A mucin hydrogel loaded with cerium-dihydromyricetin nanoszyme, a preparation method and application thereof

Abstract

The application discloses a mucin hydrogel loaded with cerium-dihydromyricetin nanoszyme and a preparation method and application thereof, and relates to the technical field of biological medicines. 4+ The preparation method comprises the following steps: dihydromyricetin and Ce are mixed to carry out a coordination reaction, so as to obtain cerium-dihydromyricetin nanoszyme; hyaluronic acid is coupled with dopamine to obtain hyaluronic acid-dopamine; mucin is coupled with aminophenylboronic acid to obtain mucin-aminophenylboronic acid; and the cerium-dihydromyricetin nanoszyme, the hyaluronic acid-dopamine and the mucin-aminophenylboronic acid are subjected to a mixing reaction to obtain the mucin hydrogel loaded with the cerium-dihydromyricetin nanoszyme. The mucin hydrogel loaded with the cerium-dihydromyricetin nanoszyme can effectively treat ulcerative colitis and has important clinical application value.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a mucin hydrogel loaded with cerium-dihydromyricetin nanozyme, its preparation method, and its application. Background Technology

[0002] Ulcerative colitis (UC) is a type of inflammatory bowel disease (IBD), a nonspecific chronic inflammatory disease with lesions primarily affecting the colonic and rectal mucosa and submucosa. UC is characterized by its chronic, difficult-to-cure, and recurrent nature. Its main pathological changes are diffuse inflammatory infiltration and ulcerative lesions of the colonic mucosa and submucosa. Clinical manifestations include weight loss, chronic diarrhea, abdominal pain, and bloody, mucous stools. In advanced stages, it can significantly increase the risk of colorectal cancer. Current clinical treatments for IBD mainly include aminosalicylic acid, antibiotics, corticosteroids, and immunosuppressants. However, long-term and repeated use of these drugs can easily lead to various serious adverse reactions: for example, long-term use of sulfapyridine to treat UC can induce oxidative stress damage, hematological abnormalities, and even infertility; long-term use of corticosteroids can lead to metabolic disorders. Furthermore, most of the aforementioned drugs are administered orally. However, after oral administration, the active ingredients are easily absorbed in large quantities by the gastrointestinal tract or degraded and inactivated due to the acidic environment of the stomach. Ultimately, only a small amount of the drug can be delivered to the lesion site in the colon, greatly limiting the therapeutic efficacy. Therefore, developing novel treatment strategies that combine highly effective colon-targeting with good safety is of significant clinical value for improving the treatment effect of UC and reducing adverse reactions, and has become a current research hotspot and urgent need in the field of UC. Summary of the Invention

[0003] The purpose of this invention is to provide a mucin hydrogel loaded with cerium-dihydromyricetin nanozyme, its preparation method, and its application, to solve the problems existing in the prior art. This mucin hydrogel loaded with cerium-dihydromyricetin nanozyme can effectively treat ulcerative colitis.

[0004] To achieve the above objectives, the present invention provides the following solution: This invention provides a method for preparing a mucin hydrogel loaded with cerium-dihydromyricetin nanozyme, comprising the following steps: Dihydromyricetin and Ce 4+ A coordination reaction was carried out to obtain cerium-dihydromyricetin nanozyme; Hyaluronic acid is coupled with dopamine to obtain hyaluronic acid-dopamine; Mucin was coupled with aminophenylboronic acid to obtain mucin-aminophenylboronic acid; The cerium-dihydromyricetin nanozyme, the hyaluronic acid-dopamine, and the mucin-aminophenylboronic acid were mixed and reacted to obtain the mucin hydrogel loaded with the cerium-dihydromyricetin nanozyme.

[0005] Furthermore, the dihydromyricetin and the Ce 4+ The molar ratio is 2:1.

[0006] Furthermore, the hyaluronic acid is coupled to the dopamine using the EDC / NHS method.

[0007] Furthermore, the mucin is coupled to the aminophenylboronic acid using the EDC / NHS method.

[0008] Furthermore, the pH value of the mixture reaction is 7.5-7.8.

[0009] The present invention also provides a mucin hydrogel loaded with cerium-dihydromyricetin nanozyme prepared according to the above preparation method.

[0010] The present invention also provides the application of the above-mentioned mucin hydrogel loaded with cerium-dihydromyricetin nanozyme in the preparation of a medicament for treating ulcerative colitis.

[0011] The present invention also provides a medicament for treating ulcerative colitis, comprising the above-mentioned mucin hydrogel loaded with cerium-dihydromyricetin nanozyme.

[0012] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0013] Furthermore, the excipients include stabilizers, humectants, or preservatives.

