A dual-network hydrogel for treating inflammatory bowel disease and a preparation method and application thereof

Abstract

This invention relates to the field of biomedical technology, disclosing a dual-network hydrogel for treating inflammatory bowel disease, its preparation method, and its application. The preparation method of the dual-network hydrogel is as follows: glycyrrhizic acid is dissolved in deionized water, and then β-lactoglobulin is added to obtain solution A; zinc chloride and 2,3,4-trihydroxybenzoic acid are dissolved together in deionized water to obtain solution B; solutions A and B are mixed in a certain proportion and stirred to obtain the dual-network hydrogel. The main framework of the dual-network hydrogel in this invention consists of a first-level network formed by self-crosslinking of glycyrrhizic acid and zinc ions, and a second-level network formed by crosslinking of β-lactoglobulin and 2,3,4-trihydroxybenzoic acid through Schiff bases and hydrogen bonds. This invention provides a dual-crosslinked hydrogel whose own framework is the therapeutic active ingredient, possessing good plasticity and adhesion, resisting harsh external environments, exhibiting strong anti-inflammatory capabilities, and effectively alleviating colitis in mice.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a dual-network hydrogel for treating inflammatory bowel disease, its preparation method, and its application. Background Technology

[0002] Inflammatory bowel disease (IBD) is an autoimmune disease characterized by excessive activation of the intestinal immune system. It manifests as recurrent intestinal inflammation and ulcers, with symptoms that are difficult to control, earning it the nickname "the cancer that doesn't kill." The incidence of IBD has been steadily increasing in recent years, becoming a major clinical challenge. Treatment options for IBD include systemic anti-inflammatory drugs (such as 5-aminosalicylic acid), systemic immunosuppressants, and monoclonal antibodies targeting inflammatory factors, but the efficacy is generally unsatisfactory. Even if the disease is initially controlled, secondary non-response or drug resistance can lead to treatment failure. Furthermore, due to the strict dietary restrictions caused by chronic intestinal intolerance, IBD patients commonly experience weight loss, malnutrition, and muscle atrophy, severely impacting their quality of life and increasing the likelihood of poor prognosis. To address these challenges, researchers are actively exploring novel treatment methods and nutritional supplementation strategies.

[0003] Hydrogels, when administered orally, offer promising potential for treating digestive system diseases due to their excellent shapeability and flexibility, allowing them to pass through the tubular system of the gastrointestinal tract. However, current systems mostly focus on using hydrogels as common drug carriers, which cannot escape the limitations of existing drugs themselves, and there is limited attention paid to developing gel strategies that combine new treatment methods and approaches.

[0004] Traditional Chinese medicine (TCM) is a natural treasure trove of medicinal resources, boasting advantages such as abundant yield, readily available materials, and natural ingredients. However, its application in inflammatory bowel disease (IBD) faces several limitations, including drug stability, bioavailability, resistance to the harsh gastrointestinal environment, and insufficient targeting of treatment sites, thus restricting its practical application. Furthermore, nutritional supplementation for IBD is a highly challenging issue. Patients experience recurrent abdominal pain and diarrhea, resulting in a hypersensitive gastrointestinal tract, decreased appetite, limited food choices, and reduced absorption capacity, rendering conventional nutritional supplementation methods ineffective. Currently, the development and application of hydrogels for multi-faceted treatment of IBD have not been fully realized. Therefore, developing an oral hydrogel system that bypasses existing drug treatments and provides multi-faceted therapy for IBD has become an urgent problem to be solved. Summary of the Invention

[0005] To overcome the shortcomings of existing technologies, this invention provides a "component-based treatment" hydrogel system that fully combines the stability of the hydrogel with the inherent advantages of each component to achieve excellent therapeutic effects. One component, glycyrrhizic acid (Ga), is a bioactive ingredient extracted from traditional Chinese medicine and is widely used in the treatment of diseases such as gastric ulcers and autoimmune hepatitis due to its outstanding anti-inflammatory properties. Simultaneously, the other component, β-lactoglobulin (BLG), is a highly nutritious whey protein that effectively promotes the absorption of essential elements such as calcium ions, improving the body's physiological environment through multiple pathways. Under the action of zinc ions and THBA, the two components form a dual-network structure, capable of resisting gastrointestinal damage and targeting intestinal tissue, demonstrating excellent therapeutic effects in colitis.

