Use of IL-17A inhibitors in the manufacture of therapeutic drugs for inflammatory diseases

Abstract

This invention provides the use of IL-17A inhibitors in the manufacture of therapeutic drugs for inflammatory diseases. [Solution] This invention demonstrates that tangeretin has remarkable anti-inflammatory activity in vivo and in vitro, inhibiting the production of pro-inflammatory factors (TNF-α and IL-6) and mitigating inflammatory responses by regulating the IL-23 / STAT3 / IL-17A signaling pathway. Furthermore, as a natural drug, tangeretin can inhibit IL-17A production more safely and stably, making it an excellent IL-17A inhibitor.

Description

[Technical Field]

[0001] This invention relates to the biomedical technology field, and more particularly to the use of IL-17A inhibitors in the manufacture of therapeutic drugs for inflammatory diseases. [Background technology]

[0002] The interleukin-17 (IL-17) family is a subgroup of cytokines, comprising IL-17(AF), and is produced by various cells, including macrophages, Th17 cells, neutrophils, and NK cells. Since the discovery of IL-17A (also known as IL-17 or CTLA8) in 1993, five other 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. Currently, IL-17A, the most extensively studied cytokine in its family, is a pro-inflammatory cytokine that plays a crucial role in host defense against microbial infections and is closely associated with various inflammatory and autoimmune diseases such as ankylosing spondylitis, multiple sclerosis, colitis, asthma, psoriasis, scleroderma, and rheumatoid arthritis. On the one hand, IL-17A induces the expression of cytokines that promote the repair of related tissues, thereby accelerating the body's recovery. On the other hand, IL-17A is highly expressed in a range of inflammatory and autoimmune diseases and simultaneously induces the massive accumulation of various inflammatory factors, potentially leading to an excessive immune response. Therefore, excessively high levels of IL-17A may exacerbate the pathological progression of related diseases. When IL-23 release increases during the onset of various autoimmune and inflammatory diseases, immune cells such as Th17 cells and macrophages (which are abundant in various autoimmune and inflammatory diseases) are further stimulated, activating the JAK-STAT3 signaling pathway. After STAT3 phosphorylation, these cells are stimulated to release IL-17A, which in turn promotes the massive secretion of pro-inflammatory factors (TNF-α, IL-6) and pro-inflammatory mediators (such as NO), thereby inducing a localized inflammatory response and accelerating the onset and progression of a series of inflammatory or autoimmune diseases.

[0003] Currently, research into IL-17A inhibitors has made significant progress. These drugs, including brodalumab, bimekizumab, CJM112, izokibep, and secukinumab, inhibit IL-17A or its receptors through different mechanisms, reducing inflammatory responses and demonstrating good therapeutic efficacy in diseases closely associated with IL-17A, such as ankylosing spondylitis, rheumatoid arthritis, and psoriasis. At the same time, IL-17A inhibitors show better therapeutic efficacy and lower immunogenicity than conventional TNF-α inhibitors in clinical practice. While IL-17A inhibitors, as biological agents, have remarkable therapeutic effects as drugs for treating a range of autoimmune or inflammatory diseases such as ankylosing spondylitis, psoriasis, and rheumatoid arthritis, they are often accompanied by a range of side effects, including gastrointestinal reactions, skin reactions, and respiratory infections. Therefore, the search for and screening of more efficient, safe, and stable anti-IL-17A drugs is crucial for the treatment of a range of autoimmune and inflammatory diseases, and currently, natural drugs with fewer toxic side effects and good biological activity are offering new avenues for the development of anti-IL-17A drugs.

[0004] Chenpi (Citri Reticulatae Pericarpium) is the mature peel of the citrus tree Citrus reticulate Blanco and its cultivated varieties. Chenpi contains many chemical components, mainly flavonoids, as well as limonoids, alkaloids, and volatile oils. Existing pharmacological reports on Chenpi and its compounds mainly mention its digestive system functions, such as regulating the movement of smooth muscles in the gastrointestinal tract, promoting the secretion of digestive juices and enzymes, and protecting the liver. As for its anti-respiratory effects, the volatile oils of Chenpi have anti-asthmatic and antitussive effects, as well as effects such as strengthening the heart, vasodilation and blood pressure reduction, anti-inflammatory, antioxidant, antiviral, antitumor, blood lipid reduction, antibacterial, and immune system enhancement. Among these, the active ingredients of dried tangerine peel have shown great potential for anti-inflammatory effects. Related research by Li Xue et al. showed that hesperidin can inhibit abnormal proliferation, incomplete differentiation, and local inflammatory responses of keratinocytes via the IRS-1 / ERK1 / 2 signaling pathway. Research by Yang GuLiang et al. showed that nobiletin can significantly inhibit the differentiation of CD4+ T cells and the expression of transcription factors Ki-67 and PCNA. However, the anti-IL-17A effect of tangeretin (Tan), an active ingredient of dried tangerine peel, has not been studied.

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

[0006] Macrophages are crucial immune cells deeply involved in the initiation of inflammatory responses. They participate in the processes of various inflammatory and autoimmune diseases, playing a vital role in the immune response through phagocytosis, antigen presentation, and cytokine secretion. Lipopolysaccharides (LPS) are one of the main components of the cell wall of Gram-negative bacteria and consist of three parts: lipid A, core polysaccharide, and O-antigen. LPS can activate various immune cells, including macrophages, T lymphocytes, B lymphocytes, and natural killer cells. After LPS treatment, macrophage activation transitions to an inflamed state, with a significant increase in inflammatory target mediators (such as NO) and pro-inflammatory cytokines (TNF-α and IL-6). These substances can promote the inflammatory process. The LPS-induced RAW264.7 cell inflammation model is an in vitro cell model widely used in the study of various diseases, including, but not limited to, autoimmune diseases such as psoriasis, rheumatoid arthritis, and systemic lupus erythematosus, in which macrophage activation and inflammatory factor production in these diseases are closely related to the onset and progression of the disease; infectious diseases such as inflammatory responses caused by bacterial or viral infections, in which 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, in which the role of inflammation in the neurodegenerative process; and tumors, in which the RAW264.7 cell model is also used to study the polarization state of macrophages in the tumor microenvironment and how it affects tumor growth and metastasis. Therefore, in the study of inflammatory infiltration or immunoinflammation, the LPS-induced RAW264.7 cell inflammation model is a typical model for screening novel drugs for the treatment of inflammatory diseases. Thus, a method for screening drugs for the treatment of a wider variety of inflammatory diseases using the RAW264.7 cell inflammation model and enriching the variety of conventional drugs for the treatment of inflammatory diseases is an urgent problem that those skilled in the art need to solve.