[0014] The present invention discloses the following technical effects: This invention develops a mucin hydrogel loaded with cerium-dihydromyricetin nanozyme for the treatment of ulcerative colitis. First, dihydromyricetin (DMY) and cerium ions are coordinated to self-assemble into a metal polyphenol nanozyme (Ce-DMY). Then, mucin is modified with aminophenylboronic acid (APBA) to obtain mucin-aminophenylboronic acid. Simultaneously, hyaluronic acid (HA) is functionalized with dopamine (DA) to prepare hyaluronic acid-dopamine. The dynamic phenylboronic acid ester bond formed by the catechol groups in mucin-aminophenylboronic acid and hyaluronic acid-dopamine enables the cross-linking assembly of the two modified materials, constructing an intestinal adhesive hydrogel suitable for rectal injection, and loading Ce-DMY to form a composite hydrogel. The synergistic mechanism of this composite hydrogel system in treating ulcerative colitis is mainly reflected in two aspects: Firstly, when the hydrogel is delivered rectally to the site of colonic inflammation, the hydrogel, based on mucin and HA, can form a biomimetic mucus layer on the surface of the damaged intestinal mucosa, creating a physical barrier to reduce pathogen invasion. Furthermore, its adhesive properties enable sustained and controlled drug release. Secondly, the hydrogel releases Ce-DMY loaded with oxidative stress to efficiently remove excess ROS and alleviate oxidative stress damage. The synergistic effect of both components leads to more effective treatment of colitis. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 16 metal ion-DMY complexes and DMY-O2 - A comparison chart of the scavenging abilities of (A), DPPH· (B), ·OH (C), and GSH (D); Figure 2 A statistical chart showing the total antioxidant activity scores of 16 metal ion-DMY complexes and DMY. Figure 3 Transmission electron microscopy image of Ce-DMY; Figure 4 The UV-vis (A), FTIR (B), XRD (C), and XPS (D) spectra of Ce-DMY are shown. Figure 5 Ce-DMY for O2 - A comparison chart of the scavenging abilities of (A), DPPH· (B), ·OH (C), and GSH (D); Figure 6A comparison of the scavenging abilities of Ce-DMY and DMY for DPPH· and ·OH, as well as their SOD-like and GPx-like enzyme activities; Figure 7 A comparison of the viability of NCM460 cells (A) and RAW264.7 cells (B) after treatment with different concentrations of Ce-DMY and DMY; Figure 8 Fluorescence positive images (Bar=20 μm) of Ce-DMY and NCM460 cells after co-incubation for 1, 3, and 6 h (A) and semi-quantitative analysis of intracellular C6 content (B); Figure 9 Figure (A) shows the comparison of the protective ability of different concentrations of Ce-DMY against H2O2 in NCM460 cells, and Figure (B) shows the comparison of the protective abilities of Ce-DMY and DMY against H2O2 in NCM460 cells. Figure 10 Fluorescent inverted image (Bar=50 μm) for detecting intracellular ROS levels in NCM460 cells using DCFH-DA staining (A) and semi-quantitative analysis of intracellular ROS levels (B); Figure 11 Inverted fluorescence image of mitochondrial membrane potential damage in NCM460 cells detected by JC-1 staining (Bar=50 μm). Figure 12 Inverted fluorescence image of NCM460 cells co-stained with Calcein-AM / PI (Bar=100 μm). Figure 13 Schematic diagram of the preparation process of MH Gel (A) and Ce-DMY / MH Gel (B); Figure 14 Scanning electron microscope images (Bar=20μm) of MH Gel (A), MH Gel (B), Ce-DMY / MH Gel (C) and Ce-DMY / MH Gel (D). Figure 15 Infrared spectra of HA, Mucin, HA-DA, Mucin-APBA, MH Gel, and Ce-DMY / MH Gel; Figure 16 Swelling curves (A) and in vitro degradation diagrams (B) for MH Gel and Ce-DMY / MH Gel; Figure 17 Rheological property analysis diagrams for MH Gel and Ce-DMY / MH Gel; Figure 18 The in vitro drug release curve for Ce-DMY / MH Gel; Figure 19The image shows the results of the cell compatibility test for Ce-DMY / MH Gel. Figure 20 The weight change rate (A) and DAI score (B) of each group of mice are shown. Figure 21 Images of the colon of mice in each group; Figure 22 Statistical graphs of colon length (A), intestinal weight index (B), and spleen weight index (C) for mice in each group; Figure 23 Statistical graph showing the expression levels of TNF-α (A) and IL-1β (B) in the colon tissue of mice in each group; Figure 24 The graph shows the detection results of MPO activity in the colon tissue of mice in each group; Figure 25 The images show histopathological analysis of the colon tissue of mice in each group. Figure 26 Alcian blue-glycogen staining images of colon tissue from each group of mice; Figure 27 Immunohistochemical images of CD86 and CD206 in the colon tissue of mice in each group (Bar=100 μm). Figure 28 Immunofluorescence images of Occludin and ZO-1 in colon tissues of mice in each group (Bar=100 μm). Figure 29 H&E staining images of major organs of mice in each group (Bar=200 μm); Figure 30 The graph shows the results of the detection of blood biochemical indicators ALT (A), AST (B), LDH (C), BUN (D), and Cre (E) in mice of each group. Detailed Implementation

[0017] Example 1: Screening of metal ions 1. Preparation of metal ion-dihydromyricetin complex Preparation of the mother liquor: Dissolve 2 mg (6.25 mM) dihydromyricetin (DMY) in 1 mL of ethanol to prepare a 2 mg / mL DMY mother liquor. Weigh out a certain mass of strontium chloride hexahydrate, nickel dichloride, chromium(III) chloride hexahydrate, cadmium chloride hemihydrate (pentahydrate), anhydrous magnesium chloride, barium chloride dihydrate, aluminum chloride, molybdenum pentachloride, ferrous sulfate heptahydrate, ferric chloride, cobalt chloride hexahydrate, zinc chloride, copper sulfate pentahydrate (II), manganese sulfate monohydrate, cerium(IV) sulfate tetrahydrate, and anhydrous calcium chloride to prepare a 12.5 mM Sr 2+ Ni 2+ Cr 3+Cd 2+ Mg 2+ Ba 2+ Al 3+ Mo 5+ Fe 2+ Fe 3+ Co 2+ Zn 2+ Cu 2+ Mn 2+ Ce 4+ and Ca 2+ Mother liquor.

[0018] A simple one-step blending method was used to prepare the metal ion-DMY complex: the DMY mother liquor and the metal ion mother liquor were mixed at a volume ratio of 1:1 and magnetically stirred for 5 min to obtain the final product.

[0019] 2. Metal ion screening results The antioxidant activity of 16 metal ion-DMY complexes was evaluated to screen for the most suitable metal ion complex with DMY for subsequent studies. The ·O2 content of each metal ion-DMY complex was measured. - The scavenging rates of DPPH·, ·OH, and GSH were set as A1, A2, A3, and A4, respectively. The scores of A1, A2, A3, and A4 for the 16 complexes were simply added together to obtain the total score A.

[0020] The results of the antioxidant activity assays for 16 metal ion-DMY complexes are shown below. Figure 1 The total score A is obtained by adding the scores A1, A2, A3, and A4 of the 16 complexes. The higher the score A, the stronger the antioxidant capacity of the complex. For example... Figure 2 As shown, except for Ca 2+ and Ba 2 + Most metal ion-DMY complexes, compared to DMY alone, enhance its antioxidant activity, such as Mo... 5+ Cu 2+ and Ce 4+ Its antioxidant capacity is particularly outstanding, with scores all above 2.5. Considering the combined activities of various enzymes and the final overall score, this invention selected Ce... 4+ Metal polyphenol nanozymes were prepared by complexing the final metal ions with DMY.

[0021] Example 2: Preparation, characterization, and functional evaluation of cerium-dihydromyricetin nanozyme 1. Preparation of cerium-dihydromyricetin nanozyme Dissolve 30 mg (0.094 mmol) of DMY in 1 mL of ethanol to prepare a 30 mg / mL (94 mM) DMY solution. Then, dissolve 30 mg of polyvinylpyrrolidone (PVP-K30) in 6 mL of ethanol to prepare a PVP-K30 solution. Then, proceed according to Ce... 4+ 1 mL of 188 mM Ce(SO4)2·4H2O solution was prepared with DMY at a molar ratio of 2:1. First, the PVP-K30 solution was placed on a magnetic stirrer. Then, the Ce(SO4)2·4H2O solution was added dropwise, and the mixture was stirred thoroughly for 5 min. Next, the DMY solution was added dropwise to initiate the self-assembly process, and the mixture was stirred at room temperature for 30 min to obtain stable Ce-DMY. Excess substances were removed by high-speed centrifugation (5000 r / min, 10 min), yielding Ce-DMY, which was then dispersed in ultrapure water for further utilization.

[0022] 2. Characterization of cerium-dihydromyricetin nanozyme The morphology of Ce-DMY was observed and photographed using a JEOL JSM-IT900 transmission electron microscope. The results are shown in the figure. Figure 3 The particle size and potential of DMY and Ce-DMY were determined using a Malvern laser particle size analyzer, and the results are shown in Table 1.

