Application of chalcone derivative psoralen B and GPR84 antagonist or medicine
By using the chalone derivative psoralein as the GPR84 receptor antagonist, the problem of single structure of GPR84 antagonist in the prior art is solved, and effective treatment of Crohn's disease, non-alcoholic fatty liver disease and reflux esophagitis is achieved.
Patent Information
- Application Number
- CN202510774742.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-11
- Publication Date
- 2025-08-19
AI Technical Summary
In the prior art, GPR84 antagonists have low structural diversity and lack of effective natural-derived compounds, resulting in insufficient treatment of diseases such as Crohn's disease, non-alcoholic fatty liver disease and reflux esophagitis.
The chalone derivative psoralein was used as the GPR84 receptor antagonist to verify its antagonism activity through cellular and animal experiments, and was used to prepare related disease drugs.
Psoralein significantly reduces colon shortening in the TNBS-induced Crohn's disease model, improves weight loss, and has concentration-dependent GPR84 receptor antagonism activity, providing therapeutic potential for Crohn's disease, non-alcoholic fatty liver disease and reflux esophagitis.
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Abstract
Description
Technical Field
[0001] The present invention relates to a chalcone derivative psoralen, which can be used as a GPR84 (G protein-coupled receptor 84) receptor antagonist and has a new application in treating GPR84 receptor-related diseases, and relates to applications in the medical field. Background Art
[0002] GPR84 (G protein-coupled receptor 84) is an orphan G protein-coupled receptor whose endogenous ligands are medium-chain fatty acids, such as capric acid, undecanoic acid, and lauric acid. GPR84 is primarily expressed in myeloid cells that constitute the innate immune system, including neutrophils, monocytes, and macrophages. Studies have shown that the GPR84 receptor is associated with diseases such as Crohn's disease (GPR84 signaling promotes intestinal mucosal inflammation via enhancing NLRP3 inflammasome activation in macrophages. Acta Pharmacol Sin. 2022; 43(8): 2042-2054.), non-alcoholic fatty liver disease (The Medium-Chain Fatty Acid Receptor GPR84 Mediates Myeloid Cell Infiltration Promoting Steatohepatitis and Fibrosis. 2020; 9(4): 1140.), and reflux esophagitis (GPR84 and TREM-1 Signaling Contribute to the Pathogenesis of Reflux Esophagitis. Mol Med. 2016; 21(1): 1011-1024.). Currently, the structural diversity of GPR84 antagonists reported in the literature is low, and there are also few reported natural GPR84 antagonists. Therefore, the discovery of new GPR84 ligands not only has potential significance for the treatment of diseases such as Crohn's disease, non-alcoholic fatty liver disease and reflux esophagitis, but also enriches the structural diversity of GPR84 ligands, providing a reference for future research on new and efficient GPR84 ligands.
[0003] Currently, there are no reports on whether psoralen has GPR84 antagonist activity and the application of this compound in the prevention and treatment of Crohn's disease, non-alcoholic fatty liver disease and reflux esophagitis. Summary of the Invention
[0004] The purpose of the present invention is to provide the use of psoralen in the preparation of GPR84 antagonists and drugs for receptor-related diseases. Specifically, one of the purposes of the present invention is to provide a target of psoralen that is the GPR84 receptor; a second purpose of the present invention is to provide the use of psoralen in the treatment of Crohn's disease, non-alcoholic fatty liver disease, and reflux esophagitis. Cell experiments have shown that the compound is an antagonist of GPR84, and animal experiments have shown that the compound can reduce colon shortening and improve weight loss in a Crohn's disease model induced by 2,4,6-trinitrobenzenesulfonic acid (TNBS), providing a basis for the development of anti-Crohn's disease drugs. The specific contents are as follows:
[0005] Use of psoralen in preparing GPR84 antagonists and drugs for receptor-related diseases.
[0006] The GPR84-related diseases are diseases or conditions selected from one or more of Crohn's disease, non-alcoholic fatty liver disease and reflux esophagitis.
[0007] The medicine comprises one or more of psoralen or its pharmaceutically acceptable solvates as active ingredients.
[0008] The application is a pharmaceutical composition prepared by using psoralen as an active ingredient and a pharmaceutical carrier.
[0009] The pharmaceutical composition is in the form of tablets, capsules, granules, suspensions, dripping pills, injections or aerosols.
[0010] Animal experiments have shown that the compound has significant efficacy in a TNBS-induced Crohn's disease colitis model, reducing TNBS-induced colon shortening and improving weight loss. The compound can be used to prepare GPR84 antagonists and prevent and treat GPR84-related diseases such as Crohn's disease, non-alcoholic fatty liver disease, and reflux esophagitis.