[0007] Psoriasis is an immune-mediated chronic inflammatory disease characterized by a synergistic inflammatory response involving various immune cells (macrophages, T cells, neutrophils, etc.) and abnormal proliferation and incomplete differentiation of keratinocytes. IL-17A plays a central role in the pathogenesis of psoriasis, not only influencing keratinocyte proliferative activity and cellular function, but also exhibiting important regulatory effects on immune cells and associated cytokines in the immunopathological environment of psoriasis. By binding to the IL-17 receptor, IL-17A promotes keratinocyte activation and proliferation, stimulating the production of other pro-inflammatory cytokines and chemokines, and triggering a positive feedback loop of inflammation within psoriatic skin lesions. Therefore, biological agents targeting IL-17A have shown remarkable efficacy in the treatment of psoriasis, effectively inhibiting the development of skin lesions and improving 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 internationally, demonstrating remarkable therapeutic effects in clinical applications. Furthermore, research has revealed that autoactivation of the IL-17A signaling pathway can sustain inflammation and accelerate disease progression, providing a new direction for research in the treatment of psoriasis. Therapeutic strategies targeting IL-17A are highly anticipated in the field of psoriasis treatment, and are expected to provide patients with more efficient and safer treatment options. Since IL-17A plays a crucial role in the pathogenesis of psoriasis, this study first clarified the anti-IL-17A effect of tangeretin in vitro, then conducted in vivo animal experiments to establish a psoriasis mouse model using imiquimod (IMQ), and further clarified the anti-IL-17A activity of tangeretin in vivo. After confirming the anti-psoriatic effect of tangeretin in IMQ psoriasis mice, the researchers of this invention applied a tangeretin ointment formulation to psoriasis patients for clinical use and further clarified its clinical effects.

[0008] Angiogenesis refers to the process by which new blood vessels are formed from existing blood vessels. This process occurs under various physiological and pathological conditions, including normal growth, wound healing, the menstrual cycle, and tumor growth. Angiogenesis is a complex biological process involving the interaction of various growth factors and signaling pathways, among which vascular endothelial growth factor (VEGF) is one of the most well-known angiogenic factors. Angiogenesis is a pathological feature of psoriasis. The clinical pathogenesis of psoriasis mainly involves two categories: excessive proliferation and incomplete differentiation of keratinocytes and infiltration of inflammatory cells. Excessive proliferation of keratinocytes inevitably leads to increased angiogenesis. Therefore, angiogenesis is merely a clear pathological manifestation of psoriasis, and there is no direct relationship between angiogenesis and the pathogenesis of psoriasis.

[0009] Patent Document 1 - The use of flavonoid compounds contained in dried tangerine peel in the manufacture of drugs that inhibit angiogenesis: Using the XTT method to detect endothelial cell proliferation and the cell cycle of endothelial cells, and observing morphological experiments of internodal and dorsal longitudinal vessels of zebrafish with a fluorescence confocal microscope, it was discovered that nobiletin, tangeretin, and sweet orange flavonoids, which are active ingredients contained in dried tangerine peel, inhibit endothelial cell proliferation and thus inhibit angiogenesis by regulating the cell cycle of vascular endothelial cells. Therefore, the patent applicant reasoned that flavonoid compounds contained in dried tangerine peel can be used in the manufacture of drugs for the treatment of diseases associated with excessive angiogenesis. Therefore, the patent applicant conducted only relevant angiogenesis experiments and found that tangeretin can significantly inhibit angiogenesis and cell proliferation, and thus reasoned that it may have some antipsoriasis activity. This does not negate the experimental results or speculations of the patent applicant, nor does it deny that there is a certain correlation between angiogenesis and psoriasis. There may be differences due to different research fields and starting points. However, tracing back the source, the patent applicant linked angiogenesis and psoriasis because they understood and speculated that one of the mechanisms of psoriasis development is the abnormal proliferation and incomplete differentiation of keratinocytes, which leads to the pathological manifestation of angiogenesis. However, there is no experimental data from psoriasis-related cell models or animal models to support this. Therefore, the "use of flavonoid compounds contained in dried tangerine peel in the manufacture of drugs for the treatment of psoriasis" disclosed in the patent application is essentially impossible to implement and inaccurate. [Prior art documents] [Patent Documents]

[0010] [Patent Document 1] CN101947215A [Overview of the project] [Problems that the invention aims to solve]

[0011] The present invention aims to provide the use of IL-17A inhibitors in the manufacture of therapeutic drugs for inflammatory diseases. The invention aims to develop novel anti-IL-17A drugs, clarify the anti-inflammatory activity and anti-IL-17A effects of tangeretin (Tan), thereby inhibiting inflammatory responses, improving disease symptoms, and providing new therapeutic means for a range of autoimmune or inflammatory diseases such as ankylosing spondylitis, multiple sclerosis, colitis, asthma, scleroderma, and rheumatoid arthritis. At the same time, because tangeretin is a natural drug, it is expected to have lower toxicity and side effects, thus resolving the range of side effects caused by conventional IL-17A inhibitors. In summary, the present invention aims to develop a novel anti-IL-17A drug, elucidate the anti-IL-17A effect of tangeretin, and improve the physical and mental health and quality of life of patients suffering from diseases closely related to IL-17A. [Means for solving the problem]

[0012] To achieve the objectives of the above invention, the present invention provides the following technical solutions.