[0006] To achieve the above objectives, the present invention provides a method for preparing a dual-network hydrogel for treating inflammatory bowel disease, comprising the following steps:

[0007] Step 1: Dissolve glycyrrhizic acid (Ga) in deionized water, then add β-lactoglobulin (BLG) to obtain solution A;

[0008] Step 2: Dissolve zinc chloride and 2,3,4-trihydroxybenzoic acid (THBA) together in deionized water to obtain solution B;

[0009] Step 3: Mix solution A and solution B in a certain proportion and stir to react, thus obtaining a double-network hydrogel.

[0010] Furthermore, in solution A of step 1, the concentration of glycyrrhizic acid Ga is 40 mg / mL, and the concentration of β-lactoglobulin BLG is 80 mg / mL.

[0011] Furthermore, in solution B of step 2, the concentration of zinc chloride is 1 mg / mL, and the concentration of 2,3,4-trihydroxybenzoic acid (THBA) is 10 mg / mL.

[0012] Furthermore, in step 3, the volume ratio of solution A to solution B is 1:1.

[0013] To achieve the above objectives, the present invention also provides a dual-network hydrogel for treating inflammatory bowel disease, which is prepared by the above preparation method.

[0014] Furthermore, the dual-network hydrogel comprises a first network and a second network; the first network is formed by self-crosslinking of zinc ions in glycyrrhizic acid and zinc chloride, and the second network is formed by crosslinking of β-lactoglobulin and 2,3,4-trihydroxybenzoic acid (THBA) through Schiff bases and hydrogen bonds; the crosslinking density of the first and second networks is enhanced by the interaction forces formed between 2,3,4-trihydroxybenzoic acid (THBA) and β-lactoglobulin and glycyrrhizic acid.

[0015] Furthermore, in the dual-network hydrogel, the mass ratio of glycyrrhizic acid, zinc chloride, β-lactoglobulin, and 2,3,4-trihydroxybenzoic acid (THBA) is 200:5:400:50.

[0016] Furthermore, the present invention also provides the application of the above-mentioned dual-network hydrogel in the preparation of oral medications for treating inflammatory bowel disease.

[0017] To achieve the above objectives, the present invention also provides an oral medication for treating inflammatory bowel disease, the active ingredient of which is the aforementioned dual-network hydrogel.

[0018] The first network (primary network) in the dual-network hydrogel of this invention is composed of Ga and Zn. 2+ The second network (secondary network) is formed through metal coordination bonds and hydrogen bonding, with THBA and BLG connected via Schiff bases and hydrogen bonds. THBA plays a dual role in the network: it crosslinks BLG and enhances the crosslinking density of Ga through hydrogen bonding. Some THBA molecules also form bridges between BLG and Ga to strengthen the hydrogel, successfully constructing a Ga / BLG / Zn network with higher matrix stiffness and viscoelasticity than the Ga mononet. 2+ / THBA dual-network hydrogel.

[0019] Practice has shown that Ga / BLG / Zn 2+ The mixed solution of each component of / THBA can rapidly gel within 10 seconds, and compared with colorless and transparent single-component Ga and Ga / Zn... 2+ (GZ) group, Ga / BLG / Zn 2+ The / THBA (GZTB group) hydrogel is light yellow, which is due to the formation of numerous coordination bonds, hydrogen bonds, and Schiff bases between its components. Benefiting from its dual-network structure, Ga / BLG / Zn... 2+ The elastic modulus of / THBA is 40 times higher than that of the GZ group (GZ: G' 8749 Pa, G'' 555.933 Pa; GZTB: G' 326834 Pa, G'' 43782.4 Pa). Meanwhile, Ga / BLG / Zn... 2+ / THBA retains good plasticity and injectability. In summary, the dual-network hydrogel provided by this invention effectively improves the stability of the hydrogel, which is beneficial for its resistance to the abrasive impact of gastrointestinal peristalsis.

