Selenium-coated zinc / cerium-based nano-enzyme and application thereof in regulating intestinal microenvironment to treat inflammatory bowel disease

Abstract

The invention discloses a selenium-coated zinc / cerium-based nano-enzyme and an application thereof in regulating intestinal microenvironment to treat inflammatory bowel disease, the selenium-coated zinc / cerium-based nano-enzyme can realize effective multiple active oxygen removal and regulate intestinal flora, a selenium layer outside the zinc / cerium-based nano-enzyme promotes excitation of an Nrf2 endogenous antioxidant pathway, efficient and lasting oxidation resistance is realized, and the selenium-coated zinc / cerium-based nano-enzyme can be used for treating inflammatory bowel disease. The strong antioxidant activity of the nano enzyme can effectively relieve damage caused by oxidative stress and promote generation of glutathione peroxidase 4 at the same time, so that ferroptosis of intestinal barrier cells is inhibited. The nano-enzyme disclosed by the invention can efficiently remove active oxygen, regulate intestinal flora and recover intestinal barrier tissues, so that effective treatment on inflammatory bowel diseases is realized.

Description

Technical Field

[0001] The present invention belongs to the technical field of nanomaterials, and specifically relates to a selenium-coated zinc / cerium-based nanozyme and an application thereof in regulating the intestinal microenvironment to treat inflammatory bowel disease. Background Art

[0002] Inflammatory bowel disease (IBD) is a chronic disease that mainly includes ulcerative colitis (UC) and Crohn's disease (CD). In recent years, changes in dietary habits and environmental factors, as well as various intrinsic and extrinsic influences, have led to a significant increase in the incidence of IBD. During the pathogenesis of IBD, the accumulation of excessive reactive oxygen species (ROS) at the site of disease causes oxidative stress, leading to tissue protein denaturation, lipid peroxidation, DNA damage, and ferroptosis. At the same time, oxidative stress causes mechanical damage to the intestinal barrier, leading to pathogen invasion and activation of immune cells in the intestinal lamina propria. In this case, the level of inflammatory factors is upregulated, causing an inflammatory cascade and exacerbating the production of ROS. Therefore, removing excess reactive oxygen has become a proven strategy for the treatment of IBD. A large number of studies have shown that cerium dioxide nanoparticles (CeO2NPs) have been used to remove excess ROS in IBD due to their excellent antioxidant properties and good biocompatibility. However, the antioxidant capacity of metal-based nanozymes will be weakened to varying degrees in the gastrointestinal tract. Similarly, existing zinc / cerium-based nanozymes have a reversible CeO2NPs. 3+ / Ce 4+ The active site confers antioxidant properties, and further modifications to further improve the antioxidant capacity and durability of zinc / cerium-based nanozymes are expected to promote the treatment of IBD.

[0003] The nuclear factor E2 p45-related factor 2 (Nrf2) signaling pathway is the main pathway for the production of endogenous antioxidants and continuously activates Nrf2 during inflammation, thereby promoting the extension of endogenous antioxidants. However, individuals have limited levels of raw materials in the body to support the activation of antioxidant synthesis of the Nrf2 pathway. Therefore, enhancing the supply of essential precursors for the Nrf2 pathway is a feasible strategy to improve the sustainability of IBD treatment. Selenoprotein glutathione peroxidase 4 (GPx4) is one of the antioxidants produced by Nrf2 activation, which can effectively inhibit oxidative stress-induced cell ferroptosis and alleviate the mechanical damage of oxidative stress to the intestinal barrier. However, selenium, which is involved in selenoprotein synthesis, is an essential trace element for the human body. Studies have shown that there is a significant negative correlation between selenium levels and the severity of UC and CD, which is due to insufficient selenium levels to maintain the sustained activation of Nrf2 during inflammation. Therefore, selenium supplementation provides a way to achieve a lasting antioxidant effect by continuously promoting the activation of the Nrf2 signaling pathway. However, targeting oxidative stress alone may not address the complex pathogenesis of IBD.

[0004] There is increasing evidence that excessive ROS can diffuse from the epithelium to the intestinal lumen by reacting with organ sulfides and tertiary amines to produce corresponding S-oxides and N-oxides. These products act as terminal electron acceptors, promote the growth of pathogens, and compete with other commensal bacteria for sites and nutrients. This leads to an imbalance in the intestinal flora, resulting in disordered levels of its metabolites, further promoting inflammatory responses and exacerbating the continued development of IBD. Therefore, a combined strategy to reduce oxidative stress, balance the intestinal flora, and restore intestinal barrier tissue is urgently needed. Summary of the invention

[0005] In order to improve the durability and high efficiency of the antioxidant effect of zinc / cerium-based nanozymes in the body, regulate the level of intestinal flora, and restore the intestinal barrier tissue, the present invention provides a selenium-coated zinc / cerium-based nanozyme for regulating the intestinal microenvironment to treat inflammatory bowel disease, as well as a preparation method and application thereof.

