Application of quercetin in preparation of medicine for treating eosinophilic esophagitis

Abstract

The invention provides application of quercetin in preparation of a medicine for treating eosinophilic oesophagitis, and belongs to the technical field of biological medicine. The invention provides application of quercetin in preparation of a medicine for treating eosinophilic esophagitis, and intervention on the EoE pathological process is realized by systematically relieving clinical symptoms of diseases, inhibiting esophageal tissue pathological injury, blocking the fibrosis process, regulating local inflammation and immune balance and down-regulating biomarker expression. The application has the core value that quercetin is applied to EoE treatment for the first time, the treatment effect of quercetin is defined, a brand new choice is provided for an existing acidophilic cell esophagitis treatment method, and meanwhile treatment indications of quercetin are perfected.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and particularly relates to application of quercetin in preparation of a drug for treating eosinophilic esophagitis. BACKGROUND

[0002] Eosinophilic esophagitis (EoE) is a chronic esophageal inflammatory disease mediated by immunity, which is characterized by a large number of eosinophilic granulocyte infiltration in the esophageal mucosa layer, and causes clinical symptoms such as progressive dysphagia, food impaction and retrosternal pain. The disease is closely related to genetic susceptibility, environmental allergen exposure and abnormal activation of Th2 type immune response, and patients often have allergic rhinitis, asthma and other atopic diseases. With the clear definition of diagnostic criteria and the popularization of pathological detection technology, the incidence of EoE shows a significant upward trend, and has become one of the main causes of esophageal dysfunction worldwide, which continuously damages the quality of life of patients.

[0003] At present, clinical treatment mainly depends on two types of drugs: proton pump inhibitors (PPIs) can reduce esophageal mucosa stimulation by inhibiting gastric acid secretion, but about half of the patients have no histological response to double dose treatment, and there is a limitation of insufficient response rate; although glucocorticoids (such as budesonide suspension) can effectively inhibit local inflammatory response, long-term use can easily induce oral and esophageal candida infection, mucosal atrophy and even adrenal function inhibition and other adverse reactions. In recent years, the monoclonal antibody dupilumab targeting IL-4 receptor alpha subunit is approved for EoE indication in Europe and the United States by blocking IL-4 / IL-13 signaling pathway, which significantly improves the histopathology score, but the high treatment cost greatly limits its wide application, especially for chronic patients who need lifelong intervention, which constitutes a heavy economic burden. Therefore, developing new therapies with reliable efficacy, long-term safety and economic accessibility has become a key direction for current clinical breakthrough. SUMMARY

[0004] The purpose of the present application is to provide application of quercetin in preparation of a drug for treating eosinophilic esophagitis, which provides an effective means for clinical application.

[0005] In order to achieve the above-mentioned purpose of the application, the present application provides the following technical solutions:

[0006] The present application provides application of quercetin in preparation of a drug for treating eosinophilic esophagitis.

[0007] The present application also provides application of quercetin in preparation of a drug for preventing onset or recurrence of eosinophilic esophagitis.

[0008] The present application also provides application of quercetin in preparation of a drug for inhibiting eosinophilic granulocyte infiltration in the esophagus.

[0009] The application also provides application of quercetin in preparation of a drug for blocking esophageal fibrosis.

[0010] The application also provides application of quercetin in preparation of an esophageal biomarker SERPINB10 expression regulator.

[0011] The application also provides application of quercetin in preparation of a product for relieving pathological reactions related to eosinophilic esophagitis, including reducing weight loss, eating obstruction or abnormal anal temperature.

[0012] Preferably, the drug or product comprises quercetin and a pharmaceutically acceptable carrier.

[0013] Preferably, the dosage form of the drug is oral preparation, injection or spray.

[0014] The application also provides application of a pharmaceutical composition in preparation of a drug for treating eosinophilic esophagitis, wherein the pharmaceutical composition comprises quercetin and other components having therapeutic activity on eosinophilic esophagitis.

[0015] Preferably, the other components having therapeutic activity on eosinophilic esophagitis include glucocorticoid drugs, proton pump inhibitors or monoclonal antibodies.