[0020] The advantages and positive effects of the dual-network hydrogel for treating inflammatory bowel disease, its preparation method, and its application described in this invention are as follows:

[0021] 1. This invention provides an orally administered Ga / BLG / Zn compound with Ga and BLG as the main components. 2+ / THBA dual-network hydrogel, constructed via hierarchical assembly: Zn 2+As anchor points, BLG forms a primary network with Ga, while BLG crosslinks with THBA to form a secondary chemical network. The two networks are bridged by THBA to form a physical-chemical hybrid dual-network hydrogel. This design allows the hydrogel to retain injectability and shapeability while significantly enhancing its tolerance to the harsh gastrointestinal environment, achieving intestinal-targeted hydrogel adhesion.

[0022] 2. In this invention, Ga / BLG / Zn 2+ / THBA can also regulate the secretion of chemokines and cytokines to achieve intestinal microenvironment balance, and promote the transformation of macrophages from M1 to M2 phenotype in the context of colitis to exert immunomodulatory functions, which is crucial for the treatment of inflammatory bowel disease. Therefore, Ga / BLG / Zn 2+ / THBA dual-network hydrogels are easy to prepare, have natural ingredients, excellent performance, and are readily available. Their therapeutic effects are comparable to first-line anti-inflammatory drugs, demonstrating great potential as a novel treatment strategy for IBD.

[0023] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0024] Figure 1 In the embodiments of the present invention, Ga / BLG / Zn 2+ / Schematic diagram of the construction mechanism of THBA dual-network hydrogel;

[0025] Figure 2 In the embodiments of the present invention, Ga / BLG / Zn 2+ / THBA dual-network hydrogel with pure Ga, Ga / Zn 2+ Zn 2+ / THBA component gelation time comparison chart, where GZ represents Ga / Zn 2+ Group, GZTB represents Ga / BLG / Zn 2+ / THBA dual-network hydrogel assembly;

[0026] Figure 3 In the embodiments of the present invention, Ga / BLG / Zn 2+ / THBA dual-network hydrogel (GZTB), Ga / Zn 2+ (GZ), FT-IR spectra of pure Ga, BLG, and THBA components;

[0027] Figure 4 In the embodiments of the present invention, Ga / BLG / Zn 2+ SEM and EDS images of the THBA dual-network hydrogel, where A is the SEM image and B is the EDS result.

[0028] Figure 5In the embodiments of the present invention, Ga / BLG / Zn 2+ / THBA dual-network hydrogel (GZTB), Ga / Zn 2+ (GZ), simple Ga hydrodynamic test results, where A is the storage modulus (G') and loss modulus (G'') of each gel under different angular velocities, and B is the storage modulus of each gel and inter-group statistical analysis under an angular frequency of 100 rad / s;

[0029] Figure 6 In the embodiments of the present invention, Ga / BLG / Zn 2+ / Injectability and plasticity studies of THBA dual-network hydrogel, where A represents the results of the injectability study and BD represents the results of the plasticity study;

[0030] Figure 7 In this embodiment of the invention, A represents Ga / BLG / Zn. 2+ / THBA dual-network hydrogel (GZTB) retention was assessed using IVIS at 4, 12, and 24 hours after gavage. B represents the reactivity with Ga / Zn. 2+ Quantitative data for (GZ) and PBS solution control groups;

[0031] Figure 8 In the embodiments of the present invention, Ga / BLG / Zn 2+ / THBA dual-network hydrogel loss in simulated digestive fluid and Ga, Zn 2+ Release curves, where A is a macroscopic image of GZTB dual-network hydrogel after immersion in various liquids from 0 to 48 hours, B is the percentage of remaining gel mass of GZTB gel at each time point after immersion, and C is the percentage of glycyrrhizic acid released by GZTB gel at each time point after immersion. In the figure, SGF is simulated gastric juice, SIF is simulated small intestinal juice, and SCF is simulated colonic juice.

