a proinflammatory cytokine that plays an important role in host defense against microbial infection and is closely related to a variety of inflammatory diseases or autoimmune diseases, such as ankylosing spondylitis, multiple sclerosis, colitis, asthma, psoriasis, scleroderma, and rheumatoid arthritis. On the one hand, IL-17A can induce the expression of cytokines that increase related tissue repair, thereby accelerating the body's recovery; on the other hand, IL-17A is highly expressed in a series of inflammatory diseases or autoimmune diseases. At the same time, because it can induce a large number of inflammatory factors to accumulate and lead to excessive immune responses, excessive IL-17A levels may worsen the pathological development of related diseases. During the onset of various autoimmune or inflammatory diseases, the increased release of IL-23 further stimulates immune cells such as Th17 cells and macrophages (which are present in large numbers in various autoimmune and inflammatory diseases), activating the JAK-STAT3 signaling pathway. After STAT3 phosphorylation, these cells are stimulated to release IL-17A, which in turn promotes the secretion of large amounts of proinflammatory factors (TNF-α, IL-6) and proinflammatory mediators (such as NO), thereby inducing local inflammatory responses and exacerbating the occurrence and development of a series of inflammatory or autoimmune diseases.

[0003] At present, IL-17A inhibitors have made some research progress, including Brodalumab, Bimekizumab, CJM112, Izokibep and Secukinumab. These drugs inhibit IL-17A or its receptors through different mechanisms of action to reduce inflammatory responses. They have shown good efficacy in the treatment of diseases closely related to IL-17A, such as ankylosing spondylitis, rheumatoid arthritis and psoriasis. At the same time, IL-17A inhibitors have shown better efficacy and lower immunogenicity than traditional TNF-α inhibitors in clinical practice. As drugs for the treatment of a series of autoimmune diseases or inflammatory diseases such as ankylosing spondylitis, psoriasis and rheumatoid arthritis, although biological IL-17A inhibitors have significant efficacy, they often have a series of side effects such as gastrointestinal reactions, skin reactions and respiratory infections. Therefore, it is of great significance to find and screen more efficient, safe and stable anti-IL-17A drugs for the treatment of a series of autoimmune diseases or inflammatory diseases. At this time, natural drugs with low toxicity and good biological activity provide a new path for the development of anti-IL-17A drugs.

[0004] Tangerine peel (Citri Reticulatae Pericarpium) is the mature peel of the small tree plant Citrus reticulate Blanco and its cultivars of the Rutaceae family. The chemical components contained in tangerine peel are relatively large, mainly flavonoids, and also contain limonoids, alkaloids and volatile oils. The existing pharmacological reports on tangerine peel and its compounds mainly include: the digestive system effects include regulating gastrointestinal smooth muscle motility, promoting digestive juice and digestive enzyme secretion, protecting the liver, etc., and anti-respiratory diseases. Tangerine peel volatile oil has the effects of relieving asthma and antitussive, cardiotonic effect, vasodilation to reduce blood pressure, anti-inflammatory, anti-oxidation, antiviral, anti-tumor, hypolipidemic, antibacterial, and improving immunity. Among them, the active ingredients of dried tangerine peel show great potential in anti-inflammatory effects. Li Xue et al.'s related research shows that hesperidin can inhibit the abnormal proliferation, incomplete differentiation and local inflammatory response of keratinocytes through the IRS-1 / ERK1 / 2 signaling pathway. Yang Guliang et al.'s research shows that nobiletin can significantly inhibit CD4 + However, the anti-IL-17A effect of Tangeretin (Tan), an active ingredient of tangerine peel, has not been studied.

[0005] Tangeretin (Tan) is a natural flavonoid compound widely found in citrus peels. Related studies have shown that tangeretin has multiple biological activities such as anti-inflammatory (inhibiting the production of pro-inflammatory factors TNF-α and IL-6), antioxidant, neuroprotective and anti-asthmatic. In addition, this compound has also shown great potential in the treatment of cancers including ovarian cancer, breast cancer and gastric cancer.

[0006] Macrophages are important immune cells closely related to the initiation of inflammatory responses. They participate in a variety of inflammatory diseases or autoimmune diseases. They participate in immune responses by phagocytosis, antigen presentation, and cytokine secretion, and play a key role in the process of inflammatory responses. Lipopolysaccharide (LPS) is one of the main components of the cell wall of Gram-negative bacteria, consisting of three parts: lipid A, core polysaccharide, and O-antigen. LPS can activate a variety of immune cells, such as macrophages, T lymphocytes, B lymphocytes, and natural killer cells. After LPS treatment, macrophage activation shifts to a pro-inflammatory state, significantly increasing the levels of inflammatory target mediators (such as NO) and inflammatory cytokines (TNF-α and IL-6), which can promote the inflammatory process. The LPS-induced RAW264.7 cell inflammation model is an in vitro cell model widely used to study a variety of diseases, including but not limited to autoimmune diseases: such as psoriasis, rheumatoid arthritis, systemic lupus erythematosus, etc. The activation of macrophages and the production of inflammatory factors in these diseases are closely related to the occurrence and development of the disease; infectious diseases: such as inflammatory responses caused by bacterial or viral infections, the RAW264.7 cell model can be used to study how pathogens activate immune cells and the host's immune response; metabolic diseases: for example, the role of inflammatory responses in metabolic diseases such as obesity and diabetes; neurodegenerative diseases: such as Alzheimer's disease and Parkinson's disease, to study the role of inflammation in neurodegenerative processes; tumors: RAW264.7 cell models are also used to study the polarization state of macrophages in the tumor microenvironment and how they affect tumor growth and metastasis. Therefore, in the study of inflammatory infiltration or immune inflammation, the LPS-induced RAW264.7 cell inflammation model is a classic model for screening new drugs for the treatment of inflammatory diseases. Therefore, how to use the RAW264.7 cell inflammation model to screen out more types of drugs for treating inflammatory diseases to enrich the types of drugs for treating inflammatory diseases in the prior art is an urgent problem that technicians in this field need to solve.

