Manganese-based nano-enzyme composite material as well as preparation method and application thereof

Abstract

The invention discloses a manganese-based nano-enzyme composite material as well as a preparation method and application thereof. The preparation method comprises the following steps: (1) preparing Mn3O4-PVP from Mn3O4 NPs and PVP10, and then loading the Mn3O4-PVP into a ZIF-8 structure in the process that Zn < 2 + > and 2-MI react to form ZIF-8, so as to obtain Mn3O4-coated ZIF-8 NPs; (2) covering the surface of the Mn3O4 ZIF-8 with HA through ion coordination, and synthesizing MZH; and (3) carrying out clathration on MZH through ES100, and synthesizing MZH coated ES100. By inhibiting a TLR4 / NF-kappa B pathway, activating an Nfr2 / Keap1 pathway and increasing expression of intestinal tight junction protein, mouse colitis induced by DSS is resisted, and symptoms of the mouse colitis are relieved.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical application technology, and in particular to a manganese-based nanoenzyme composite material, its preparation method, and its application. Background Technology

[0002] Increased reactive oxygen species (ROS) are a crucial mechanism in the development of intraepithelial neoplasia (IBD). Superoxide anion (O2·-), hydrogen peroxide (H2O2), and hydroxyl radicals (·OH) are naturally occurring oxygen-containing substances with strong oxidizing capabilities, belonging to the ROS category. ROS participate in various biochemical reactions through the production and clearance of natural enzymes in the body. Under normal physiological conditions, they are usually in dynamic equilibrium, but when this equilibrium is disrupted, the concentration of ROS in the body becomes abnormal. When the rate of ROS clearance by the endogenous antioxidant system is lower than the rate of ROS production in the body, excessive ROS accumulation can induce tissue protein denaturation, lipid peroxidation, DNA damage, apoptosis, iron degeneration, and pyroptosis, affecting normal physiological structure and function, ultimately promoting the development of various diseases. Under the pathological conditions of IBD, activated neutrophils can be recruited and accumulate in the intestine. Neutrophils produce reactive oxygen species (ROS). Excessive ROS disrupts redox homeostasis, further causing oxidative damage to epithelial cell communication and related proteins and DNA, thus exacerbating IBD symptoms and hindering treatment efficiency. Increasing research indicates that antioxidant therapy, utilizing external antioxidants to consume excess ROS in IBD treatment, is useful. However, the limitations of natural antioxidant enzymes (such as instability and short half-life) greatly restrict their practical application in IBD treatment. Nanozymes, with their biomimetic antioxidant properties, high stability, and large-scale production capabilities, can replace natural antioxidant enzymes as external antioxidants to consume excess ROS, showing great potential for IBD treatment.

[0003] Manganese-based nanozymes are nanomaterials containing manganese metal and its oxides with enzyme-like activities. They exert anti-inflammatory effects by scavenging ROS, regulating the immune system and endogenous antioxidant system, without damaging normal tissue cells. They aggregate in hypoxic acidic environments, reaching a local pro-oxidation threshold, achieving functional switching, promoting oxygen and ROS production, and causing oxidative stress, making them suitable for antibacterial and antitumor applications. Related studies have shown that Mn3O4NPs possess free radical scavenging and various enzyme-like activities, including superoxide dismutase (SOD)-like activity, catalase (CAT)-like activity, and glutathione peroxidase (GPX)-like activity, and are widely used in anti-inflammatory and antitumor research, especially in the study of intraepithelial neoplasia (IBD).

[0004] Chinese invention patent CN119235914A, entitled "A Multi-Enzyme HMPB / Mn3O4 Nanozyme with Multi-Enzyme Activity, Its Preparation Method and Application," discloses a multi-enzyme HMPB / Mn3O4 nanozyme with multi-enzyme activity, its preparation method and application. This nanozyme uses HMPB with a hollow mesoporous structure as a carrier, and loads nano-Mn3O4 onto its inner and outer surfaces via a self-assembly method. The HMPB / Mn3O4 nanozyme provided by this invention exhibits good multi-enzyme activity, shows no significant cytotoxicity to NCM460 and RAW264.7 cells, and can improve the related symptoms of DSS-induced ulcerative colitis in mice, showing promise as a novel potential drug for treating ulcerative colitis. However, its specificity is limited, and its constituent materials cannot accumulate more extensively in the inflamed parts of the intestine. Summary of the Invention

[0005] The purpose of this invention is to provide a manganese-based nanozyme composite material, its preparation method, and its application.

[0006] To address the problems existing in the prior art, the technical solution adopted in this invention is: In a first aspect, the present invention provides a method for preparing a manganese-based nanozyme composite material, comprising the following steps: (a) Synthesis of Mn3O4@ZIF-8 NPs: Mn3O4-PVP was prepared by using Mn3O4NPs and PVP10, and then Mn3O4-PVP was subjected to Zn 2+ During the reaction with 2-MI to form ZIF-8, Mn3O4@ZIF-8 NPs were loaded into the ZIF-8 structure to obtain Mn3O4@ZIF-8 NPs; (ii) MZH was synthesized by coating HA onto the surface of Mn3O4@ZIF-8 through ion coordination; (iii) MZH is included in ES100 to synthesize MZH@ES100.

[0007] The MZH@ES100 prepared using the method of this invention exhibits enteric coating properties, enabling oral administration; it accumulates more at sites of inflammation; and it disintegrates under the slightly acidic conditions of the inflamed site, allowing Mn3O4 to exert its effects primarily at the inflammatory site. This invention utilizes PVP10, where C=O and Zn... 2+ The weak coordination and the hydrophobic interaction between the polar group and 2-MI in polar solvents make the Mn3O4 prepared by this method more stable in polar solvents, less prone to aggregation, and better loaded onto ZIF-8.

[0008] Furthermore, step (a) includes the following steps: (1) Dissolve 400±1 mg Mn3O4NPs in 10±0.1 mL of methanol, add 70±1 mL of methanol containing 800±1 mg PVP10, stir vigorously for 24±1 h, wash the resulting precipitate with methanol to obtain Mn3O4-PVP, and dry it for later use. (2) Disperse 20±0.1 mg Mn3O4-PVP in 20±1 mL methanol, sonicate for 2±0.1 min, then add 10±0.1 mL of Zn(NO3)2·6H2O methanol solution with a concentration of 45±1 mg / mL, stir magnetically for 15±1 min at room temperature, and sonicate for 15±1 min; then add 10±1 mL of 2-MI methanol solution with a concentration of 12.5±1 mg / mL, continue to sonicate for 15±1 min, and stir magnetically for 15±1 min; centrifuge, wash the precipitate with methanol to obtain Mn3O4@ZIF-8 NPs, and dry for later use.

[0009] Furthermore, the Mn3O4NPs are synthesized according to the following method: (1) Dissolve 5.69±0.1 g Mn(AC)2·4H2O in 279±1 mL of anhydrous ethanol and stir until the solution is clear and transparent; (2) The mixed solution was transferred to a high-pressure reactor with a polytetrafluoroethylene liner and reacted at a constant temperature of 120±1℃ for 24±1 h. (3) After the reaction is complete, centrifuge to collect the precipitate, purify the product by extraction, and wash the precipitate with anhydrous ethanol and distilled water. (4) The precipitate was collected and dried in a constant temperature drying oven to obtain Mn3O4NPs.

[0010] Furthermore, step (ii) includes the following steps: 200±1 mg Mn3O4@ZIF-8 was dispersed in 65 mL H2O; 250±1 mg hyaluronic acid was dispersed in 500 mL H2O; the two were mixed and magnetically stirred at room temperature for 30±1 min, centrifuged, and the resulting precipitate was washed with H2O to obtain MZH, which was then dried for later use.

[0011] Furthermore, step (iii) includes the following steps: (1) Disperse 10±0.1 mg MZH in 2.5±0.1 mL H2O, then add 75±1 μL Tween-20, sonicate for 2±0.1 min to obtain the aqueous phase; (2) Dissolve 300±1 mg of ES100 in small amounts and multiple times in 5±0.1 mL of organic solvent to obtain an organic phase; (3) Slowly add 0.5±0.1 mL of aqueous phase to 5±0.1 min of organic phase, sonicate on ice for 1±0.1 min to obtain primary emulsion; add 50±1 mL of 2±0.1% polyvinyl alcohol dropwise to the above primary emulsion while stirring, and sonicate on ice for 4±0.1 min; stir the mixed solution in a fume hood for 20±1 h to allow the organic solvent to evaporate, centrifuge, wash the obtained precipitate with H2O to obtain MZH@ES100, and dry it for later use.

[0012] Further, the organic solvent in step (2) is composed of the following substances in the following volume ratio: dichloromethane: ethanol: isopropanol = 4-5: 3-4: 5-6.

[0013] Secondly, the present invention provides a manganese-based nanoenzyme composite material, which is prepared by the method described in the first aspect above.

[0014] Thirdly, the present invention provides the application of the manganese-based nanozyme composite material described in the second aspect above in the preparation of a drug for treating a DSS-induced mouse colitis model.

[0015] In a fourth aspect, the present invention provides a medicament for preparing a treatment for a DSS-induced mouse colitis model, comprising the manganese-based nanozyme composite material described in the second aspect above.

[0016] Furthermore, the therapeutic dose of the manganese-based nanoenzyme composite material is 2.5-40 mg / kg.