[0016] Advantages of the application:

[0017] The application provides application of quercetin in preparation of a drug for treating eosinophilic esophagitis (EoE), which realizes intervention on pathological progress of EoE by systemically relieving clinical symptoms of the disease, inhibiting pathological damage of esophageal tissue, blocking fibrosis process, regulating local inflammation and immune balance and down-regulating expression of biomarkers. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1This diagram demonstrates that quercetin can inhibit OVA-induced EoE symptoms, including: A. Schematic diagram of EoE mouse modeling and intervention with dexamethasone and quercetin treatment; B. Percentage change in body weight of mice in different treatment groups from day 14 to day 27 (n=7); C. Change in rectal temperature of mice in different treatment groups within 40 minutes after OVA gavage challenge intervention on day 26 (n=7); D. Allergic reaction scores and symptom quantification tables of mice in different treatment groups (n=7); E. Line graph of food intake of mice in different treatment groups from day 14 to day 26 (n=7). The # symbol is used to indicate the difference between EoE group mice and blank control group mice. The symbols are used to represent the differences between the EoE group mice and the two groups of EoE mice treated with drugs. # indicates the difference between the EoE disease group and the blank control group. This indicates the difference between the drug treatment group and the EoE disease group; P<0.05， <0.01, #### and <0.0001, no significant difference in ns;

[0019] Figure 2 This study demonstrates that quercetin intervention can alleviate OVA-induced esophageal eosinophil infiltration and degranulation. A. H&E staining of esophageal mucosa in mice from different treatment groups; black arrows indicate infiltrating eosinophils (upper image ×400, lower image ×800); B. Immunofluorescence staining of eosinophils in esophageal mucosa tissue from mice from different treatment groups (CD170 antibody, red, ×400); C. Number of eosinophils per high-power field in esophageal mucosal epithelium of mice from different treatment groups (n=7); D. Electron microscopic images of eosinophils in esophageal epithelium of mice in the EoE disease group, dexamethasone treatment group, and quercetin treatment group; yellow boxes indicate intracellular crystal granules within eosinophils, blue arrows indicate intracellular cavities formed by degranulation, and red boxes indicate the tracks formed by eosinophil degranulation (scale bar: 1 μm); # indicates differences between the EoE disease group and the blank control group. Indicates the differences between the drug treatment group and the EoE disease group; ## and <0.01, no significant difference in ns;

[0020] Figure 3This study demonstrates the inhibition of quercetin on esophageal mast cell activation and collagen deposition in EoE mice, including: A. Representative images of esophageal tissue stained with toluidine blue from different treatment groups, with yellow arrows indicating mast cells (×400); B. Mast cell count per high-power field in esophageal tissue from different treatment groups (n=7); C. Tpsab1 expression in esophageal tissue from different treatment groups detected by qRT-PCR (n=7); D. Tgfb1 expression in esophageal tissue from different treatment groups detected by qRT-PCR (n=7); E. Representative images of Masson staining in esophageal tissue from different treatment groups (×100); F. Proportion of collagen fiber deposition in esophageal tissue from different treatment groups (n=7). # indicates differences between the EoE disease group and the blank control group. This indicates the difference between the drug treatment group and the EoE disease group; <0.05, #### and <0.0001, no significant difference in ns;

[0021] Figure 4 This study demonstrates that quercetin can regulate the Th1 / Th2 immune imbalance in EoE mice. Specifically: AD. qRT-PCR was used to detect the gene expression of cytokines (IL-4, IL-5, IL-13, IFN-γ) in the esophageal tissue of mice in different treatment groups (n=7); EF. qRT-PCR was used to detect the gene expression of transcription factors (T-bet, GATA3, FOXP3) in the esophageal tissue of mice in different treatment groups (n=7); # indicates the difference between the EoE disease group and the blank control group. This indicates the difference between the drug treatment group and the EoE disease group; <0.05, ##P<0.01, ### and <0.001, #### and <0.0001, no significant difference in ns;

[0022] Figure 5 This study demonstrates the inhibition of SERPINB10 protein expression in the esophagus of EoE mice by quercetin, including: A. Representative immunohistochemical images of SERPINB10 in the esophagus of mice in different treatment groups (×400); B. Quantitative analysis of SERPINB10 expression in the esophagus of mice in different treatment groups (n=7), where # indicates the difference between the EoE disease group and the blank control group. This indicates the difference between the drug treatment group and the EoE disease group; <0.01,#### P<0.0001。 Detailed Implementation

[0023] This invention provides the use of quercetin in the preparation of a medicament for treating eosinophilic esophagitis.