[0007] Psoriasis is an immune-mediated chronic inflammatory disease, characterized by inflammatory responses mediated by a variety of immune cells (such as macrophages, T cells, and neutrophils) and abnormal proliferation and incomplete differentiation of keratinocytes. IL-17A plays a central role in the pathogenesis of psoriasis. It not only affects the proliferation activity and cell function of keratinocytes, but also has an important regulatory effect on immune cells and related cytokines in the immunopathological environment of psoriasis. IL-17A promotes the activation and proliferation of keratinocytes by binding to IL-17 receptors, and stimulates the production of other proinflammatory cytokines and chemokines, leading to a positive feedback loop of inflammation in psoriasis lesions. Therefore, biological agents targeting IL-17A have shown significant efficacy in the treatment of psoriasis, can effectively inhibit the occurrence of lesions, and improve disease symptoms. In recent years, monoclonal antibodies targeting the IL-17A signaling pathway, such as secukinumab, ixekizumab, and brodalumab, have been launched in China and abroad, and have shown significant efficacy in clinical applications. In addition, the study also found that the self-activation of the IL-17A signaling pathway can maintain inflammation and promote disease progression, which provides a new research direction for the treatment of psoriasis. The treatment strategy targeting IL-17A is highly anticipated in the field of psoriasis treatment and is expected to provide patients with more efficient and safe treatment options. Since IL-17A plays an important role in the pathogenesis of psoriasis, this study conducted in vivo animal experiments to establish a psoriasis mouse model using imiquimod (IMQ) after clarifying the anti-IL-17A activity of tangeretin in vitro.

[0008] Angiogenesis refers to the process of forming new blood vessels from existing blood vessels. This process occurs under a variety of physiological and pathological conditions, such as normal development, wound healing, menstrual cycle, and tumor growth. Angiogenesis is a complex biological process that involves the interaction of multiple growth factors and signaling pathways, among which vascular endothelial growth factor (VEGF) is one of the most famous angiogenesis-promoting factors. Angiogenesis is a pathological feature of psoriasis. The clinical pathogenesis of psoriasis mainly includes two major types of keratinocyte hyperproliferation and incomplete differentiation and inflammatory cell infiltration. With the hyperproliferation of keratinocytes, there will inevitably be an increase in angiogenesis. Therefore, angiogenesis is only an apparent pathological manifestation of psoriasis, and there is no direct connection between angiogenesis and the pathogenesis of psoriasis.

[0009] CN101947215A-Application of flavonoids in tangerine peel in the preparation of drugs for inhibiting angiogenesis The XTT method was used to detect endothelial cell proliferation, endothelial cell cell cycle detection, and observation of zebrafish intersegmental blood vessels and dorsal longitudinal axis blood vessel morphology under a fluorescence confocal microscope system. It was found that the active ingredients of tangerine peel, tangerine peel and sweet orange flavonoids in tangerine peel inhibit the proliferation of endothelial cells by regulating the cell cycle of vascular endothelial cells, thereby inhibiting angiogenesis. Therefore, the patent applicant inferred that the flavonoids in tangerine peel can be used to prepare drugs for treating diseases related to excessive angiogenesis, so the patent applicant only conducted related angiogenesis experiments and found that tangerine peel can significantly inhibit angiogenesis and cell proliferation, thereby inferring that it may have certain anti-psoriatic activity. We do not deny the experimental results and speculations of the patent applicant, nor do we deny that angiogenesis and psoriasis have a certain connection, and there may be deviations due to different research fields and starting points. However, tracing back to the source, the reason why the patent applicant linked angiogenesis with psoriasis was that one of the pathogenesis of psoriasis was that abnormal proliferation and incomplete differentiation of keratinocytes would lead to pathological manifestations of angiogenesis, and thus speculated, without any experimental data from psoriasis-related cell models and animal models. Therefore, the "application of flavonoid compounds in tangerine peel in the preparation of drugs for the treatment of psoriasis" disclosed in the patent application is essentially unattainable and inaccurate. Summary of the invention

[0010] The purpose of the present invention is to provide the use of IL-17A inhibitors in the preparation of drugs for treating inflammatory diseases. The present invention aims to develop new anti-IL-17A drugs, clarify the anti-inflammatory activity and anti-IL-17A effect of Tangeretin (Tan), thereby inhibiting inflammatory reactions, improving disease symptoms, and providing new treatment approaches for a series of autoimmune diseases or inflammatory diseases such as ankylosingspondylitis, multiple sclerosis, colitis, asthma, scleroderma, rheumatoid arthritis, etc. At the same time, as a natural medicine, Tangeretin has less toxicity and side effects, and is expected to solve a series of side effects caused by traditional IL-17A inhibitors. In general, the present invention aims to develop new anti-IL-17A drugs, clarify the anti-IL-17A effect of Tangeretin, so as to improve the physical and mental health and quality of life of patients suffering from diseases closely related to IL-17A.