[0023] Table 1. Statistical table of nanoparticle size, PDI, and ζ-potential ( n =3)

[0024] The UV-Vis spectra of DMY and Ce-DMY were scanned in the 200-500 nm range using a UV spectrophotometer, and the results are as follows: Figure 4 As shown in Figure A. When DMY and Ce 4+ After chelation, compared with the characteristic peak of DMY at 291 nm, the characteristic peak of Ce-DMY at this point showed a significant red shift, indicating a change in their coordination relationship and confirming the interaction between DMY and Ce. 4+ There are coordination structures between them.

[0025] Infrared spectral results as follows Figure 4 As shown in Figure B. Comparing the FTIR data of DMY and Ce-DMY, it was found that Ce-DMY has a range of values ​​between 3000 and 3700 cm⁻¹. -1 The characteristic hydroxyl stretching vibration peak is broad and strong at 1612 cm⁻¹. The carbonyl vibration frequency in the DMY molecule is located at 1612 cm⁻¹. -1 When DMY and Ce 4+ When coordination complexes are formed, their vibrational frequency shifts to 1635 cm⁻¹. -1 This indicates that the carbonyl oxygen in DMY participates in coordination. Ce-DMY at 574 cm⁻¹-1 The new absorption peak at this point can be attributed to the stretching vibration of Ce-O, indicating that Ce... 4+ Coordination occurred between Ce and DMY. This confirms the successful synthesis of Ce-DMY.

[0026] Ce-DMY was analyzed using X-ray diffraction. Figure 4 As shown in Figure C, Ce-DMY did not detect any cerium-containing crystalline phases and exhibited an amorphous structure.

[0027] The chemical composition and elemental valence states of Ce-DMY were analyzed using X-ray photoelectron spectroscopy. Figure 4 As can be seen from D, Ce-DMY contains characteristic peaks of Ce3d, O1s, N1s and C1s, confirming the successful synthesis of Ce-DMY.

[0028] The DMY content in Ce-DMY was determined to be approximately 20% by ultraviolet spectroscopy.

[0029] 3. Evaluation of the antioxidant capacity of cerium-dihydromyricetin nanozyme (1) Determination of SOD-like enzyme activity To confirm that Ce-DMY possesses SOD-like enzyme activity and can effectively scavenge O2 - This invention investigates the effect of Ce-DMY on ·O2 using the NBT method. - Its clearing ability. For example... Figure 5 As shown in Figure A, concentrations ranging from 50 to 800 µg / mL were detected. -1 Ce-DMY versus O2 - The scavenging ability of Ce-DMY was observed to increase in a concentration-dependent manner. Experiments showed that even at a low concentration of 50 μg / mL, Ce-DMY still exhibited good scavenging ability for O2. - The ability of Ce-DMY to inhibit ·O2 at a concentration of 800 μg / mL. - The clearance rate reached approximately 87%, indicating that Ce-DMY possesses good SOD-like enzyme activity and can effectively remove ·O2. - .

[0030] (2) Determination of DPPH· scavenging capacity DPPH· is a stable nitrogen-centered free radical with a single electron. Its dissolved purple-red solution exhibits a strong absorption peak at 517 nm. The presence of antioxidants can pair with this single electron, thus reducing the absorbance at 517 nm and causing the solution to fade to pale yellow or colorless. This characteristic is used to test the scavenging ability of Ce-DMY on DPPH·. Figure 5As shown in Figure B, Ce-DMY achieved a DPPH· scavenging rate of over 20% at a concentration of only 25 μg / mL, and over 95% of DPPH· was scavenged at a concentration of 400 μg / mL, demonstrating that Ce-DMY possesses excellent DPPH· scavenging capabilities.

[0031] (3) Determination of ·OH scavenging ability The Fenton reaction is the most common chemical reaction that generates ·OH. ·OH can oxidize salicylic acid to 2,3-dihydroxybenzoic acid, which has strong absorption near 510 nm. The absorbance value is directly proportional to the amount of ·OH. This characteristic is used to detect the scavenging ability of Ce-DMY for ·OH. For example... Figure 5 As shown in Figure C, the scavenging rate of Ce-DMY showed a significant concentration-dependent increasing trend with increasing Ce-DMY concentration (62.5-1000 μg / mL). At a concentration of 1000 μg / mL, the scavenging rate exceeded 60%, indicating that Ce-DMY has a good ability to scavenge ·OH.

[0032] (4) Determination of GPx-like enzyme activity GSH reacts with DTNB colorimetric solution in an oxidation reaction to generate a stable yellow 5-thiodinitrobenzoic acid anion. The concentration of this anion is determined by detecting the absorbance at 412 nm, thereby calculating the scavenging rate of GSH. Figure 5 As shown in Figure D, the scavenging rate of Ce-DMY showed a significant concentration-dependent increasing trend with increasing Ce-DMY concentration (62.5-1000 μg / mL). At a concentration of 1000 μg / mL, the scavenging rate exceeded 60%, indicating that Ce-DMY has a good ability to scavenge GSH.

[0033] (5) Comparison of antioxidant activities of Ce-DMY and DMY This invention tested the effects of Ce-DMY and DMY on scavenging DPPH· and ·OH, as well as on SOD-like enzyme activity and GPx-like enzyme activity. Figure 6 As shown, Ce-DMY exhibits significantly higher SOD-like and GPx-like enzyme activities, as well as greater DPPH· and ·OH scavenging capabilities compared to DMY. This indicates that Ce-DMY synthesized through coordination has stronger free radical scavenging activity and superior antioxidant capacity compared to DMY.

[0034] Example 3 1. Experimental Methods 1.1 Cytotoxicity assay RAW264.7 and NCM460 cells were seeded into 96-well plates at a density of 1 × 10⁶ cells per well. 4After incubating overnight to allow cells to adhere, the cells were co-cultured with Ce-DMY and DMY at concentrations of 6.25, 12.5, 25, 50, 100, and 200 μg / mL for 24 h. The effect of Ce-DMY on the proliferation of RAW264.7 and NCM460 cells was assessed using the MTT assay.

[0035] 1.2 Cell uptake experiment Take appropriate amounts of coumarin 6 and Ce-DMY, and add them to a solution containing Ce(SO4)2·4H2O at a mass ratio of 1:10. 4+ C6-labeled Ce-DMY (C6@Ce-DMY) was obtained by magnetically stirring a solution of C6@Ce-DMY in ethanol (2:1 molar ratio of C6@Ce-DMY) at room temperature in the dark for 24 h, followed by centrifugation at 6000 r / min for 10 min. The precipitate was collected and washed three times with ultrapure water. NCM460 cells were seeded into 6-well cell culture plates containing sterile climbing sheets and incubated overnight until cell attachment. The original culture medium was then discarded, and C6@Ce-DMY at a concentration of 100 μg / mL was added. The cells were incubated for 1, 3, and 6 h, respectively. After incubation, the cells were washed three times with PBS and fixed in 4% paraformaldehyde fixative for 15 min. The fixative was discarded, and the cells were washed three times with PBS. DAPI staining solution was added and incubated for 10 min. Unbound free dye was washed away with PBS. After washing three times, the cell slides were removed and mounted. The fluorescence intensity of the cells at different incubation times was observed using an upright fluorescence microscope. The fluorescence intensity was quantified and the results were analyzed using ImageJ image analysis software.

