Application of xanthohumol in preparation of medicine for improving or treating migraine
By using chlorhexidol to activate the PPARA/AMPK pathway, the problem of difficult to effectively improve or treat migraine in the prior art is solved, and migraine-like behavior and central sensitization are significantly alleviated, and effective treatment of migraine is achieved.
Patent Information
- Application Number
- CN202510049315.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-13
- Publication Date
- 2025-05-06
AI Technical Summary
The prior art is difficult to effectively improve or treat migraine, and its pathogenesis is still unclear.
Using yoxhumpol as the active ingredient, by activating the PPARA/AMPK pathway, central sensitization in migraine model mice was alleviated and migraine-like behavior was significantly alleviated.
Xanthulphenol significantly increased the mechanical and cold pain thresholds, improved photophobia, reduced spontaneous nociceptive behavior, and reduced expression of CGRP and c-Fos, reducing central sensitization in migraine model mice.
Smart Images
Figure CN119925320A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of biomedicine, and in particular to the application of xanthohumol in preparing a medicine for improving or treating migraine. Background Art
[0002] Migraine is a common, recurrent, disabling neurological disease characterized by recurrent unilateral throbbing headaches, often accompanied by symptoms such as nausea, vomiting, photophobia, and phonophobia. According to the 2021 Global Burden of Disease Study (GBD), age-standardized disability-adjusted life years (DALYs) for migraine rank third among neurological diseases. Migraine patients have an increased risk of cardiovascular and cerebrovascular diseases, and have comorbidities such as depression, epilepsy, and circadian rhythm disorders. It has a huge impact on the patient's quality of life and imposes a huge burden on the patient's family and socioeconomics.
[0003] The pathogenesis of migraine is still unclear. Currently, it is mainly believed that the pathogenesis of migraine involves central sensitization and neuroinflammation of the trigeminovascular system. It is characterized by increased excitability and synaptic plasticity of sensory neurons, mainly in the TNC and thalamus. Activation of the trigeminovascular pathway leads to the release of proinflammatory, vasodilation or pain-producing neuropeptides, such as calcitonin gene-related peptide (CGRP) and pituitary adenylate cyclase-activating polypeptide (PACAP). CGRP is abundant in trigeminal ganglion neurons and is secreted in the trigeminal ganglion. It may promote central sensitization of secondary neurons by interacting with adjacent neurons and satellite glial cells. Some studies have shown that AMPK is involved in the central sensitization of migraine. In a nitroglycerin-induced recurrent chronic migraine mouse model, activation of AMPK can alleviate central sensitization.
[0004] Peroxisome proliferator-activated receptor α (PPARα / PPARA) is a ligand-activated transcription factor that belongs to the NR1C nuclear receptor subfamily along with PPARγ and PPARβ / δ. PPARA can bind to sequence-specific target elements in the promoter region of target genes and drive multiple aspects of normal cellular physiology and pathology, including lipid metabolism, glucose homeostasis, cell differentiation, proliferation, obesity, cancer, and inflammation. Several studies have shown that PPARA agonists have analgesic properties in several models of visceral pain, inflammatory pain, and neuropathic pain. Fenofibrate is a PPARA agonist approved for the reduction of NTG-induced migraine-like symptoms in rats by inhibiting the CGRP / p-CREB / P2X3 and NGF / PKC / ASIC3 signaling pathways.
[0005] Therefore, it is necessary to develop a drug for improving or treating migraine. Summary of the invention
[0006] The purpose of the present invention is to provide the use of xanthohumol in the preparation of drugs for improving or treating migraine. The present invention finds for the first time that xanthohumol can improve the migraine-like behavior of migraine model mice, indicating that xanthohumol has great application prospects in the preparation of drugs for improving or treating migraine.
[0007] To achieve the above object, the present invention adopts the following technical solution:
[0008] The invention provides application of xanthohumol in preparing a medicine for improving or treating migraine.
[0009] Furthermore, the medicine also includes pharmaceutically acceptable excipients and carriers.
[0010] Furthermore, the auxiliary material includes at least one of a filler, a disintegrant, a binder, an excipient, a lubricant, a sweetener or a colorant.
[0011] Furthermore, the dosage form of the drug includes at least one of granules, tablets, pills, capsules, and injections.
[0012] Furthermore, the effective dosage of the xanthohumol is 1.0 to 50 mg / kg.
[0013] Furthermore, the xanthohumol significantly alleviates the migraine-like behavior of migraine model mice, including increasing the mechanical pain threshold and cold pain threshold, improving the photophobia behavior of mice and reducing the spontaneous nociceptive behavior of mice.
[0014] The present invention provides a potential mechanism of xanthohumol in treating migraine.