[0032] Figure 9 In the embodiments of the present invention, Ga / BLG / Zn 2+ The efficacy of oral administration of THBA dual-network hydrogel (GZTB) in the treatment of inflammatory bowel disease (IBD) is shown in Figures A, B, C, DSS, and G. Figures A, B, C, DSS, DSS, and GZTB are presented. Figures B, C, DSS, DSS, and GZTB are presented. Figures D, E, F, G, G, H, I, I, and J are presented. Figures A, B, C, DSS, DSS, GZTB, GZTB are presented. Figures B, C, DSS, GZTB, GZTB are presented. Figures A, B, C, DSS, GZTB, GZTB are presented. Figures B, C, DSS, GZTB ... 2+ (GZ), BLG;

[0033] Figure 10 In this embodiment of the invention, A represents Ga / BLG / Zn. 2+ Principal component analysis of bulk-RNA seq sequencing of intestinal tissues from the THBA dual-network hydrogel group (GZTB), the healthy group (Control), and the colitis model group (DSS+0.9%NaCl) suggests that the gene transcriptome expression of the tissues after GZTB treatment is more similar to that of healthy tissues. B is a bar chart of bulk-RNA seq sequencing deconvolution analysis, and C is the ratio of pro-inflammatory M1 cells and anti-inflammatory M2 cells.

[0034] Figure 11 The results of flow cytometry analysis of colon cells from the healthy control group, the DSS+0.9%NaCl group, and the GZTB treatment group are shown. A represents total immune cells (CD45+). + B represents the proportion of all living cells; B represents the total myeloid cells (CD45). + CD11b + The proportion of total immune cells is 0; C represents neutrophils (CD45). + CD11b + Ly6G + The proportion of myeloid cells to total cells; D represents pro-inflammatory macrophages (CD45). + CD11b + Ly6G-SiglecF-CD11c-CD64 + Ly6C + The proportion of intestinal macrophages is represented by E; E represents anti-inflammatory macrophages (CD45). + CD11b + Ly6G-SiglecF-CD11c-CD206 + MHC-II + The proportion of intestinal macrophages;

[0035] Figure 12 A bar chart showing the statistical analysis results of the percentage of each immune cell group in the total immune cells, where A represents the total immune cells (CD45). + B represents the proportion of all living cells; B represents the total myeloid cells (CD45). + CD11b + The proportion of total immune cells is 0; C represents neutrophils (CD45). + CD11b + Ly6G + The proportion of myeloid cells to total cells; D represents pro-inflammatory macrophages (CD45). + CD11b + Ly6G-SiglecF-CD11c-CD64 + Ly6C +The proportion of intestinal macrophages is represented by E; E represents anti-inflammatory macrophages (CD45). + CD11b + Ly6G-SiglecF-CD11c-CD206 + MHC-II + The proportion of intestinal macrophages;

[0036] Figure 13 In the embodiments of the present invention, Ga / BLG / Zn 2+ GO enrichment analysis of differentially regulated genes in intestinal tissues of the THBA dual-network hydrogel group (GZTB), healthy group and colitis model group (DSS), where A is DSSvsControl and B is DSSvsGZTB.

[0037] Figure 14 In the embodiments of the present invention, Ga / BLG / Zn 2+ / Detection and analysis of inflammatory factors and immune cell chemokines in intestinal tissues of THBA dual-network hydrogel group (GZTB), healthy group and colitis model group (DSS), where A is the detection and analysis of inflammatory factors and B is the detection and analysis of immune cell chemokines.

[0038] Figure 15 In the embodiments of the present invention, Ga / BLG / Zn 2+ Immunohistochemical staining results and statistical analysis of intestinal IL-10 protein in the THBA dual-network hydrogel group (GZTB) and colitis model group (DSS), where A represents the immunohistochemical staining results and B represents the statistical analysis of the immunohistochemical staining results. Detailed Implementation

[0039] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0040] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0041] Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national standards. Experimental instruments, equipment, and reagents in the following embodiments that do not specify their sources are all commercially available materials.

[0042] Unless otherwise defined or stated, all technical and scientific terms used in this invention have the same meaning as those skilled in the art.

[0043] Example 1 Ga / BLG / Zn 2+Preparation of THBA dual-network hydrogel

[0044] ① Preheat 5 mL of deionized water to 60 °C, then add 200 mg of GA to dissolve it to obtain a 40 mg / mL Ga solution, and then add 400 mg of BLG to obtain solution A;

[0045] ② Dissolve 5 mg ZnCl2 and 50 mg THBA in 5 mL of deionized water to obtain solution B;

[0046] ③ Before use, take 5 mL of solution A and 5 mL of solution B and quickly stir to obtain Ga / BLG / Zn. 2+ The synthesis diagram of the THBA dual-network hydrogel is shown below. Figure 1 As shown, solutions A and B can rapidly gel within 10 seconds, as... Figure 2 As shown.