[0011] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0012] The present invention provides use of an IL-17A inhibitor in preparing a drug for treating inflammatory diseases, wherein the IL-17A inhibitor is hesperidin.

[0013] Preferably, the drug further comprises a medically acceptable excipient.

[0014] Preferably, the drug is in the form of tablets, liquids, ointments, gel preparations, emulsions, capsules, suppositories or granules.

[0015] Preferably, the drug for treating inflammatory diseases is a drug for treating ankylosing spondylitis, a drug for treating multiple sclerosis, a drug for treating colitis, a drug for treating asthma, a drug for treating psoriasis, a drug for treating scleroderma or a drug for treating rheumatoid arthritis.

[0016] Preferably, the concentration of the IL-17A inhibitor in the drug for treating inflammatory diseases is 5 to 40 μM (of course, other effective concentrations are also included).

[0017] Preferably, the inflammation-related factors of the inflammatory disease include IL-23, IL-17A, TNF-α and IL-6.

[0018] Preferably, the inflammatory disease is an inflammatory disease induced by lipopolysaccharide.

[0019] Preferably, when the drug for treating inflammatory diseases is a drug for treating psoriasis, hesperidin has the effect of reducing skin thickness and the severity of scaling.

[0020] Preferably, the tangeretin reduces the inflammatory response by inhibiting the production of pro-inflammatory factors and regulating the IL-23 / STAT3 / IL-17A signaling pathway.

[0021] Preferably, the drug for treating inflammatory diseases is a drug that inhibits the content of cellular inflammatory factor NO.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] This study explored the anti-IL-17A effect of tangeretin in the LPS-induced RAW264.7 cell inflammation model and the imiquimod-induced psoriasis mouse model. It was found that in the LPS-induced RAW264.7 cell inflammation model, tangeretin showed good anti-inflammatory activity and effectively inhibited the production of IL-23 and IL-17A, indicating that tangeretin can inhibit the inflammatory response by regulating the IL-23 / IL-17A inflammatory axis, and can be used as a new IL-17A inhibitor. It also provides a new path for the treatment of a series of autoimmune diseases or inflammatory diseases such as psoriasis, ankylosing spondylitis, colitis, scleroderma and rheumatoid arthritis. Given that the IL-23 / IL-17A inflammatory axis occupies an important position in a series of inflammatory diseases or autoimmune diseases, and to further clarify the mechanism of action of tangeretin in inhibiting IL-17A in macrophages, this study traced the upstream pathway JAK / STAT3 of IL-17A secretion in cells. Western-blot data showed that in activated macrophages, tangeretin treatment group significantly inhibited the phosphorylation of STAT3, and had a significant inhibitory effect on the phosphorylation of two STAT3 phosphorylation sites (Tyr 705 and Ser 727), among which the inhibition of p-STAT3 (Ser 727) was more significant. The above experimental results indicate that tangeretin may inhibit the secretion of IL-17A by inhibiting IL-23-mediated STAT3 phosphorylation. It also shows that tangeretin exerts anti-inflammatory activity and inhibits the production of IL-17A by regulating the IL-23 / STAT3 / IL-17A signaling pathway.

[0024] In the psoriasis mouse model induced by imiquimod, we found that tangeretin can significantly improve the back skin lesions of psoriasis mice and effectively reduce the back erythema and scales. The results of H&E staining showed that the thickness of the epidermal tissue of the back skin of mice was significantly reduced, the hyperkeratosis was reduced, and the infiltration of inflammatory cells was reduced after tangeretin treatment, and the anti-psoriatic activity was obviously dose-dependent. Immunohistochemical detection showed that tangeretin can significantly inhibit the expression levels of p-STAT3 and IL-17A in the back skin of psoriasis mice, which is consistent with the experimental results of the LPS-induced RAW264.7 cell verification model, and more strongly illustrates the anti-IL-17A potential of tangeretin, and tangeretin may exert anti-psoriatic activity by regulating the IL-23 / STAT3 / IL-17 signaling pathway. At the same time, the results of immunohistochemistry showed that tangeretin can significantly inhibit the expression level of PCNA in the back skin of mice, suggesting that tangeretin may exert anti-psoriatic activity by inhibiting abnormal proliferation and incomplete differentiation of keratinocytes.

[0025] For example, CN201610107672.1 also discloses the use of Panax notoginseng saponin IVa butyl ester as an inhibitor of the IL-6 / STAT3 signaling pathway, wherein the diseases associated with the sustained activation of the IL-6 / STAT3 signaling pathway include but are not limited to autoimmune diseases, inflammatory diseases, bone diseases, metabolic diseases, neurological and neurodegenerative diseases, cardiovascular diseases, allergies and asthma, Alzheimer's disease and cancer. Based on the relevance of the pathway of the present invention to the prior art, hesperidin should also have the potential to prepare drugs for treating the above diseases.