[0017] The advantages and beneficial effects of this invention are: This invention combines Mn3O4NPs with multiple enzyme activities, a zeolite-like imidazole metal framework ZIF-8, hyaluronic acid (HA), an inflammatory cell-targeting material, and Eudragit S100 (ES100), an oral enteric-coated material, to prepare an orally bioavailable manganese-based nanoenzyme composite material, MZH@ES100, which was then used to treat a DSS-induced mouse colitis model. First, Mn3O4@ZIF-8 was synthesized in a one-pot process. Then, HA was coated onto the surface of Mn3O4@ZIF-8 via ion coordination to synthesize Mn3O4@ZIF-8@HA (MZH). Finally, to prevent MZH from being inactivated in the stomach during oral administration, MZH was encapsulated with ES100 to synthesize MZH@ES100. Because ES100 is enteric-coated, upon reaching the colon, the MZH@ES100 structure disintegrates, releasing MZH. The HA on the MZH surface can specifically bind to CD44, allowing MZH to accumulate more at the site of inflammation. The relatively acidic environment of the inflamed site further causes the ZIF-8 framework to disintegrate, releasing Mn3O4NPs to exert enzyme-like activity and scavenge ROS, while simultaneously releasing Zn. 2+ Ions can promote the repair of the intestinal barrier.

[0018] This invention encapsulates ES100 onto the surface of MZH via emulsification to form water-in-oil ES100 microspheres, resulting in more targeted targeting. ES100 exhibits enteric solubility, HA acts as a CD44 receptor, and ZIF-8 disintegrates under inflammatory acidic conditions, all of which allow the material to accumulate more in inflamed areas of the intestine. The MZH@ES100 prepared according to this invention was used in a study of a dextran sulfate sodium salt (DSS)-induced mouse colitis model, and its anti-inflammatory, antioxidant, and colonic barrier maintenance effects were evaluated. Results showed that the synthesized MZH@ES100 remained intact after treatment with simulated gastric and small intestinal fluids, but underwent significant deformation after treatment with simulated colonic fluids. Furthermore, its related free radical scavenging activity and enzyme-like activity were enhanced compared to before treatment. MZH@ES100 can combat DSS-induced mouse colitis and alleviate symptoms by inhibiting the TLR4 / NF-κB pathway, activating the Nfr2 / Keap1 pathway, and increasing the expression of intestinal tight junction proteins. Attached Figure Description

[0019] Figure 1 The figure shows the results of the material characterization experiments: Figure 1 A is the TEM image of Mn3O4; Figure 1 B is the TEM of ZIF-8; Figure 1 C is the TEM of Mn3O4@ZIF-8; Figure 1D is the TEM of MZH; Figure 1 E represents the XRD characterization; Figure 1 F represents the Zeta potential, where the Zeta potential is 6.71±0.62 mV for ZIF-8, 11.90±0.26 mV for Mn3O4, 21.16±0.19 mV for Mn3O4@ZIF-8, and -22.08±0.48 mV for MZH. Figure 1 G represents TEM-mapping characterization; the distribution of Mn (yellow), Zn (red), N (cyan), and C (dark blue) elements on the surface of Mn3O4@ZIF-8; Figure 2 The chemical composition and structural characteristics of Mn3O4NPs and Mn3O4@ZIF-8 are shown in the figure: Figure 2 A represents the XPS characterization of Mn3O4 and Mn3O4@ZIF-8; Figure 2 B and E are XPS characterizations of Mn 2p in Mn3O4 and Mn3O4@ZIF-8; Figure 2 C and F are XPS characterizations of O 1s in Mn3O4 and Mn3O4@ZIF-8; Figure 2 D represents the XPS characterization of Zn 2p in Mn3O4@ZIF-8; Figure 3 The figure shows the results of experiments on the stability of the material in simulated gastrointestinal fluid—free radical scavenging activity and enzyme-like activity: Figure 3 A shows the TEM characterization images of Mn3O4NPs and MZH@ES100 before and after treatment with SGF, SGF+SIF, and SGF+SIF+SCF. Figure 3 BF represents the free radical scavenging ability of Mn3O4NPs and MZH@ES100 (1 mg / mL) before and after treatment with SGF, SGF+SIF, and SGF+SIF+SCF, as well as the activities of SOD-like, GPX-like, and CAT-like enzymes. The scavenging ability against ·OH was detected by the MB method, the total antioxidant capacity was detected by the ABTS+ method, the SOD-like enzyme activity was detected by the NBT method, the GPX-like enzyme activity was detected by the DNTB method, and the CAT-like enzyme activity was detected by the ammonium molybdate method. Figure 3 GI represents the GPX, SOD, and CAT enzyme activities of Mn3O4NPs at different concentrations; Figure 4 The figure shows the results of an experiment in which MZH@ES100 alleviated DSS-induced colitis symptoms in mice. Figure 4 A represents the modeling method; Figure 4 B represents the mouse DAI index; Figure 4 C represents the change in mouse body weight; Figure 4 D represents the statistical value of mouse colon length; Figure 4 E represents the mouse colon; Figure 4F represents H&E staining of mouse colon; Figure 5 The figure shows the experimental results of MZH@ES100 inhibiting colonic oxidative stress and activating the Nrf2 / Keap1 pathway to counteract DSS-induced colitis. Figure 5 AD represents the levels of T-SOD, T-AOC, MDA, and GSH in the colon of mice; Figure 5 E represents the mRNA expression levels of mouse colonic antioxidant genes CuZn-SOD, Mn-SOD, CAT, and GPx analyzed by qRT-PCR. Figure 5 F represents Western blotting analysis of the expression of Nrf2 / Keap1 pathway-related proteins in the mouse colon. Figure 5 F(a) represents the protein band. Figure 5 F(b) is a bar chart (n=3; data are shown as mean ± SD; multiple comparisons were performed using one-way ANOVA and LSD test; compared with the healthy control group: * P <0.05, ** P <0.01, *** P <0.01; compared with the DSS model group: # P <0.05, ## P <0.01, ### P <0.01; when there is no significance P >0.05 (not shown in the graph); Figure 6 The figure shows the experimental results of MZH@ES100 inhibiting the inflammatory response and suppressing the TLR4 / NF-κB pathway to combat DSS-induced colitis. Figure 6 AB represents the serum and colonic levels of the relevant inflammatory cytokines IL-6, IL-10, TNF-α, and IL-1β in mice (n=6; data are presented as mean ± SD; multiple comparisons were performed using one-way ANOVA and LSD test; compared with healthy controls:* P <0.05,** P <0.01, *** P <0.01; compared with the DSS model group: # P <0.05, ## P <0.01, ### P <0.01. When there is no significance. P >0.05 (not shown in the graph); Figure 6C represents the mRNA expression levels of mouse colitis genes IL-6, IL-10, TNF-α, and IL-1β analyzed by qRT-PCR. Figure 6 D represents the expression of TLR4 / NF-κB pathway-related proteins in the mouse colon using Western blotting analysis (n=3). (Figure 1) Figure 6 D(a) represents the protein band. Figure 6 D(b) is a bar chart; Figure 7 The figure shows the experimental results of MZH@ES100 maintaining the intestinal barrier and increasing the expression of tight junction proteins to combat DSS-induced colitis: Figure 7 A represents the serum levels of D-LA, DAO, and LPS in mice (n=6; data are presented as mean ± SD; multiple comparisons were performed using one-way ANOVA and LSD test; compared with healthy controls:* P <0.05,** P <0.01, *** P <0.01; compared with the DSS model group: # P <0.05, ## P <0.01, ### P <0.01. When there is no significance. P >0.05 (not shown in the graph); Figure 7 B represents the mRNA expression levels of mouse colonic tight junction proteins ZO-1, OCLN, and CLDN1 genes analyzed by qRT-PCR. Figure 7 C represents the expression of mouse colonic tight junction proteins ZO-1, OCLN, and CLDN1 by Western Blot analysis (n=3. OCLN: Occludin, CLDN1: Claudin1). Figure 8 For safety evaluation, changes in body weight (A) and organ indices (B) of mice in each group were measured; Note: n=6; Data are presented as mean ± SD; Multiple comparisons were performed using one-way ANOVA and LSD test; Compared with the blank control group: * P <0.05,** P <0.01, *** P <0.01, ns: P >0.05.

[0020] Figure 9 The figure shows the serum biochemical parameters of mice in each group for safety evaluation experiments: Figure 9 A represents the serum AST and ALT activities in mice; Figure 9B represents the concentrations of TBil, Crea, and Urea. Figure 9 C represents ALP, CK, and LDH activity (C); Note: (n=6; data are presented as mean ± SD; multiple comparisons were performed using one-way ANOVA and LSD test; compared with the blank control group:*) P <0.05,** P <0.01, *** P <0.01, ns: P >0.05); Figure 10 For safety evaluation, the liver, spleen, kidney, and colon of mice in each group were stained with H&E. Note: 200 X; scale bar: 200 μm. Detailed Implementation

[0021] The technical solutions of the embodiments of this application will be clearly described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this application. All other embodiments obtained by those skilled in the art based on the embodiments of this application are within the scope of protection of this application.