[0011] Beneficial effects of the present invention
[0012] The present invention discloses the use of psoralen in the preparation of a GPR84 receptor antagonist and a drug for receptor-related diseases. The present invention confirms that cell experiments show that psoralen is a GPR84 antagonist, psoralen has concentration-dependent GPR84 receptor antagonist activity, and is a GPR84 antagonist in a concentration-dependent manner. Animal experimental models have demonstrated that psoralen has the effect of treating Crohn's disease, and the present invention has good prospects for drug development.
[0013] Animal studies have shown that the compound exhibits significant efficacy in a TNBS-induced colitis model, mitigating TNBS-induced colon shortening, improving weight loss, and alleviating inflammatory responses. The compound can be used to prepare GPR84 antagonists and prevent and treat GPR84-related diseases such as Crohn's disease, non-alcoholic fatty liver disease, and reflux esophagitis. BRIEF DESCRIPTION OF THE DRAWINGS
[0014] Figure 1 A: DMR responses induced by different concentrations of psoralen in HEK293T-GPR84 cells; Figure 1 B: Concentration-effect curve of GTPL5846 after pretreatment with different concentrations of psoralen.
[0015] Figure 2 A: The trend of weight changes of mice in each group over time;
[0016] Figure 2 B: Schematic diagram of the effect of psoralen on colon length in TNBS-induced Crohn's disease mice;
[0017] Figure 2 C: Statistical graph of colon length in each group of mice;
[0018] Figure 2 D: Typical tissue section staining results of mice in each group (H&E, PSR and F4 / 80 staining). Scale bar: 100 μm;
[0019] Figure 2 EK: qPCR was used to detect the levels of inflammatory cytokines in colon tissue.
[0020] Figure 2 LN: Western blot was used to detect the expression levels of E-cadherin and occludin in colonic mucosa. DETAILED DESCRIPTION
[0021] The present invention will now be further described with reference to examples, which are intended to illustrate the present invention but not to limit it.
[0022] The present invention is described below by means of specific examples, but the present invention is not limited thereto.
[0023] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and biological materials described are commercially available unless otherwise specified.
[0024] Isobavachalcone, also known as isobavachalcone;
[0025] Example 1 Psoralen is a GPR84 antagonist
[0026] HEK293T-GPR84 cells were derived from transfection. First, 24 μL of Lipofectamine 3000 transfection reagent (Lipofectamine TM 3000 transfection reagent, Thermo Fisher Scientific) and 8 μg of GPR84 plasmid pcDNA3.1 (General Biotechnology (Anhui) Co., Ltd.) were diluted in 750 μL of Opti-MEM medium (Thermo Fisher Scientific) and mixed. After standing at room temperature for 15 minutes, the mixture was added dropwise to HEK293T cells cultured in a 10 cm inner diameter cell culture dish with 70%-80% cell coverage. HEK293T cells were cultured at 37°C in DMEM medium (Thermo Fisher Scientific) supplemented with a final concentration of 10% (v / v) FBS (Thermo Fisher Scientific). 24 hours after transfection, HEK293T-GPR84 cells were transferred from the 10 cm inner diameter culture dish to five 15 cm inner diameter culture dishes at concentrations of 1 / 50, 1 / 200, 1 / 500, 1 / 800, and 1 / 1000, respectively. The 15 cm dishes were cultured in DMEM medium supplemented with 600 μg / mL zeocin. After one day of growth, the medium was replaced with 21 mL of medium supplemented with 200 μg / mL Zeocin. The medium was changed every 2-3 days (2 days in this case) until cell clusters began to form. The selected cell population was transferred to a 96-well plate for culture (100 μL culture medium containing 200 μg / mL Zeocin per well). After the cells were confluent, they were transferred to a 24-well plate for culture (200 μL culture medium containing 200 μg / mL Zeocin per well). After the cells were confluent, they were transferred to a 6-well plate for culture (2 mL culture medium containing 400 μg / mL G418 per well) for 3 days, and a stably transfected cell line was successfully obtained.
[0027] The probe molecule GTPL5846 and GPR84 antagonist 8 were purchased from MedChemExpress. Psoralen B was purchased from Shanghai Taoshu Biotechnology Co., Ltd.
