Double-network hydrogel for treating inflammatory bowel disease as well as preparation method and application of double-network hydrogel

Abstract

The invention relates to the technical field of biological medicine, and discloses dual-network hydrogel for treating inflammatory bowel disease as well as a preparation method and application of the dual-network hydrogel. The preparation method of the double-network hydrogel comprises the following steps: dissolving glycyrrhizic acid into deionized water, and then adding beta-lactoglobulin to obtain a solution A; dissolving zinc chloride and 2, 3, 4-trihydroxybenzoic acid in deionized water to obtain a solution B; mixing the solution A and the solution B in proportion, and stirring for reaction to obtain the double-network hydrogel. A main framework of the double-network hydrogel is composed of a first-stage network formed by glycyrrhizic acid and zinc ions through self-crosslinking and a second-stage network formed by beta-lactoglobulin and 2, 3, 4-trihydroxybenzoic acid through Schiff base and hydrogen bond crosslinking. The invention provides the dual-crosslinked hydrogel with the skeleton being a therapeutic effective component and good plasticity and adhesion, and the dual-crosslinked hydrogel has the advantages of resistance to external severe environment and strong anti-inflammatory ability, and can effectively relieve mouse colitis.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a double-network hydrogel for treating inflammatory bowel disease and a preparation method and application thereof. BACKGROUND

[0002] Inflammatory bowel disease (IBD) is an autoimmune disease characterized by excessive activation of intestinal immunity, which is manifested as recurrent intestinal inflammation and ulceration, symptoms are difficult to control, and is called "incurable cancer". In recent years, the incidence of IBD has been increasing, which has become a major challenge in clinical treatment. The treatment options for inflammatory bowel disease include systemic anti-inflammatory drugs (such as 5-aminosalicylic acid), systemic immunosuppressants, and monoclonal antibodies targeting inflammatory factors, but the therapeutic effect is not ideal. Even if the initial condition is controlled, secondary non-response or drug resistance may still occur, leading to treatment failure. In addition, due to strict dietary restrictions caused by chronic intestinal intolerance, inflammatory bowel disease patients generally have weight loss, malnutrition and muscle atrophy, which seriously affect the quality of life and are more likely to lead to adverse prognosis. In the face of these challenges, researchers are actively exploring new treatment methods and nutritional supplement strategies.

[0003] Oral administration of hydrogel has good plasticity and flexibility, and can pass through the gastrointestinal tract system, showing new prospects in the field of digestive system disease treatment. However, most current systems focus on using hydrogel as a common drug carrier, which cannot escape the limitations of existing drugs, and there is limited attention on developing gel strategies that combine new treatment methods and new treatment ideas.

[0004] Chinese herbal medicine is a natural treasure trove of medicine, with the advantages of abundant production, easy availability of materials, and natural substances. However, its application in inflammatory bowel disease has several limitations, including drug stability, bioavailability, severe gastrointestinal environment resistance, and lack of treatment site targeting, which limit its practical application. In addition, nutritional supplementation for inflammatory bowel disease is a highly difficult problem. Due to repeated abdominal pain and diarrhea, the gastrointestinal tract is in a hypersensitive state, which reduces the desire to eat, limits food choices, and reduces gastrointestinal absorption capacity, so ordinary nutritional supplementation methods have limited effect. At present, the development of hydrogels that simultaneously achieve multi-angle treatment in inflammatory bowel disease has not been fully developed and applied. Therefore, how to develop an oral hydrogel system that escapes existing treatment drugs and treats inflammatory bowel disease from multiple angles has become a problem to be solved. SUMMARY

[0005] In order to overcome the deficiencies of the prior art, the present application provides a "composition is treatment" hydrogel system, which fully combines the stability of the hydrogel and the advantages of the components themselves, and plays a good therapeutic effect. One of the components, glycyrrhizic acid (Ga), is a bioactive component extracted from traditional Chinese medicine, which is widely used in the treatment of gastric ulcer, autoimmune hepatitis and other diseases due to its excellent anti-inflammatory effect. At the same time, the other component, beta-lactoglobulin (BLG), is a whey protein with high nutritional value, which can effectively promote the absorption of essential elements such as calcium ions and improve the physiological environment of the body in multiple ways. The two form a double network structure under the action of zinc ions and THBA, which can resist the impact of the gastrointestinal tract, target the intestinal tissue, and show excellent efficacy in colitis.