[0036] 1.3 Therapeutic Experiments on Hydrogen Peroxide Damage Cell Model NCM460 cells were seeded into 96-well plates at a density of 1 × 10⁶ cells per well. 4 After incubating overnight to allow cells to adhere, 0, 25, 50, 100, and 200 μg / mL of Ce-DMY (at the same concentration of DMY) were added to each well. After co-culturing for 24 h, the supernatant was discarded. Except for the control group, all other groups were treated with DMEM containing 600 μM H2O2 for 4 h to establish an oxidative stress injury model. Cell viability was then assessed using the MTT assay.

[0037] 1.4 Intracellular ROS scavenging assay Intracellular ROS clearance was assessed using DCFH-DA. NCM460 cells were seeded into 12-well plates at a density of 1 × 10⁶ cells per well. 4Cells were incubated overnight and allowed to adhere to the culture vessel. Then, the following groups were added: Control group, H2O2 group, and 100 μg / mL Ce-DMY (DMY at the same concentration) + H2O2 group. After 24 h of incubation, the supernatant was discarded. Except for the Control group, all other groups were added to DMEM containing 600 μM H2O2 and incubated in a cell culture incubator for 4 h. After discarding the supernatant, the cells were washed once with PBS, followed by the addition of DCFH-DA working solution and incubation in the dark for 1 h. Cells were washed 1 to 2 times with PBS, and then observed and analyzed using an inverted fluorescence microscope. The fluorescence intensity was quantitatively assessed using ImageJ.

[0038] 1.5 Mitochondrial membrane potential detection JC-1 staining was used to detect changes in mitochondrial membrane potential. NCM460 cells were seeded into 12-well plates at a density of 1 × 10⁶ cells per well. 4 Cells were incubated overnight and allowed to adhere to the culture vessel. Then, the following groups were added: Control group, H2O2 group, and 100 μg / m³ Ce-DMY (DMY at the same concentration) + H2O2 group. After 24 h of incubation, the supernatant was discarded. Except for the Control group, all other groups were added to DMEM containing 600 μM H2O2 and incubated in a cell culture incubator for 4 h. The supernatant was discarded, and the cells were washed once with PBS. Then, JC-1 working solution was added, and the cells were incubated in the dark for 1 h. The cells were washed 1 to 2 times with PBS, and then the mitochondrial membrane potential damage was observed using an inverted fluorescence microscope.

[0039] 1.6 Cell Viability Staining Assay This invention uses Calcein-AM and PI for double staining of live and dead cells to analyze the in vitro anti-inflammatory effect of Ce-DMY. NCM460 cells were seeded into 12-well plates at a density of 1 × 10⁻⁶ cells per well. 4 Cells were incubated overnight and allowed to adhere to the culture vessel. Then, the following groups were added: Control group, H2O2 group, and 100 μg / mL Ce-DMY (DMY of equal concentration) + H2O2 group. After 24 h of incubation, the supernatant was discarded. Except for the Control group, all other groups were added to DMEM containing 600 μM H2O2 and incubated in a cell culture incubator for 4 h. The supernatant was discarded, and the cells were washed once with PBS. Then, AM / PI double staining working solution was added, and the cells were incubated in the dark for 1 h. The cells were washed 1 to 2 times with PBS, and then the cell viability was observed using an inverted fluorescence microscope.

[0040] 2. Experimental Results 2.1 Cytotoxicity evaluation of Ce-DMY Having demonstrated that Ce-DMY possesses excellent ROS scavenging ability, this invention further investigates the cytotoxicity of Ce-DMY. For example... Figure 7 As shown, NCM460 and RAW264.7 cells were selected to evaluate the effects of different concentrations of Ce-DMY and DMY on cell viability. MTT results showed that even at a concentration increased to 200 μg / mL, Ce-DMY did not exhibit significant cytotoxicity to either NCM460 or RAW264.7 cells, with cell viability remaining above 80%, indicating that Ce-DMY has good cell safety within the concentration range of 0–200 μg / mL. When treating NCM460 and RAW264.7 cells with the same concentration of DMY, good cell compatibility was observed within the concentration range of 0–50 μg / mL. However, when the concentration exceeded 100 μg / mL, cell viability decreased significantly, and at a concentration of 200 μg / mL, cell viability decreased to approximately 40%, indicating that Ce-DMY at the same concentration has higher cell compatibility than DMY alone.

[0041] 2.2 Cellular uptake of Ce-DMY To verify whether Ce-DMY can be taken up by cells and thus exert its antioxidant effect, the cell internalization performance of Ce-DMY was assessed using an upright fluorescence microscope. To visualize Ce-DMY, C6 was used to label Ce-DMY, resulting in C6@Ce-DMY, and DAPI was used to label the cell nucleus. The results are as follows: Figure 8 As shown in Figure A, green fluorescence represents C6@Ce-DMY, and blue fluorescence represents the cell nucleus. It can be seen that in addition to the blue fluorescence of the cell nucleus, there is also obvious green fluorescence, which increases with the extension of cell uptake time, indicating that the prepared Ce-DMY can be well taken up by NCM460 cells. Figure 8 Image B is a semi-quantitative analysis of C6 content in NCM460 cells, and the results are consistent with the fluorescence images.

[0042] 2.3 Assessment of the intracellular antioxidant capacity of Ce-DMY The antioxidant capacity of Ce-DMY was further evaluated at the cellular level by utilizing its ROS scavenging and endocytosis activities. The protective effect of Ce-DMY on cells was assessed using the H2O2-induced cell damage mechanism combined with the MTT assay. Figure 9As shown in Figure A, the cell viability of NCM460 cells gradually increased with increasing Ce-DMY concentration, exhibiting a dose-dependent relationship. At a concentration of only 25 μg / mL, the cell viability reached approximately 77%. When the concentrations reached 100 μg / mL and 200 μg / mL, compared to the H2O2 group, the cell viability increased by approximately 33% and 30%, respectively, reaching approximately 88% and 85%, indicating that Ce-DMY at these two concentrations could effectively protect against H2O2-induced cell damage. Therefore, a concentration of 100 μg / mL was selected for subsequent cell experiments to further evaluate its antioxidant capacity. Figure 9 As shown in Figure B, the cell survival rate of the Ce-DMY group was approximately 28% higher than that of the DMY group, indicating a stronger cell protection effect.

[0043] 2.4 DCFH-DA staining Cells produce excessive ROS under H2O2 stimulation, leading to oxidative stress damage. To investigate the ROS scavenging function of Ce-DMY at the cellular level, DCFH-DA (2,7-dichlorofluorescein diacetate) was used to specifically label ROS. DCFH-DA can enter cells and be hydrolyzed into DCFH by esterases within the cell. Subsequently, intracellular ROS oxidizes DCFH, generating green fluorescent DCF. The fluorescence intensity is proportional to the intracellular ROS level.