[0028] The HEK293T-GPR84 model constructed using the above method was tested for psoralen by DMR (dynamic mass resetting) analysis. HEK293T-GPR84 cells were seeded in 384-well biosensor microplates at a density of 25,000 cells per well overnight until approximately 95% confluence was achieved. Cell culture conditions were cultured in DMEM medium (Thermo Fisher Scientific) at 37°C and 5% (v / v) CO2 air, and the DMEM medium was supplemented with a final concentration of 10% (v / v) FBS (Thermo Fisher Scientific). The growth medium was replaced with a buffer solution consisting of 1×HBSS and 10mM HEPES, the pH was adjusted to 7.4, the buffer volume ratio was: HEPES:HBSS: ultrapure water = 1:10:89, and it was stabilized for 1 hour. Use The DMR assay was performed using the system. First, cells were pretreated with psoralen (final concentrations of 100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.125 μM, and 1.563 μM) for 1 hour before establishing a 2-minute baseline. Subsequently, 10 μL of GTPL5846 (final concentration of 40 nM) was added and the signal was recorded for 1 hour. The results are shown in Figure 2. Figure 1 A and Figure 1 B. Psoralen itself does not generate DMR signals, but can inhibit the DMR signals of GTPL5846 in a concentration-dependent manner. The IC 50 The value was 17.94±1.03μM. The above results showed that psoralen was a GPR84 antagonist.
[0029] Example 2: Improvement of Psoralen on TNBS-induced Crohn's disease
[0030] Male BALB / c mice were purchased from Jicui Yaokang (Nanjing, China, Animal License No.: SCXK(Su)2023-0009) at 8 weeks of age and weighing 23-28 g. All animals were housed in an SPF-grade sterile environment at the Ganjiang Traditional Chinese Medicine Innovation Center (License No.: SYSK(Gan)2024-0013) and followed a 12-hour day-night cycle. The experimental procedures strictly followed the "Guidelines for the Care and Use of Laboratory Animals" and were approved by the Animal Ethics Committee of the Ganjiang Traditional Chinese Medicine Innovation Center (License No.: GJCMIC2024-003, Approval Date: November 18, 2024). After the mice arrived, they were first acclimated to the environment for one week, and then the colitis induction experiment was started. The weight and health of the mice were monitored at least once a day during the study. Male BALB / c mice were randomly divided into five groups (n=8 in each group), including: blank control group, TNBS model group, TNBS+5-aminosalicylic acid (200 mg / kg) group, TNBS+psoralen (10 mg / kg) group and TNBS+psoralen (25 mg / kg) group. Colitis was induced in Balb / c mice according to the method of Wirtz, S., Popp, V., Kindermann, M., Gerlach, K., Weigmann, B., Fichtner-Feigl, S. & Neurath, MF, 2017. Chemically induced mouse models of acute and chronic intestinal inflammation. Nat Protoc, 12(7), 1295-1309. After fasting for 24 hours, the mice were lightly anesthetized with tribromoethanol (Beijing Jitian Biotechnology Co., Ltd.) intraperitoneally (200 μL / 10 g). A 3.5F catheter was carefully inserted into the colon, with the tip of the catheter 4 cm away from the anus. The TNBS model group was slowly infused with 100 μL of 5% (v / v) ethanol solution (50% volume of water and 50% volume of ethanol, ethanol is used to destroy the intestinal epithelial barrier) containing 5% mass concentration of 2,4,6-trinitrobenzenesulfonic acid (TNBS) (Sigma-Aldrich, Germany) through a catheter connected to a 1 mL syringe.The control group mice were perfused with 100 μL of 50% ethanol (the operation was the same as that of the TNBS model group); the TNBS + 5-aminosalicylic acid (200 mg / kg) group was perfused with 100 μL of 5% TNBS in 50% (v / v) ethanol solution (prepared as described above), to which aminosalicylic acid (200 mg / kg mouse) was added (the operation was the same as that of the TNBS model group); the TNBS + psoralen (10 mg / kg) group was perfused with 100 μL of 5% TNBS in 50% ethanol solution (prepared as described above), to which psoralen (10 mg / kg mouse) was added (the operation was the same as that of the TNBS model group); the TNBS + psoralen (25 mg / kg) group was perfused with 100 μL of 5% TNBS 50% ethanol solution (prepared as described above) was added with psoralen (25 mg / kg mouse) (the operation was the same as the TNBS model group). After perfusion treatment, the mice were kept in an upright position for 30 seconds and then returned to the cage. After 2 hours of TNBS treatment (TNBS model group, TNBS + 5-aminosalicylic acid (200 mg / kg) group, TNBS + psoralen (10 mg / kg) group and TNBS + psoralen (25 mg / kg) group), mice were randomly treated with 0.9% NaCl, 5-aminosalicylic acid (200 mg / kg) or psoralen (10 mg / kg or 25 mg / kg) by gavage (100 μL / 10 g) once a day for 3 days. The mice were killed on the 12th day of the experiment, and colon tissues were collected for histological evaluation, quantitative PCR (qPCR) and Western blotting analysis.For histological examination, the transverse colon section was fixed with 4% paraformaldehyde and embedded in paraffin, and the following process was performed: Hematoxylin-eosin (H&E) staining (for details, please refer to the H&E staining service of Wuhan Sevier Biotechnology Co., Ltd., item number: GP1031): After the paraffin sections were dewaxed and hydrated, hematoxylin stained the nucleus (3-5 minutes), hydrochloric acid ethanol differentiation, blueing, eosin stained the cytoplasm (1-3 minutes), gradient ethanol dehydration, xylene transparent, and neutral gum sealing; Sirius red (PSR) staining (for details, please refer to the PSR staining service of Wuhan Sevier Biotechnology Co., Ltd., item number: GP1138): section After dewaxing and hydration, stain with Sirius red stain (0.1% in saturated picric acid solution) for 1 hour, differentiate with acidic ethanol, and mount the slides directly or after dehydration and transparency. Observe collagen fibers under polarized light. F4 / 80 immunohistochemical staining (for details, please refer to the F4 / 80 staining detection service of Wuhan Saiwei Biotechnology Co., Ltd., catalog number: GDP1001): antigen retrieval (citrate buffer, high pressure or microwave), block endogenous peroxidase, incubate with F4 / 80 primary antibody (clone BM8, 1:100-1:200) (4°C overnight), bind secondary antibody (HRP-labeled), develop color with DAB, counterstain nuclei with hematoxylin, dehydrate and mount.