[0006] In order to solve the technical problem, the present invention adopts the following technical solution:

[0007] The present invention first discloses a method for preparing a selenium-coated zinc / cerium-based nanozyme, which is characterized in that zinc / cerium-based nanozyme powder is first synthesized, and then selenium nanoparticles are further coated to obtain selenium-coated zinc / cerium-based nanozyme. Specifically, the method comprises the following steps:

[0008] Step 1, 100-200 mg CTAB, 4-6 mg ascorbic acid, 10-15 mg HMTA and 24-28 mg Zn(NO3)2 are mixed in 30-40 mL deionized water and stirred evenly; the obtained mixture is placed in an oil bath pot at 75-85°C and magnetically stirred for 9-11 hours; after the reaction is completed, centrifuge, wash with water, wash with alcohol, and dry to obtain zinc-based nanomaterial powder;

[0009] Step 2, dissolving zinc-based nanomaterial powder and Ce(NO3)4 in deionized water and stirring until uniformly mixed; placing the resulting mixture in an oil bath at 75-85°C with magnetic stirring for 11-12 hours; after the reaction, centrifuging, washing with water, washing with alcohol, and drying to obtain zinc / cerium-based nanozyme powder;

[0010] Step 3, dissolve 10 mg of zinc / cerium-based nanozyme powder in 20 mL of deionized water containing 20-30 mg of Na2SeO3, stir at room temperature for 0.5-1 h, slowly add 10 mL of deionized water containing 40-60 mg of ascorbic acid, and then continue stirring for 1-2 h; after the reaction is completed, centrifuge, wash with water, wash with alcohol, and freeze-dry to obtain selenium-coated zinc / cerium-based nanozyme powder.

[0011] The particle size of the selenium-coated zinc / cerium-based nanozyme prepared by the present invention is 140-180 nm, and can be used to prepare a drug for regulating the intestinal microenvironment to treat inflammatory bowel disease. The selenium-coated zinc / cerium-based nanozyme of the present invention can be added to a sodium alginate solution for oral delivery, and further oral delivery of a CaCl2 aqueous solution can be prepared into a drug encapsulated by a sodium alginate hydrogel in vivo. Alternatively, the drug can be directly prepared into a drug encapsulated by a sodium alginate hydrogel and then delivered orally.

[0012] The beneficial effects of the present invention are embodied in:

[0013] 1. The present invention prepares selenium-coated zinc / cerium-based nanozymes for the first time. The method is simple, the obtained products have uniform morphology and low production cost.

[0014] 2. The selenium-coated zinc / cerium-based nanozyme of the present invention is a new type of nanozyme that can regulate the intestinal microenvironment and relieve enteritis. It significantly alleviates the occurrence of enteritis in the DSS (dextran sulfate)-induced prevention and delay of colitis models and the TNBS (trinitrobenzene sulfonic acid)-induced mouse Crohn's disease model. Its mechanism of action includes:

[0015] (1) The selenium-coated zinc / cerium-based nanozyme has multiple types of enzyme activities, which further improves the ability and types of scavenging reactive oxygen species compared to zinc / cerium-based nanozymes. It can scavenges multiple reactive oxygen species, including superoxide radicals, hydrogen peroxide, and ABTS, and has glutathione peroxidase function.

[0016] (2) The selenium element contained in the selenium-coated zinc / cerium-based nanozyme can promote the activation of the endogenous Nrf2 antioxidant channel in the body, while promoting the expression of GPx4 protein, reducing the ferroptosis of intestinal barrier tissue cells caused by oxidative stress, and achieving efficient and lasting in vivo antioxidant effect.

[0017] (3) The selenium element contained in the selenium-coated zinc / cerium-based nanozyme can inhibit lipid peroxidation of intestinal epithelial cells and restore the intestinal barrier.

[0018] (4) The selenium-coated zinc / cerium-based nanozyme can inhibit the polarization of M0 macrophages to M1 macrophages and reduce the inflammatory response mediated by M1 macrophages.