[0044] like Figure 10 As shown in Figure A, cells treated with H2O2 exhibited a significant increase in green fluorescence, indicating high expression of ROS within the cells, with the fluorescence intensity significantly higher than that of the Control group. Pretreatment with Ce-DMY and DMY resulted in varying degrees of decrease in green fluorescence intensity. Ce-DMY showed the weakest green fluorescence intensity, closer to that of the Control group, demonstrating a more significant ROS scavenging effect than DMY alone. Figure 10 Image B shows a semi-quantitative analysis of intracellular ROS levels in each group of NCM460 cells, and the results are consistent with the fluorescence images. These results indicate that Ce-DMY can effectively scavenge intracellular ROS and maintain redox balance.

[0045] 2.5 JC-1 staining Mitochondria are the primary site of reactive oxygen species (ROS) production within cells. Excessive ROS accumulation can lead to a decrease in mitochondrial membrane potential, increasing inner mitochondrial membrane permeability and resulting in insufficient ATP production, causing mitochondrial dysfunction and ultimately inducing apoptosis. JC-1 is a fluorescent dye that specifically accumulates in the mitochondrial matrix. Under normal mitochondrial membrane potential, JC-1 aggregates and exhibits red fluorescence. When the membrane potential decreases, JC-1 becomes monomeric and exhibits green fluorescence. JC-1 was used in this experiment to detect mitochondrial membrane potential damage. Figure 11 As shown, the Control group exhibited a strong red fluorescence signal, indicating that the mitochondrial membrane potential was at a normal level and the cells were in good condition. When cells were treated with only H2O2, their green fluorescence significantly increased while their red fluorescence significantly decreased. This suggests that H2O2-induced cellular inflammatory responses lead to a decrease in mitochondrial membrane potential, impaired mitochondrial function, and ultimately, apoptosis or necrosis. However, after pretreatment with Ce-DMY and DMY, both red and green fluorescence showed varying degrees of enhancement and reduction. Furthermore, Ce-DMY exhibited stronger red fluorescence and weaker green fluorescence compared to DMY, demonstrating a better protective effect on mitochondria and a greater ability to alleviate oxidative stress within mitochondria. These results indicate that Ce-DMY can significantly reduce mitochondrial oxidative stress and effectively inhibit mitochondrial damage.

[0046] 2.6 Calcein AM / PI staining To more intuitively assess the protective effect of Ce-DMY against H2O2-induced cell damage, the experiment employed a CalceinAM / PI double staining method to detect cell viability. This method is based on the difference in membrane integrity between living and dead cells: living cells contain metabolic enzymes such as esterases, which can convert specific dyes into fluorescent substances. After Calcein-AM enters living cells, it is hydrolyzed by esterases within the cells, generating green fluorescence. Dead cells lack active esterases, so Calcein-AM cannot be converted and therefore does not emit fluorescence. However, propidium iodide (PI) can only enter cells with damaged membrane structures, binding to nucleic acids and emitting red fluorescence, while living cells are not stained by PI due to the barrier effect of the cell membrane. Observation under a fluorescence microscope clearly distinguishes between living cells exhibiting green fluorescence and apoptotic or necrotic cells exhibiting red fluorescence. Figure 12As shown, after H2O2 treatment, NCM460 cells showed a significant increase in red fluorescence and a significant decrease in green fluorescence compared to the Control group, indicating that H2O2 treatment caused extensive ROS damage and cell death. In the Ce-DMY and DMY groups, red fluorescence was significantly reduced and green fluorescence was significantly increased compared to the H2O2 group. However, compared to DMY alone, the Ce-DMY treatment group showed stronger green fluorescence and weaker red fluorescence, exhibiting a superior protective effect. These results demonstrate that Ce-DMY can effectively reduce ROS-induced cell damage and has a protective effect on cells.

[0047] Example 4: Preparation and characterization of cerium-dihydromyricetin nanoenzyme mucin hydrogel 1. Preparation of cerium-dihydromyricetin nanoenzyme mucin hydrogel Synthesis of hyaluronic acid-dopamine (HA-DA): 1 g of hyaluronic acid (HA) was completely dissolved in 100 mL of deionized water. Then, 1.48 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 0.9 g of N-hydroxysuccinimide (NHS) were slowly added to the HA solution, and the mixture was stirred vigorously for 30 min to activate the carboxyl groups of HA. Subsequently, 1.48 g of dopamine (DA) was added to the solution, and the pH was adjusted to between 5.0 and 5.5. The reaction was carried out for 8 h in the dark. The final solution was then dialyzed in pure water for 48 h to remove unreacted reagents and salts, resulting in a molecular weight cutoff of 8000-14000 Da. The resulting dialyzed solution was freeze-dried and stored for later use.

[0048] Synthesis of mucin-aminophenylboronic acid (Mucin-APBA): 2.4 g of mucin was completely dissolved in 30 mL of deionized water, followed by the addition of 1.15 g of EDC and 0.69 g of NHS. The pH of the solution was controlled at 5-6, and after vigorous stirring for 20 min, 0.8 g of aminophenylboronic acid was added to the reaction system, and the pH was adjusted to 5-6 with HCl. The reaction was continued overnight at room temperature. The resulting solution was then dialyzed in pure water for 72 h, yielding a molecular weight cutoff of 8000-14000 Da. The resulting dialyzed solution was freeze-dried and stored for later use.

[0049] Preparation of cerium-dihydromyricetin nanoenzyme mucin hydrogel: Ce-DMY powder was added to 5wt% Mucin-APBA solution and mixed thoroughly to obtain a Ce-DMY-Mucin-APBA solution with a Ce-DMY concentration of 4 mg / mL. This Ce-DMY-Mucin-APBA solution was then mixed with 2wt% HA-DA solution at a volume ratio of 1:1. The pH was adjusted to 7.5-7.8 with NaOH, and the mixture was stirred rapidly to ensure homogeneity, yielding a cerium-dihydromyricetin nanoenzyme mucin hydrogel (Ce-DMY / MH Gel). To prepare a blank hydrogel (MH Gel), Ce-DMY was omitted.

[0050] like Figure 13 As shown, after synthesizing MH Gel and Ce-DMY / MH Gel gel precursors, the pH was adjusted to 7.5 to prepare homogeneous gels. Ce-DMY / MH Gel was stained with Rhodamine B.

[0051] 2. Characterization of cerium-dihydromyricetin nanozyme mucin hydrogel SEM images of cross-sections of MH Gel and Ce-DMY / MH Gel as follows Figure 14 As shown, both MH Gel and Ce-DMY / MH Gel exhibit a multi-level porous structure with relatively uniform pore distribution and good connectivity between pores, which is beneficial for drug release.