[0006] To achieve the above purpose, the present application provides a preparation method of a double network hydrogel for treating inflammatory bowel disease, comprising the following steps: Step 1, dissolve glycyrrhizic acid Ga into deionized water, then add beta-lactoglobulin BLG to obtain solution A; Step 2, dissolve zinc chloride and 2,3,4-trihydroxybenzoic acid THBA in deionized water to obtain solution B; Step 3, mix solution A and solution B in proportion, stir and react to obtain a double network hydrogel.

[0007] Further, in solution A of step 1, the concentration of glycyrrhizic acid Ga is 40 mg / mL, and the concentration of beta-lactoglobulin BLG is 80 mg / mL.

[0008] Further, 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.

[0009] Further, in step 3, the volume ratio of solution A to solution B is 1:1.

[0010] To achieve the above purpose, the present application also provides a double network hydrogel for treating inflammatory bowel disease, which is prepared by the above preparation method.

[0011] Further, the double network hydrogel comprises a first network and a second network; the zinc ions in glycyrrhizic acid and zinc chloride form a first network through self-crosslinking, and beta-lactoglobulin and 2,3,4-trihydroxybenzoic acid THBA form a second network through Schiff base and hydrogen bond crosslinking; the first network and the second network enhance the crosslinking density through the interaction force formed by 2,3,4-trihydroxybenzoic acid THBA and beta-lactoglobulin and glycyrrhizic acid.

[0012] Further, in the double network hydrogel, the mass ratio of glycyrrhizic acid, zinc chloride, beta-lactoglobulin and 2,3,4-trihydroxybenzoic acid THBA is 200:5:400:50.

[0013] 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.

[0014] 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.

[0015] 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.

[0016] 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.

[0017] 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: 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 the anchor point and Ga form the primary network, BLG and THBA cross-link to form a chemical secondary network, and the two are bridged by THBA to form a physical-chemical hybrid double network hydrogel. This design significantly enhances the tolerance of the hydrogel to the harsh environment of the gastrointestinal tract while retaining the ability to be injected and shaped, achieving intestinal-targeted hydrogel adhesion.

[0018] 2、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 environment of colitis to play an immunoregulatory function, which is crucial for the treatment of inflammatory bowel disease. Therefore, Ga / BLG / Zn 2+ / THBA double network hydrogel is simple to prepare, has natural ingredients, excellent performance and is easy to obtain, and its therapeutic effect is comparable to that of first-line anti-inflammatory drugs, showing great potential as a new treatment strategy for IBD.