[0022] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0023] Manganese acetate tetrahydrate (Mn(AC)2·4H2O, AR 99%, CAS:6156-78-1, MACKLIN); Anhydrous ethanol (Ethanol, AR, CAS: 64-17-5); Isopropanol (AR, CAS: 67-63-0, KESHI); Anhydrous methanol (AR, CAS: 67-56-1, KESHI); 2-Methylimidazole (2-MeIM, CAS: 693-98-1, MACKLIN); Zinc nitrate hexahydrate (Zn(NO3)2·6H2O, AR 99%, CAS: 10196-18-6, aladdin); Dichloromethane (AR 99%, CAS: 75-09-2, CHRON); Polyvinyl alcohol 1788 (CAS: 9002-89-5, MACKLIN); Polyvinylpyrrolidone PVP10 (CAS: 9003-39-8, MACKLIN); Poly(methyl methacrylate-co-methacrylic acid) (Eudragit S100 / ES100 / Eudragit S100, CAS: 25086-15-1, MACKLIN); Tween 20 (CAS: 9005-64-5, Adamas Life); Hyaluronic acid (HA, CAS: 9004-61-9, MACKLIN).

[0024] Nitric acid (68-70%, CAS: 7697-37-2, CHRON); Perchloric acid (GR, CAS: 7601-90-3, CHRON).

[0025] Example 1 This embodiment provides a method for preparing a manganese-based nanoenzyme composite material (MZH@ES100), including the following steps: One-pot synthesis of Mn3O4@ZIF-8 NPs: (1) Synthesis of Mn3O4: 5.69 g of Mn(AC)2·4H2O was dissolved in 279 mL of anhydrous ethanol and stirred magnetically until the solution was clear and transparent. The mixture was then transferred to a high-pressure reactor with a polytetrafluoroethylene liner and reacted at a constant temperature of 120℃ for 24 h. After the reaction was completed, the precipitate was collected by centrifugation at 4200 r / min for 5 min and purified by extraction (the precipitate was washed 3 times with anhydrous ethanol and distilled water). The precipitate was collected and dried in a constant temperature drying oven at 60℃ for 12 h to obtain Mn3O4NPs. Mn3O4NPs refers to Mn3O4 particles with a size in the nanoscale (1-100 nm) range. The Mn3O4NPs synthesized by this method have various enzyme activities.

[0026] (2) Synthesis of Mn3O4-PVP: 400 mg of Mn3O4NPs were dissolved in 10 mL of methanol, and 70 mL of methanol containing 800 mg of PVP10 was added. The mixture was stirred vigorously for 24 h. The resulting precipitate was washed three times with methanol (4200 r / min, 15 min) to obtain Mn3O4-PVP, which was then dried at 60 °C for later use.

[0027] (3) Synthesis of Mn3O4@ZIF-8 NPs: 10 mL of 12.5 mg / mL 2-methylimidazole (2-MI) methanol solution and 10 mL of 45 mg / mL Zn(NO3)2·6H2O methanol solution were prepared for later use. 20 mg of Mn3O4-PVP was dispersed in 20 mL of methanol and sonicated for 2 min. Then, the above 10 mL of 45 mg / mL Zn(NO3)2·6H2O methanol solution was added, and the mixture was magnetically stirred for 15 min and sonicated for 15 min at room temperature. Then, 10 mL of 12.5 mg / mL 2-MI methanol solution was added, and the mixture was sonicated for 15 min and magnetically stirred for 15 min. The mixture was centrifuged at 4200 r / min for 5 min, and the resulting precipitate was washed three times with methanol to obtain Mn3O4@ZIF-8 NPs, which were then dried at 60℃ for later use.

[0028] In step (one), Zn 2+ It coordinates with the N atom on 2-MI to form a Zn-N coordinate bond, wherein Zn 2+ Provides one 4S and three 4P orbitals, one deprotonated N provides one lone pair of electrons, Zn 2+ Mn3O4 undergoes sp3 hybridization with deprotonated N, and further forms a cage-like coordination compound with a hexahedral crystal structure, namely the ZIF-8 framework structure, through intermolecular forces. The ZIF-8 framework structure has numerous pores, a large specific surface area, and a large number of adsorption sites, thus allowing Mn3O4 to be loaded into these pores. PVP 10 can prevent the aggregation of Mn3O4 in the reaction system, as aggregation would be detrimental to its loading.

[0029] (II) Preparation of Mn3O4@ZIF-8@HA NPs (MZH): 200 mg of Mn3O4@ZIF-8 NPs were dispersed in 65 mL of H2O; 250 mg of hyaluronic acid (HA) was dispersed in 500 mL of H2O. The two were mixed and magnetically stirred at room temperature for 30 min, then centrifuged at 4200 r / min for 5 min. The resulting precipitate was washed three times with H2O to obtain MZH, which was then dried at 37℃ for later use.

[0030] (III) Preparation of Mn3O4@ZIF-8@HA@ES-100 (MZH@ES100): (1) Aqueous phase: 10 mg MZH was dispersed in 2.5 mL of H2O, and then 75 μL of Tween-20 was added and sonicated for 2 min.

[0031] (2) Organic phase: 300 mg of ES100 was dissolved in 5 mL of organic solvent in small amounts several times. The organic solvent was composed of the following substances in the following volume ratio: dichloromethane: ethanol: isopropanol = 5: 4: 6.

[0032] (3) Preparation of 2% polyvinyl alcohol: Add 2 g of polyvinyl alcohol 1788 in small amounts several times while stirring 100 mL of warm water at about 40℃. Stir continuously at room temperature with magnetic force overnight to completely dissolve the solid powder and obtain 2% polyvinyl alcohol.

[0033] (4) Slowly add 0.5 mL of the aqueous phase to the organic phase for 5 min, and sonicate on ice for 1 min to obtain a primary emulsion; add 50 mL of 2% polyvinyl alcohol dropwise to the above primary emulsion while stirring, and sonicate on ice for 4 min. Stir the mixed solution in a fume hood for 20 h to allow the organic solvent to evaporate, centrifuge at 10000 r / min for 5 min, wash the resulting precipitate 3 times with H2O to obtain MZH@ES100, and dry at 37℃ for later use.

[0034] All methanol used in this application is anhydrous methanol.

[0035] Experimental Example 1. Test materials Sodium dextran sulfate (DSS, CAS: 9011-18-1, MV 40000, Maclean's); Sodium carboxymethyl cellulose (CMC-Na, CAS: 9004-32-4, 89132AA, Adamas); Tribromoethanol (Avertin, Y81407C, Adamas). RIPA lysis buffer (G2002-100ML, Xavier); phosphatase inhibitor (G2007-1ML, Xavier); PMSF (G2008-1ML, Xavier); ZO-1 rabbit antibody (ZO-1 Polyclonal antibody, 21773-1-AP, Sanying Biotech); Claudin1 rabbit antibody (Claudin1 Polyclonal antibody, AF0127, Anke Biotech); Occludin rabbit antibody (Occludin Polyclonal antibody, 27260-1-AP, Sanying Biotech); IκBα rabbit antibody (IκBα Alpha Polyclonal antibody, 10268-1-AP, Sanying Biotech); p-NF-κB rabbit antibody (Phospho-NF-κB P65(Ser536) Recombinant antibody, 80379-2-RR, Sanying Biotech); TLR4 rabbit antibody (TLR4 Ployclonal antibody (19811-1-AP, Sanying Bio); keap1 (keap1 Ployclonal antibody, AF5266, Anke Bio); HO-1 (HO-1 Ployclonal antibody, AF5393, Anke Bio); NQO1 (NQO1 Ployclonal antibody, DF6437, Anke Bio); Nrf2 (Nrf2 Ployclonal antibody, AF0639, Anke Bio); Goat anti-rabbit IgG (HRP-conjugated Goat Anti-Rabbit IgG (H+L), SA00001-2, Sanying Bio); Membrane regeneration solution (SW3021, Baisha); Universal antibody diluent (BL1060A, Baisha); Color pre-stained protein marker (BL712A, Baisha); High-sensitivity ECL chemiluminescent substrate (BL520A, Baisha); Direct-labeled internal control antibody β-ation (Recombinant Anti-beta Actin antibody (HRPConjugated), ZB15001-HRP-100, Xavier).

[0036] Nanjing Jiancheng Biochemical Reagent Kits and Enzyme Immunosorbent Assay (ELISA) Kits: Total Antioxidant Capacity Assay Kit (T-AOC, A015-2-1), Interleukin-6 Cytokine Assay Kit (IL-6, MM-0163M2), Total Superoxide Dismutase Assay Kit (SOD, A001-1-1), Tumor Necrosis Factor-α Assay Kit (TNF-α, MM-0132M2), Malondialdehyde Assay Kit (MDA, A003-4-1), Interleukin-1β Assay Kit (IL-1β, MM-0040M2), Reduced Glutathione Assay Kit (GSH, A006-2-1), Interleukin-10 Cytokine Assay Kit (IL-10, MM-0176M2), Bacterial Endotoxin Lipopolysaccharide Assay Kit (LPS, MM-4580M2), D-Lactate Assay Kit (D-LA, MM-43853M2), Diamine Oxidase Assay Kit (DAO, MM-43853M2), MM-0228M2).

[0037] Healthy male Kunming rats (KM, Dashuo, SCXK (chuang) 2020-030, Chengdu, China), aged 4 weeks and weighing 20-22 g, were used for the experiment after a 7-day acclimatization period. The use of experimental animals was approved by the Animal Protection and Use Committee of Sichuan Agricultural University, approval number: 20230558, date: September 2023. All mice were housed in a standard SPF facility, fed with standard pelleted feed, and provided with free access to water.