[0052] Infrared spectral results as follows Figure 15 As shown, the following are the infrared spectra of HA, Mucin, HA-DA, Mucin-APBA, MH Gel, and Ce-DMY / MH Gel. HA-DA has a lower infrared spectrum than HA at 1727 cm⁻¹. -1 The appearance of a new absorption peak at 1375 cm⁻¹ can be attributed to the amide I band (C=O stretching), confirming the formation of an amide bond between the amino group of DA and the carboxyl group of HA, representing the successful synthesis of HA-DA. Mucin-APBA shows a higher absorption peak at 1375 cm⁻¹ compared to Mucin. -1 The discovery of the new BO stretching vibration (a characteristic of phenylboronic acid) confirms the successful grafting of mucin onto APBA, leading to the successful synthesis of mucin-APBA. MH gel at 1080 cm⁻¹ -1 The appearance of a new absorption peak can be attributed to the COB characteristic peak (phenylboronic ester bond vibration), confirming the formation of a phenylboronic ester bond between HA-DA and Mucin-APBA, indicating the successful preparation of MH Gel. Ce-DMY / MH Gel retains the vibrational characteristics of the Ce-O bond and incorporates the skeletal peaks of MH Gel, indicating that Ce-DMY forms a stable composite system with MH Gel after being compounded.

[0053] 3. Analysis of swelling and in vitro degradation behavior like Figure 16 As shown in Figure A, both MH Gel and Ce-DMY / MH Gel exhibit excellent swelling properties. They rapidly absorb water and swell within 4 hours, reaching swelling equilibrium within 24 hours, with final swelling rates reaching 2900% and 2300%, respectively. This characteristic allows them to expand sufficiently in the rectum, better filling the intestinal space, adhering to the mucosal surface, increasing the contact area between the drug and the colitis lesions, and facilitating the drug's efficacy. Figure 16 As shown in Figure B, both MH Gel and Ce-DMY / MH Gel can gradually degrade over time. At a degradation time of 24 h, the weight retention rates were 47% and 30%, respectively, and at 72 h, they were 8% and 4%, respectively. The gels exhibit a moderate degradation rate, enabling both long-term drug release and reduced dosing frequency, while also facilitating rapid metabolism and excretion, thus lowering the risk of intestinal obstruction caused by long-term residue.

[0054] 4. Rheological property analysis The rheological properties of MH gel and Ce-DMY / MH gel were tested using a rheometer, and the frequency sweep is as follows: Figure 17 As shown, G' is the storage modulus; a higher G' indicates higher hydrogel strength and stronger resistance to deformation. G" is the loss modulus; a higher G" indicates that the hydrogel is more prone to deformation and has a stronger energy dissipation capacity. When the strain is fixed (≤1%, in the linear viscoelastic region) and scanned within the frequency range of 0.1~40 Hz, the G' of both gels is consistently higher than G", indicating that both gels have stable structures and viscoelastic properties with elasticity as the main characteristic, making them suitable for rectal drug delivery.

[0055] 5. Injectability analysis When the gelled MH Gel and Ce-DMY / MH Gel were injected into PBS solution at 37°C, hydrogel filaments were generated without diffusion and the gel remained stable, indicating that they can be used for rectal administration.

[0056] 6. Analysis of in vitro drug release behavior like Figure 18As shown, the release behavior of Ce-DMY in Ce-DMY / MH gel is time-dependent, with the release amount positively correlated with time. Ce-DMY is gradually released from Ce-DMY / MH gel over time, reaching a plateau around 12 hours. Within 0-12 hours, the cumulative release rate rapidly increases from 0 to 68%. After 12 hours, the release rate significantly decreases, reaching a cumulative release rate of 78% at 48 hours. Throughout the process, the 0-12 hour release phase is beneficial for rapidly increasing local drug concentration and improving therapeutic efficacy; the 12-48 hour release phase maintains long-term effective drug concentration, continuously modulates inflammation, and reduces the frequency of administration.

[0057] 7. Cell compatibility of Ce-DMY / MH Gel The cytotoxicity of Ce-DMY / MH gel against NCM460 cells was detected by the MTT assay. Results are as follows: Figure 19 As shown, after treatment with hydrogel extracts of different concentrations (312.5–5000 μg / mL), the cell viability remained above 95%, and there was no significant difference compared with the control group. This indicates that the hydrogel extract has no obvious cytotoxicity within the tested concentration range and has excellent cell compatibility.

[0058] Example 4: In vivo pharmacodynamic study of dihydromyricetin nanoenzyme mucin hydrogel 1. Experimental Methods 1.1 Construction and Treatment of UC Model Thirty healthy male SPF-grade C57BL / 6J mice (8 weeks old, weighing 20–22 g) were selected as experimental animals. Each mouse was ear-tagged with ear-tag pliers, weighed, and the weight was recorded. Based on the weight data, the 30 mice were randomly divided into 6 groups of 5 mice each to ensure no significant difference in average weight between groups. The specific groups were: Normal group, DSS (sodium dextran sulfate) model group, DMY group, Ce-DMY group, MH Gel group, and Ce-DMY / MH Gel group.

[0059] The colitis modeling period was 9 days. From day 1 to day 7, except for the Normal group, the drinking water of the other 5 groups of mice was replaced with 3% DSS aqueous solution. Mice had free access to water, and the DSS aqueous solution was changed daily. On days 8 and 9, they were fed normal drinking water. Simultaneously, on days 1, 3, 5, and 7, DMY solution, Ce-DMY solution, MH gel, and Ce-DMY / MH gel were administered rectally, with a volume of 200 μL for each. The Ce-DMY solution contained 20 mg / kg of Ce-DMY, and the DMY solution contained 3.8 mg / kg of DMY (corresponding to the DMY content in Ce-DMY). The Normal group and the DSS modeling group were given the same volume of physiological saline. The mice's weight, fecal condition, and fecal blood were recorded daily. Animal samples were collected on day 9.

[0060] Collection of colon, blood, and major organ samples: After euthanasia, the colonic tissue from the cecum to the anus was quickly removed, its length measured, photographed, and recorded. The colonic segment was also weighed and recorded. A 1 cm segment of colon was then fixed in tissue fixative (4% paraformaldehyde) for section staining. The remaining colon was flash-frozen in liquid nitrogen and immediately transferred to -80°C for later use. Simultaneously, the mouse heart, liver, spleen, lungs, and kidneys were collected and fixed in tissue fixative. Whole blood was also collected, allowed to coagulate at room temperature, centrifuged, and the supernatant serum was collected and stored at -80°C.

[0061] 1.2 Indicator Testing (1) The mice were scored according to their weight change, fecal state and bloody stool. The formula is as follows: DAI = weight change score + fecal state score and bloody stool score. The scoring criteria are shown in Table 2.

[0062] Table 2 DAI Scoring Criteria

[0063] (2) Measure the length of the mouse colon and weigh and record the weight of the mouse spleen.

[0064] Intestinal weight index (mg / cm) = colon weight / colon length; spleen weight index (mg / g) = spleen weight / body weight.

[0065] (3) Detect the levels of inflammatory cytokines TNF-α and IL-1β in colon tissue.

[0066] (4) Detect myeloperoxidase (MPO) activity in colon tissue.