[0024] The applications described in this invention encompass the use of quercetin in the preparation of therapeutic agents for all interventions in the course of eosinophilic esophagitis (EoE). These treatments may include intervention strategies targeting different stages of the disease, such as: early inflammation control (e.g., initial diagnosis), symptom relief during active phases (dysphagia exacerbations), maintenance therapy for chronic inflammation (long-term disease management), complication prevention (avoiding esophageal stricture), postoperative / dilation adjuvant therapy (delaying recurrence and fibrosis progression), and salvage therapy for refractory EoE. Its therapeutic activity is manifested in multiple therapeutic endpoints, including reducing esophageal eosinophil counts, repairing mucosal damage, improving swallowing function, reversing tissue remodeling, or delaying disease progression.

[0025] This invention also provides the use of quercetin in the preparation of medicaments for the prevention of eosinophilic esophagitis (EoE) attacks or relapses. This invention includes the use of quercetin in the preparation of medicaments for the prevention of new-onset EoE (e.g., in high-risk populations) or relapses during remission. Application scenarios include intermittent maintenance dosing, pre-exposure prophylaxis, or long-term low-dose dosing strategies after symptom relief, etc.

[0026] This invention also provides the application of quercetin in the preparation of drugs that inhibit eosinophil infiltration in the esophagus. This invention relates to an inhibitory drug prepared by quercetin targeting the core pathological step of EoE—eosinophil (Eos) infiltration. It can be used in scenarios such as drug combination therapy research and model development.

[0027] The present invention also provides the use of quercetin in the preparation of drugs that block esophageal fibrosis.

[0028] This invention also provides the application of quercetin in the preparation of esophageal biomarker SERPINB10 expression regulator.

[0029] The present invention also provides the use of quercetin in the preparation of products that alleviate pathological reactions associated with eosinophilic esophagitis, including weight loss, food obstruction, or abnormal rectal temperature.

[0030] This invention covers the use of quercetin in the preparation of products that improve direct / secondary pathological responses to EoE (Electroesophageal Erectile dysfunction). These pathological responses include, but are not limited to: weight loss due to insufficient nutrient intake, malabsorption, or increased metabolic consumption caused by swallowing pain / obstruction; food obstruction due to inflammatory edema, smooth muscle dysfunction, or luminal narrowing, resulting in a feeling of solid / liquid food retention, delayed swallowing, or reflux; and abnormal rectal temperature as a marker of systemic inflammatory response (e.g., low-grade fever) or an indication of secondary infection. The products aim to alleviate these symptoms by reducing local inflammation, improving esophageal motility, repairing the mucosal barrier, or modulating nerve sensitivity.

[0031] Preferably, the drug or product comprises quercetin and a pharmaceutically acceptable carrier.

[0032] In a preferred embodiment of the present invention, the quercetin drug / product needs to be compatible with a pharmaceutical carrier, which includes, according to its function, a solubilizer (such as cyclodextrin, phospholipid complex), a stabilizer (antioxidant BHT / ascorbic acid), an absorption promoter (bile salts, chitosan), a sustained-release material (HPMC, PLGA) or a taste improver (sweetener, flavoring agent) to meet the needs of different dosage forms.

[0033] Preferably, the dosage form of the drug is an oral preparation, an injection, or a spray. Specifically, the dosage forms include:

[0034] Oral preparations: enteric-coated tablets, sustained-release capsules, oral liquids, chewing gums, or suspensions;

[0035] Injectable preparations: intravenous injection solutions (containing solubilizing excipients), subcutaneous injection suspensions, or intravenous infusion powders for injection;

[0036] Sprays: oral mucosal spray, posterior pharyngeal wall targeted spray, or nebulized inhalation solution (acting on the esophagus after swallowing).

[0037] The present invention also provides the use of a pharmaceutical composition in the preparation of a medicament for treating eosinophilic esophagitis, the pharmaceutical composition comprising quercetin and other components having therapeutic activity against eosinophilic esophagitis.

[0038] This invention relates to a synergistic combination of quercetin and other active ingredients, comprising:

[0039] Glucocorticoids: localized (e.g., budesonide suspension) or systemic (e.g., prednisone);

[0040] Proton pump inhibitors (PPIs): Omeprazole, esomeprazole, etc. (used for PPI-responsive EoEs);

[0041] Monoclonal antibodies: targeting IL-4Rα (duprilmab), IL-5 (meplenumab) or TSLP (teplenumab).

[0042] Other: mast cell stabilizers (sodium cromoglycate), dietary intervention adjuvants (amino acid formulations), or JAK inhibitors.

[0043] Combination therapies include improving efficacy (synergistic anti-inflammatory effects), overcoming drug resistance (multi-pathway blockade), reducing monotherapy toxicity, or simplifying dosing regimens, among others.