[0019] The technical solutions of the present application will be further described in detail below with the help of the accompanying drawings and examples. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 The construction mechanism of Ga / BLG / Zn 2+ / THBA double network hydrogel in the embodiments of the present application is shown in the figure; Figure 2 Ga / BLG / Zn 2+ / THBA double network hydrogel, simple Ga, Ga / Zn 2+ , Zn 2+ / THBA component gelation time comparison chart, wherein GZ represents Ga / Zn 2+ group, GZTB represents Ga / BLG / Zn 2+ / THBA double network hydrogel group; Figure 3 Ga / BLG / Zn 2+ / THBA double network hydrogel (GZTB), Ga / Zn 2+ (GZ), simple Ga, BLG, THBA component FT-IR spectrum in the embodiments of the present application; Figure 4 Ga / BLG / Zn 2+ / THBA double network hydrogel in the embodiments of the present application is shown in the figure, wherein A is a scanning electron microscope SEM graph, and B is an EDS result; Figure 5 Ga / BLG / Zn 2+ / THBA double network hydrogel (GZTB), Ga / Zn 2+(GZ), the simple Ga fluid mechanics detection results, wherein A is the storage modulus (G') and the loss modulus (G'') of each gel under different angular velocity conditions, B is the storage modulus of each gel and the statistical analysis between groups under the condition of angular frequency of 100 rad / s; Figure 6 Ga / BLG / Zn 2+ / THBA double network hydrogel in the embodiment of the present application Figure 7 A is Ga / BLG / Zn 2+ / THBA double network hydrogel (GZTB) in the embodiment of the present application, B is the retention condition after intragastric administration for 4 hours, 12 hours and 24 hours, and IVIS is used for detection, and C is the quantitative data of Ga / Zn 2+ (GZ) and PBS solution control group; Figure 8 Ga / BLG / Zn 2+ / THBA double network hydrogel in the embodiment of the present application 2+ release curve, wherein A is the macroscopic general picture of GZTB double network hydrogel after immersion in each liquid for 0 to 48 hours, B is the remaining gel mass percentage of GZTB gel at each time point after immersion, C is the glycyrrhizic acid release percentage of GZTB gel at each time point after immersion, and SGF is simulated gastric juice, SIF is simulated small intestinal juice, and SCF is simulated colon juice; Figure 9 Ga / BLG / Zn 2+ / THBA double network hydrogel (GZTB) in the embodiment of the present application 2+ (GZ), BLG; Figure 10 A is Ga / BLG / Zn 2+Figure 6 shows the results of principal component analysis of bulk-RNA seq of intestinal tissue of GZTB group (GZTB), healthy group (Control) and colitis model group (DSS+0.9% NaCl), suggesting that the expression of tissue gene transcriptome after GZTB treatment is more similar to that of healthy tissue. B is the bar chart of bulk-RNA seq deconvolution analysis, and C is the proportion of pro-inflammatory M1 cells and anti-inflammatory M2 cells; Figure 11 Figure 7 shows the results of flow cytometry analysis of colon cells of healthy control group, DSS+0.9% NaCl group and GZTB treatment group. A is the proportion of total immune cells (CD45 + ) to all live cells; B is the proportion of total myeloid cells (CD45 + CD11b + ) to total immune cells; C is the proportion of neutrophils (CD45 + CD11b + Ly6G + ) to total myeloid cells; D is the proportion of pro-inflammatory macrophages (CD45 + CD11b + Ly6G-SiglecF-CD11c-CD64 + Ly6C + ) to intestinal macrophages; E is the proportion of anti-inflammatory macrophages (CD45 + CD11b + Ly6G-SiglecF-CD11c-CD206 + MHC-Ⅱ + ) to intestinal macrophages; Figure 12 Figure 8 shows the statistical analysis results of the proportion of each group of immune cells to total immune cells. A is the proportion of total immune cells (CD45 + ) to all live cells; B is the proportion of total myeloid cells (CD45 + CD11b + ) to total immune cells; C is the proportion of neutrophils (CD45 + CD11b + Ly6G + ) to total myeloid cells; D is the proportion of pro-inflammatory macrophages (CD45 + CD11b + Ly6G-SiglecF-CD11c-CD64 + Ly6C + ) to intestinal macrophages; E is the proportion of anti-inflammatory macrophages (CD45 + CD11b + Ly6G-SiglecF-CD11c-CD206 +MHC-II + ) the proportion of intestinal macrophages; Figure 13 Ga / BLG / Zn 2+ / THBA double network hydrogel group (GZTB), healthy group and colitis model group (DSS) intestinal tissue up-regulated and down-regulated differential genes GO enrichment analysis, wherein A is DSS vs Control, B is DSS vs GZTB; Figure 14 Ga / BLG / Zn 2+ / THBA double network hydrogel group (GZTB), healthy group and colitis model group (DSS) intestinal tissue inflammatory factor and immune cell chemokine detection analysis, wherein A is inflammatory factor detection analysis, B is immune cell chemokine detection analysis; Figure 15 Ga / BLG / Zn 2+ / THBA double network hydrogel group (GZTB) and colitis model group (DSS) intestinal IL-10 protein immunohistochemical staining results and statistical analysis, wherein A is immunohistochemical staining results, B is immunohistochemical staining results statistical analysis. DETAILED DESCRIPTION

[0021] The technical solutions of the present application are further described below through the drawings and examples.

[0022] Unless otherwise defined, the technical terms or scientific terms used in the present application shall have the usual meanings understood by those skilled in the art to which the present application belongs.

[0023] Based on the examples in the present application, all other examples obtained by those of ordinary skill in the art without making creative efforts fall within the scope of protection of the present application. The experimental methods not specified in the following examples are generally determined according to national standards. The experimental instruments, equipment and reagents not specified in the following examples are all commercially available raw materials.