[0067] (5) Hematoxylin and eosin (H&E) were used to stain colon sections, and the pathological changes of colon tissue were observed under an upright microscope. The histopathological scores of each group of mice were determined according to Table 3.

[0068] Table 3. Pathological Histological Scoring

[0069] (6) Alcian blue-glycogen staining to detect mucin expression.

[0070] (7) Immunohistochemical staining of colon tissue sections with CD86 and CD206 was performed to detect the polarization of macrophages in the colon of each group.

[0071] (8) Immunofluorescence analysis was performed on colon tissue to detect the expression levels of tight junction proteins Occludin and ZO-1 in the colon.

[0072] 1.3 Biosafety of the drug (1) The major organs (heart, liver, spleen, lungs and kidneys) of the mice were removed from the paraformaldehyde fixative, H&E staining was performed, and their pathological changes were observed under an upright microscope.

[0073] (2) The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), blood urea nitrogen (BUN) and creatinine (Cre) in plasma samples from each group of mice were measured to assess the levels of blood biochemical indicators.

[0074] 2. Experimental Results 2.1 Changes in mouse body weight and DAI index like Figure 20 As shown in Figure A, during the experimental period, the body weight of mice in the Normal group showed a steady upward trend, reaching approximately 106% of the initial weight (100%) on day 8. Mice in the DSS group experienced a significant decrease in body weight starting from day 4, dropping to approximately 80% of the initial weight on day 8, a significant difference compared to the Normal group. Among the drug-treated groups, the weight loss in the DMY, Ce-DMY, and MH Gel groups was less severe than in the DSS group, with body weights maintained at approximately 84%, 87%, and 91% on day 8, respectively. The Ce-DMY / MH Gel group showed the smallest weight loss, maintaining approximately 94% of the initial weight on day 8, significantly lower than the DSS group and exhibiting better relief than other drug-treated groups, indicating that Ce-DMY / MH Gel has a certain therapeutic effect on colitis. Figure 20As shown in Figure B, the DAI score of mice in the Normal group was 0 throughout the entire experimental period, with no colitis-related symptoms. The DAI score of mice in the DSS group increased significantly from day 4, and the DAI score was the highest among the other groups on day 8. Among the drug administration groups, the DAI score increase trend of the DMY group, Ce-DMY group, and MHGel group was slower than that of the DSS group. The DAI score increase trend of the Ce-DMY / MH Gel group was significantly lower than that of the DSS group and other drug administration groups, and the DAI score on day 8 was significantly lower than that of other drug administration groups, indicating that the Ce-DMY / MH Gel group had a better therapeutic effect on colitis.

[0075] 2.2 Colon length, intestinal weight index, and spleen weight index in mice Shortened colon length is a typical manifestation of intestinal fibrosis and inflammation in ulcerative colitis (UC). An elevated intestinal weight index reflects edema and inflammatory infiltration of the colonic mucosa, while an elevated spleen weight index indicates systemic immune activation and inflammation. Figure 21 As shown, the colon length in the DSS group was significantly shorter than that in the Normal group and other drug-treated groups. The Ce-DMY / MH Gel group had the longest colon length compared to the other drug-treated groups. Colon length data are shown below. Figure 22 As shown in Figure A, the results are consistent with the colon image. Figure 22 As shown in Figures B and C, the intestinal weight and spleen weight indices of the DSS group were significantly higher than those of the other groups. Among the drug-treated groups, the Ce-DMY / MH Gel group had the lowest intestinal weight and spleen weight indices, indicating that it can improve colitis by inhibiting local intestinal inflammation and has the best therapeutic effect among all groups.

[0076] 2.3 Changes in inflammatory cytokines in mouse colon tissue High expression of pro-inflammatory factors is a prominent feature of ulcerative colitis (UC). TNF-α and IL-1β are core pro-inflammatory cytokines in the pathogenesis of UC: both are mainly secreted by activated macrophages and neutrophils, and can directly damage colonic mucosal epithelial cells and disrupt tight junction structures. Simultaneously, they amplify the inflammatory cascade by activating the NF-κB signaling pathway, making them key factors in the persistent progression of intestinal inflammation; elevated expression levels indicate aggravated intestinal inflammatory damage. Therefore, this invention used RT-qPCR technology to measure the expression levels of TNF-α and IL-1β. The results are as follows: Figure 23As shown, the expression levels of TNF-α and IL-1β in the Normal group remained at extremely low levels. Compared with the Normal group, the expression levels of TNF-α and IL-1β in the DSS group were significantly upregulated, indicating that DSS-induced colitis triggered a strong release of pro-inflammatory factors. The expression levels of TNF-α and IL-1β in the drug-treated groups were lower than those in the DSS group. Among them, the expression levels of TNF-α and IL-1β in the Ce-DMY / MH Gel group were the lowest, dropping to levels close to those in the Normal group. This indicates that the synergistic agent has the best clearance effect on pro-inflammatory factors TNF-α and IL-1β and can best alleviate colitis inflammation.

[0077] 2.4 Changes in MPO activity in mouse colon tissue Myeloperoxidase (MPO) is a specific marker of neutrophils; elevated MPO activity directly reflects the degree of neutrophil infiltration, representing the severity of inflammation. Figure 24 As shown, the significant increase in MPO activity in the DSS group confirms the large-scale recruitment and activation of neutrophils in DSS-induced colitis, representing an exacerbation of intestinal inflammation. The MPO activity in each treatment group was lower than that in the DSS group, with Ce-DMY / MH Gel exhibiting the lowest MPO activity and the strongest ability to reduce MPO activity. This indicates that Ce-DMY / MH Gel can effectively inhibit neutrophil infiltration and activation, and effectively reduce MPO activity.

[0078] 2.5 Histopathological analysis of colon tissue Colonic histopathology is an important standard for assessing tissue damage in colitis. To thoroughly evaluate the histopathological characteristics of the colon in each group of mice, colonic tissue was stained with H&E. Figure 25 As shown, the colonic mucosa in the Normal group was intact and continuous, with numerous and tightly packed crypts, and no obvious inflammatory cell infiltration was observed. In contrast, the DSS group showed significant mucosal damage, severe crypt destruction, and abundant inflammatory cell infiltration in the lamina propria and submucosa, indicating that the DSS model successfully induced typical colitis pathological damage. The integrity of the mucosal layer, the number of crypts, and inflammatory cell infiltration all recovered to varying degrees in each treatment group. The Ce-DMY / MH Gel group showed a mucosal structure, number and arrangement of crypts highly similar to the Normal group, with minimal inflammatory cell infiltration and clear boundaries between the intestinal wall layers, indicating that the Ce-DMY / MH Gel group can effectively treat colitis.