[0026] For example, CN202311554027.0 discloses the use of crocodile mouth flower extract in the preparation of drugs for preventing or treating ulcerative colitis. The crocodile mouth flower extract inhibits the expression of inflammatory pathway proteins such as IKKβ / IKKα / NF-κB, JAK2 / STAT3, and plays a therapeutic role in ulcerative colitis. Hesperidin also works through this pathway, so hesperidin also has the potential to treat colitis.

[0027] For example, CN202210436187.4 discloses the use of Tripterygium wilfordii root extract in the preparation of drugs for treating rheumatoid arthritis. Tripterygium wilfordii root extract regulates IL-6 / STAT3 / IL-17 axis, regulates Th17 differentiation and Th17 / Treg balance, and inhibits inflammatory response, which is a new way to treat RA. Hesperidin also works through this pathway, so hesperidin also has the potential to treat rheumatoid arthritis. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0029] Figure 1 It is the chemical structural formula of Tangeretin (Tan).

[0030] Figure 2 The effects of tangeretin and LPS on the viability of RAW264.7 cells; A: The effects of tangeretin on the viability of RAW264.7 cells; B: The effects of tangeretin and LPS on the viability of RAW264.7 cells. Compared with the Control group, * P<0.05, ** P<0.01, *** P<0.001.

[0031] Figure 3The effect of tangeretin on NO production in RAW264.7 cells induced by LPS; A: The effect of tangeretin on NO production in RAW264.7 cells induced by LPS (24h), B: The effect of tangeretin on NO production in RAW264.7 cells induced by LPS (48h). Compared with the Control group, ## P<0.001 compared with LPS group * P<0.05, ** P<0.01, *** P<0.001.

[0032] Figure 4 A: The effect of tangeretin on the secretion level of TNF-α in RAW264.7 cells induced by LPS; B: The effect of tangeretin on the secretion level of IL-6 in RAW264.7 cells induced by LPS. Compared with the Control group, ## P<0.001, compared with LPS group * P<0.05, ** P<0.01, *** P<0.001.

[0033] Figure 5 A: The effect of tangeretin on the expression of IL-23mRNA in RAW264.7 cells induced by LPS; B: The effect of tangeretin on the expression of IL-17A mRNA in RAW264.7 cells induced by LPS; C: The effect of tangeretin on the expression of TNF-α mRNA in RAW264.7 cells induced by LPS; D: The effect of tangeretin on the expression of IL-6 mRNA in RAW264.7 cells induced by LPS. Compared with the Control group, ## P<0.001, compared with LPS group * P<0.05, ** P<0.01, *** P<0.001.

[0034] Figure 6 A: Western blotting analysis to detect the expression levels of STAT3, p-STAT3 (Tyr 705) and p-STAT3 (Ser 727); B, C, D: semi-quantitative analysis of STAT3, p-STAT3 (Ser 727) and p-STAT3 (Tyr 705). Compared with the Control group, ## P<0.001, compared with LPS group * P<0.05, ** P<0.01, *** P<0.001.

[0035] Figure 7Representative photos of the back skin of mice in the preventive and therapeutic administration groups of tangeretin.

[0036] Figure 8 These are the results of H&E staining of skin sections of the prophylactic and therapeutic administration groups of tangeretin, scale bar = 100 μm, magnification 100×.

[0037] Fig. 9 The expression level of p-STAT3 in the back skin of mice in the preventive and therapeutic administration groups of tangeretin, scale bar = 200 μm, magnification 200×.

[0038] Fig.10 The expression level of IL-17A in the back skin of mice in the preventive and therapeutic administration groups of tangeretin, scale bar = 200 μm, magnification 200×.

[0039] Fig.11 The expression levels of PCNA in the back skin of mice in the preventive and therapeutic administration groups of tangeretin, scale bar = 200 μm, magnification 200×.

[0040] Fig.12 It is the technical roadmap of the present invention. DETAILED DESCRIPTION

[0041] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0042] The present invention uses LPS to stimulate RAW264.7 cells to establish a cell inflammation model and an imiquimod-induced psoriasis mouse model, and systematically explores the anti-inflammatory activity and mechanism of action of Tangeretin (Tan). Fig.12 As shown. The cell inflammation model was established by stimulating RAW264.7 cells with LPS and the psoriasis mouse model was established by imiquimod to study the anti-inflammatory activity and mechanism of action of tangeretin.

[0043] RAW264.7 cell culture: Mouse mononuclear macrophages (RAW264.7, ATCC) were cultured in complete medium containing 10% fetal bovine serum (FBS), 1% double antibiotics (penicillin and streptomycin) and 89% DMEM at 37°C and 5% CO2. This experiment used 3-8 generations of RAW264.7 cells for related experiments.

[0044] LPS (100 ng / mL) was added for stimulation, with or without hesperidin, and the cell supernatant was collected to detect the contents of NO, IL-23, IL-17A, TNF-α and IL-6 in the cell supernatant. The cell lysate was collected and Western blot was used to detect protein (STAT3, p-STAT3 (Ser727), p-STAT3 (Tyr705)) expression. Total cell RNA was extracted and real-time fluorescence quantitative polymerase chain reaction was used to detect the expression levels of IL-23, IL-17A, TNF-α, and IL-6 mRNA.