[0038] 2. Experimental Methods 2.1 Experimental Materials: 1. MZH prepared in step (ii) of Example 1; 2. MZH@ES100 prepared in step (iii) of Example 1; 3. Mn3O4NPs prepared in step (1) of step (one) of Example 1; 4. ZIF-8.

[0039] 2.2 Synthesis method of ZIF-8: Mix 10 mL of 12.5 mg / mL 2-MI methanol solution and 10 mL of 45 mg / mL Zn(NO3)2·6H2O methanol solution, add the mixture to 20 mL of methanol, and stir magnetically for 15 min, sonicate for 30 min, and stir magnetically for 15 min in sequence. Centrifuge at 4200 r / min for 5 min, wash the resulting precipitate three times with methanol, and dry it at 60℃ for later use.

[0040] 2.3 Characterization of materials Characterization parameters (morphology, size, etc.) were obtained using transmission electron microscopy (TEM) and energy dispersive spectroscopy (TEM-mapping); elemental composition was determined using X-ray photoelectron spectroscopy (XPS); the crystal structure of the product was characterized using X-ray diffraction (XRD); and the surface charge of the nanoparticles before and after modification was analyzed using a Zeta potential analyzer. The Mn content in MZH and MZH@ES100 was determined using inductively coupled plasma mass spectrometry (ICP-MS).

[0041] 2.4 Stability in simulated gastrointestinal fluids Mn3O4NPs and MZH@ES100 were treated with simulated gastric fluid (SGF, pH 1.2) for 4 h, simulated intestinal fluid (SIF, pH 6.8) for 6 h, and simulated colonic fluid (SCF, pH 7.4) for 40 h, respectively. Morphological changes were then observed by TEM characterization. The specific treatment method was as follows: 10 mg each of Mn3O4NPs and MZH@ES100 were soaked sequentially in 20 mL of SGF, SIF, and SCF at 37℃, and stirred at 37℃ for 2 h, 6 h, and 40 h, respectively. After the treatment, the samples were washed three times with H2O (centrifuged at 4200 rpm for 5 min).

[0042] 2.5 Free radical scavenging activity and enzyme-like activity of materials before and after treatment with simulated gastrointestinal fluid To investigate the changes in free radical scavenging activity and enzyme-like activity of MZH@ES100 and Mn3O4NPs after treatment with simulated gastrointestinal fluid SF (SGF+SIF+SCF), the free radical scavenging ability and enzyme-like activity of 1 mg / mL untreated Mn3O4NPs, untreated MZH@ES100, and Mn3O4NPs and MZH@ES100 after SF (SGF+SIF+SCF) treatment were measured. The pretreatment methods for the materials are as follows: Mn3O4 NPs and MZH@ES100 were treated with simulated gastric fluid (SGF, pH 1.2) for 4 h, simulated intestinal fluid (SIF, pH 6.8) for 6 h, and simulated colonic fluid (SCF, pH 7.4) for 40 h, respectively. The specific treatment methods are as follows: 10 mg of each of Mn3O4 NPs and MZH@ES100 were soaked in 20 mL of SGF, SIF, and SCF at 37℃, respectively, and stirred at 37℃ for 2 h, 6 h, and 40 h, respectively. After the treatment, the materials were washed three times with H2O (centrifuged at 4200 rpm for 5 min).

[0043] The enzyme-like activities of Mn3O4NPs at different concentrations (0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7 mg / mL) were also detected. The specific methods are as follows: 2.5.1 Detection of Free Radical Scavenging Ability (1) 2,2′-Hydrazine-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) method: ABTS generates green free radical ABTS·+ under the oxidation of potassium persulfate (K2S2O8), which has a characteristic absorption peak at 734 nm. When antioxidants are present in the system, the generation of ABTS·+ is inhibited, resulting in a lighter solution color. The degree of decrease in absorbance at 734 nm is positively correlated with the ability of the antioxidant to scavenge free radicals. Therefore, the activity of the sample in scavenging ABTS free radicals can be assessed by measuring the change in absorbance.

[0044] Preparation of ABTS stock solution: Mix 1.1025 mL of 7.4 mmol / L ABTS aqueous solution with 3.575 mL of 2.6 mmol / L K2S2O8 aqueous solution, let stand in the dark at room temperature for 12 h, and then dilute 50 times with anhydrous ethanol.

[0045] Take 20 μL of each of the four different treated materials (1 mg / mL Mn3O4NPs untreated, MZH@ES100 untreated, and Mn3O4NPs and MZH@ES100 treated with SF(SGF+SIF+SCF)) (control group with distilled water) and add them to 530 μL of anhydrous ethanol. Then add 1.45 mL of the above ABTS stock solution and mix thoroughly. UV-Vis full wavelength scanning is performed.

[0046] (2) Methylene Blue (MB) Method: This method is used to detect the scavenging ability of materials against hydroxyl radicals (·OH). MB has a characteristic absorption peak at 664 nm, and MB can be bleached by ·OH. Therefore, the absorbance value of the remaining MB can be used to evaluate the material's ability to scavenge ·OH. The specific method is as follows: First, ·OH was generated via the Fenton reaction, i.e., 600 μL of 5 mmol / L FeCl2·4H2O was mixed with 400 μL of 5 mmol / L hydrogen peroxide (H2O2) and reacted in the dark for 5 min. After 5 min, 200 μL of four different treated materials (1 mg / mL untreated Mn3O4NPs, untreated MZH@ES100, and Mn3O4NPs and MZH@ES100 treated with SF (SGF+SIF+SCF)) and 800 μL of 1 mmol / L MB were added sequentially, mixed well, and reacted in the dark for another 50 min. UV-Vis full-wavelength scanning was performed.

[0047] 2.5.2 Detection of Glutathione peroxidase (GSH-Px / GPX) activity The dithio-p-nitrobenzoic acid (DTNB) method is used to detect GPX-like enzyme activity in materials. Hydrogen peroxide (H₂O₂) reacts with reduced glutathione (GSH) to produce oxidized glutathione (GSSG), and GPX promotes this reaction. Simultaneously, GSH reacts with 5,5'-dithio-p-nitrobenzoic acid (DTNB) under GPX catalysis to form a yellow 5-thio-2-nitrobenzoic acid anion, which has a maximum absorption peak at 412 nm. Therefore, if the material exhibits GPX-like enzyme activity, it catalyzes the formation of GSSG from GSH, leading to a decrease in GSH and thus a reduction in the yellow product. The amount of GSH reduction can be calculated, and this reduction reflects the GPX-like enzyme activity.

[0048] Four different treatment materials (untreated Mn3O4NPs (1 mg / mL), untreated MZH@ES100, and Mn3O4NPs and MZH@ES100 treated with SF(SGF+SIF+SCF)) and different concentrations (0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7 mg / mL) of Mn3O4NPs were mixed with 2.7 mL of PBS buffer (the control group was replaced with an equal volume of distilled water) and incubated in a water bath at 37°C for 5 min. Then, 400 μL of GSH and 200 μL of H2O2 (10 mmol / L and 15 mmol / L, respectively) were added sequentially. After mixing, the mixture was allowed to stand at room temperature for 10 min, and then 500 μL of DTNB colorimetric solution was added, and the mixture was reacted at room temperature for 1 min. Finally, a full-wavelength scan was performed using UV-Vis, and the GPX-like enzyme activity in the sample was calculated based on the absorbance value at 412 nm (the change in absorbance was positively correlated with enzyme activity):

[0049] (G1 is the final absorbance value of the material group, and G0 is the final absorbance value of the control group.) 2.5.3 Detection of Superoxide Dismutase (SOD) Activity The nitrotetrazole blue (NBT) method was used to detect the SOD-like enzyme activity of materials. Riboflavin and L-methionine generate superoxide anions (O2·-) under ultraviolet irradiation. O2·- can reduce nitrotetrazole blue (NBT) to blue formazan, which has a characteristic absorption peak at 560 nm. If the material exhibits SOD-like enzyme activity, i.e., it decomposes O2·-, the amount of formazan produced will decrease, and the absorbance value will decrease. This allows for comparison of the strength of the SOD-like enzyme activity of the materials. The specific operation is as follows: Add 150 μL each of untreated Mn3O4NPs, untreated MZH@ES100, and Mn3O4NPs and MZH@ES100 treated with SF(SGF+SIF+SCF) to 1.35 mL of phosphate buffer (30 mM). Then add 300 μL of 100 nmol / mL EDTA solution, 400 μL of 100 μmol / mL L-methionine, 300 μL of 200 nmol / mL riboflavin, and 500 μL of 460 nmol / mL NBT. After mixing, irradiate at 25 °C and 4000 lx for 20 min, followed by UV-Vis full-wavelength scanning.

[0050]

[0051] (S1 is the final absorbance value of the material group, and S0 is the final absorbance value of the control group.) 2.5.4 Detection of Catalase (CAT) Activity The ammonium molybdate method was used to detect CAT-like enzyme activity in materials. CAT can decompose H₂O₂, and H₂O₂ can combine with ammonium molybdate to form a pale yellow complex with absorbance at 405 nm. If the material exhibits CAT-like enzyme activity, the amount of this pale yellow complex will be reduced. The activity of the CAT-like enzyme in the material can be compared by comparing the absorbance values.