[0019] (5) The selenium-coated zinc / cerium-based nanozymes can positively regulate the relative level of intestinal flora. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a schematic diagram of the finished product of the selenium-coated zinc / cerium-based nanozyme prepared in Example 1;

[0021] Figure 2 This is a transmission electron microscopy image of the selenium-coated zinc / cerium-based nanozyme prepared in Example 1;

[0022] Figure 3 This is the scavenging ABTS graph of the zinc / cerium-based nanozyme and the selenium-coated zinc / cerium-based nanozyme in Example 2;

[0023] Figure 4 This is a diagram of the removal of hydrogen peroxide by the zinc / cerium-based nanozyme and the selenium-coated zinc / cerium-based nanozyme in Example 2;

[0024] Figure 5 The removal of O2 by the zinc / cerium-based nanozymes and selenium-coated zinc / cerium-based nanozymes in Example 2 - picture;

[0025] Figure 6 This is a graph showing the GPx-like enzyme activity detection of the zinc / cerium-based nanozyme in Example 2;

[0026] Figure 7 This is a graph showing the GPx-like enzyme activity detection of selenium-coated zinc / cerium-based nanozymes in Example 2;

[0027] Figure 8 This is the cytotoxicity graph of HT-29 cells of selenium-coated zinc / cerium-based nanozymes in Example 3;

[0028] Fig. 9 This is the cell active oxygen staining image of the selenium-coated zinc / cerium-based nanozyme in Example 4;

[0029] Fig.10This is a diagram of the promotion of mouse intestinal epithelial cell migration by selenium-coated zinc / cerium-based nanozymes in Example 5;

[0030] Fig.11 The colon length statistics of mice in different treatment groups after DSS-induced preventive treatment in Example 6;

[0031] Fig.12 The flow cytometry analysis of the M1 macrophage content in the colon lymphoid tissue of different treatment groups after DSS-induced preventive treatment in Example 6;

[0032] Fig.13 This is a statistical graph of colon length of mice in different treatment groups after delayed treatment induced by DSS in Example 6;

[0033] Fig.14 The expression of Nrf2 and GPx4 proteins in the colon of mice in each treatment group after delayed DSS treatment was analyzed by Western blot in Example 6;

[0034] Fig.15 This is a statistical graph of the colon length of mice in different treatment groups after Crohn's disease modeling in Example 6;

[0035] Fig.16 is a Venn diagram of common and unique gut microbiota species in the intestines of mice in different treatment groups after DSS-induced delayed treatment in Example 6;

[0036] Fig.17 This is a heat map of the microbial species composition in the intestines of mice in different treatment groups after DSS-induced delayed treatment in Example 6. DETAILED DESCRIPTION

[0037] In order to make the above-mentioned purposes, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail in combination with the embodiments below. The following content is only an example and explanation of the concept of the present invention. The technicians in the relevant technical field make various modifications or supplements to the specific embodiments described or replace them in a similar manner. As long as they do not deviate from the concept of the invention or exceed the scope defined by the claims, they should all fall within the protection scope of the present invention.

[0038] Example 1

[0039] In this example, selenium-coated zinc / cerium-based nanozymes were prepared as follows:

[0040] (1) 131.6 mg CTAB, 4.2 mg ascorbic acid, 12.62 mg HMTA and 26.78 mg Zn(NO3)2 were mixed in 30 mL deionized water and stirred until the mixture was uniformly mixed; the resulting mixture was placed in an oil bath and magnetically stirred at 85°C for 10 h; after the reaction, the mixture was centrifuged, washed with water and ethanol, and dried at 60°C to obtain zinc-based nanomaterial powder.

[0041] (2) Dissolve 30 mg of zinc-based nanomaterial powder in 30 mL of deionized water containing 30 mg of Ce(NO3)4 and stir until the mixture is uniformly mixed; place the resulting mixture in an oil bath and react with magnetic stirring at 85°C for 12 h; after the reaction, centrifuge, wash with water, wash with ethanol, and dry at 60°C to obtain zinc / cerium-based nanozyme powder (denoted as ZCO).

[0042] (3) Dissolve 10 mg of zinc / cerium-based nanozyme powder in 20 mL of deionized water containing 30 mg of Na2SeO3, stir at room temperature for 0.5 h, slowly add 10 mL of deionized water containing 60 mg of ascorbic acid, and then continue stirring for 2 h; after the reaction, centrifuge, wash with water, wash with alcohol, and freeze-dry to obtain selenium-coated zinc / cerium-based nanozyme powder (denoted as ZCSO).

[0043] Figure 1 The selenium-coated zinc / cerium-based nanozyme powder obtained in this example and the photo of the nanozyme dissolved in water are shown.

[0044] Figure 2 This is a transmission electron micrograph of the selenium-coated zinc / cerium-based nanozyme obtained in this example. From the figure, it can be seen that the nanozyme has a spherical structure with a diameter of 140 to 180 nm.

[0045] Example 2

[0046] This example verifies the ability of the selenium-coated zinc / cerium-based nanozyme ZCSO prepared in Example 1 to scavenge reactive oxygen species, and uses ZCO as a comparison.