[0045] Statistical methods: The experimental data were analyzed using Excel 2021 and GraphPadPrism 9.5 software, and the experimental results were expressed as Mean ± SD values. For comparisons between multiple groups, student's T test (two-tailed) was used for statistical analysis, P < 0.05 was considered to be statistically significant, P < 0.01 was considered to be a significant difference, and P < 0.001 was considered to be a very significant difference.

[0046] Experimental drugs and reagents: Tangeretin (Tangeretin, Tan, purity ≥ 98.0%, chemical structure see Figure 1 ) was purchased from Shanghai Yuanye Biotechnology Co., Ltd. and dissolved in DMSO (Mei Lun, cell culture grade) to prepare a 100 mM stock solution.

[0047] Lipopolysaccharide (LPS) and clobetasol propionate (Clo) were purchased from Shanghai Aladdin Company and dissolved in DMSO to prepare 1 mg / mL and 50 mM stock solutions, respectively. Imiquimod cream (Mingxinlidi), clobetasol propionate cream (Clo) (Fuyuan Pharmaceutical). Cell proliferation and toxicity detection (CCK-8) kit and BCA quantitative kit (Meilun), NO detection kit (Biyuntian), ELISA (TNF-α and IL-6) kit (Lianke Biotechnology), RNA extraction kit (Jianshi Biotechnology), reverse transcription kit and real-time fluorescence quantitative PCR kit (abm), protein lysate (Solebo), GAPDH and STAT3 antibodies (Sanying), p-STAT3, IL-17A, p-STAT3 (Ser727) and p-STAT3 (Tyr 705) antibodies (CST).

[0048] Experimental instruments: cell culture incubator (Thermo), centrifuge (Eppendorf), analytical balance (Sartoroius Stedim Biotech), microplate reader (BioTek), gene amplifier (Dongsheng International Trading Company), real-time fluorescence quantitative PCR instrument (Thermo), vertical electrophoresis instrument and protein transfer system (BioRad), gel imaging system (Senxi Saizhi Technology Co., Ltd.)

[0049] Example 1 Detection of the cytotoxic effects of tangeretin and LPS on RAW264.7 cells by CCK-8 method

[0050] RAW264.7 cells in the logarithmic growth phase were inoculated into a 96-well plate and the cell density was adjusted to 5 × 10 3 / well, cultured for 24 h until the cells stably adhered to the wall, removed the original complete culture medium, added different concentrations (5, 10, 20, 40, 80, 160, 320 μM) of tangeretin, with or without LPS (100 ng / mL) for 24 h, added 10 μL of CCK-8 solution to each well, and continued to incubate at 37 °C for 1 h. The OD value of each sample at 450 nm was detected by a microplate reader, and the cell viability was calculated.

[0051] (Cell viability (%) = [A (drug addition) - A (blank)] / [A (0 drug addition) - A (blank)] × 100%, Note: A (drug addition): OD value of wells with cells, CCK-8 solution and drug solution, A (0 drug addition): OD value of wells with cells, CCK-8 solution but no drug solution, A (blank): OD value of wells without cells.)

[0052] Results: Effects of tangeretin and LPS on the viability of RAW264.7 cells

[0053] CCK-8 results showed ( Figure 2 There was no significant difference in the effects of AB and LPS (100ng / mL) on the viability of RAW264.7 cells (P>0.05). Hesperidin did not produce significant toxicity to RAW264.7 cells at a concentration of 0-40μM for 24h (P>0.05), but the viability of RAW264.7 cells decreased when the concentration of hesperidin was higher than 40μM (P<0.05). Since the subsequent experiments were carried out to study the anti-inflammatory activity and anti-IL-17A effect of hesperidin in the RAW264.7 cell inflammation model, it was necessary to use a non-cytotoxic drug concentration to treat cells, so we determined that the concentration of hesperidin in the subsequent experiments was 0-40μM and the concentration of LPS in the subsequent experiments was 100ng / mL.

[0054] Example 2 Griess method to detect the effect of tangeretin on LPS-induced NO production in RAW264.7 cells

[0055] RAW264.7 cells in the logarithmic growth phase were inoculated into a 48-well plate and the cell density was adjusted to 1.0 × 10 5After culturing for 24 hours, the cells were stably attached to the wall. The original culture medium was removed and different concentrations (5, 10, 20, 40 μM) of tangeretin were added for pretreatment for 1 hour. LPS (100 ng / mL) was added and incubated for another 24 hours. The cell supernatant was collected. The OD540nm value of the cell supernatant was detected according to the instructions of the Griess kit, and the NO content in the cell supernatant was calculated.