[0044] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0045] Example

[0046] I. Materials

[0047] 1. Experimental apparatus

[0048] Tissue dehydrator (ASP300, Leica Instruments GmbH, Germany); Paraffin embedding machine (EG1150C, Leica Instruments GmbH, Germany); Paraffin microtome (RM2016, Leica Instruments GmbH, Germany); Tissue spreader (KD-P, Zhejiang Jinhua Kedi Instrument Equipment Co., Ltd., China); Freezing stage (JB-L5, Wuhan Junjie Electronics Co., Ltd., China); Oven (DHG-9140A, Shanghai Huitai Instrument Manufacturing Co., Ltd., China); Microwave oven (MKJ-J1N, Qingdao Maikewei Microwave Innovation Technology Co., Ltd., China); Decolorizing shaker (WD-9405A, Beijing Liuyi Instrument Factory, China); Vortex mixer (TYXH-II, Tianyue Electronics, China); Pipette (Eppendorf, Germany); Histochemistry pen (GT1001, Genetech, USA); Upright optical microscope (NIKON ECLIPSE CI, Nikon, Japan); Inverted fluorescence microscope (NIKON ECLIPSE). TI-SR, Nikon, Japan; Imaging system (NIKON DS-U3, Nikon, Japan); Mouse weight balance (B10500, Yuyan Instruments Co., Ltd., China).

[0049] 2. Experimental reagents and consumables

[0050] 10ul, 200ul, and 1000ul pipette tips (Axygen, USA); 15ml and 50ml centrifuge tubes (NEST, China); 1.5ml colorless sterile EP tubes (Axygen, USA); glass slides and coverslips (Merck, Germany); anhydrous ethanol, hydrochloric acid, ammonia, and neutral resin (Sinopharm Chemical Reagent Co., Ltd., China); xylene (Sinopharm Chemical Reagent Co., Ltd., China); EDTA antigen retrieval solution, citrate antigen retrieval solution, and PBS buffer (Shanghai Ruibaohe Biotechnology Co., Ltd., China); DEPC Water (R0021, Shanghai Beyotime Biotechnology Co., Ltd., China); Hydrogen peroxide (10011218, Sinopharm Chemical Reagent Co., Ltd., China); Goat serum for blocking (SB-IHC100, Shanghai Sangon Biotech Co., Ltd., China); BSA (A8020, Beijing Solarbio Technology Co., Ltd., China); Primary antibody diluent (CST, USA); Secondary antibody (immunohistochemistry) (Long Island Biotechnology Co., Ltd., China); Hematoxylin staining solution (C0107, Shanghai Beyotime Biotechnology Co., Ltd., China); Hematoxylin-eosin staining kit (C0105S, Shanghai Beyotime Biotechnology Co., Ltd., China); Toluidine blue staining kit (E670105, Sangon Biotech, China); DAPI staining solution (C1005, Shanghai Beyotime Biotechnology Co., Ltd.); DAB horseradish peroxidase chromogenic kit (P0203, Shanghai Beyotime Biotechnology Co., Ltd., China); Masson Trichrome staining kit (G1340, Beijing Solarbio Science & Technology Co., Ltd., China); Anti-fluorescence quenching mounting solution (HY-K1042, MCE, USA); Anti-SERPINB10 antibody (immunohistochemistry, 21719-1-AP, Proteintech, China); Anti-CD170 antibody (immunofluorescence, 14-1702-82, Invitrogen, USA); Calcipotriol MC903 (HY-10001, MCE, USA); Ovalbumin (A5503, Sigma-Aldrich, USA); Dexamethasone (HY-14648, MCE, USA); Quercetin (HY-18085, MCE, USA); Sodium carboxycarboxylate powder (HY-Y0703, MCE, USA); 4% paraformaldehyde (abs9179, Univita, China); RNALater (R0118, Shanghai Beyotime Biotechnology Co., Ltd., China). Gluta fixative (for electron microscopy, BL911A, Biosharp, China); Trizol reagent (15596018CN, Invitrogen, USA); Reverse transcription kit (RR037A, Takara, Japan); TB Green® Premix Ex Taq™ (Tli RNaseHPlus), ROX plus (RR42LR, Takara, Japan).