[0047] Example 2 on Ga / BLG / Zn 2+ Characterization of THBA dual-network hydrogel

[0048] (1) The chemical interactions between different hydrogels were analyzed by FT-IR (Nicolet 6700, USA). Specifically, the interactions between Ga / BLG / Zn hydrogels were analyzed. 2+ The formation of the THBA dual-network hydrogel was achieved using Zn 2+ Through metal-coordinate bonds and hydrogen bonds, THBA interacts with the carboxyl and hydroxyl groups of Ga to form a multidentate coordination structure. The secondary network is constructed by THBA providing aldehyde groups and numerous amino groups of BLG through Schiff base formation and hydrogen bonding. Simultaneously, THBA plays a dual role in the network: not only does it form a stable structure with BLG, but it also enhances the crosslinking density with the GA network through hydrogen bonding. Some THBA molecules also form bridges between BLG and GA to strengthen the hydrogel. FT-IR has verified this process, such as... Figure 3 As shown, Zn 2+ The introduction did not change the structural integrity of the GA at 1725 cm. -1 (Free carboxyl group), 1658 cm -1 (Hydrogen-bonded carboxyl groups) and 1041 cm -1 Characteristic absorption peaks were observed at the (CO stretching vibration) region; however, after the addition of BLG and THBA, a peak was observed at 1600 cm⁻¹. -1 Nearby asymmetric stretching vibration changes confirm Zn 2+ Coordination crosslinking with the carboxylate groups of each component (GA / BLG / THBA) and the increased peak width in this region indicate the formation of a broad multi-hydrogen bond network within the quaternary system, thereby enhancing the stability of the supramolecular structure, confirming the Ga / BLG / Zn... 2+ / THBA was successfully built.

[0049] (2) The Ga and Ga / Zn ratios were analyzed by scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS) (JSM-7800F, JEOL). 2+ Ga / Zn 2+ / THBA and Ga / BLG / Zn 2+ The microstructure of the THBA hydrogel was characterized, such as... Figure 4 SEM images showed that all hydrogels exhibited a porous structure, with Ga / BLG / Zn... 2+ The pore structure of the / THBA hydrogel is more uniformly distributed and more tightly interconnected. EDS analysis shows that the GZTB hydrogel is mainly composed of C, N, and O elements as its basic framework, and contains trace amounts of Cl, S, and Zn elements, confirming the successful synthesis of the hydrogel.

[0050] (3) Ga / BLG / Zn 2+ Mechanical testing of THBA dual-network hydrogel:

[0051] Rheological tests were performed on cylindrical hydrogel samples (10 mm in diameter) using a Discovery DHR-2 rheometer (TA Instruments). The experiments were conducted at room temperature in stress-controlled mode, with a frequency scan range of 0.1–100 rad / s, to detect the mechanical properties of the hydrogels. The results showed ( Figure 5 Thanks to its dual-network structure, Ga / BLG / Zn 2+ The elastic modulus of / THBA is greater than that of Ga / Zn 2+ The group's performance improved by tens of times.

[0052] (4) Ga / BLG / Zn 2+ / Injectability and plasticity of THBA dual-network hydrogel:

[0053] First, the injectability of the hydrogel was evaluated by injecting the hydrogel using a 1 mL syringe. The gel formed the letter "IBD" marking. Figure 6 (A) indicates Ga / BLG / Zn 2+ The THBA dual-network hydrogel exhibits excellent injectability, allowing it to pass smoothly through tubing without obstruction—a crucial characteristic for oral administration. Secondly, the hydrogel was poured into cruciate and star-shaped models, and its shape was observed after gel formation. The results showed ( Figure 6 (B) The hydrogel can completely fill the grooves in containers of different shapes. This discovery demonstrates the excellent shape adaptability of the dual-network hydrogel in this invention.

[0054] (5) Ga / BLG / Zn 2+ / THBA Dual-Network Hydrogel Intestinal Adhesion Capacity Assay:

[0055] Eight-week-old wild-type male C57BL / 6 mice were fasted for 24 hours and then administered fluorescein-conjugated materials via gavage. Colon tissue samples were collected at 4, 12, and 24 hours post-gavage, and residual fluorescence intensity was detected using a small animal in vivo imaging system. Results are as follows: Figure 7 The results showed that Ga / BLG / Zn ratio was observable 12 hours after administration. 2+ / THBA showed significantly enhanced adhesion within the colon.