[0013] The present invention provides the use of an IL-17A inhibitor in the manufacture of a drug for the treatment of inflammatory diseases, wherein the IL-17A inhibitor is tangeretin.

[0014] Preferably, the drug also includes medically acceptable excipients.

[0015] Preferably, the drug is in the form of a tablet, liquid, ointment, gel, emulsion, capsule, suppository, or granule.

[0016] Preferably, the drug for treating the inflammatory disease 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.

[0017] Preferably, the concentration of the IL-17A inhibitor in the drug for treating inflammatory diseases is 5 to 40 μM (of course, other effective use concentrations are also included).

[0018] Preferably, the inflammatory factors related to the inflammatory disease include IL-23, IL-17A, TNF-α, and IL-6.

[0019] Preferably, the inflammatory disease is an inflammatory disease caused by lipopolysaccharide.

[0020] Preferably, when the drug for treating the inflammatory disease is a drug for treating psoriasis, tanghin has the effect of reducing the skin thickness and the degree of scales.

[0021] Preferably, the tanghin inhibits the production of inflammatory inducers and reduces the inflammatory response by regulating the IL-23 / STAT3 / IL-17A signaling pathway.

[0022] Preferably, the drug for treating the inflammatory disease is a drug that inhibits the content of the cellular inflammatory factor NO.

Advantages of the Invention

[0023] Compared with the prior art, the present invention has the following beneficial effects. This study investigated the anti-IL-17A effects of tangeretin in an LPS-induced RAW264.7 cell inflammation model and an imiquimod-induced psoriasis mouse model. In the LPS-induced RAW264.7 cell inflammation model, tangeretin was found to exhibit excellent anti-inflammatory activity, effectively inhibiting the production of IL-23 and IL-17A. This suggests that tangeretin inhibits the inflammatory response by modulating the IL-23 / IL-17A inflammatory axis and can be used as a novel IL-17A inhibitor. It also offers a new approach to treating a range of autoimmune or inflammatory diseases, including psoriasis, ankylosing spondylitis, colitis, scleroderma, and rheumatoid arthritis. Considering the important role of the IL-23 / IL-17A inflammatory axis in a range of inflammatory and autoimmune diseases, this study tracked the upstream IL-17A secretion pathway JAK / STAT3 in cells to further elucidate the mechanism by which tangeretin inhibits IL-17A in macrophages. Western-blot data showed that in activated macrophages, the tangeretin-treated group significantly inhibited STAT3 phosphorylation, exhibiting a marked inhibitory effect on phosphorylation at two STAT3 phosphorylation sites (Tyr 705 and Ser 727), with even more pronounced inhibition of p-STAT3 (Ser 727). These experimental results suggest that tangeretin may inhibit IL-17A secretion by inhibiting IL-23-mediated STAT3 phosphorylation. Furthermore, tangeretin has been shown to exert anti-inflammatory activity by regulating the IL-23 / STAT3 / IL-17A signaling pathway and inhibiting IL-17A production.

[0024] In this invention, we discovered that tangeretin significantly improved dorsal skin lesions in psoriasis mice in an imiquimod-induced psoriasis mouse model, effectively reducing dorsal erythema and scaling. H&E staining results showed that after treatment with tangeretin, the thickness of the epidermal tissue of the dorsal skin of mice decreased significantly, excessive keratinization was reduced, and inflammatory cell infiltration was reduced. The anti-psoriasis activity was significantly dose-dependent. Immunohistochemical analysis showed that tangeretin significantly inhibited the expression levels of p-STAT3 and IL-17A in the dorsal skin of psoriasis mice, which is consistent with experimental results in an LPS-induced RAW264.7 cell validation model, demonstrating a more potent anti-IL-17A potential of tangeretin. Furthermore, tangeretin may exert its anti-psoriasis activity by modulating the IL-23 / STAT3 / IL-17 signaling pathway. Simultaneously, immunohistochemical results showed that tangeretin can significantly inhibit PCNA expression levels in mouse dorsal skin, suggesting that tangeretin may exert its antipsoriasis activity by inhibiting abnormal proliferation and incomplete differentiation of keratinocytes. When the present invention was used in clinical patients with psoriasis, the erythema of scaling in the patients was significantly reduced after use, and the scaling almost completely disappeared 10 days after the start of use. After using tangeretine ointment, the patients' overall PASI scores decreased significantly, with a very significant difference compared to non-medicated areas of the patient's body, indicating a very remarkable anti-psoriasis effect.

[0025] For example, CN201610107672.1 also discloses the use of tangeretine saponin IVa butyl ester as an inhibitor of the IL-6 / STAT3 signaling pathway, among which diseases associated with 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 correlation between the pathway of the present invention and the prior art, tangeretine should also have the potential to produce drugs for the treatment of the above diseases.

[0026] For example, CN202311554027.0 discloses the use of Clinacanthus nutans extract in the manufacture of drugs for the prevention or treatment of ulcerative colitis. Clinacanthus nutans extract inhibits the expression of inflammatory pathway proteins such as IKKβ / IKKα / NFκB and JAK2 / STAT3, thereby exerting a therapeutic effect on ulcerative colitis. Tangeretin also acts through this pathway and therefore has the potential to treat colitis.