[0056] Results: Effects of tangeretin on NO production in RAW264.7 cells induced by LPS

[0057] Griess results show that ( Figure 3 AB), compared with the Control group, the NO content in the cell supernatant of the LPS group was significantly increased (P < 0.001), indicating that LPS stimulated RAW264.7 cells to successfully establish a cell inflammation model. The NO content in the cell supernatant of the 24h treatment group of tangeretin was significantly reduced (P < 0.001) and was dose-dependent. The inhibitory effect of tangeretin on NO at a concentration of 40 μM was equivalent to that of the positive control clobetasol propionate (Clo). In the 48h treatment group of tangeretin, the NO content in the cell supernatant of each group increased overall, but tangeretin still showed a strong inhibitory effect on the production of NO by RAW264.7 cells. It was preliminarily judged that tangeretin had certain anti-inflammatory activity.

[0058] Example 3 ELISA to detect the effect of tangeretin on the secretion level of pro-inflammatory factors in RAW264.7 cells induced by LPS

[0059] RAW264.7 cells in the logarithmic growth phase were inoculated into 6-well plates and the cell density was adjusted to 5.0×10 5 After culturing for 24 hours, the cells were stably attached to the wall. The original culture medium was removed and different concentrations (5, 10, 20, 40 μM) of tangeretin were added for pretreatment for 1 hour. LPS (100 ng / mL) was added and incubated for another 24 hours. The cell supernatant was collected. The OD value of the cell supernatant at 450 nm was detected according to the instructions of the TNF-α and IL-6 ELISA kits, and the content of pro-inflammatory factors (TNF-α and IL-6) in the cell supernatant was calculated.

[0060] Results: The ELISA results showed that the effect of tangeretin on the secretion level of pro-inflammatory factors in RAW264.7 cells induced by LPS Figure 4AB), compared with the Control group, the levels of proinflammatory factors (TNF-α and IL-6) in the supernatant of the LPS group were significantly increased (P<0.001), and after treatment with tangeretin, the levels of proinflammatory factors (TNF-α and IL-6) in the supernatant of the cells were significantly decreased (P<0.001), and showed a dose-dependent manner. When the concentration of tangeretin was 40μM, the inhibitory effect on the secretion of proinflammatory factors (TNF-α and IL-6) was equivalent to that of the positive control clobetasol propionate (Clo), indicating that tangeretin can inhibit the production of proinflammatory factors in RAW264.7 cells in a dose-dependent manner, thereby reducing the inflammatory response.

[0061] Example 4 Real-time fluorescence quantitative polymerase chain reaction to detect the effect of tangeretin on LPS-induced IL-23, IL-17A, TNF-α and IL-6 mRNA expression in RAW264.7 cells

[0062] RAW264.7 cells were seeded into 6-well plates and the cell density was adjusted to 5.0×10 6 The cells were cultured for 24 h until they stably attached to the wall. The original culture medium was removed and different concentrations (5, 10, 20, 40 μM) of tangeretin were added for pretreatment for 1 h. LPS (100 ng / mL) was added for further incubation for 24 h. The commercial TRlcom lysis buffer provided by Jianshi Biotechnology was added to extract total RNA from the cells using the centrifugal column method. The RNA was immediately reverse transcribed into c-DNA. The fluorescence signal value of each sample was detected according to the instructions of the real-time fluorescence quantitative PCR kit and specific primers (Table 1) (cycle conditions: enzyme activation at 95°C, 3 min, 1 cycle; denaturation at 95°C, 15 s, annealing / extension at 60°C, 1 min, 40 cycles). The mRNA expression level of each group was normalized with the GAPDH expression level, and 2 -ΔΔCt The relative expression of mRNA was calculated by the method.

[0063] Table 1 Real-time fluorescence quantitative polymerase chain reaction system

[0064]

[0065]

[0066] Table 2 Real-time fluorescence quantitative PCR primer sequences

[0067]

[0068] As shown in SEQ ID NOs: 1-10.

[0069] Results: Effects of tangeretin on the mRNA expressions of IL-23, IL-17A, TNF-α and IL-6 in RAW264.7 cells induced by LPS

[0070] Real-time fluorescence quantitative polymerase chain reaction results showed that ( Figure 5 AD), compared with the Control group, the relative expression levels of IL-23, IL-17A, TNF-α and IL-6 mRNA were significantly increased after LPS stimulation (P < 0.001), and the relative expression levels of IL-23, IL-17A, TNF-α and IL-6 mRNA in the experimental cells were significantly decreased after treatment with tangeretin (P < 0.001), and were dose-dependent. When the concentration of tangeretin was 40 μM, the inhibitory effect on the mRNA of proinflammatory factors (IL-23, IL-17A, TNF-α and IL-6) was equivalent to that of the positive control clobetasol propionate (Clo). Experimental results ( Figure 5 B) showed that tangeretin exhibited a strong inhibitory effect on IL-17A, and tangeretin inhibited the inflammatory response by regulating the IL-23 / IL-17A inflammatory axis.