[0024] Unless otherwise defined or explained, all professional and scientific terms used in the present application have the same meaning as familiar to those skilled in the art.

[0025] Example 1 Preparation of Ga / BLG / Zn 2+ / THBA double network hydrogel ① 5 mL of deionized water was preheated to 60℃, then 200 mg of GA was dissolved to obtain a Ga solution of 40 mg / mL, and then 400 mg of BLG was added to obtain solution A; (2) 5 mg ZnCl2 and 50 mg THBA were dissolved in 5 mL deionized water to obtain solution B; (3) 5 mL solution A and 5 mL solution B were quickly mixed and stirred before use to obtain Ga / BLG / Zn 2+ / THBA double network hydrogel. The synthesis mode chart is shown in Figure 1 , and the solution A and solution B can be quickly gelled within 10 seconds, as shown in Figure 2 .

[0026] Example 2 Characterization of Ga / BLG / Zn 2+ / THBA double network hydrogel (1) The chemical interactions between different hydrogels were analyzed by FT-IR (Nicolet 6700, USA) spectroscopy. For the formation of Ga / BLG / Zn 2+ / THBA double network hydrogel, Zn 2+ forms a multi-ligand coordination structure with the carboxyl and hydroxyl groups of Ga through metal coordination bonds and hydrogen bonds, and the secondary network is constructed by the Schiff base and hydrogen bonds formed between the aldehyde groups of THBA and the numerous amino groups of BLG. At the same time, THBA has a dual function in the network: it not only forms a stable structure with BLG, but also enhances the crosslinking density of the GA network through hydrogen bonds, and some THBA molecules also form bridges between BLG and GA to strengthen the hydrogel. FT-IR verifies this process, as shown in Figure 3 , the introduction of Zn 2+ does not change the structural integrity of GA, and characteristic absorption peaks can be seen at 1725 cm -1 (free carboxyl group), 1658 cm -1 (hydrogen-bonded carboxyl group), and 1041 cm -1 (C-O stretching vibration); after the addition of BLG and THBA, the asymmetric stretching vibration near 1600 cm -1 confirms that Zn 2+ coordinates with the carboxylate groups of each component (GA / BLG / THBA), and the increased peak width in this region indicates the formation of a wide multi-hydrogen bond network within the quaternary system, thereby enhancing the stability of the supramolecular structure, confirming the successful construction of Ga / BLG / Zn 2+ / THBA.

[0027] (2) The microstructure of Ga, Ga / Zn 2+ , Ga / Zn 2+ / THBA, and Ga / BLG / Zn 2+ / THBA hydrogels was characterized by scanning electron microscopy SEM and energy dispersive spectroscopy EDS (JSM-7800F, JEOL), as shown in Figure 4SEM images show that all hydrogels exhibit porous structure, in which Ga / BLG / Zn 2+ / THBA hydrogel has more uniform pore structure and more closely interconnected pores. EDS shows that GZTB hydrogel is mainly composed of C, N and O elements as the basic skeleton, and contains trace amounts of Cl, S and Zn elements, confirming the successful synthesis of the hydrogel.

[0028] (3) Mechanical testing of Ga / BLG / Zn 2+ / THBA double network hydrogel: The rheological properties of the cylindrical hydrogel samples (diameter 10 mm) were tested using a Discovery DHR-2 rheometer (TA Instruments). The experiment was carried out at room temperature using a stress control mode, and the frequency scanning range was set to 0.1-100 rad / s to detect the mechanical properties of the hydrogel. The results show (Fig. Figure 5 ), thanks to the double network structure, the elastic modulus of Ga / BLG / Zn 2+ / THBA is tens of times higher than that of Ga / Zn 2+ .

[0029] (4) Injectability and plasticity of Ga / BLG / Zn 2+ / THBA double network hydrogel: First, the injectability of the hydrogel was evaluated by pushing the hydrogel through a 1 mL syringe, and the gel could form the "IBD" letter mark (Fig. Figure 6 A), indicating that the Ga / BLG / Zn 2+ / THBA double network hydrogel has good injectability and can smoothly pass through the lumen without obstruction, which is an important requirement for oral administration. Second, the hydrogel was poured into cross-shaped and star-shaped models, and the shape of the gel was observed after formation. The results show (Fig. Figure 6 B), the hydrogel can completely fill the grooves in different shaped containers. This finding shows that the double network hydrogel in the present application has excellent shape adaptability.