[0027] For example, CN202210436187.4 discloses the use of Tetrastigma hemsleyanum Diels et Gilg root extract in the manufacture of therapeutic drugs for rheumatoid arthritis. Tetrastigma hemsleyanum Diels et Gilg root extract modulates the IL-6 / STAT3 / IL-17 axis, regulates Th17 differentiation and Th17 / Treg balance, and inhibits inflammatory responses, making it a novel method for treating RA. Tangeretin also acts through this pathway and therefore has potential to treat rheumatoid arthritis. [Brief explanation of the drawing]

[0028] To more clearly illustrate embodiments of the present invention or technical solutions in the prior art, the following drawings, which are necessary for describing embodiments or the prior art, are briefly introduced below. Clearly, the drawings in the following description are merely embodiments of the present invention, and those skilled in the art can obtain other drawings based on the provided drawings without any creative effort.

[0029] [Figure 1] This is the chemical structure of tangeretin (Tan). [Figure 2]The effects of tangeretin and LPS on RAW264.7 cell viability are shown. A shows the effect of tangeretin on RAW264.7 cell viability. B shows the effect of tangeretin and LPS on RAW264.7 cell viability. Compared to the control group, *P<0.05, **P<0.01, and ***P<0.001. [Figure 3] This shows the effect of tangeretin on NO production in LPS-induced RAW264.7 cells. A shows the effect of tangeretin on NO production in LPS-induced RAW264.7 cells (24 hours). B shows the effect of tangeretin on NO production in LPS-induced RAW264.7 cells (48 hours). Compared to the control group, ##P<0.001, and compared to the LPS group, *P<0.05, **P<0.01, and ***P<0.001. [Figure 4] A shows the effect of tangeretin on TNF-α secretion levels in LPS-induced RAW264.7 cells. B shows the effect of tangeretin on IL-6 secretion levels in LPS-induced RAW264.7 cells. Compared to the control group, ##P<0.001, and compared to the LPS group, *P<0.05, **P<0.01, and ***P<0.001. [Figure 5] A shows the effect of tangeretin on IL-23 mRNA expression in LPS-induced RAW264.7 cells. B shows the effect of tangeretin on IL-17A mRNA expression in LPS-induced RAW264.7 cells. C shows the effect of tangeretin on TNF-α mRNA expression in LPS-induced RAW264.7 cells. D shows the effect of tangeretin on IL-6 mRNA expression in LPS-induced RAW264.7 cells. Compared to the control group, ##P<0.001; compared to the LPS group, *P<0.05, **P<0.01, and ***P<0.001. [Figure 6]A is the detection of STAT3, p-STAT3(Tyr 705), and p-STAT3(Ser 727) expression levels by Western blot analysis. B, C, and D are semi-quantitative analyses of STAT3, p-STAT3(Tyr 705), and p-STAT3(Ser 727), respectively. Compared to the control group, ##P<0.001, and compared to the LPS group, *P<0.05, **P<0.01, and ***P<0.001. [Figure 7] These are representative photographs of the dorsal skin of mice in the prophylactic and therapeutic tangeretin administration groups. [Figure 8] These are the results of H&E staining of skin sections from the prophylactic and therapeutic tangeretin administration groups. The scale is Bar = 100 μm, and the magnification is 100 ×. [Figure 9] This shows the expression levels of p-STAT3 in the dorsal skin of mice in the prophylactic and therapeutic tangeretin administration groups. Scale: Bar = 200 μm, magnification: 200 ×. [Figure 10] This shows the expression levels of IL-17A in the dorsal skin of mice in the prophylactic and therapeutic tangeretin administration groups. Scale: Bar = 200 μm, magnification: 200 ×. [Figure 11] This shows the expression levels of PCNA in the dorsal skin of mice in the prophylactic and therapeutic tangeretin administration groups. Scale: Bar = 200 μm, magnification: 200 ×. [Figure 12] This is a comparison chart of patients with severe psoriasis before and after treatment with tangeretin ointment. Arm scaling was significantly reduced, and erythema subsided. 12a is before treatment, 12b-h are after treatment, and 12h is 14 days after treatment. [Figure 13] These are the PASI score results for psoriasis patients undergoing treatment. After treatment with tangeretine ointment, the PASI scores of psoriasis patients decreased significantly. Compared to day 0, **P<0.05, ***P<0.01, and ****P<0.001. [Figure 14] This is the technical roadmap for the present invention. [Modes for carrying out the invention]

[0030] The technical solutions provided by the present invention will be described in detail below with reference to examples, but these should not be understood as limiting the scope of protection of the present invention.

[0031] This invention systematically investigates the anti-inflammatory activity and mechanism of action of tangeretin (Tan) by stimulating RAW264.7 cells using LPS to establish a cellular inflammation model and an imiquimod-induced psoriasis mouse model. The technology roadmap is shown in Figure 14. A cellular inflammation model was established by stimulating RAW264.7 cells using LPS, and a psoriasis mouse model was established using imiquimod to systematically study the anti-inflammatory activity and mechanism of action of tangeretin. Clinical trials were conducted to evaluate the anti-psoriasis pharmacodynamics of tangeretin.

[0032] RAW264.7 cell culture: Mouse mononuclear macrophages (RAW264.7, ATCC) were cultured at 37°C and 5% CO2 in complete medium containing 10% fetal bovine serum (FBS), 1% biantibodies (penicillin and streptomycin), and 89% DMEM. In this experiment, RAW264.7 cells from 3 to 8 generations were used in related experiments.

[0033] Cells were stimulated with LPS (100 ng / mL) regardless of the presence or absence of tangeretin, and the cell supernatant was collected. The content of NO, IL-23, IL-17A, TNF-α, and IL-6 in the cell supernatant was detected. Cell lysates were collected, and the expression of proteins (STAT3, p-STAT3 (Ser727), p-STAT3 (Tyr705)) was detected by Western blotting. Total RNA was extracted from the cells, and the expression levels of IL-23, IL-17A, TNF-α, and IL-6 mRNA were detected by real-time fluorescence quantitative polymerase chain reaction.

[0034] Statistical Methods: Experimental data were analyzed using Excel 2021 and GraphPad Prism 9.5 software, and experimental results were expressed as Mean ± SD values. For comparisons between multiple groups, Student's T-test (two-tailed) was used for statistical analysis. A P<0.05 was considered statistically significant, a P<0.01 was considered significant, and a P<0.001 was considered very significant.