[0031] The results showed that compared with the blank control group, the mice in the TNBS-treated group had a significant decrease in body weight, shortened colon, and had colon damage, indicating that TNBS successfully induced Crohn's disease-like colitis. In the psoralen-treated group, psoralen (10 and 25 mg / kg) improved the weight loss in a dose-dependent manner ( Figure 2 A) and shortened colon ( Figure 2 BC). Histological analysis by H&E staining showed that psoralen treatment significantly alleviated colonic damage, as demonstrated by reduced mucosal ulceration, preserved crypt architecture, and decreased inflammatory cell infiltration ( Figure 2 D). Sirius red staining showed that collagen deposition in the TNBS group increased compared with the blank control group, while psoralen treatment reduced this deposition ( Figure 2 D). F4 / 80 is a macrophage surface marker (brown in the figure). The analysis results show that psoralen B reduces the accumulation of macrophages in the mucosal area ( Figure 2 D). Macrophages can produce a variety of cytokines associated with colitis, including TNF-α, IL-6, IL-1α, IL-1β, and IL-23. We tested the effects of psoralen on these cytokines and found that psoralen reduced their mRNA expression levels ( Figure 2 Given that Crohn's disease also involves the immune response of Th1 / Th17 cells (secreting IFN-γ and IL-17, respectively), we evaluated the expression of these factors and found that psoralen could reduce the expression of IFN-γ and IL-17 at the mRNA level ( Figure 2 JK). Finally, we investigated the protective effects of psoralen on adherens junction / tight junction proteins, including E-cadherin and occludin, which are crucial for maintaining the intestinal epithelial barrier. Compared with the blank control group, the expression of E-cadherin and occludin in TNBS-induced colitis mice was significantly decreased, while psoralen could upregulate their expression levels ( Figure 2 These results indicate that psoralen (25 mg / kg) can improve disease phenotypes, reduce inflammatory factors, and improve the intestinal barrier, demonstrating its efficacy in treating Crohn's disease and surpassing the therapeutic effect of 5-aminosalicylic acid (200 mg / kg), suggesting potential development prospects.
Claims
1. Use of a chalcone derivative, psoralen, in the preparation of a GPR84 antagonist or in the preparation of a drug for treating GPR84 receptor-related diseases.
2. The use according to claim 1, characterized in that: The GPR84 receptor-related disease refers to one or more of Crohn's disease, non-alcoholic fatty liver disease or reflux esophagitis.
3. A GPR84 antagonist or a drug for treating a GPR84 receptor-related disease, characterized in that: The antagonist or drug comprises one or more of psoralen or its pharmaceutically acceptable solvates as active ingredients.
4. The GPR84 antagonist or the drug for treating GPR84 receptor-related diseases according to claim 3, characterized in that: The GPR84 antagonist or drug for treating GPR84 receptor-related diseases is a pharmaceutical composition prepared with one or more of psoralen or its pharmaceutically acceptable solvates as active ingredients and a pharmaceutically acceptable carrier.
5. The GPR84 antagonist or the drug for treating GPR84 receptor-related diseases according to claim 3 or 4, characterized in that: The GPR84 receptor-related disease refers to one or more of Crohn's disease, non-alcoholic fatty liver disease or reflux esophagitis.
6. The GPR84 antagonist or the drug for treating GPR84 receptor-related diseases according to claim 3 or 4, characterized in that: The pharmaceutical composition is any one of tablets, capsules, granules, suspensions, dripping pills, injections or aerosols.