[0052] 3% H2O2 was mixed with untreated Mn3O4NPs (1 mg / mL), untreated MZH@ES100, and Mn3O4NPs and MZH@ES100 treated with SF(SGF+SIF+SCF), as well as Mn3O4NPs at different concentrations (0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7 mg / mL). The mixtures were incubated in the dark for 20 min, and the reaction was terminated by adding ammonium molybdate. UV-Vis full-wavelength scanning was then performed immediately afterward. The CAT-like enzyme activity was reflected by the H2O2 scavenging rate, which was measured at 405 nm.

[0053] (C1 is the final absorbance value of the material group, and C0 is the final absorbance value of the blank control group.) 2.6 Determination of safe oral dose in MZH@ES100 mice Thirty male KM mice (20-22 g) were divided into 5 groups of 6 mice each. Different doses of MZH@ES100 (0, 10, 20, 40, 80 mg / kg body weight) were administered by gavage daily for 9 consecutive days (materials were suspended in 0.5% CMC-Na, freshly prepared and used). On the 10th day, the mice were anesthetized, weighed, and blood was collected from the orbital region. The mice were then dissected, and the heart, liver, spleen, lungs, and kidneys were removed, weighed, and organ indices calculated. Liver, spleen, kidney, and colon tissue samples were collected, and H&E staining was performed on the sections. Serum levels of AST, ALT, ALP, TBIL, CK, LDH, Urea, and Crea were measured using an automated biochemical analyzer.

[0054] 2.7 Therapeutic effect of MZH@ES100 on DSS-induced ulcerative colitis in mice Eighty male KM mice weighing 20–22 g were randomly divided into five groups of 16 mice each (Table 1). These groups were: a healthy control group (Control), a DSS model group (DSS), and three MZH@ES100 treatment groups (2.5, 10, and 40 mg / kg BW; DSS+2.5, DSS+10, and DSS+40). The specific treatment methods are as follows: (1) Modeling methods: such as Figure 4As shown in Figure A, a mouse colitis model was established by adding 4% DSS to the drinking water of mice in the DSS model group and the MZH@ES100 treatment group for 9 consecutive days. No DSS was added to the normal drinking water of mice in the healthy treatment group.

[0055] (2) Treatment methods: During the 9-day experiment, mice in the MZH@ES100 treatment group were treated once daily by gavage at doses of 2.5, 10, and 40 mg / kg BW. The MZH@ES100 material was suspended in 0.5% CMC-Na before gavage, and was prepared fresh each time. The mixture was ultrasonically homogenized before gavage to ensure uniform dispersion of MZH@ES100 in 0.5% CMC-Na. The healthy control group and the DSS model group received the same volume of blank 0.5% CMC-Na. Each mouse was weighed daily, and the severity of colitis was quantified using the Disease Activity Index (DAI), which included the following parameters: weight loss (0% loss, 0 points; 1-3% loss, 1 point; 3-6% loss, 2 points; 6-9% loss, 3 points; >9% loss, 4 points); fecal consistency (normal, 0 points; loose stool, 2 points; diarrhea, 4 points); rectal bleeding (normal, 0 points; visible blood in stool, 2 points; gross hemorrhage, 4 points). These parameters were summed to obtain the DAI score.

[0056] (3) On day 10, mice were anesthetized, blood was collected from the eyeballs, colon length was measured, and colon slices, tissues, and gene samples were collected.

[0057] Table 1. Grouping and Treatment of Mice

[0058] Note: "+" indicates that the process should be executed; "-" indicates that the process should not be executed.

[0059] 2.8 Processing of serum and intestinal tissue samples All mice were anesthetized with tribromoethanol. Before blood collection, the whiskers of the mice were trimmed, and the eyeballs were removed using curved forceps to allow blood to flow out. Blood was then collected in enzyme-free EP tubes. Samples were incubated at room temperature for 1 hour and then centrifuged at 4°C and 3500 rpm for 5 minutes to separate the serum. The separated serum was collected in new enzyme-free EP tubes for subsequent testing. The obtained serum should be a pale yellow, transparent liquid.

[0060] Thoroughly rinse the colon tissue sample with pre-cooled distilled water. Take 0.1 g of colon tissue into an enzyme-free homogenizing tube, add homogenizing beads and 0.9 mL of 0.9% NaCl solution, and homogenize three times at 4℃, 60 Hz, and 1 min. Centrifuge the homogenized tissue at 4℃ and 3500 r / min for 5 min. Collect the supernatant into a new enzyme-free EP tube for subsequent analysis.

[0061] 2.9 Colonic biochemical marker detection The total antioxidant capacity (T-AOC), GSH content, SOD activity, MDA content, and total protein (TP) content in the treated colon supernatant were detected according to the instructions of Nanjing Jiancheng Biotechnology Reagent Kit. The results of T-AOC, SOD, GSH, and MDA assays were quantified using TP.

[0062] 2.10 Cytokine Detection The levels of inflammation-related cytokines IL-6, IL-10, IL-1β, and TNF-α in the processed colon supernatant were detected according to the instructions of the enzyme immunoassay ELISA kit; and the levels of intestinal barrier-related cytokines D-LA, DAO, and LPS in the serum were detected.

[0063] 2.11 Histological Analysis Liver, spleen, kidney, and colon tissues were stained with hematoxylin and eosin (H&E). After collection, the tissues were fixed with 4% paraformaldehyde, dehydrated and cleared with 75%, 80%, 95%, and 100% ethanol, embedded in paraffin, and cut into 4 μm thick sections according to standard procedures. Each tissue was stained with HE, dried, and then imaged using a Leica Aperio Versa 8 microscope.

[0064] 2.12 RNA extraction and quantitative real-time PCR (qRT-PCR) Total RNA was extracted using the TRIzol method. The solution was stored at -80℃ for later use. cDNA was synthesized according to the Super Mix Perfect for qPCR kit instructions. Specific primers were designed and synthesized on the SYBR Redremix Ex Tag™ II platform (Table 2). qRT-PCR was performed according to the SYBR Redremix Ex Tag™ II specification. The mRNA expression levels of CuZn-SOD, Mn-SOD, CAT, GPX, ZO-1, Occludin, and Claudin-1 were detected, with β-actin as an internal reference gene. -△△Ct The method was analyzed.

[0065] 2.13 Western Blot Analysis The expression of proteins related to the TLR4 / NF-κB pathway, Nrf2 / Keapl pathway, and tight junctions in mouse colon was detected by Western blotting. Proteins were extracted from colon tissue using RIPA lysis buffer, and protein concentrations were determined according to the BCA kit instructions. Proteins were separated using 10% SDS-PAGE (8% for ZO-1) and transferred to PVDF membranes pre-activated with methanol. The membranes were blocked with 5% skim milk on a shaker at room temperature, followed by incubation with primary antibodies against the target proteins (overnight at 4°C) and HRP-labeled secondary antibodies (shakerically at room temperature for 1 h). Protein bands were visualized using an ECL detection kit on a Bio-Rad ChemiDoc XRS+ system (Bio-Rad Laboratories, Inc., Hercules, USA). Differences in protein expression were detected using ImageJ 2x, and grayscale values ​​were calculated. Results from the healthy control group were normalized.

[0066] 2.14 Statistical Analysis TEM images were processed using ImageJ 2x, XPS images using Avantage software, and XRD images using MDI Jade6 software, respectively. Statistical analysis and graphing were performed using Origin 2021 software. Data for each group were first subjected to the Shapiro-Wilk normality test, followed by the Levene homogeneity of variance test. Multiple comparisons were performed using one-way ANOVA and LSD tests. All data are presented as mean ± standard deviation (x ± SE). P <0.05 indicates that the difference is significant. P <0.01 indicates that the difference is highly significant. P <0.001 indicates that the difference is highly significant. P >0.05 indicates no significance.

[0067] 3. Experimental Results and Discussion 3.1 Characterization of materials To verify the successful synthesis of MZH, the Mn3O4NPs were first characterized by TEM. Figure 1 A) ZIF-8 ( Figure 1 B), Mn3O4@ZIF-8 ( Figure 1 C), MZH ( Figure 1 Based on the morphological characteristics of D), a large number of Mn3O4NPs were found to be attached to the surface of ZIF-8. The Mn3O4NPs were uniformly distributed spherical particles with a particle size ranging from 6.0 to 10.5 nm. The ZIF-8 particles had a diameter of approximately 50 nm. Figure 1C, the morphological profile of the synthesized Mn3O4@ZIF-8 still exhibits relatively obvious ZIF-8 characteristics. Figure 1 B) Basic characteristics: The particle size is approximately 300-500 nm, and its surface has a large number of Mn3O4NPs distributed; while MZH (Mn3O4NPs) synthesized by further loading hyaluronic acid (HA) Figure 1 D) Morphologically similar to Mn3O4@ZIF-8 in TEM characterization Figure 1 C) There is little difference.

[0068] XRD results are derived from Figure 1 As can be seen from E, the diffraction peaks of Mn3O4@ZIF-8 simultaneously possess the main characteristic peaks of both Mn3O4NPs (gray dashed line) and ZIF-8 (red dashed line), further indicating that Mn3O4NPs were successfully loaded onto ZIF-8, and that Mn3O4@ZIF-8 retained the crystal structures of both Mn3O4NPs and ZIF-8 after loading. Analysis of the Zeta potential of the material at each stage revealed ( Figure 1 F), ZIF-8 and Mn3O4NPs have Zeta potentials of 6.71±0.62 mV and 11.90±0.26 mV, respectively. After recombination, the Zeta potential rises to 21.16±0.19 mV (Mn3O4@ZIF-8). Although Mn3O4@ZIF-8 and MZH show little difference in morphology and size in TEM characterization, the Zeta potential of Mn3O4@ZIF-8 loaded with HA (MZH) decreases from 21.6 mV to -22.08 mV. This indicates that the negatively charged HA covers the originally positively charged Mn3O4@ZIF-8, further proving the successful binding of MZH.