[0035] Experimental drugs and reagents: Tangeretin (Tan, purity ≥ 98.0%, chemical structure shown in Figure 1) was purchased from Shanghai Yuanye Biotechnology Co., Ltd. and dissolved in DMSO (Meilon, cell culture grade) to prepare a 100 mM stock solution. Clinical trial tangeretin ointment preparation (Preparation of tangeretin petrolatum ointment: 9.616 g of petrolatum was weighed into an evaporating dish, heated and melted in a water bath, 0.384 g of tangeretin powder was added while stirring, and after homogeneous stirring, it was cooled and solidified to obtain the ointment).

[0036] Lipopolysaccharide (LPS) and clobetasol propionate (Clo) were all purchased from Shanghai Aladdin and dissolved in DMSO to prepare stock solutions of 1 mg / mL and 50 mM, respectively. Imiquimod cream (Mingxin Lidi), clobetasol propionate cream (Clo) (Fuyuan Pharmaceutical). Cell proliferation and toxicity detection (CCK-8) kit and BCA quantification kit (Meilon), 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 (Ser 727), p-STAT3 (Tyr 705) antibodies (CST).

[0037] Laboratory equipment: Cell culture incubator (Thermo), centrifuge (Eppendorf), analytical balance (SartoroiusStedim Biotech), microplate reader (BioTek), gene amplifier (Dongsheng International Trading Company), real-time fluorescence quantitative PCR system (Thermo), vertical electrophoresis system and protein transfer system (BioRad), gel imaging system (Senxi Saizhi Technology Co., Ltd.)

[0038] Example 1: Detection of toxic effects of tangeretin and LPS on RAW264.7 cells using the CCK-8 method.

[0039] Logarithmically growing RAW264.7 cells were inoculated into a 96-well plate, and the cell density was increased to 5 × 10⁶. 3 Cells were adjusted to the number of cells per well and cultured for 24 hours until the cells were stably attached to the wall. Then the original complete medium was removed, and tangeretin at different concentrations (5, 10, 20, 40, 80, 160, 320 μM) was added to each well, with or without LPS (100 ng / mL), for 24 hours. After that, 10 μL of CCK-8 solution was added to each well, and the wells were incubated at 37°C for 1 hour. The OD value at 450 nm for each sample was detected using a microplate reader, and cell viability was calculated.

[0040] (Cell viability (%) = [A (drug added) - A (blank)] / [A (no drug added) - A (blank)] × 100%, Note: A (drug added): OD value of wells containing cells, CCK-8 solution, and drug solution; A (no drug added): OD value of wells containing cells and CCK-8 solution but no drug solution; A (blank): OD value of wells without cells.)

[0041] Results: Effects of tangeretin and LPS on RAW264.7 cell viability

[0042] The results of CCK-8 (Figures 2A-B) showed no statistically significant difference in the effect of LPS (100 ng / mL) on RAW264.7 cell viability (P>0.05). Tangeretin did not show significant toxicity to RAW264.7 cells when treated at concentrations of 0-40 μM for 24 hours (P>0.05), but RAW264.7 cell viability decreased when the tangeretin concentration exceeded 40 μM (P<0.05). Subsequent experiments were conducted to investigate the anti-inflammatory activity and anti-IL-17A effect of tangeretin in a RAW264.7 cell inflammation model. Therefore, it was necessary to use non-cytotoxic drug concentrations to treat the cells, and the subsequent experimental concentrations of tangeretin were set to 0-40 μM and LPS to 100 ng / mL.

[0043] Example 2: Effect of tangeretin on NO production in LPS-induced RAW264.7 cells detected by the Griess method. RAW264.7 cells in the logarithmic growth phase were inoculated into a 48-well plate, and the cell density was increased to 1.0 × 10⁶. 5 Cells were adjusted to the required number of cells per well and cultured for 24 hours until the cells were stably attached to the cell wall. The original medium was then removed, and tangeretin at different concentrations (5, 10, 20, 40 μM) was added for 1 hour of pretreatment. LPS (100 ng / mL) was then added, and incubation continued for 24 hours. The cell supernatant was then collected. The OD540nm value of the cell supernatant was detected according to the Griess kit instructions, and the NO content in the cell supernatant was calculated.

[0044] Results: Effect of tangeretin on NO production in LPS-induced RAW264.7 cells

[0045] Griess's results (Figures 3A-B) showed that the NO content in the cell supernatant of the LPS group was significantly increased compared to the control group (P<0.001). This indicated that LPS stimulated RAW264.7 cells and successfully established a cellular inflammation model. The NO content in the cell supernatant of the 24-hour tangeretin treatment group was significantly decreased (P<0.001), and this was dose-dependent. The inhibitory effect on NO at a tangeretin concentration of 40 μM was equivalent to that of the positive control clobetasol propionate (Clo). In the 48-hour tangeretin treatment group, the NO content in the cell supernatant increased overall in all groups, but tangeretin still showed a strong inhibitory effect on NO production by RAW264.7 cells. Tangeretin was preliminaryly judged to have some anti-inflammatory activity.

[0046] Example 3: Detection of the effect of tangeretin on the secretion levels of pro-inflammatory factors in LPS-induced RAW264.7 cells by enzyme-linked immunosorbent assay (ELISA). Logarithmically growing RAW264.7 cells were inoculated into a 6-well plate, and the cell density was increased to 5.0 × 10⁶. 5 Cells were prepared in wells and cultured for 24 hours until the cells were stably attached to the cell wall. The original medium was then removed, and tangeretin was added at different concentrations (5, 10, 20, 40 μM) for 1 hour of pretreatment. LPS (100 ng / mL) was added, and incubation continued for 24 hours, after which the cell supernatant was collected. The OD value at 450 nm of the cell supernatant was detected according to the instructions of the TNF-α and IL-6 ELISA kit, and the content of pro-inflammatory factors (TNF-α and IL-6) in the cell supernatant was calculated.