[0051] II. Experimental Methods

[0052] 1. Animal experiments

[0053] 1) Purchase and raising of laboratory animals

[0054] SPF-grade male BALB / c mice aged 8-12 weeks were purchased from Shanghai Bikaikeyi Biotechnology Co., Ltd. and housed in the animal facility of Shanghai Ruitaimos Biotechnology Co., Ltd. The experimental animals were managed under standardized conditions in an SPF-grade barrier environment. Their housing system was equipped with independent ventilation systems to maintain air cleanliness, a 12-hour diurnal light cycle, and their food intake was changed daily and recorded. Bedding and water were changed twice a week. The animal research protocol has been approved by the Animal Welfare and Ethics Review Committee of Shanghai Ruitaimos Biotechnology Co., Ltd., IACUC approval number: 20241112-11.

[0055] 2) Construction of EoE animal models

[0056] Male BALB / c mice aged 8-12 weeks were randomly assigned to a control group, an EoE disease group, a dexamethasone treatment group, and a quercetin treatment group. Mice were treated daily with 2 nmol MC903 (dissolved in 20 μL of 100% ethanol) and PBS solution containing 100 μg OVA, applied to both inner and outer sides of the ear for 14 consecutive days. On days 14, 17.5, 18, 20, 22, 24, and 26, mice were administered 50 mg OVA via gavage, while continuously receiving 1.5 g / L OVA in drinking water. The control group received the same amount of PBS solution for skin stimulation and gavage.

[0057] 3) Drug intervention in EoE mice

[0058] The drug solution used for oral administration to mice was prepared using a 0.5% sodium carboxymethyl cellulose solution (dissolved in physiological saline to form a homogeneous suspension). The solution was prepared fresh for each use, and ultrasound-assisted dissolution could be used if necessary. Starting from day 14, mice in the treatment group were administered dexamethasone at a dose of 0.5 mg / kg and quercetin at a dose of 50 mg / kg daily by gavage until sacrifice. The blank control group and the EoE disease group received an equal volume of 0.5% sodium carboxymethyl cellulose solution by gavage.

[0059] 4) Recording of functional indicators in mice

[0060] During the allergic reaction provocation phase in the latter half of the modeling process (days 14-26), the weight changes and food intake of the mice were recorded daily. Each mouse's weight was measured three times a day, and the average of the three measurements was recorded. The rectal temperature of the mice was recorded immediately and 40 minutes after the last OVA provocation intervention (day 26). The symptoms of the mice during the modeling process were observed and recorded to establish a systemic allergic reaction score (scoring criteria: no obvious allergic reaction symptoms = 0 points; scratching the nose or scalp = 1 point; diarrhea, perioral edema, and decreased activity = 2 points; cyanosis of the lips and tail, difficulty breathing, and significantly decreased activity = 3 points; no response or only mild response to external stimuli, limb tremors, spasms, and hypothermia = 4 points; death = 5 points).

[0061] 5) Mouse euthanasia and tissue collection

[0062] The animal experiment ended on day 27 after modeling. Mice were euthanized by cervical dislocation, and esophageal tissue was collected under a microscope. The esophageal tissue was isolated, and a portion was fixed and preserved in a general tissue fixative (4% paraformaldehyde); another portion was immersed in RNALater reagent and stored overnight at 4°C, then stored at -80°C; and a third portion was preserved in electron microscopy fixative.

[0063] 2. Paraffin embedding of esophageal tissue

[0064] Fresh esophageal tissue was fixed in 4% paraformaldehyde for more than 24 hours. After being removed from the fixative, the tissue was trimmed and placed in a dehydration box. It was then dehydrated with a series of alcohols, embedded in an embedding machine, cooled on a freezing stage, and the paraffin block was removed and trimmed. The paraffin block was placed on a paraffin sectioner, and paraffin sections with a thickness of 4 μm were cut. The floating sections were flattened on a 40°C warm water plate, picked up with a glass slide, and placed in a 60°C oven for baking. They were then stored at room temperature for subsequent staining.

[0065] 3. H&E staining

[0066] Paraffin sections were sequentially immersed in xylene I for 20 minutes, xylene II for 20 minutes, anhydrous ethanol I for 10 minutes, anhydrous ethanol II for 10 minutes, 95% ethanol for 5 minutes, 80% ethanol for 5 minutes, and 70% ethanol for 5 minutes, followed by washing with distilled water. The sections were then stained with hematoxylin for 5 minutes, rinsed with tap water to remove excess stain, differentiated with 1% hydrochloric acid alcohol for a few seconds, rinsed again with tap water, and then blued with 0.6% ammonia solution, followed by rinsing with running water. The sections were then stained with eosin for 2 minutes. Finally, the sections were sequentially immersed in 95% ethanol I for 5 minutes, 95% ethanol II for 5 minutes, anhydrous ethanol I for 5 minutes, anhydrous ethanol II for 5 minutes, xylene I for 5 minutes, and xylene II for 5 minutes to complete dehydration. After air-drying, the sections were mounted with neutral resin. Microscopic observation and image acquisition were performed; the nuclei appeared blue, and the cytoplasm appeared pink or red.