[0030] (5) Intestinal adhesion detection of Ga / BLG / Zn 2+ / THBA double network hydrogel: After 24 hours of fasting, eight-week-old wild-type male C57BL / 6 mice were given a fluorescent conjugated material by gavage. At 4 hours, 12 hours and 24 hours after gavage, the colon tissue was collected, and the residual fluorescence intensity was detected using a small animal live imaging system. The results show (Fig. Figure 7 that the intestinal adhesion of Ga / BLG / Zn 2+ / THBA is significantly enhanced 12 hours after administration.

[0031] (6) Ga / BLG / Zn 2+ Degradation characteristics of Ga / BLG / Zn / THBA double-network hydrogel: Ga / BLG / Zn / THBA double-network hydrogel was immersed in simulated gastrointestinal fluids to analyze its degradation characteristics. As shown in A and B of Figure 8 2+ 2+ Ga / BLG / Zn / THBA showed excellent tolerance in the strong acid environment of simulated gastric fluid (SGF) and maintained structural stability for more than 48 hours, while Ga / Zn hydrogel degraded by about 30% within 6 hours. In the near-neutral condition (pH = 6.8) and presence of protease environment of simulated intestinal fluid (SIF), the Ga / BLG / Zn / THBA gradually disintegrated within 6-24 hours, while the GZ formulation rapidly degraded within 6 hours and completely decomposed within 24 hours. Finally, Ga / BLG / Zn / THBA achieved rapid release in the weakly alkaline environment after reaching the colon. 2+ 2+ 2+ 2+ The anti-inflammatory component Ga in Ga / BLG / Zn / THBA released only 1.144% in SGF after 4 hours, and about 90% remained even at 48 hours; about 24.70% was released in SIF after 4 hours; and in the 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. The release behavior of zinc ions was similar to that of Ga (C of Figure 8

[0032] Example 3 Verification of therapeutic effect of Ga / BLG / Zn / THBA double-network hydrogel in mouse colitis 2+ (1) Construction of mouse colitis model and treatment of Ga / BLG / Zn / THBA double-network hydrogel: 2+ A mouse colitis model induced by dextran sulfate sodium (DSS) was used to evaluate its in vivo therapeutic effect. DSS, as a toxin, can destroy colon epithelial cells, allowing luminal antigens to penetrate the mucosal layer and even reach the submucosal tissue, thereby triggering widespread inflammation.

[0033] The modeling process was as follows: 8-week-old male C57BL / 6 mice were randomly divided and then fed for 2 days to adapt to the environment, after which DSS was provided through free drinking water for 7 consecutive days.

[0034] Ga / BLG / Zn / THBA showed excellent tolerance in the strong acid environment of simulated gastric fluid (SGF) and maintained structural stability for more than 48 hours, while Ga / Zn hydrogel degraded by about 30% within 6 hours. In the near-neutral condition (pH = 6.8) and presence of protease environment of simulated intestinal fluid (SIF), the Ga / BLG / Zn / THBA gradually disintegrated within 6-24 hours, while the GZ formulation rapidly degraded within 6 hours and completely decomposed within 24 hours. Finally, Ga / BLG / Zn / THBA achieved rapid release in the weakly alkaline environment after reaching the colon. 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.

[0035] 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).

[0036] (2) via Ga / BLG / Zn 2+ Tissue manifestations of THBA dual-network hydrogel treatment for IBD: 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).

[0037] 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.