[0047] Results: Effects of tangeretin on pro-inflammatory factors in LPS-induced RAW264.7 cells According to the ELISA results (Figure 4 A-B), the content of pro-inflammatory factors (TNF-α and IL-6) in the cell supernatant of the LPS group was significantly increased compared to the control group (P<0.001). After tangeretin treatment, the content of pro-inflammatory factors (TNF-α and IL-6) in the cell supernatant decreased significantly in a dose-dependent manner (P<0.001). Furthermore, at a tangeretin concentration of 40 μM, the inhibitory effect on the secretion of pro-inflammatory factors (TNF-α and IL-6) was equivalent to that of the positive control clobetasol propionate (Clo). This indicates that tangeretin can dose-dependently inhibit the production of pro-inflammatory factors in RAW264.7 cells and mitigate inflammatory responses.

[0048] Example 4: Detection of the effect of tangeretin on the expression of IL-23, IL-17A, TNF-α, and IL-6 mRNA in LPS-induced RAW264.7 cells by real-time fluorescence quantitative polymerase chain reaction. RAW264.7 cells were inoculated into a 6-well plate, and the cell density was set to 5.0 × 10⁶. 6 After adjusting the cells / well and culturing for 24 hours until the cells were stably attached to the wall, the original medium was removed, tangeretin was added at different concentrations (5, 10, 20, 40 μM) for 1 hour of pretreatment, LPS (100 ng / mL) was added, and incubation continued for 24 hours. Then, commercially available TRlcom lysis solution provided by Jianshi Biotechnology was added, and total RNA was extracted from the cells using centrifugal column chromatography. Immediately, it was reverse transcribed into cDNA, and the fluorescence signal value of each sample was detected according to the instructions and specific primers (Table 1) of the real-time fluorescence quantitative PCR kit (cycle conditions: enzyme activation 95°C, 3 min, 1 cycle; denaturation 95°C, 15 sec; annealing / extension 60°C, 1 min, 40 cycles). After normalizing the mRNA expression level of each group to the GAPDH expression level, 2 -ΔΔCt The relative expression levels of mRNA were calculated using the method.

[0049] Real-time fluorescence quantitative polymerase chain reaction system [Table 1]

[0050] Real-time fluorescence quantification PCR primer sequences [Table 2] As shown in SEQ ID NO:1~10.

[0051] Results: Effects of tangeretin on IL-23, IL-17A, TNF-α, and IL-6 mRNA expression in LPS-induced RAW264.7 cells. Real-time fluorescence quantitative polymerase chain reaction detection results (Figure 5 A-D) showed that, compared to the control group, the relative expression levels of IL-23, IL-17A, TNF-α, and IL-6 mRNA significantly increased after LPS stimulation (P<0.001). After tangeretin treatment, the relative expression levels of IL-23, IL-17A, TNF-α, and IL-6 mRNA in experimental cells decreased significantly in a dose-dependent manner (P<0.001). This indicated that at a tangeretin concentration of 40 μM, the inhibitory effect on mRNA of pro-inflammatory factors (IL-23, IL-17A, TNF-α, IL-6) was equivalent to the inhibitory effect of the positive control clobetasol propionate (Clo). The experimental results (Figure 5B) showed that tangeretin exhibited a strong inhibitory effect on IL-17A, indicating that tangeretin inhibits the inflammatory response by regulating the IL-23 / IL-17A inflammatory axis.

[0052] Example 5: Effect of tangeretin on STAT3 protein phosphorylation levels in LPS-induced RAW264.7 cells Logarithmic growth phase RAW264.7 cells were inoculated into a 100 mm culture dish, and the cell density was increased to 1.6 × 10⁶. 6Cells were prepared in a dish and cultured for 24 hours until the cells were stably attached to the wall. Then, tangeretin was added at different concentrations (5, 10, 20, 40 μM) for 2 hours for pretreatment. LPS (100 ng / mL) was added, and the culture was incubated for 24 hours. The original medium was removed, and the dish was washed twice with pre-cooled PBS buffer. Protein lysis solution (300 μL per dish) was added, and the mixture was dissolved on ice for 30 minutes. The supernatant was collected by centrifugation at 12,000 rpm at 4°C for 5 minutes, and the protein concentration was measured by BCA. A total protein sample of 60 μg was loaded onto a 10% SDS-PAGE gel, separated, and then transferred to a PVDF membrane by electrophoresis. The sample was 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 mixture was incubated overnight at 4°C. 5 mL of horseradish peroxidase-labeled secondary antibody was added, and the mixture was incubated at room temperature for 1 hour. Chromogenic detection was performed using ECL, and protein band quantification was analyzed using ImageJ software.

[0053] Results: Effect of tangeretin on STAT3 phosphorylation in LPS-induced RAW264.7 cells In the results of Western blot (Figure 6 A - D), there were no statistically significant changes in STAT3 protein expression among each treatment group (P > 0.05). Regarding the phosphorylated expression of STAT3 protein, compared with the Control group, the expression of STAT3 phosphorylation (Tyr 705 and Ser 727) proteins at two sites in the LPS group 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 (Ser727) proteins in a dose - dependent manner (P < 0.001). Especially in the expression of p - STAT3 (Ser 727), tangeretin showed a more significant inhibitory effect than p - STAT3 (Tyr 705). The inhibitory effect of tangeretin at a concentration of μM on the phosphorylation level of STAT3 protein was equivalent to that of the positive control clobetasol propionate (Clo), effectively inhibiting the phosphorylation of STAT3 protein, and further indicating that tangeretin alleviates the inflammatory response by regulating the IL - 23 / STAT3 / IL - 17A signaling pathway.