[0015] Preferably, the therapeutic mechanism refers to: xanthohumol can activate the PPARA / AMPK pathway in the TNC region of migraine mice, thereby alleviating the central sensitization of migraine. Xanthohumol can increase the expression of PPARA, activate the AMPK pathway, and increase its phosphorylation level. In addition, xanthohumol can also reduce the levels of indicators related to central sensitization of migraine, such as CGRP and c-FOS.
[0016] The present invention also provides a medicine for improving or treating migraine, wherein the active ingredient of the medicine comprises xanthohumol.
[0017] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:
[0018] (1) Xanthohumol can significantly alleviate the migraine-like behavior of migraine model mice, including increasing the mechanical pain threshold and cold pain threshold, improving the photophobia of mice, and reducing the spontaneous nociceptive behavior of mice.
[0019] (2) Xanthohumol can reduce the expression of CGRP and c-Fos in the TNC region and alleviate central sensitization in migraine model mice.
[0020] (3) Xanthohumol can bind to PPARA protein, and its binding ability with PPARA was verified by molecular docking technology.
[0021] (4) The PPARA / AMPK pathway is the target of xanthohumol in the treatment of migraine. Xanthohumol can significantly increase the protein expression level of PPARA in the TNC region. At the same time, xanthohumol can activate AMPK and increase its phosphorylation level.
[0022] (5) The compound is safe and effective, and no obvious toxic side effects were observed during the entire experimental process of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings in the following description are some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative work.
[0024] Figure 1 This is a diagram of the administration pattern of xanthohumol in treating migraine model mice;
[0025] Figure 2 The results of evaluating the effect of xanthohumol (Xn) on improving pain-related behaviors in migraine model mice; (a) is the periorbital pain threshold, (b) is the periorbital cold pain threshold, (c) is the freezing time, and (d) is the number of head scratching within 5 minutes;
[0026] Figure 3 To evaluate the photophobia behavior of xanthohumol (Xn) in improving migraine mice; (a) is the number of holes in the light chamber, (b) is the time the mouse stays in the dark box, and (c) is a representative trajectory diagram and heat map;
[0027] Figure 4 The effect of xanthohumol (Xn) on the protein expression of c-Fos and CGRP in TNC mice with migraine was detected by WB. (a) is the protein immunoblot of c-Fos and CGRP in each group of mice, and (b) is the quantitative statistical graph of proteins in each group of mice;
[0028] Figure 5 Immunofluorescence detection of the expression of c-Fos and CGRP in TNC of migraine mice treated with xanthohumol (Xn); (a) is the immunofluorescence image of c-Fos and CGRP in each group of mice, and (b) is the statistical analysis result of immunofluorescence in each group of mice.
[0029] Figure 6 The molecular docking and interaction diagram of xanthohumol (Xn) and PPARA;
[0030] Figure 7 The effect of xanthohumol (Xn) on the protein expression of PPARA, AMPK, and p-AMPK in migraine mice TNC was detected by WB. (a) is the protein immunoblot of PPARA, AMPK, and p-AMPK in each group of mice, and (b) is the quantitative statistical graph of proteins in each group of mice;
[0031] Figure 8 Immunofluorescence images and quantitative images of PPARA and AMPK in TNC mice with migraine treated with xanthohumol (Xn).
[0032] (a) is the immunofluorescence image of PPARA and AMPK in each group of mice, and (b) is the statistical analysis result of immunofluorescence in each group of mice. DETAILED DESCRIPTION
[0033] The present invention will be described in detail below in conjunction with specific implementations and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementations and examples are used to illustrate the present invention, rather than to limit the present invention.
[0034] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.
[0035] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or obtained by existing methods.
[0036] The present application will be described in detail below with reference to embodiments and experimental data.
[0037] Example 1
[0038] 1. Experimental Materials
[0039] 1.1 Experimental animals
[0040] This study included 6-8 week old female C57BL / 6 mice purchased from the Experimental Animal Center of Renmin Hospital of Wuhan University. All mice were housed under specific pathogen-free (SPF) conditions with a 12-h light / dark cycle and free access to food and water. Animal experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Renmin Hospital of Wuhan University and performed in accordance with the ARRIVE guidelines.
[0041] 1.2 Experimental drugs and reagents
[0042] 3% isoflurane (provided by the Animal Experiment Center of Renmin Hospital of Wuhan University);
[0043] 1.5% isoflurane (provided by the Animal Experiment Center of Renmin Hospital of Wuhan University);
[0044] Xanthohumol was purchased from Aladdin Biochemical Technology Co., Ltd. with a purity of ≥96%. When used, it was dissolved in corn oil containing 0.5% dimethyl sulfoxide (DMSO) at a concentration of 25 mg / kg.