[0038] (3) Ga / BLG / Zn 2+ Modulation of intestinal immune microenvironment by Ga / BLG / Zn RNA sequencing analysis was performed on the intestinal tissues of 0.9% NaCl gavage control group, Ga / BLG / Zn 2+ / THBA double-network hydrogel treatment group after 7 days of DSS-induced, and healthy mice without induction of intestinal inflammation. Although the three groups showed different transcriptional characteristics, the transcriptional characteristics of GZTB-treated mice were closer to that of healthy mice ( Figure 10 A). The impact of hydrogel treatment on immune cell subsets, including lymphocytes, macrophages, and neutrophils, was studied by Cibersort, an algorithm that infers the relative proportion of specific cell types in tissues from gene expression data. The 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-type macrophages increased significantly. GZTB treatment effectively reversed this change and exhibited a characteristic shift by suppressing M1 polarization while promoting anti-inflammatory M2-type macrophage differentiation ( Figure 10 B, C). These changes were confirmed by flow cytometry ( Figures 11-12 ). Compared with the DSS group, the proportion of total immune cells (65.48% vs 31.41%) and myeloid-derived cells (14.56% vs 9.74%) in the colon tissue after hydrogel treatment decreased significantly. Among them, the proportion of neutrophils and Ly6C + pro-inflammatory intestinal macrophages in total immune cells was only half of that in the DSS group (neutrophils: 1.77% vs 2.50%; Ly6C + macrophages: 4.27% vs 2.51%). Conversely, the proportion of CD206 + MHCII + anti-inflammatory intestinal macrophages increased significantly, even slightly higher than the healthy control group (GA-BLG vs DSS: 5.01% vs 1.99%; GA-BLG vs healthy control: 5.01% vs 4.21%).

[0039] (4) Ga / BLG / Zn 2+ / THBA double-network hydrogel inhibits excessive immune response: Based on the differentially expressed genes of Ga / BLG / Zn 2+ / THBA treatment group, DSS-induced colitis mice, and healthy control group, gene ontology (GO) enrichment analysis was performed. The results are as follows Figure 13The 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.

[0040] (5) Conclusion: 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.

[0041] Therefore, the present application provides a double cross-linked hydrogel with good plasticity and adhesion, which is the therapeutic effective component itself, has strong anti-inflammatory ability, can effectively relieve mouse colitis, and can be used for preparing a drug preparation for treating inflammatory bowel disease.

[0042] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application, but not to limit them. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can still be modified or replaced by equivalents, and these modifications or replacements cannot make the modified technical solutions deviate from the spirit and scope of the technical solutions of the present application.

Claims

1. A method of preparing a dual-network hydrogel for the treatment of inflammatory bowel disease, characterized in that, The method comprises the following steps: Step 1: Dissolve glycyrrhizin Ga into deionized water, then add beta-lactoglobulin BLG to obtain solution A; Step 2: Dissolve zinc chloride and 2,3,4-trihydroxybenzoic acid THBA into deionized water to obtain solution B; Step 3: Mix solution A and solution B in a certain proportion, stir and react to obtain a double-network hydrogel.

2. The method of claim 1, wherein: In solution A of step 1, the concentration of glycyrrhizin Ga is 40 mg / mL, and the concentration of beta-lactoglobulin BLG is 80 mg / mL.

3. The method of claim 1, wherein: 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.

4. The method of claim 1, wherein: In step 3, the volume ratio of solution A to solution B is 1:

1.

5. A dual network hydrogel for the treatment of inflammatory bowel disease, characterized in that: Prepared by the preparation method of any one of claims 1-4.

6. The dual-network hydrogel of claim 5, wherein: The double-network hydrogel comprises a first network and a second network; the first network is formed by self-crosslinking of glycyrrhizin and zinc ions in zinc chloride, and the second network is formed by Schiff base and hydrogen bond crosslinking of beta-lactoglobulin and 2,3,4-trihydroxybenzoic acid THBA; the first network and the second network are crosslinked with increased crosslinking density through the interaction force of 2,3,4-trihydroxybenzoic acid THBA with beta-lactoglobulin and glycyrrhizin.

7. The dual-network hydrogel of claim 6, wherein: In the double-network hydrogel, the mass ratio of glycyrrhizin, zinc chloride, beta-lactoglobulin and 2,3,4-trihydroxybenzoic acid THBA is 200:5:400:

50.

8. Use of the double-network hydrogel prepared by the preparation method of any one of claims 1-4 or the double-network hydrogel of any one of claims 5-7 in the preparation of an oral drug for treating inflammatory bowel disease.

9. An oral medicament for treating inflammatory bowel disease, characterized by: The effective component is the double-network hydrogel prepared by the preparation method of any one of claims 1-4 or the double-network hydrogel of any one of claims 5-7.

Images