[0054] Example 6 Effect of Tangeretin on the Back Skin Lesions of Imiquimod (IMQ) - induced Psoriasis Mice Balb / c mice (female, 6 - 8 weeks old, 20 ± 2 g), certificate number [SCXK(Xiang) 2021 - 0002], were purchased from Slake Jingda Company (Changsha, China). All mice were housed in an SPF - graded animal room with a 12 - hour light / 12 - hour dark cycle. After the mice got used to the feeding for one week, the experiment was started. 96 mice were randomly divided into 12 groups (8 mice per group): Control group, Control solvent (white petrolatum) group, IMQ group, IMQ solvent (white petrolatum) group, preventive administration group of the positive drug clobetasol propionate (Clo) (IMQ + Clo 55 mg), therapeutic administration group of the positive drug (IMQ + Clo(A) 55 mg), low - dose preventive administration group of Tan (IMQ + Tan 40 μg / cm 2 ), medium - dose group (IMQ + Tan 160 μg / cm 2 ), high - dose group (IMQ + Tan 640 μg / cm 2), Tan treatment low-dose group (IMQ+Tan(A) 40μg / cm³) 2 ), middle dose group (IMQ+Tan(A) 160μg / cm 2 ), high dose group (IMQ+Tan(A)640 μg / cm 2 After anesthetizing the mice, a 2cm x 3cm area on the back was selected and hair removal was performed. After 48 hours of hair removal, modeling was started, and all model mice received topical application of imiquimod cream 65mg (55mg on the back and 5mg in each ear) every morning for 10 days. In the tangeretin prophylactic administration group, the low-dose group (40μg / cm³) 2 ), medium dose group (160 μg / cm 2 ), high-dose group (640 μg / cm 2 ) was administered locally to the back and both ears every afternoon for 10 days starting from the first day of modeling. In the tangeretin treatment group, the low-dose group (40 μg / cm³) 2 ), medium dose group (160 μg / cm 2 ), high-dose group (640 μg / cm 2 The drug was administered topically to the back and both ears every afternoon for 10 days starting from the third day of modeling. Mice in the prophylactic group receiving the positive drug were treated with clobetasol propionate (55 mg per mouse, 45 mg on the back and 5 mg in each ear) from the first day of modeling, while mice in the therapeutic group receiving the positive drug were treated with clobetasol propionate (55 mg per mouse, 45 mg on the back and 5 mg in each ear) from the third day of modeling.

[0055] Results: Tangeretin significantly inhibited dorsal skin lesions in psoriasis mice. As shown in Figure 7, mice in the IMQ group showed more pronounced erythema and scaling on their backs than mice in the control group. Compared to the IMQ group, the prophylactic and therapeutic tangeretin administration groups showed significant improvement in the pathological symptoms of psoriasis, and erythema and scaling were significantly reduced in a dose-dependent manner. The prophylactic and high-dose therapeutic tangeretin administration groups showed improvement in the skin lesions on the backs of psoriasis mice comparable to that of the positive control group (Clo). Scaling and erythema on the back skin of mice in the prophylactic administration group almost disappeared, and their skin condition was similar to that of the control group. After treatment with the tangeretin therapeutic administration group, the skin pathological symptoms of psoriasis mice improved significantly, with only slight scaling remaining on the backs. These results indicate that tangeretin has the effect of reducing skin thickness and the severity of scaling (Figure 7).

[0056] Example 7: Histological evaluation of tangeretin in dorsal skin lesions of imiquimod (IMQ)-induced psoriasis mice. Mouse dorsal skin tissue was fixed in a 4% paraformaldehyde solution for 30 days and then detected with H&E staining. The detailed procedure was as follows: The fixed tissue was sequentially dehydrated in a concentration gradient ethanol solution, then cleared with xylene for 30 minutes each time, and the samples were embedded in paraffin to prepare 3-5 μm serial sections. After dewaxing and rehydration, the sections were stained with hematoxylin-eosin reagent to identify skin tissue structure and cell nuclei, and then dried and sealed with neutral resin. Images were acquired using an optical microscope (DM500, Leica, Germany) at a magnification of 100x during observation.

[0057] Results: Tangeretin significantly improved keratinocyte thickening, incomplete differentiation, and inflammatory cell infiltration in the dorsal skin of psoriasis mice. Compared to the control group, the IMQ group showed clear epidermal thickening, parakeratosis, and significant inflammatory cell infiltration. In the IMQ group, compared to the prophylactic Tan group, the epidermal tissue thickness of the dorsal skin of mice in the prophylactic Tan group decreased, keratinization was reduced, and inflammatory cell infiltration was minimal, indicating a clear dose-dependent activity. In the IMQ group, compared to the therapeutic Tan group, the therapeutic Tan group also showed stronger pharmacological activity, particularly in the high-dose group, with decreased epidermal tissue thickness, reduced keratinization, and minimal inflammatory cell infiltration, also showing a clear dose-dependent activity. In summary, H&E staining results of mouse skin lesions showed that tangeretin exhibited potent antipsoriasis activity regardless of whether it was administered prophylactically or therapeutically, and that this activity was significantly dose-dependent (Figure 8).

[0058] Example 8: Effect of tangeretin on the expression levels of p-STAT3, IL-17A, and PCNA in the dorsal skin of imiquimod (IMQ)-induced psoriasis mice. Mouse dorsal skin samples were collected, paraffin sections were prepared, dewaxed with xylene, incubated in H2O2 solution at room temperature for 10 minutes after dewaxing, blocked with BSA at room temperature for 1 hour to inhibit endogenous peroxidase activity and remove nonspecific background adsorption, p-STAT3, IL-17A, and PCNA antibodies were added, incubated overnight at 4°C, horseradish peroxidase-labeled secondary antibody was added, incubated at room temperature for 30 minutes, DAB was added dropwise for color development, hematoxylin was used for counterstaining, a negative control was established, parameters were standardized, and photographs were taken and recorded. The magnification during observation was 200x.