[0067] 4. Immunohistochemical staining

[0068] Esophageal FFPE samples from each group of the validation cohort were placed in a -20°C freezer for approximately 2 hours. Paraffin tissue sections with a thickness of 4 μm were then cut using a paraffin microtome. The sections were fixed onto glass slides and baked in a 60°C oven for approximately 1 hour. The sections were then immersed sequentially in xylene I and xylene II for 15 minutes each; in anhydrous ethanol I and anhydrous ethanol II for 5 minutes each; and in 85% ethanol and 75% ethanol for 5 minutes each, followed by rinsing with distilled water three times for 5 minutes each time. The sections were then placed in a retrieval cassette, completely submerged in citrate buffer (pH 6.0), and subjected to antigen retrieval in a microwave oven, avoiding excessive evaporation of the buffer. After retrieval, the sections were allowed to cool naturally and washed three times with PBS buffer (pH 7.4) for 5 minutes each time on a shaker. Finally, they were immersed in 3% H2O2 solution at room temperature in the dark for 25 minutes, followed by washing with PBS buffer three times as before. After drying the sections, use a hydrophobic histochemical pen to delineate the tissue area and add 10% sheep serum for blocking, ensuring complete coverage of the sample. Block at room temperature for 30 minutes. Gently shake off the blocking solution, add the pre-prepared primary antibody dilution solution according to the instructions to the sections, place them flat in a humidified chamber, and incubate overnight at 4°C. After incubation at 4°C, bring the sections to room temperature, shake off the primary antibody dilution solution, and wash them three times in PBS solution (pH 7.4), 5 minutes each time. After drying the sections, add universal secondary antibody to the circle to cover the tissue and incubate at room temperature for 50 minutes. Gently shake off the secondary antibody on the surface of the sections, wash them three times in PBS solution (pH 7.4), 5 minutes each time, shake off the sections, and add the prepared DAB staining solution to the circle. Observe and control the staining time under a microscope. When a distinct brownish-yellow color appears under the microscope, rinse with tap water to stop the staining and record the staining time. Stain the tissue sections with hematoxylin for 3 minutes, rinse off excess staining solution with running water, and treat with 1% hydrochloric acid alcohol for 10 minutes. After rinsing with running water for 6 seconds, the slides were treated with ammonia for blueing. The slides were then sequentially immersed in 75% ethanol and 85% ethanol for 6 minutes each, followed by anhydrous ethanol I and anhydrous ethanol II for 6 minutes each, and xylene I for 5 minutes for dehydration and clearing. After air drying, the slides were mounted with neutral resin. Observation was performed under a high-power optical microscope. Due to the extremely small size of esophageal biopsy tissue, three fields of view were randomly selected from each slide for image acquisition, ensuring that each image filled the entire field of view. Image-Pro Plus software was used to quantify the integrated option density (IOD) value of each image, and the average value of multiple fields of view was taken for each sample.

[0069] 5. Immunofluorescence staining

[0070] The steps for dewaxing paraffin sections to water, blocking with BSA, and adding primary and secondary antibodies are the same as for immunohistochemical staining and will not be detailed here. For antigen retrieval, EDTA antigen retrieval buffer at pH 9.0 is used. Slides are placed in PBS buffer and washed three times on a decolorizing shaker for 5 minutes each time. After drying the sections, DAPI staining is added, and the sections are incubated at room temperature for 10 minutes in the dark. The washing steps are repeated, and after drying, the sections are mounted with anti-fluorescence quenching mounting medium. Images are observed and acquired under an inverted fluorescence microscope. DAPI staining of cell nuclei appears blue, and red fluorescent pigment labeling indicates relevant positive expression.

[0071] 6. Masson staining

[0072] Paraffin sections were routinely dewaxed to water, stained with Weigert iron hematoxylin staining solution from the Masson trichrome staining kit for 5 minutes, differentiated with 1% hydrochloric acid alcohol, washed with water to regain blue color, stained with Ponceau S and fuchsin for 10 minutes, rinsed with distilled water, washed with phosphomolybdic acid solution for 1-2 minutes, counterstained directly in aniline blue staining solution for 5 minutes, and treated with 1% glacial acetic acid for 1 minute to complete differentiation, sections were routinely dehydrated and mounted, observed under a microscope and images were acquired. Blue indicates collagen fiber deposition, and the results were quantified using ImageJ FIJI software.