[0045] Inflammation soup (containing 0.1 mM prostaglandin E2, 1 mM serotonin, 1 mM histamine and 1 mM bradykinin) was purchased from Solebao Biotechnology Co., Ltd.
[0046] 1.3 Experimental instruments and equipment
[0047] Heating pad, brain stereotaxic instrument (Shenzhen Ruiwode Technology Life Science Co., Ltd.), surgical instruments, cotton balls, 5 μL pipettes, disposable syringes, dental drills and drill bits (Shenzhen Ruiwode Technology Life Science Co., Ltd.), guide cannulas, dental cement, Von Frey fiber filaments (North Coast Medical, USA), light / dark box testing equipment, etc.
[0048] 2. Experimental Methods
[0049] 2.1 Experimental groups: 8 mice in each group, including sham operation + saline (Sham + Saline) group, sham operation + xanthohumol (Sham + Xn) group, inflammatory soup + saline (IS + Saline) group, inflammatory soup + xanthohumol (IS + Xn) group.
[0050] 2.2 Construction of IS migraine mouse model
[0051] Mice were induced with 3% isoflurane and then maintained with 1.5% isoflurane. The mice were fixed on a stereotaxic apparatus and a skin incision was made in the central midline of the head to expose the skull. A small hole with a diameter of 1 mm was drilled on the skull 1 mm posterior and 1 mm lateral to the bregma, taking care not to penetrate the dura mater. The catheter was implanted in the drilled hole and fixed with dental cement. The incision was closed with sutures after surgery. After surgery, 20 μL of inflammatory soup or saline was given through the catheter every day for 7 consecutive days.
[0052] 2.3 Behavioral testing
[0053] 2.3.1 Pain behavior test:
[0054] 2.3.1.1 Spontaneous pain behaviors include head scratching and freezing behavior. Head scratching refers to the scratching of the scalp and orbital skin area by the mouse's forepaws, which is generally considered to be an aversive behavior caused by craniofacial pain. Freezing behavior is the most prominent behavior in the traditional IS model, defined as complete immobility except for breathing. Freezing behavior is expressed as the ratio (%) of freezing time to total recording time.
[0055] 2.3.1.2 Von Frey test: For the assessment of mechanical hyperalgesia, a series of standardized Von Frey filaments, starting with 0.008 g filaments, were applied continuously to the orbital region with an interval of 1-2 minutes between consecutive stimulations. If the mouse stroked the face with the ipsilateral forepaw, quickly retracted the head from the stimulation or vocalized, it was considered a positive response. If the response was negative, the mechanical stimulation was gradually increased until a positive response was obtained, and then the stimulation was reduced until a negative result was observed. The minimum force that produced a positive response was recorded as the threshold force. Each mouse was tested at least three times, and the average threshold was measured.
[0056] 2.3.2 Photophobia behavior test:
[0057] Photophobia was assessed using the light / dark box test. The light / dark box consisted of a bright and a dark compartment with a passage between the two compartments (each compartment measured 30 × 30 × 20 cm). The box was placed in a dedicated room where mice could acclimate to the environment before testing. At the beginning of the experiment, the mouse was placed in the center of the box and allowed to explore freely for a total of 5 minutes. The movement trajectory of the mouse was automatically detected. The time the mouse spent in the dark compartment was recorded to assess photophobia.
[0058] 2.4 Index detection
[0059] 2.4.1WB detection:
[0060] Brain tissue was lysed in RIPA buffer containing PMSF, protease inhibitors, and phosphatase inhibitors for 30 minutes. Protein concentration was determined using a BCA protein assay kit. Proteins were separated by SDS-PAGE gel method and transferred to PVDF membranes. After blocking the membrane with blocking solution containing 5% skim milk at room temperature for 1 hour, the membrane was incubated with the following primary antibodies overnight at 4°C: c-Fos (1:500), CGRP (1:1000), PPARA (1:3000), AMPK (1:500), p-AMPK (1:500), COX2 (1:1000), GAPDH (1:5000). Then HRP-conjugated secondary antibodies were incubated at room temperature for 1 hour. Finally, protein bands were exposed using chemiluminescence software.
[0061] 2.4.2 Immunofluorescence detection:
[0062] Frozen sections were fixed with paraformaldehyde for 15 minutes. Then, the sections were rinsed with PBS for 5 minutes and blocked with 3% bovine serum albumin (BSA) and 0.3% TritonX-100 for 1 hour. Incubated overnight at 4°C with mouse anti-c-Fos (1:200), mouse anti-CGRP (1:500), mouse anti-AMPKα1 (1:200), mouse anti-PPARA (1:200) and mouse anti-COX2 (1:200). The primary antibody was combined with an appropriate fluorescent secondary antibody. Fluorescent secondary antibodies included FITC or Cy3-labeled donkey anti-rabbit IgG (1:200) and FITC or Cy3-labeled donkey anti-mouse IgG (1:200). The sections were rinsed three times with PBS, mounted with mounting media, and observed under a fluorescence microscope.