[0069] To further confirm the distribution of Mn3O4 NPs, Mn3O4@ZIF-8 was characterized using energy-dispersive X-ray spectroscopy (TEM-mapping). The results are presented by... Figure 1 As shown in Figure G, Zn, N, and C elements constitute the main framework of ZIF-8, while Mn elements are distributed on ZIF-8. This further confirms the integration of Zn and Mn elements in the composite material, and Mn3O4NPs were successfully loaded into ZIF-8.

[0070] ICP-MS detection of Mn content in MZH and MZH@ES100: The Mn contents in MZH and MZH@ES100 are 5.20±0.2433% and 1.28±0.0632%, respectively (Table 2). Calculations show that the MZH content in MZH@ES100 is 24.6%.

[0071] Table 2. Mn content in MZH and MZH@ES100

[0072] XPS was used to further elucidate the chemical composition and structural characteristics of Mn3O4NPs and Mn3O4@ZIF-8. The Mn, Zn, C, and O elements present in the samples were identified through comprehensive spectral analysis. Figure 2 A). Further analysis of the elemental peak spectrum. Mn 2p exists in Mn3O4@ZIF-8. 3 / 2 (653.27 eV) and Mn 2p 1 / 2 (641.27 eV) orbital peak ( Figure 2 B, E), this indicates that Mn element exists in the oxidized state Mn. 2 + Mn 3+ Both forms exist. Simultaneously, Zn 2p exists in Mn3O4@ZIF-8. 3 / 2 (1044.86 eV) and Zn 2p 1 / 2 The (1021.82 eV) orbital peak indicates that the Zn element is in the Zn orbital position. 2+ oxidation state ( Figure 2 D). XPS spectrum O1s peak diagram shows ( Figure 2 After modification with ZIF-8 (C, F), the lattice oxygen content (Mn-O-Mn) ratio decreased, while the corresponding surface adsorbed oxygen (Mn-OH) and oxygen vacancy (HOH) contents increased. Oxygen vacancies can provide catalytic active sites for enzyme reactions; therefore, the increase in oxygen vacancies may help promote the enzyme-like activity of the composite material, indicating that the loaded Mn3O4@ZIF-8 may be more conducive to the enzyme-like activity of Mn3O4NPs. Related studies have shown that the HA surface contains a large number of carboxyl groups, while the ZIF-8 surface has a large number of Zn groups. 2+ Therefore, the mechanism by which HA binds to ZIF-8 may be related to ion coordination.

[0073] 3.3 Stability of the material in simulated gastrointestinal fluid—free radical scavenging activity and enzyme-like activity ES100 material exhibits enteric coating properties, maintaining its structural integrity even at low pH levels in the stomach. Therefore, the enteric coating properties of the composite material were verified by simulating the gastrointestinal environment. The results are presented by… Figure 3 As shown in Figure A, TEM characterization of MZH after inclusion in ES100 revealed that it was spherical with a particle size of approximately 500 nm. ES100 typically disintegrates at pH > 7; the pH in the stomach is approximately 1.2, in the small intestine approximately 6.8, and in the colon approximately 7.4. TEM results after treatment in a simulated gastrointestinal environment showed… Figure 3A) MZH@ES100 maintained its structural integrity in distilled water (pH 7.0), simulated gastric juice (SGF, pH 1.2), and simulated small intestinal juice (SIF, pH 6.8). However, it underwent significant deformation after treatment with simulated colonic juice (SCF, pH 7.4). TEM characterization showed no significant change in morphology after treatment with Mn3O4NPs. This further demonstrates the successful inclusion of ES100, indicating that MZH@ES100 can deliver MZH into the colon for release and exert its effect. It also suggests that for long-term storage in solution, the solution pH should be <7 to maintain its stability, but short-term exposure to distilled water does not cause deformation.

[0074] Increased ROS is an important mechanism in the development of IBD. The body's endogenous antioxidant system (such as SOD, GPX, and CAT enzymes) can effectively remove ROS produced in the body. For example, superoxide dismutase (SOD) catalyzes the formation of H2O2 from O2·-; glutathione peroxidase (GPX) catalyzes the reaction of H2O2 with reduced glutathione (GSH) to produce oxidized glutathione (GSSG); and catalase (CAT) decomposes H2O2 into H2O and O2. When the ROS concentration exceeds its regulatory capacity, external antioxidants can be used to consume excess ROS and combat inflammation. Therefore, to test whether the material synthesized in this study can be used to assist in the consumption of excess ROS, the ABTS free radical scavenging method was used to detect the total antioxidant capacity of the material, the MB method was used to detect the ·OH scavenging capacity, and the DTNB, NBT, and ammonium molybdate methods were used to detect its GPX-like, SOD-like, and CAT-like enzyme activities, respectively. The results showed that compared with the untreated state ( Figure 3 BI), MZH@ES100, after being treated with SF, has a ROS removal capability ( Figure 3 B, C) and SOD-like substances Figure 3 D), GPX ( Figure 3 E), CAT Figure 3 F) Enzyme activities were significantly increased, possibly related to their deformation. Conversely, the related activities of Mn3O4NPs were greatly reduced after SF treatment, indicating that the gastrointestinal environment may lead to partial inactivation of Mn3O4NPs. Furthermore, after SF treatment, the related free radical scavenging activity and enzyme-like activity of MZH@ES100 (MZH@ES100+SF in the figure) were stronger than those of Mn3O4 (Mn3O4+SF in the figure). Figure 3 GI). Since Mn3O4NPs are the main substances exhibiting the relevant activities, the relevant activities of different concentrations of Mn3O4NPs were further investigated. Figure 3GI studies found that when the concentration of Mn3O4NPs reached 0.4 mg / mL, it exhibited good CAT, SOD, and GPX-like enzyme activities; when the concentration of Mn3O4NPs was 0.5 mg / mL, the GPX-like enzyme activity / GSH scavenging rate was 66.35%, the SOD-like activity / NBT scavenging rate was 83.23%, and the CAT-like enzyme activity / H2O2 scavenging rate was 45.56%; when the concentration of Mn3O4NPs was 0.6 mg / mL, the GPX-like activity / GSH scavenging rate was 81.21%, the SOD-like activity / NBT scavenging rate was 83.34%, and the CAT-like enzyme activity / H2O2 scavenging rate was 49.35%; when the concentration of Mn3O4NPs was 0.7 mg / mL, the GPX-like activity / GSH scavenging rate was 82.84%, the SOD-like activity / NBT scavenging rate was 84.16%, and the CAT-like enzyme activity / H2O2 scavenging rate was... ２ O ２ The clearance rate was 49.90%; therefore, the inclusion of ES100 can protect the relevant activities of the material, so that the composite material can remain stable in the gastric environment and release more of its relevant activities in the colonic environment.

[0075] In vitro stability tests in simulated gastrointestinal fluid demonstrated the material's enteric solubility. Furthermore, the ES100-encapsulated material exhibited enhanced enzyme activity after passing through the simulated gastrointestinal fluid, while unprocessed Mn3O4 showed decreased enzyme activity after treatment with the simulated gastrointestinal fluid. Therefore, it is speculated that Mn3O4 is highly likely to be inactivated in the stomach or, due to a lack of protection, will be significantly depleted during this process, resulting in a low concentration at the final detection level.

[0076] 3.4 MZH@ES100 alleviates DSS-induced colitis symptoms in mice The biosafety of MZH@ES100 in mice was first evaluated at oral doses of 0, 10, 20, 40, and 80 mg / kg daily for 9 consecutive days. As shown in Figures 8-10, compared with the blank control group, different concentrations (0, 10, 20, 40, and 80 mg / kg BW) of MZH@ES100 administered by gavage for 9 consecutive days showed no significant toxicity in mice. During the 9-day evaluation period, mice did not exhibit symptoms of poisoning such as diarrhea, vomiting, or mental abnormalities. After anesthesia and euthanasia, relevant liver and kidney indicators in their serum were measured, and major organs such as the liver, spleen, kidney, and colon were collected for H&E staining. The results indicate that different oral doses of MZH@ES100 did not cause weight gain in mice. Figure 8 A) Organ Index ( Figure 8 B) Abnormalities will not lead to abnormalities in serum liver and kidney related indicators in mice. Figure 9 H&E staining of liver, spleen, kidney, and colon tissues from mice in groups A, B, and C at different concentrations is shown below. Figure 10As shown, compared with the blank control group, there were no obvious pathological changes in the liver, kidney, spleen and colon of mice in different concentrations of the drug administration group.