[0059] Results: Tangeretin significantly inhibited the expression levels of p-STAT3, IL-17A, and PCNA in the dorsal skin of psoriasis mice. Compared to the control group, the expression levels of p-STAT3, IL-17A, and PCNA were significantly increased in the IMQ group (Figures 9, 10, and 11). The expression levels of p-STAT3, IL-17A, and PCNA in the dorsal skin of mice in the tangeretin prophylactic and therapeutic groups were significantly decreased in a dose-dependent manner. This demonstrated that tangeretin significantly inhibits the production of p-STAT3, IL-17A, and PCNA, thereby reducing inflammatory responses, keratinocyte abnormal proliferation, and incomplete differentiation, exhibiting antipsoriasis activity and demonstrating its superior anti-IL-17A inhibitor properties.

[0060] In summary, this study demonstrates that tangeretin possesses significant anti-inflammatory activity, potentially mitigating inflammatory responses by inhibiting the production of pro-inflammatory factors (TNF-α and IL-6) and regulating the IL-23 / STAT3 / IL-17A signaling pathway. As a natural drug, tangeretin can inhibit IL-17A production more safely and stably, making it an excellent IL-17A inhibitor.

[0061] Example 9: Typical treatment case of a psoriasis patient with a tangeretin ointment formulation. Mr. Ning, male, 35 years old. Fifteen years ago, after catching a cold, the patient developed itchy, scaly erythematous lesions on the trunk. They visited a local hospital and were diagnosed with psoriasis vulgaris. The patient received treatment with topical clobetasol propionate, retinoic acid, and calcipotriol, but the rash recurred and gradually spread to the trunk and limbs. Psoriatic lesions now cover more than 50% of the skin, and the current clinical diagnosis is severe psoriasis vulgaris. The tangeretin ointment of this invention is applied topically once daily for treatment (the amount of ointment used per application is 6 mg / cm³). 2 The dosage is 230 μg / cm³. 2 After 4 days of treatment, the symptoms improved significantly, and after 10 days of continued use, the symptoms improved dramatically, and the scaling almost completely disappeared (Figure 12). For the protection of patient privacy, the patient's personal information will not be disclosed here. The patient's personal information and medical records are stored at Shonan Gakuin Hospital and are available for viewing. Results: Tangeretin significantly improved the symptoms of scaly erythema on the arms of psoriasis patients.

[0062] As shown in Figure 12, Ms. Ning, a psoriasis patient, had very thick and numerous scales on her arm. After using tangeretine ointment for two days, the scales began to decrease. After using the experimental drug for four days, the scales on the patient's arm decreased significantly and almost completely disappeared. The effects became more pronounced with continued use of the drug, and by the 10th day of use, the scales had completely disappeared. By the 14th day, the erythema in the affected skin area had significantly faded, the erythematous lesions had shrunk considerably, and healthy skin had appeared.

[0063] Example 10 Clinical trial of the efficacy of tangeretin ointment formulation Five patients clinically diagnosed with psoriasis were observed, and tangeretin ointment was applied to the affected area once daily for 14 consecutive days. Day 0 was defined as the day before application of tangeretin ointment, and Day 1 was defined as the day after the first day of application. During this period, the therapeutic effect of tangeretin on psoriasis patients was monitored by scoring patients using the Psoriasis Area and Severity Index (PASI). The severity score for skin lesions included erythema and scaling, and each parameter was evaluated on a score from 0 to 4 (0: no clinical symptoms, no scaling, no erythema; 1: mild clinical symptoms, mild and pale redness; 2: moderate clinical symptoms, mild erythema and scaling; 3: prominent clinical symptoms, prominent erythema and many scales; 4: very severe clinical symptoms). During the treatment process, the tangeretin ointment preparation described in the present invention (apply the tangeretin ointment preparation to the affected area once a day, with a single application amount of 6 mg / cm³ of ointment) is used. 2 The dosage is 230 μg / cm³. 2 After treatment with tangeretine ointment, all psoriasis patients showed significant improvement on day 4, and by day 9-10, the scaling had almost completely disappeared. Continued use resulted in reduced skin redness and a significant alleviation of symptoms. PASI score monitoring data showed a substantial decrease in the overall PASI scores of patients after treatment with tangeretine ointment, indicating a very pronounced antipsoriatic effect (Figure 13). Results: Tangeretin significantly improved the pathological symptoms of psoriasis patients, and PASI scores in psoriasis patients decreased significantly after using the tangeretin ointment formulation of the present invention.

[0064] As shown in Figure 13, when five psoriasis patients used the tangeretin ointment formulation, their PASI scores began to decrease from the second day of administration, and significantly decreased from the fourth day of administration. Subsequently, the PASI scores continued to decrease with the extension of the administration period, demonstrating a significant improvement in the pathological symptoms of the psoriasis patients in the experimental group.

[0065] The above describes only preferred embodiments of the present invention, and it should be noted that those skilled in the art can make several improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The use of an IL-17A inhibitor in the manufacture of a drug for the treatment of inflammatory diseases, wherein the IL-17A inhibitor is tangeretin.

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

3. The use according to claim 2, characterized in that the drug is in the form of a tablet, liquid, ointment, gel, emulsion, capsule, suppository, or granule.

4. The use according to claim 1, characterized in that the drug for treating the inflammatory disease is a drug for treating psoriasis, 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 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 the treatment of inflammatory diseases is 5 to 40 μM.

6. The use according to claim 1, characterized in that the inflammatory factors associated with the aforementioned 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 caused by lipopolysaccharide.

8. The use according to claim 4, characterized in that, when the drug for treating the inflammatory disease is a drug for treating psoriasis, tangeretin has the effect of reducing skin thickness and the degree of scaling.

9. The use of tangeretin according to claim 1, characterized in that it 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 the inflammatory disease is a drug that inhibits the content of the cellular inflammatory factor NO.

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