[0056] (6) Ga / BLG / Zn 2+ / THBA dual-network hydrogel degradation characteristics:

[0057] Using simulated digestive fluids to mimic the human gastrointestinal environment, Ga / BLG / Zn 2+ The degradation characteristics of THBA dual-network hydrogels were analyzed by immersion in them. Figure 8 As shown in Figures A and B, Ga / BLG / Zn 2+ / THBA exhibits excellent tolerance to the highly acidic environment of simulated gastric juice (SGF), maintaining structural stability for over 48 hours, while Ga / Zn 2+ The hydrogel degraded by approximately 30% within 6 hours. Under near-neutral conditions (pH=6.8) of simulated small intestinal fluid (SIF) and in the presence of proteases, this Ga / BLG / Zn... 2+ / THBA gradually disintegrates within 6-24 hours, while the GZ formulation degrades rapidly within 6 hours and completely decomposes within 24 hours. Ultimately, Ga / BLG / Zn... 2+ / THBA is rapidly released in a weakly alkaline environment upon reaching the colon. Ga / BLG / Zn 2+ The anti-inflammatory component Ga in THBA showed only 1.144% release after 4 hours of immersion in SGF, with approximately 90% remaining even after 48 hours; after 4 hours of treatment in SIF, approximately 24.70% was released; however, in a simulated colon environment, the cumulative release rate of Ga reached 13.98% at 1 hour, 30.00% at 2 hours, 63.47% at 4 hours, and 91.83% at 12 hours. Zinc ion release behavior was also observed to be similar to that of Ga. Figure 8 (C) further confirmed the controlled-release capability of hydrogel in different segments of the gastrointestinal tract—releasing very little in gastric juice and rapidly in the intestinal environment.

[0058] Example 3 Ga / BLG / Zn 2+ Validation of the efficacy of THBA dual-network hydrogel in mouse colitis

[0059] (1) Establishment of mouse colitis model and Ga / BLG / Zn 2+ / THBA dual-network hydrogel therapy:

[0060] The efficacy of this treatment in vivo was evaluated using a mouse model of colitis induced by sodium dextran sulfate (DSS). DSS, as a toxin, can damage colonic epithelial cells, allowing intraluminal antigens to penetrate the mucosal layer and even reach the submucosal tissue, thereby triggering widespread inflammation.

[0061] The modeling process was as follows: 8-week-old male C57BL / 6 mice were randomly divided into groups and fed environmental adaptation for 2 days. Then, DSS was provided through free drinking water for 7 consecutive days.

[0062] Ga / BLG / Zn 2+ / THBA dual-network hydrogel treatment began one day after DSS administration via drinking water, administered by gavage at a dose of 200 μL once daily. The control groups (Ga / Zn) 2+ BLG, 5-ASA, and 0.9% NaCl were also administered via daily gavage at a dose of 200 μL. Figure 9 (A). After gavage, the mice were observed daily, and their weight, fecal morphology, and fecal blood were recorded.

[0063] The results showed that, compared with the untreated group, Ga / BLG / Zn 2+ / THBA dual-network hydrogel can effectively maintain body weight, reduce disease activity index, and relieve rectal bleeding, with efficacy comparable to first-line drug 5-ASA. Figure 9 (BD).

[0064] (2) via Ga / BLG / Zn 2+ Tissue manifestations of THBA dual-network hydrogel treatment for IBD:

[0065] Seven days after DSS-induced enteritis, intestinal and spleen samples were collected from mice to assess histopathological findings. Colonic shortening is a typical pathological manifestation of inflammation, and the measurement data indicated Ga / BLG / Zn... 2+ / THBA dual-network hydrogel treatment significantly improved DSS-induced colonic shortening ( Figure 9 (E, F).