[0077] DSS is a water-soluble, negatively charged sulfate polysaccharide, often added to drinking water to induce colitis in mice. Related studies have shown that DSS-induced colitis is most similar to human ulcerative colitis (UC). Therefore, this study investigated the potential therapeutic effects and possible mechanisms of MZH@ES100 on UC by adding DSS to the drinking water of mice to induce colitis. Figure 4 A). DAI is an important indicator for quantifying the degree of colitis, superficially reflecting the severity of DSS-induced colitis in mice. Figure 4 As shown in Figure B, the DAI index of mice in the DSS model group was significantly higher than that of the healthy control group and the MZH@ES100 treatment group after day 3, and their body weight decreased significantly. Figure 4 C), at day 9, the DAI index of mice in the DSS model group reached approximately 5. Compared to the DSS model group, the MZH treatment group significantly reduced the DAI index in mice ( ### P <0.001). Meanwhile, the colon in the DSS model group became shorter ( Figure 4 D, E), H&E staining revealed histopathological changes in the colon, including cell necrosis, mucosal damage, increased inflammatory cell infiltration, and destruction of crypt structures. Figure 4 As shown in Figure F, compared with the healthy control group, the DSS model group mice showed colon cell necrosis, severe mucosal damage, increased inflammatory cell infiltration, and destruction of crypt structure (extensive intestinal villus dissolution, colon cell necrosis, and loss of normal structure were visible in black). Amyloidosis and extensive inflammatory cell infiltration (black) were observed in the intestinal lumen. ); crypt atrophy and tortuosity, with increased distance between the base and the muscularis mucosa (black) ), abnormal shape (black) The treatment groups with different concentrations of MZH@ES100 all showed improvement compared to the DSS model group: in the DSS+2.5 treatment group, necrosis and atrophy of the colonic mucosal epithelium were observed (red). ), inflammatory cell infiltration with necrosis (red) ), local loss of crypt structure, intestinal gland atrophy (red) The number of goblet cells was increased compared to the DSS model group; local necrosis (green) was observed in the colonic mucosa of the DSS+10 treatment group. ), inflammatory cell infiltration with necrosis (green) The number of goblet cells was increased in the DSS model group, while the number of inflammatory cells was decreased in the DSS+2.5 treatment group. No inflammatory cell infiltration was found in the colonic mucosa, muscularis mucosae, submucosa, muscularis propria, and serosa of the DSS+40 treatment group, and no significant pathological changes were observed, showing a normal histological structure comparable to the healthy control group. Therefore, the DSS+40 treatment group showed significant improvement compared to the DSS model group.

[0078] Compared with the DSS model group, the symptoms of mice treated with different concentrations of MZH@ES100 were all alleviated. Figure 4 (DF). This further demonstrates the successful establishment of the DSS-induced mouse colitis model, and that MZH@ES100 can alleviate the symptoms of DSS-induced colitis.

[0079] 3.5 MZH@ES100 inhibits colonic oxidative stress and activates the Nrf2 / Keap1 pathway to combat DSS-induced colitis. Oxidative stress is one of the main causes of IBD. This study evaluated the effect of MZH@ES100 on DSS-induced oxidative stress in the colon of mice by detecting colonic antioxidant biochemical indicators in different groups, analyzing the mRNA expression levels of colonic antioxidant genes, and analyzing the expression levels of Nrf2 / Keap1-related proteins in the antioxidant pathway.

[0080] T-AOC reflects the overall level of various antioxidant macromolecules, small molecules, and enzymes in the body; SOD is an important antioxidant enzyme that plays a crucial role in the body's oxidation-antioxidant balance and participates in the first line of defense against ROS within cells; GSH is a non-enzymatic low-molecular-weight scavenger often used to measure the body's antioxidant capacity; oxidative stress produces ROS, triggering lipid peroxidation, which further produces lipid peroxides such as MDA, so the degree of cell damage can be indirectly reflected by detecting the amount of MDA. Figure 5 As shown, compared with the healthy control group, the colonic T-SOD in the DSS model group ( Figure 5 A), T-AOC ( Figure 5 B) GSH significantly decreased ( Figure 5 D)( *** P <0.001), MDA increased significantly ( Figure 5 C)( *** P <0.001). MZH@ES100 significantly inhibited the changes caused by DSS ( Figure 5 AD)( * P <0.05). The mRNA expression levels of CuZn-SOD, Mn-SOD, CAT, and GPx in the colon are as follows: Figure 5As shown in E. Compared with the healthy control group, the expression of CuZn-SOD, Mn-SOD, CAT and GPx genes in the colon of mice in the DSS model group was significantly downregulated (E). *** P <0.001). Compared with the DSS model group, the expression of CuZn-SOD, Mn-SOD, CAT and GPx genes was significantly upregulated in different concentrations of MZH@ES100 treatment groups. ### P <0.001), even close to that of the healthy control group.

[0081] The Nrf2 / Keap1 pathway plays a crucial role in the development and maintenance of normal function of the gastrointestinal tract (GI). Increasing evidence suggests that activation of the Nrf2 / Keap1 pathway in IBD has potential therapeutic benefits, having been shown to reduce intestinal inflammation, restore intestinal barrier integrity, and enhance antioxidant defense mechanisms. The structure of the Keap1 protein changes under oxidative stress. As a repressor of Nrf2, Keap1 normally binds to and promotes the ubiquitination and degradation of Nrf2. Therefore, when the Keap1 structure changes, undegraded Nrf2 is released and further translocated to the nucleus, leading to the activation of related antioxidant genes. This promotes the expression of related proteins such as HO-1 and NQO1, activating endogenous cellular antioxidant defense mechanisms. Western blot analysis of Nrf2 / Keap1-related proteins in the antioxidant pathway is shown below. Figure 5 As shown in F. Compared with the healthy control group, the expression of Nrf2 and Keap1 proteins upstream of the Nrf2 / Keap1 pathway in the colon of the DSS model group decreased ( ). *** P <0.001) and rising ( *** P <0.001), downstream HO-1 and NQO1 expression decreased ( ** P <0.01), the Nrf2 / Keap1 pathway was inhibited. However, compared to the DSS model group, MZH@ES100 treatment reversed this change and simultaneously enhanced the expression of downstream Nrf2 / Keap1 proteins HO-1 and NQO1. # P <0.05).

[0082] The above results indicate that the Nrf2 / Keap1 pathway, the colonic antioxidant defense mechanism, is inhibited in DSS-induced colitis model mice, while MZH@ES100 can activate the Nrf2 / Keap1 pathway to initiate antioxidant defense mechanisms, inhibit oxidative stress, and alleviate DSS-induced colitis. Changes in colonic biochemical indicators and the expression of antioxidant genes also suggest that MZH@ES100 alleviates DSS-induced colitis in mice by inhibiting oxidative stress.

[0083] 3.6 MZH@ES100 inhibits inflammatory responses and suppresses the TLR4 / NF-κB pathway to counteract DSS-induced colitis. Dysfunction of the TLR4 / NF-κB signaling pathway plays a crucial role in the pathogenesis of IBD. This study evaluated the effect of MZH@ES100 on DSS-induced colonic inflammation in mice by detecting serum and colonic inflammatory cytokines in different groups of mice, analyzing the mRNA expression levels of colon-associated inflammatory cytokines and the expression levels of TLR4 / NF-κB-related proteins in the inflammatory pathway.

[0084] Activation and dysfunction of both the innate and adaptive immune systems can lead to abnormal inflammatory responses in the gut of IBD patients, resulting in the influx of innate immune cells (neutrophils, macrophages, dendritic cells, and NK cells) and adaptive immune cells (B cells and T cells) into the lamina propria. With the activation of immune cells, levels of pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6 increase, while levels of anti-inflammatory factors such as IL-10 decrease. The results of this study show (…). Figure 6 AC), compared with the healthy control group, the levels of pro-inflammatory factors IL-6, TNF-α, and IL-1β in the colon and serum of mice in the DSS model group were significantly increased ( *** P <0.001), the level of the anti-inflammatory factor IL-10 decreased significantly ( *** P <0.001). The MZH@ES100 treatment group significantly inhibited this change ( ## P <0.01). Meanwhile, the mRNA expression levels of TNF-α, IL-1β, and IL-6 were significantly increased in the DSS model group ( *** P <0.001), the mRNA expression level of IL-10 was significantly reduced ( *** P <0.001). Compared with the DSS model group, the mRNA expression levels of TNF-α, IL-1β, IL-6 and IL-10 in the MZH@ES100 treatment groups of different concentrations were closer to those in the healthy control group.

[0085] TLR4 is a pattern recognition receptor that recognizes pathogen-associated molecular patterns (PAMPs) entering the bloodstream and activates the downstream NF-κB signaling pathway, leading to the phosphorylation and degradation of IκBα protein. IκBα is an inhibitory protein that binds to NF-κB, preventing NF-κB from entering the nucleus and activating the transcription of inflammatory genes. Therefore, when TLR4 / NF-κB is activated, IκBα is phosphorylated and degraded, releasing NF-κB (p-NF-κB) into the nucleus, thereby activating the transcription of inflammatory genes and initiating an inflammatory response. Western blot analysis of TLR4 / NF-κB-related proteins in this study showed that… Figure 6 (D) Compared with the healthy control group, the DSS model group showed increased expression levels of TLR4 and p-NF-κB proteins in the colon, while the expression level of IκBα protein was decreased, suggesting that the TLR4 / NF-κB pathway was activated. Conversely, treatment groups with different concentrations of MZH@ES100 inhibited the activation of the TLR4 / NF-κB pathway. Simultaneously, activation of the TLR4 / NF-κB signaling pathway led to intestinal myeloid cell infiltration and the release of inflammatory mediators such as TNF-α, IL-1β, and IL-6 in the colonic tissue. This corresponds to the aforementioned results regarding cytokines, etc. Figure 6 AC).