[0063] 3. Experimental results
[0064] like Figure 2 As shown, xanthohumol treatment significantly improved pain sensitization in migraine model mice. Figure 2 a shows that the periorbital mechanical pain threshold of IS model mice was significantly lower than that of the Sham+Saline group, and xanthohumol treatment increased the periorbital pain threshold of IS model mice. Figure 2 b shows that the cold hypersensitivity score of IS model mice was significantly higher than that of Sham+Saline group, while after xanthohumol treatment, the score of IS+Xn group mice was significantly reduced and the cold hypersensitivity was alleviated. Figure 2 c and d show that the spontaneous pain behavior of IS model mice was reduced after receiving xanthohumol treatment, as shown by the significant reduction in the number of head scratching and freezing behavior time of mice in the IS+Xn group compared with those in the IS+Saline group.
[0065] like Figure 3 As shown, xanthohumol treatment alleviated the photophobia behavior of migraine model mice. Figure 3 a shows that the number of shuttles between the light and dark boxes in the IS+Saline group mice was significantly reduced, while the number of shuttles in the IS+Xn group mice was significantly increased compared with the IS+Saline group mice. Figure 3 b shows that the mice in the IS+Saline group spent more time in the dark box, while the mice in the IS+Xn group spent less time in the dark box than the mice in the IS+Saline group. Figure 3 c shows that the activity trajectories and heat maps of mice in each group also indicate that IS model mice have obvious photophobia behavior, and xanthohumol treatment can improve this phenomenon.
[0066] like Figure 4 , Figure 5 As shown, WB and immunofluorescence experiments showed that xanthohumol treatment could reduce the protein expression levels of CGRP and c-Fos in the TNC region of IS model mice and alleviate the central sensitization of migraine model mice. Figure 4 a is a representative immunoblot image. Figure 4 b is the corresponding statistical analysis graph. Figure 5 a is a representative immunofluorescence image. Figure 5 b is the corresponding statistical analysis graph.
[0067] like Figure 6 As shown, molecular docking technology verified that xanthohumol can bind to PPARA protein.
[0068] like Figure 7 , Figure 8 As shown, WB and immunofluorescence experiments showed that the IS model mice had increased PPARA and p-AMPK protein levels in the TNC region after xanthohumol treatment, activating the PPARA / AMPK pathway. Figure 7 a shows that the expression of PPARA and p-AMPK in the TNC region of IS model mice was significantly reduced compared with that of IS+Saline group mice, and the expression levels of PPARA and p-AMPK proteins were significantly increased after xanthohumol treatment.
[0069] In summary, xanthohumol treatment can alleviate central sensitization in migraine model mice by activating the PPARA / AMPK axis.
[0070] Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements that are inherent to such process, method, article, or apparatus.
[0071] Although preferred embodiments of the present invention have been described, additional changes and modifications may be made to these embodiments by those skilled in the art once the basic inventive concepts are known. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention. Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.
Claims
1. Application of xanthohumol in the preparation of a medicine for improving or treating migraine.
2. The use according to claim 1, characterized in that: The medicine also includes pharmaceutically acceptable excipients and carriers.
3. The use according to claim 1, characterized in that: The auxiliary material includes at least one of a filler, a disintegrant, a binder, a lubricant, a sweetener or a colorant.
4. The use according to claim 1, characterized in that: The dosage form of the drug includes at least one of granules, tablets, pills, capsules and injections.
5. The use according to claim 1, characterized in that: The effective dosage of the xanthohumol is 1.0 to 50 mg / kg.
6. The use according to claim 1, characterized in that: The xanthohumol significantly alleviates the migraine-like behavior of migraine model mice.
7. The use according to claim 6, characterized in that: The migraine-like behavior includes increasing the mechanical pain threshold and the cold pain threshold, improving the photophobia behavior of mice and reducing the spontaneous injury behavior of mice.
8. A drug for improving or treating migraine, characterized in that: The active ingredient of the drug includes xanthohumol.
9. The drug for improving or treating migraine according to claim 8, characterized in that: The drug also includes pharmaceutically acceptable excipients.
10. The drug for improving or treating migraine according to claim 8, characterized in that: The medicament further comprises at least one of a compound or a composition for protecting the stability of the xanthohumol.