[0066] Pathological examination (H&E staining) of intestinal tissues in each group showed that DSS severely damaged the intestinal epithelial barrier, leading to the disappearance of crypt structures, while Ga / BLG / Zn 2+ / THBA dual-network hydrogel can effectively maintain normal intestinal structure ( Figure 9 The enhanced expression of the tight junction protein Occludin further confirms its protective role in intestinal epithelial integrity. Figure 9 (H). Meanwhile, the spleen is the largest secondary immune organ and a key indicator of systemic inflammation levels. The Ga / BLG / Zn ratio was determined by calculating the spleen / body weight ratio. 2+The THBA dual-network hydrogel treatment group had the lowest values ​​among mice receiving DSS-induced colitis, even lower than the 5-ASA group, suggesting that the systemic anti-inflammatory activity of this formulation is slightly superior to that of traditional drugs. Figure 9 (I). Furthermore, quantitative polymerase chain reaction (qPCR) detection revealed Ga / BLG / Zn 2+ The mRNA expression of cytokines (IL-6, IFN-γ, IL-1β) in the colon of the THBA group was also significantly lower than that of the DSS group. Figure 9 (J). In summary, Ga / BLG / Zn 2+ / THBA dual-network hydrogel effectively relieved enteritis in an acute enteritis model.

[0067] (3) Ga / BLG / Zn 2+ / Regulation of the intestinal immune microenvironment by THBA dual-network hydrogel:

[0068] The control group was administered 0.9% NaCl by gavage 7 days after DSS induction, and the Ga / BLG / Zn ratio was... 2+ RNA sequencing analysis was performed on intestinal tissues from the THBA dual-network hydrogel treatment group and healthy mice without induced enteritis. Although the three groups showed different transcriptional characteristics, the transcriptional characteristics of the GZTB treatment group mice were closer to those of healthy mice. Figure 10 (A). Using Cibersort, a deconvolution algorithm that infers the relative proportions of specific cell types in tissues based on gene expression data, the effects of hydrogel therapy on immune cell subsets (including lymphocytes, macrophages, and neutrophils) were investigated. Results showed that myeloid-derived cells were one of the main immune cell types in the DSS group. Among myeloid cells, neutrophils and pro-inflammatory M1 macrophages were significantly increased. GZTB treatment effectively reversed this change and exhibited a characteristic transformation by inhibiting M1 polarization while simultaneously promoting the differentiation of anti-inflammatory M2 macrophages. Figure 10 (B, C). These changes were confirmed by flow cytometry. Figures 11-12 Compared with the DSS group, the proportions of total immune cells (65.48% vs 31.41%) and myeloid-derived cells (14.56% vs 9.74%) in colon tissue were significantly reduced after hydrogel treatment. Neutrophils and Ly6C were also significantly reduced in the GZTB treatment group. + The proportion of pro-inflammatory intestinal macrophages in total immune cells was only half that of the DSS group (neutrophils: 1.77% vs 2.50%; Ly6C). + Macrophages: 4.27% vs 2.51%). Conversely, CD206 + MHCII +The proportion of anti-inflammatory intestinal macrophages was significantly increased, even slightly higher than that of the healthy control group (GA-BLG vs DSS: 5.01% vs 1.99%; GA-BLG vs healthy control: 5.01% vs 4.21%).

[0069] (4) Ga / BLG / Zn 2+ THBA dual-network hydrogel inhibits excessive immune response:

[0070] Based on Ga / BLG / Zn 2+ Differentially expressed genes in the THBA treatment group, DSS-induced colitis mice, and healthy control group were analyzed using gene ontology (GO) enrichment analysis. Results are as follows: Figure 13 The results showed that biological processes related to leukocyte recruitment and immune cytokine production were significantly inhibited after treatment, especially interleukin-6 production (P=1.7e-27) and positive regulation of cytokine production in the inflammatory response (P=1.2e-07). This also affected related inflammatory factors (…). Figure 14 (A) and immune cell chemokines ( Figure 14 Heatmap clustering analysis was performed on B) to observe Ga / BLG / Zn 2+ / THBA significantly reduced the expression levels of both, bringing them close to those of the healthy control group, indicating that hydrogel treatment inhibits inflammatory pathway activation through a dual mechanism of blocking immune cell chemotaxis and suppressing pro-inflammatory capacity. IL-10 is a classic anti-inflammatory factor associated with type II anti-inflammatory immunity, and immunohistochemical staining confirmed Ga / BLG / Zn 2+ / THBA treatment significantly enhanced IL-10 expression in the longitudinal crypt structures of the intestine (including the top, middle, and bottom regions of the crypts). Figure 15 In summary, Ga / BLG / Zn 2+ / THBA regulates the homeostasis of the immune microenvironment, thereby inhibiting the recruitment of immune cells, reducing the secretion of pro-inflammatory factors, and promoting anti-inflammatory immune responses, ultimately achieving the effect of alleviating IBD.