[0073] 7. Toluidine blue staining

[0074] Paraffin sections were routinely dewaxed to water, diluted and stained according to the kit instructions, and prepared fresh each time. Staining was performed with 0.1% toluidine blue solution at room temperature for 10 minutes; sections were rinsed with distilled water for 5 minutes; differentiation was performed using 0.5% glacial acetic acid, and cell nuclei and granules were clearly visible; after routine dehydration, xylene clearing, and mounting with neutral resin, the sections were observed under a microscope and images were acquired. Mast cells appeared purplish-red, and the background was light blue or colorless.

[0075] 8. Transmission electron microscopy

[0076] In this study, the transmission electron microscopy (TEM) examination of esophageal tissue from experimental animals was technically supported by Shanghai Ruibaohe Biotechnology Co., Ltd. The steps are briefly described below: The tissue sampling process should be carried out at low temperatures as much as possible. The tissue was rinsed with 4% pre-cooled physiological saline and placed in a penicillin vial containing pre-cooled electron microscopy fixative. The sample was then aspirated until it settled to the bottom. Stepwise dehydration (using acetone and sterile water as a dehydrating agent), infiltration embedding, and sectioning were performed sequentially. The tissue sections were stained with uranyl acetate and lead citrate, and observed using a JEOL-1230 TEM. Images were acquired using a Gatan Bioscan Camera 792.

[0077] 9. Real-Time Quantitative Reverse Transcription PCR (qRT-PCR)

[0078] 1) RNA extraction from esophageal tissue

[0079] Remove mouse esophageal tissue frozen at -80℃, thaw and discard the RNA layer; add 2 stainless steel grinding beads and 1 ml Trizol reagent to an EP tube, and homogenize the tissue using a tissue homogenizer; transfer the tissue homogenate to a sterile EP tube, add 200 μl Trizol, vortex for 10-15 seconds; let stand at room temperature for 3 minutes, then centrifuge at 12000 rpm for 15 minutes at 4℃; aspirate the clear layer liquid to a new sterile EP tube, add 600 μL isopropanol, vortex thoroughly, and place in a -20℃ freezer for 2 hours to allow complete RNA precipitation; centrifuge at 12000 rpm for 15 minutes at 4℃, the precipitate at the bottom of the tube is RNA; discard the supernatant, add 1 ml of 75% ethanol diluted with DEPC water to each tube, centrifuge at 7500 rpm for 5 minutes at 4℃; repeat the above washing steps, discarding the supernatant; aspirate as much liquid as possible from the tube, and transfer the EP tube to a sterile EP tube. Invert the tube onto filter paper and place it in the air vent of a clean bench to dry. Add DEPC water to the sterile EP tube to fully dissolve the RNA, and use Nanodrop to determine the RNA concentration.

[0080] 2) mRNA reverse transcription

[0081] Configure the reaction system according to the kit requirements, and set the PCR instrument reaction conditions as follows: 37℃, 15 minutes; 85℃, 5 seconds.

[0082] 3) Real-time quantitative PCR

[0083] We used the NCBI gene bank and the PrimerBank online platform, along with previous literature, to design conventional primers.

[0084] Prepare the reaction system according to the TB Green® Premix Ex Taq™ kit requirements.

[0085] Samples were loaded into 96-well plates, and the final PCR reaction was performed using the StepOnePlus™ Real-Time PCR system. The program was set as follows: Holding stage (95℃, 30 seconds, 1 cycle) and Cycling stage (95℃, 5 seconds, 60℃, 30 seconds, 40 cycles). β-actin was used as an internal reference gene, and the relative expression level of the target gene was calculated using 2-ΔΔCt.

[0086] III. Statistical Analysis Methods

[0087] Data were statistically analyzed and plotted using GraphPad Prism 10 software. The Shapiro-Wilk test was used to test normality; normally distributed data were described using mean ± standard deviation, while non-normally distributed data were described using the median. For component comparisons of two independent samples, if the data were normally distributed, a t-test was used, with Welch's correction applied if variances were unequal; if the data were not normally distributed, the Mann-Whitney test was used. For comparisons between multiple independent samples, if the data were normally distributed and passed the homogeneity of variance test, one-way ANOVA was used; if the data were normally distributed but did not pass the homogeneity of variance test, Welch ANOVA was used; if the data were not normally distributed, the Kruskal-Wallis test was used. P < 0.05 indicates statistical significance, and # indicates the difference between the EoE disease group and the blank control group. This is used to represent the differences between the drug treatment group and the EoE disease group. The specific meaning can be seen in the figure caption. ns represents no statistical difference.