[0086] The results above indicate that MZH@ES100 can alleviate DSS-induced colonic inflammation in mice by inhibiting the TLR4 / NF-κB pathway.

[0087] 3.7 MZH@ES100 maintains the intestinal barrier and increases the expression of tight junction proteins to combat DSS-induced colitis. The epithelial barrier in the colon is the first line of defense against luminal antigens, toxins, and microbial components. Related studies have shown that diethylstilbestrol (DSS) can disrupt this barrier by damaging epithelial cells, increasing permeability, and exposing the lamina propria to harmful substances. The sulfated polysaccharide structure of DSS interacts with the negatively charged mucosal surface, disrupting the stability of the protective mucus layer and impairing the integrity of tight junctions, allowing luminal contents to penetrate deeper into the intestinal tissue. This study evaluated the effect of MZH@ES100 on DSS-induced intestinal barrier damage in mice by detecting serum levels of intestinal barrier-related cytokines in different groups of mice and analyzing the mRNA and protein expression levels of colonic tight junction proteins.

[0088] DAO is a highly active intracellular enzyme found in the cytoplasm of intestinal mucosal cells, while LPS and DLA are metabolic products of gut microbiota. When the intestinal barrier / mucosa is damaged or permeability is altered, LPS, DLA, and DAO are released into the bloodstream. The expression of intestinal barrier-related cytokines in mouse serum is shown below. Figure 7As shown in Figure A, compared with the healthy control group, the serum levels of D-LA, DAO, and LSP in the DSS model group mice were significantly increased ( *** P <0.001). Serum D-LA, DAO, and LSP levels decreased significantly after MZH@ES100 treatment. ## P <0.01).

[0089] Impaired mechanical barrier function of intestinal epithelial cells is an unavoidable pathological feature of intestinal dysplasia (IBD). As a crucial component of this barrier, tight junctions (TJs) expressed in intestinal epithelial cells form physical barriers preventing exogenous substances from passing through the intercellular spaces. TJ structures are composed of various molecules, primarily including ocludin, claudin, and zonula occludens (ZO). In ulcerative colitis (UC), the downregulation of ocludin, claudin1, and ZO-1 leads to TJ structure disruption and increased intestinal epithelial permeability. This results in the translocation of bacterial products such as lipopolysaccharide (LPS), exacerbating epithelial damage. Simultaneously, elevated levels of related inflammatory cytokines and their mediators further exacerbate TJ structure disruption. The results of this study show that… Figure 7 B), compared with the healthy control group, the mRNA expression levels of the colonic tight junction proteins ZO-1, Claudin1, and Occludin in the DSS model group mice were significantly decreased. *** P <0.001). MZH@ES100 can alleviate the decrease in mRNA levels of colonic tight junction proteins ZO-1, Claudin1, and Occludin caused by DSS. Meanwhile, Western blot analysis of mouse colonic tight junction proteins showed... Figure 7 As shown in Figure C, compared with the healthy control group, the expression of colonic tight junction proteins ZO-1, Occludin, and Claudin1 was reduced in the DSS model group, while MZH@ES100 treatment reversed this change compared with the DSS model group.

[0090] The above results further suggest that the DSS-induced colitis model was successfully established, and that MZH@ES100 can counteract DSS-induced colitis by maintaining the intestinal barrier.

[0091] 4. Experimental Conclusions In summary, a composite material MZH@ES100 based on manganese-based nanozymes Mn3O4NPs was successfully synthesized. Its structure remained intact after treatment in simulated gastric and small intestinal environments, but deformed after treatment in a simulated colonic environment. Furthermore, the free radical scavenging ability and enzyme activity of MZH@ES100 were enhanced after treatment compared to before treatment. This may be related to the deformation of the composite material in the simulated colonic environment, releasing a large amount of MZH (mainly due to the free radical scavenging ability and enzyme activity exerted by Mn3O4NPs attached to the MZH surface). Meanwhile, the activity related to Mn3O4NPs decreased significantly. This indicates that MZH@ES100 can carry Mn3O4NPs into the colon for release and enzyme-like activity while maintaining structural integrity beforehand; Mn3O4NPs are partially inactivated after passing through the gastrointestinal tract. Simultaneously, oral administration of MZH@ES100 can counteract DSS-induced colitis in a mouse model by inhibiting intestinal inflammation, suppressing oxidative stress, and maintaining the intestinal barrier. Its main mechanisms include inhibiting the TLR4 / NF-κB pathway, activating the Nfr2 / Keap1 pathway, and increasing the expression of intestinal tight junction proteins. These findings suggest that oral administration of MZH@ES100 may be an effective way to prevent colitis and combat oxidative stress.

[0092] The embodiments of this application have been described above with reference to the accompanying drawings. However, this application is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of this application without departing from the spirit and scope of the claims, and all of these forms are within the protection scope of this application.

Claims

1. A method for preparing a manganese-based nanoenzyme composite material, characterized in that, Includes the following steps: (a) Synthesis of Mn3O4@ZIF-8 NPs: Mn3O4-PVP was prepared by Mn3O4 NPs and PVP10, and then Mn3O4-PVP was subjected to Zn 2+ During the reaction with 2-MI to form ZIF-8, Mn3O4@ZIF-8 NPs were loaded into the ZIF-8 structure to obtain Mn3O4@ZIF-8 NPs; (ii) MZH was synthesized by coating HA onto the surface of Mn3O4@ZIF-8 through ion coordination; (iii) MZH is included in ES100 to synthesize MZH@ES100.

2. The method for preparing the manganese-based nanoenzyme composite material according to claim 1, characterized in that, Step (I) includes the following steps: (1) Dissolve 400±1 mg Mn3O4 NPs in 10±0.1 mL methanol, add 70±1 mL methanol containing 800±1 mg PVP10, stir vigorously for 24±1 h, wash the resulting precipitate with methanol to obtain Mn3O4-PVP, and dry for later use. (2) Disperse 20±0.1 mg Mn3O4-PVP in 20±1 mL methanol, sonicate for 2±0.1 min, then add 10±0.1 mL of Zn(NO3)2·6H2O methanol solution with a concentration of 45±1 mg / mL, stir magnetically for 15±1 min at room temperature, and sonicate for 15±1 min; then add 10±1 mL of 2-MI methanol solution with a concentration of 12.5±1 mg / mL, continue to sonicate for 15±1 min, and stir magnetically for 15±1 min; centrifuge, wash the precipitate with methanol to obtain Mn3O4@ZIF-8 NPs, and dry for later use.

3. The method for preparing the manganese-based nanoenzyme composite material according to claim 2, characterized in that, The Mn3O4 NPs were synthesized according to the following method: (1) Dissolve 5.69±0.1 g Mn(AC)2·4H2O in 279±1 mL of anhydrous ethanol and stir until the solution is clear and transparent; (2) The mixed solution was transferred to a high-pressure reactor with a polytetrafluoroethylene liner and reacted at a constant temperature of 120±1℃ for 24±1 h. (3) After the reaction is complete, centrifuge to collect the precipitate, purify the product by extraction, and wash the precipitate with anhydrous ethanol and distilled water. (4) The precipitate was collected and dried in a constant temperature drying oven to obtain Mn3O4NPs.

4. The method for preparing the manganese-based nanoenzyme composite material according to claim 1, characterized in that, The steps (ii) Includes the following steps: 200±1 mg Mn3O4@ZIF-8 was dispersed in 65 mL H2O; 250±1 mg hyaluronic acid was dispersed in 500 mL H2O; the two were mixed and magnetically stirred at room temperature for 30±1 min, centrifuged, and the resulting precipitate was washed with H2O to obtain MZH, which was then dried for later use.

5. The method for preparing the manganese-based nanoenzyme composite material according to claim 1, characterized in that, Step (iii) includes the following steps: (1) Disperse 10±0.1 mg MZH in 2.5±0.1 mL H2O, then add 75±1 μL Tween-20, sonicate for 2±0.1 min to obtain the aqueous phase; (2) Dissolve 300±1 mg of ES100 in small amounts and multiple times in 5±0.1 mL of organic solvent to obtain an organic phase; (3) Slowly add 0.5±0.1 mL of aqueous phase to 5±0.1 min of organic phase, sonicate on ice for 1±0.1 min to obtain primary emulsion; add 50±1 mL of 2±0.1% polyvinyl alcohol dropwise to the above primary emulsion while stirring, and sonicate on ice for 4±0.1 min; stir the mixed solution in a fume hood for 20±1 h to allow the organic solvent to evaporate, centrifuge, wash the obtained precipitate with H2O to obtain MZH@ES100, and dry it for later use.

6. The method for preparing the manganese-based nanoenzyme composite material according to claim 5, characterized in that, The organic solvent in step (2) is composed of the following substances in the following volume ratios: Dichloromethane: Ethanol: Isopropanol = 4-5: 3-4: 5-6.

7. A manganese-based nanoenzyme composite material, characterized in that, It is prepared by the method described in any one of claims 1-6.

8. The use of the manganese-based nanozyme composite material of claim 7 in the preparation of a drug for treating a DSS-induced mouse colitis model.

9. A drug for preparing a therapeutic model of DSS-induced mouse colitis, characterized in that... Including the manganese-based nanoenzyme composite material as described in claim 7.

10. The medicament according to claim 9, characterized in that... The dosage of the manganese-based nanozyme composite material is 2.5-40 mg / kg.

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