[0071] Example 5 Effect of tangeretin on LPS-induced STAT3 protein phosphorylation level in RAW264.7 cells

[0072] RAW264.7 cells in the logarithmic growth phase were inoculated in a 100 mm culture dish and the cell density was adjusted to 1.6 × 10 6 Each dish was cultured for 24 hours until the cells stably adhered to the wall. After pretreatment with different concentrations (5, 10, 20, 40 μM) of tangeretin for 2 hours, LPS (100 ng / mL) was added for incubation for 24 hours, the original culture medium was removed, the cells were washed twice with pre-cooled PBS buffer, and then protein lysis buffer (300 μL per dish) was added for lysis on ice for 30 minutes. The cells were centrifuged at 4°C, 12000 rpm for 5 minutes, and the supernatant was collected and the protein concentration was determined by BCA method. 60 μg of total protein sample was loaded, separated by 10% SDS-PAGE gel, and then electrophoretically transferred to PVDF membrane, blocked with 5% skim milk at room temperature for 1 hour, 3 mL of GAPDH, STAT3, p-STAT3 (Ser727), and p-STAT3 (Tyr705) antibodies were added, and the cells were incubated overnight at 4°C. 5 mL of horseradish peroxidase-labeled secondary antibodies were added and incubated for 1 hour at room temperature. ECL method was used for color development, and protein band quantification was analyzed by Image J software.

[0073] Results Effects of tangeretin on LPS-induced STAT3 phosphorylation in RAW264.7 cells

[0074] Western blot results showed that ( Figure 6AD), and there was no significant change in STAT3 protein expression in each treatment group (P>0.05). In terms of STAT3 protein phosphorylation expression, compared with the Control group, the expression of STAT3 phosphorylation (Tyr 705 and Ser 727) at two sites in the LPS group was significantly increased (P<0.001); compared with the LPS group, the tangeretin treatment group inhibited the expression of p-STAT3 (Tyr 705) and p-STAT3 (Ser 727) proteins in a dose-dependent manner (P<0.001), especially on the expression of p-STAT3 (Ser 727), tangeretin showed a more significant inhibitory effect than p-STAT3 (Tyr 705). The inhibitory effect of tangeretin on the phosphorylation level of STAT3 protein at a concentration of 40μM was comparable to that of the positive control clobetasol propionate (Clo), which can effectively inhibit the phosphorylation of STAT3 protein, and also further indicated that tangeretin alleviated the inflammatory response by regulating the IL-23 / STAT3 / IL-17A signaling pathway.

[0075] Example 6 Effect of cisplatin on imiquimod (IMQ)-induced back lesions in psoriasis mice Balb / c mice (female, 6-8 weeks, 20±2g), certificate number [SCXK(Xiang)2021-0002] were purchased from Slake Jingda Company (Changsha, China). All mice were kept in an SPF animal room with a 12h light / 12h dark cycle, and the experiment was started after one week of adaptive feeding of the mice. 96 mice were randomly divided into 12 groups (8 / group): Control group, Control solvent (white vaseline) group, IMQ group, IMQ solvent (white vaseline) group, preventive administration of positive drug clobetasol propionate (Clo) group (IMQ+Clo 55mg), therapeutic administration of positive drug group (IMQ+Clo(A)55mg), preventive administration of Tan low-dose group (IMQ+Tan40μg / cm 2 ), medium dose group (IMQ+Tan160μg / cm 2 ), high-dose group (IMQ+Tan640μg / cm 2 ), the low-dose Tan group (IMQ+Tan(A) 40μg / cm 2 ), medium dose group (IMQ+Tan(A)160μg / cm 2 ), high-dose group (IMQ+Tan(A)640μg / cm 2 After the mice were anesthetized, a 2cm×3cm area on the back was selected for hair removal. Modeling began 48 hours after hair removal. All modeling mice were externally applied with 65mg (55mg on the back and 5mg on both ears) of imiquimod cream every morning for 10 days. 2 ), medium dose group (160 μg / cm2 ) and high-dose group (640 μg / cm 2 ) was topically administered to the back and ears every afternoon from the first day of modeling for 10 days. 2 ), medium dose group (160 μg / cm 2 ) and high-dose group (640 μg / cm 2 ) The mice in the positive drug group were given local administration to the back and ears every afternoon from the third day of modeling for 10 days. The mice in the positive drug group were given clobetasol propionate (55 mg / mouse, 45 mg on the back and 5 mg on each ear) for prevention from the first day of modeling, and the mice in the positive drug group were given clobetasol propionate (55 mg / mouse, 45 mg on the back and 5 mg on each ear) for treatment from the third day of modeling.

[0076] Results: Hesperidin significantly inhibited the back lesions of psoriasis mice

[0077] like Figure 7 As shown, the IMQ group mice had significant erythema and scales on the back compared with the Control group mice. Compared with the IMQ group, the preventive and therapeutic groups of tangeretin had a significant effect on improving the pathological symptoms of psoriasis, and the erythema and scales were significantly reduced in a dose-dependent manner. The improvement effect of the preventive and therapeutic high-dose groups of tangeretin on the back skin lesions of psoriasis mice was comparable to that of the positive control Clo. Among them, the scales and erythema on the back skin of the preventive group mice almost disappeared, and the skin condition was similar to that of the Control group. After treatment with the therapeutic group of tangeretin, the pathological symptoms of the skin of psoriasis mice were greatly improved, with only a very small amount of back scales remaining. The above research results all show that tangeretin has the effect of reducing skin thickness and the severity of scales ( Figure 7 ).