[0088] The results show:

[0089] Quercetin treatment can significantly alleviate symptoms of weight loss, food obstruction, and allergic reactions associated with eosinophilic esophagitis, with efficacy comparable to dexamethasone. (See appendix) Figure 1 Meanwhile, quercetin can effectively improve EoE-specific histological lesions, specifically manifested as reduced eosinophil infiltration in the esophageal epithelium and inhibition of eosinophil degranulation, see Appendix. Figure 2 .

[0090] In blocking the progression of fibrosis, quercetin can downregulate the expression of Tpsab1 and the cytokine TGF-β1 at the mRNA level in esophageal tissue, while significantly reducing the proportion of collagen fiber deposition. It has been confirmed that it can inhibit esophageal fibrosis formation by regulating the function of esophageal eosinophils and mast cells, reducing the production of TGF-β1, thereby alleviating the damage of inflammatory response to the normal structure and function of the esophagus and relieving its clinical symptoms. (See appendix) Figure 3 .

[0091] Furthermore, quercetin can restore the Th1 / Th2 immune balance. T-bet and GATA3 are core transcription factors mediating Th1 and Th2 cell differentiation, respectively. Quercetin treatment can significantly inhibit the high expression level of esophageal Gata3 mRNA induced by EoE and, to some extent, restore the expression level of Tbx21 mRNA, thereby correcting the Th1 / Th2 immune imbalance. In addition, quercetin can alleviate local esophageal inflammation and reverse the upward trend in the expression of inflammatory cytokines IL-4, IL-5, and IL-13 genes. (See appendix) Figure 4 .

[0092] Notably, quercetin significantly downregulated the expression of SERPINB10, an independent biomarker for EoE. SERPINB10, an EoE biomarker independent of the number of eosinophils infiltrating the esophagus and the comorbidity of reflux esophagitis, has been shown to have high specific expression in the esophageal tissue of EoE patients. The results showed that quercetin treatment significantly downregulated the expression of this protein in EoE esophageal tissue, further supporting its targeted therapeutic effect. (See appendix) Figure 5 .

[0093] The aforementioned synergistic effects through multiple pathways make quercetin a potentially safer and more economical alternative to EoE treatment than hormones.

[0094] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle 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 quercetin in the preparation of drugs for treating eosinophilic esophagitis, characterized in that, The treatment involves downregulating SERPINB10 expression in esophageal tissue.

2. The use of quercetin in the preparation of drugs for preventing the onset or recurrence of eosinophilic esophagitis, characterized in that, The treatment involves modulating the Th1 / Th2 immune balance in esophageal tissue, wherein the modulation includes increasing T-bet expression and / or decreasing GATA3 expression.

3. The application of quercetin in the preparation of drugs that inhibit esophageal eosinophil infiltration, characterized in that, The treatment includes inhibiting mast cell activation in esophageal tissue, wherein the inhibition is manifested as a reduction in Tpsab1 expression.

4. The use of quercetin in the preparation of drugs to block esophageal fibrosis, wherein the treatment includes inhibiting TGF-β1 expression and collagen deposition in esophageal tissue.

5. Application of quercetin in the preparation of esophageal biomarker SERPINB10 expression regulator.

6. The application of quercetin in the preparation of products that alleviate pathological reactions associated with eosinophilic esophagitis, characterized in that, The pathological reactions associated with eosinophilic esophagitis include weight loss, food obstruction, or abnormal rectal temperature.

7. The application according to any one of claims 1 to 6, characterized in that, The drug or product contains quercetin and a pharmaceutically acceptable carrier.

8. The application according to claim 7, characterized in that, The drug is available in oral, injectable, or spray form.

9. The use of a pharmaceutical composition in the preparation of a medicament for treating eosinophilic esophagitis, characterized in that, The pharmaceutical composition contains quercetin and other ingredients that have therapeutic activity against eosinophilic esophagitis, and the composition is used to downregulate SERPINB10 expression in esophageal tissue.

10. The application according to claim 7, characterized in that, Other ingredients with therapeutic activity against eosinophilic esophagitis include glucocorticoids, proton pump inhibitors, or monoclonal antibodies.

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