[0071] (5) Conclusion:

[0072] This invention provides a method using Zn 2+ A dual-network hydrogel crosslinked with THBA and glycyrrhizic acid (Ga) and β-lactoglobulin (BLG) exhibits enhanced mechanical strength and intestinal mucosal adhesion while maintaining injectability and shapeability. Ga / BLG / Zn 2+ / THBA exhibits remarkable anti-inflammatory properties, with its efficacy in alleviating colitis in mice comparable to that of the first-line anti-inflammatory drug 5-ASA. Notably, this therapeutic efficacy is entirely derived from Ga / BLG / Zn. 2+ / THBA hydrogel itself is composed entirely of natural plant and food-derived ingredients, requiring no external drug loading. Ga / BLG / Zn 2+ / THBA treatment reduces the recruitment of immune cells in the intestinal mucosa and exerts a potent therapeutic effect by attenuating the release of inflammatory factors and promoting macrophage polarization towards the anti-inflammatory M2 phenotype (rather than the pro-inflammatory M1 phenotype). Ga / BLG / Zn 2+ / THBA administration promotes type II immune responses and regulates the immune homeostasis of the microenvironment. In summary, Ga / BLG / Zn 2+ / THBA, as a self-healing material, revolutionizes existing food and drug delivery models by integrating a combined strategy of enhancing anti-inflammatory activity and providing nutritional support, ultimately achieving therapeutic effects through immune regulation, demonstrating significant translational potential.

[0073] Therefore, the present invention provides a double cross-linked hydrogel whose own skeleton is the therapeutic active ingredient, which has good plasticity and adhesion, resists harsh external environments, has strong anti-inflammatory ability, can effectively relieve colitis in mice, and can be used to prepare drug formulations for the treatment of inflammatory bowel disease.

[0074] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method of preparing a dual-network hydrogel for the treatment of inflammatory bowel disease, characterized in that, Includes the following steps: Step 1: Dissolve glycyrrhizic acid (Ga) in deionized water, then add β-lactoglobulin (BLG) to obtain solution A; wherein the concentration of glycyrrhizic acid (Ga) is 40 mg / mL and the concentration of β-lactoglobulin (BLG) is 80 mg / mL. Step 2: Dissolve zinc chloride and 2,3,4-trihydroxybenzoic acid (THBA) together in deionized water to obtain solution B; wherein the concentration of zinc chloride is 1 mg / mL and the concentration of 2,3,4-trihydroxybenzoic acid (THBA) is 10 mg / mL. Step 3: Mix solution A and solution B in a certain proportion and stir to react, to obtain a double-network hydrogel; wherein the volume ratio of solution A to solution B is 1:

1. The mass ratio of glycyrrhizic acid, zinc chloride, β-lactoglobulin and 2,3,4-trihydroxybenzoic acid (THBA) in the dual-network hydrogel is 200:5:400:

50.

2. A dual network hydrogel for the treatment of inflammatory bowel disease, characterized in that: It is prepared by the preparation method described in claim 1.

3. The dual-network hydrogel of claim 2, wherein: The dual-network hydrogel comprises a first network and a second network; the first network is formed by self-crosslinking of zinc ions in glycyrrhizic acid and zinc chloride, and the second network is formed by crosslinking of β-lactoglobulin and 2,3,4-trihydroxybenzoic acid (THBA) through Schiff bases and hydrogen bonds; the crosslinking density of the first and second networks is enhanced by the interaction between 2,3,4-trihydroxybenzoic acid (THBA) and β-lactoglobulin and glycyrrhizic acid.

4. The application of the double-network hydrogel prepared by the preparation method of claim 1 or the double-network hydrogel of claim 2 or 3 in the preparation of oral medications for treating inflammatory bowel disease.

5. An oral medicament for treating inflammatory bowel disease, characterized by: Its effective component is the dual-network hydrogel prepared by the preparation method described in claim 1 or the dual-network hydrogel described in claim 2 or 3.

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