[0079] 2.6 Changes in the mucus layer of the colonic mucosa The colonic mucus barrier is the first physical defense against intestinal flora invasion. Mucin secreted by goblet cells is the main functional component of this barrier, forming the structural and functional core of the colonic mucus barrier. Therefore, mucin becomes a key target for colonic histopathological evaluation. AB-PAS staining is used to specifically detect changes in mucin. Figure 26 As shown, the colonic mucosa of mice in the Normal group was covered with continuous and dense blue signals, and the number of goblet cells was large and tightly arranged, indicating that the mucin secretion in the colon was sufficient and the mucus barrier was intact. The blue signal in the DSS group was significantly reduced, the number of goblet cells was significantly reduced, the mucin secretion was severely insufficient, and the mucus barrier was severely damaged. The blue staining area in each drug administration group increased to varying degrees. Among them, Ce-DMY / MH Gel showed a continuous blue-purple mucus layer on the mucosa surface and richer signals in the goblet cells compared with other drug administration groups, which was closer to the Normal group. This proves that Ce-DMY / MH Gel can effectively restore the secretion of colonic mucin and the integrity of the mucus barrier.

[0080] 2.7 Polarization of macrophages in the colon Macrophages, as core cells of the intestinal mucosal immune system, generally exist in pro-inflammatory (M1) and anti-inflammatory (M2) phenotypes, regulating the occurrence, development, and repair of intestinal inflammation. CD86, a marker of M1 macrophages, indicates high expression, signifying macrophage activation and the release of pro-inflammatory factors such as TNF-α and IL-6, exacerbating intestinal mucosal damage. CD206, on the other hand, is a marker of M2 macrophages; M2 macrophages participate in tissue repair and inflammation reduction by secreting anti-inflammatory factors such as IL-10 and TGF-β. Immunohistochemical staining of CD86 and CD206 in colonic tissue can help determine the macrophage polarization state in the colon, thereby detecting the level of inflammation in different colonic regions. Figure 27As shown, regarding CD86 expression: the Normal group had very few CD86-positive staining areas, only sporadic distribution, indicating that M1 macrophages in the colon tissue were in a resting state; while the DSS group had diffusely distributed CD86-positive staining in the colonic mucosa and submucosa, with dense and widespread brown-yellow signals, indicating that M1 pro-inflammatory macrophages were recruited and activated in large numbers in the colitis model; the CD86-positive areas in the drug-treated groups were reduced to varying degrees compared with the DSS group, with the Ce-DMY / MH Gel group showing a significant reduction in positive staining, and the brown-yellow areas were close to the level of the Normal group compared with other drug-treated groups, indicating that Ce-DMY / MH Gel can effectively inhibit the infiltration and activation of M1 pro-inflammatory macrophages. Regarding CD206 expression: CD206 positive staining was significantly weaker in the DSS group than in the Normal group, with sparse brown-yellow areas and reduced signal intensity, indicating that the differentiation of M2 anti-inflammatory macrophages was suppressed and the immune repair function was weakened in the colitis model; the CD206 positive area in the drug-treated group was slightly increased compared to the DSS group, with Ce-DMY / MH Gel positive staining significantly enhanced. The distribution range and signal intensity of the brown-yellow area in the drug-treated group were closer to those in the Normal group than in other drug-treated groups, indicating that Ce-DMY / MH Gel can effectively promote the differentiation of M2 anti-inflammatory repair macrophages.

[0081] 2.8 Expression of colonic tight junction proteins Tight junction proteins Occludin and ZO-1 are core functional proteins of the intestinal epithelium's tight junctions. Decreased expression of these proteins leads to increased intestinal permeability, triggering microbial translocation and an inflammatory cascade, resulting in impaired intestinal barrier function. To evaluate the therapeutic effects of various drugs on the colonic barrier in mice, this invention utilizes immunofluorescence technology to detect the expression levels of ZO-1 and Occludin in colonic tissue. Figure 28 As shown, blue represents DAPI-labeled cell nuclei, while green and red represent Alexa Fluor-488 and Alexa Fluor-594-labeled Occludin and ZO-1, respectively. Compared to the Normal group, the DSS group showed a significant decrease in fluorescence signal intensity and fragmentation of Occludin and ZO-1, directly reflecting the structural damage to the tight junctions of the intestinal epithelium after DSS-induced colitis. In contrast to the DSS group, the fluorescence signals of Occludin and ZO-1 in all treatment groups were significantly enhanced, with improved linear distribution continuity. The Ce-DMY / MH Gel group exhibited the strongest fluorescence signal, indicating its superior therapeutic effect in maintaining the intestinal barrier. This demonstrates that Ce-DMY / MH Gel can maintain intestinal barrier integrity by protecting and repairing the tight junction structure. The Ce-DMY group and MH Gel followed, with the DMY-only treatment group showing the worst effect.

[0082] 2.9 Biosafety Analysis This invention investigated the safety of each formulation in mice after administration. The results are as follows: Figure 29 and Figure 30 As shown in the H&E staining images of the major organs of mice, the morphology of all organs in the normal control group and each treatment group was normal, with no obvious pathological damage. This indicates that Ce-DMY, MH Gel, and Ce-DMY / MH Gel have good in vivo biocompatibility at effective therapeutic doses and have not caused toxic effects on the major organs of the body. In addition, the blood biochemical indicators of mice in each group—alanine aminotransferase (AST), aspartate aminotransferase (ALT), lactate dehydrogenase (LDH), blood urea nitrogen (BUN), and creatinine (Cre)—were similar, with no significant differences, indicating that Ce-DMY, MH Gel, and Ce-DMY / MH Gel all have good in vivo biocompatibility.

[0083] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for preparing a mucin hydrogel loaded with cerium-dihydromyricetin nanozyme, characterized in that, Includes the following steps: dihydromyricetin and Ce 4+ carrying out a coordination reaction to obtain a cerium-dihydromyricetin nanoszyme; Hyaluronic acid is coupled with dopamine to obtain hyaluronic acid-dopamine; Mucin was coupled with aminophenylboronic acid to obtain mucin-aminophenylboronic acid; The cerium-dihydromyricetin nanozyme, the hyaluronic acid-dopamine and the mucin-aminophenylboronic acid were mixed and reacted to obtain the mucin hydrogel loaded with the cerium-dihydromyricetin nanozyme. The mucin was coupled to the aminophenylboronic acid using the EDC / NHS method.

2. The preparation method according to claim 1, characterized in that, The dihydromyricetin and the Ce 4+ The molar ratio is 2:

1.

3. The preparation method according to claim 1, characterized in that, The hyaluronic acid was coupled to the dopamine using the EDC / NHS method.

4. The preparation method according to claim 1, characterized in that, The pH value of the mixture reaction is 7.5-7.

8.

5. A mucin hydrogel loaded with cerium-dihydromyricetin nanozyme prepared by the preparation method according to any one of claims 1-4.

6. The use of a mucin hydrogel loaded with cerium-dihydromyricetin nanozyme as described in claim 5 in the preparation of a medicament for treating ulcerative colitis.

7. A drug for treating ulcerative colitis, characterized in that, The mucin hydrogel containing the cerium-dihydromyricetin nanozyme as described in claim 5.

8. The medicament according to claim 7, characterized in that, The drug also includes pharmaceutically acceptable excipients.

9. The medicament according to claim 8, characterized in that, The excipients include stabilizers, humectants, or preservatives.

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