[0078] Example 7 Histological evaluation of cisperetin on imiquimod (IMQ)-induced psoriasis back lesions in mice

[0079] The back skin tissue of mice was fixed in 4% paraformaldehyde solution for 30 days and then stained with H&E. The detailed operation steps are as follows: the fixed tissue was placed in a concentration gradient ethanol solution for dehydration, and then transparentized in xylene for 30 minutes each time, and the sample was embedded in paraffin to make 3-5μm continuous sections. After dewaxing and rehydration, the sections were stained with hematoxylin-eosin reagent to identify the skin tissue structure and cell nuclei, and the sections were dried and sealed with neutral resin. The images were collected by an optical microscope (DM500, Leica, Germany), and the magnification was 100 times during observation.

[0080] Results Hesperidin significantly improved the thickening and incomplete differentiation of keratinocytes and inflammatory cell infiltration in the back skin of psoriasis mice

[0081] Compared with the Control group, the IMQ group had obvious thickening of epidermal tissue, accompanied by incomplete keratinization, and a large number of inflammatory cell infiltrations. Compared with the preventive Tan group, the IMQ group showed reduced epidermal tissue thickness, reduced excessive keratinization, and a small amount of inflammatory cell infiltration in the back skin of mice in the preventive Tan group, and the activity was obviously dose-dependent. Compared with the therapeutic Tan group, the IMQ group also showed strong pharmacological activity in the therapeutic Tan group, especially in the high-dose group, which showed reduced epidermal tissue thickness, reduced excessive keratinization, and a small amount of inflammatory cell infiltration, which was also significantly dose-dependent. In short, the H&E staining results of the mouse skin lesions showed that tangeretin showed strong anti-psoriatic activity regardless of preventive or therapeutic administration, and there was a significant dose-dependency ( Figure 8 ).

[0082] Example 8 Effect of tangeretin on the expression levels of p-STAT3, IL-17A and PCNA in the back skin of psoriasis mice induced by imiquimod (IMQ)

[0083] Mouse back skin samples were collected and made into paraffin sections, dewaxed with xylene, and incubated in H2O2 solution at room temperature for 10 min to inhibit the activity of endogenous peroxidase. BSA was blocked at room temperature for 1 h to remove non-specific background adsorption, p-STAT3, IL-17A and PCNA antibodies were added and incubated overnight at 4°C, and horseradish peroxidase-labeled secondary antibodies were added and incubated at room temperature for 30 min. DAB was added for color development, and hematoxylin was used for counterstaining. A negative control was set up, and photos were taken with unified parameters. The magnification was 200 times during observation.

[0084] Results Hesperidin significantly inhibited the expression levels of p-STAT3, IL-17A and PCNA in the back skin of psoriasis mice

[0085] Compared with the Control group, the expression levels of p-STAT3, IL-17A and PCNA in the IMQ group were significantly increased ( Fig. 9 , Fig.10 and Fig.11 ), the expression levels of p-STAT3, IL-17A and PCNA in the back skin of mice in the preventive and therapeutic administration groups of tangeretin were significantly reduced in a dose-dependent manner, indicating that tangeretin can significantly inhibit the production of p-STAT3, IL-17A and PCNA, thereby reducing the inflammatory response and abnormal proliferation and incomplete differentiation of keratinocytes, exerting anti-psoriatic activity, and is an excellent anti-IL-17A inhibitor.

[0086] In summary, this study showed that tangeretin has significant anti-inflammatory activity, which may reduce the inflammatory response by inhibiting the production of proinflammatory factors (TNF-α and IL-6) and regulating the IL-23 / STAT3 / IL-17A signaling pathway. Tangeretin, as a natural medicine, can inhibit the production of IL-17A more safely and stably, and is a good IL-17A inhibitor.

[0087] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Use of an IL-17A inhibitor in the preparation of a drug for treating inflammatory diseases, characterized in that: The IL-17A inhibitor is hesperidin.

2. The use according to claim 1, characterized in that: The medicament also includes medically acceptable excipients.

3. The use according to claim 2, characterized in that: The medicine is in the form of tablets, liquids, ointments, gel preparations, emulsions, capsules, suppositories or granules.

4. The use according to claim 1, characterized in that: The drug for treating inflammatory diseases is a drug for treating psoriasis, a drug for ankylosing spondylitis, a drug for treating multiple sclerosis, a drug for treating colitis, a drug for treating asthma, a drug for treating scleroderma or a drug for treating rheumatoid arthritis.

5. The use according to claim 1, characterized in that: The concentration of the IL-17A inhibitor in the drug for treating inflammatory diseases is 5 to 40 μM.

6. The use according to claim 1, characterized in that: The inflammation-related factors of the inflammatory disease include IL-23, IL-17A, TNF-α and IL-6.

7. The use according to claim 1, characterized in that: The inflammatory disease is an inflammatory disease induced by lipopolysaccharide.

8. The use according to claim 4, characterized in that: When the drug for treating inflammatory diseases is a drug for treating psoriasis, hesperidin has the effect of reducing skin thickness and the severity of scales.

9. The use according to claim 1, characterized in that: The tangeretin reduces the inflammatory response by inhibiting the production of pro-inflammatory factors and regulating the IL-23 / STAT3 / IL-17A signaling pathway.

10. The use according to claim 1, characterized in that: The drug for treating inflammatory diseases is a drug for inhibiting the content of cellular inflammatory factor NO.

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