[0047] 1. Clear ABTS

[0048] The characterization method is as follows: take 3 mg of ABTS diammonium salt, add 0.735 mL of distilled water, and prepare ABTS diammonium salt stock solution. Take 1 mg of K2S2O8, add 1.43 mL of distilled water, and prepare potassium persulfate (K2S2O8) stock solution. Mix 0.2 mL of ABTS diammonium salt stock solution and 0.2 mL of K2S2O8 stock solution, place at room temperature for 12 hours in a dark environment, and dilute the mixture 10 to 20 times with pH 7.4 phosphate buffer until the absorbance is 0.70±0.02. This solution is the ·ABTS free radical working solution. Take 1.6 mL of ·ABTS free radical working solution, take 400 μL of different concentrations of ZCSO or ZCO and mix it to form a solution with a final concentration of ZCSO or ZCO of 25 to 400 μg / mL, place it in a 37°C oven for half an hour, and measure its absorbance at 734 nm.

[0049] Figure 3 The in vitro clearance ABTS diagram of the zinc / cerium-based nanozyme and selenium-coated zinc / cerium-based nanozyme prepared in Example 1 is shown in FIG. Figure 3 It can be seen that the scavenging effect of ZCSO on ·ABTS free radicals is concentration-dependent. With the increase of ZCSO concentration, the scavenging rate of ·ABTS gradually increases, and the scavenging efficiency of ZCSO is stronger than that of ZCO at the same concentration.

[0050] 2. Remove hydrogen peroxide

[0051] The characterization method is to quantitatively analyze the CAT-like enzyme activity of ZCSO or ZCO nanozymes by monitoring the oxygen produced by the decomposition of H2O2. ZCSO or ZCO nanozyme powder and H2O2 were added to 5mL PBS solution, and the H2O2 concentration was 100mM, and the ZCSO or ZCO nanozyme concentration was 0 (control group), 100, 200, 300μg / mL, and a mixed catalytic reaction was performed. The oxygen solubility (mg / L) was monitored within 30min using a dissolved oxygen meter.

[0052] Figure 4 The in vitro hydrogen peroxide removal diagram of the zinc / cerium-based nanozyme and selenium-coated zinc / cerium-based nanozyme prepared in Example 1, Figure 4 It can be seen that with the increase of ZCSO concentration, the oxygen production gradually increases, and at the same concentration, the oxygen production efficiency of ZCSO is stronger than that of ZCO.

[0053] 3. Clear O2 -

[0054] The SOD detection kit (WST-1 method) was used to measure the effect of ZCSO or ZCO on ·O2 - The inhibition rate was used to characterize its SOD-like enzyme activity.

[0055] Figure 5In vitro removal of O2 by the zinc / cerium-based nanozymes and selenium-coated zinc / cerium-based nanozymes prepared in Example 1 - As shown in the figure, with the increase of ZCSO concentration, ·O2 - The inhibition rate of ZCSO to O2 - The inhibition efficiency is stronger than that of ZCO.

[0056] 4. GPx-like enzyme activity

[0057] The in vitro GPx-like enzyme activity of ZCSO or ZCO nanozymes was evaluated using 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) as a chromogenic substrate. First, 1.8 mL of PBS buffer (pH 7.4), 15 μL of 10 mM glutathione (GSH) aqueous solution, and 30 μL of 10 μM H2O2 were mixed, and 200 μL of aqueous solutions of ZCSO or ZCO nanozymes of different concentrations were added to make the final concentration of the enzyme in the system 0-32 μg / mL, and mixed in a dark environment. After incubation at 37 ° C for 1 h, 5 μL of DTNB solution (25 mM) was added to the reaction mixture. After incubation in the dark for 5 minutes, the supernatant was collected, centrifuged at 12,000 rpm for 5 minutes, and then the absorbance was measured at a wavelength of 415 nm.

[0058] Figure 6 and Figure 7 These are the in vitro GPx-like enzyme activity detection diagrams of the zinc / cerium-based nanozymes and selenium-coated zinc / cerium-based nanozymes prepared in Example 1, respectively. It can be seen that ZCO has no GSH scavenging activity, while ZCSO has a significant ability to promote the reaction of GSH and H2O2, and the ability increases with increasing concentration.

[0059] Example 3

[0060] In this example, the cytotoxicity of ZCSO was determined using the standard CCK-8 method, and the specific steps were as follows:

[0061] HT-29 cells were incubated with different concentrations of ZCSO in a 96-well plate for 24 h. At the end of the treatment, 10 μL of CCK-8 dye was added to each well and incubated for another 1 h at 37°C. Subsequently, the absorbance at 450 nm was measured using an enzyme marker for viability analysis. This was repeated 3 times. Relative cell viability was determined by comparing treated and untreated samples. Figure 8 This is the cytotoxicity graph of HT-29 cells of the selenium-coated zinc / cerium-based nanozyme prepared in Example 1. It can be seen from the figure that there is no obvious toxicity to the cells when the ZCSO concentration reaches 40 μg / mL.

[0062] Example 4

[0063] In order to verify the scavenging effect of selenium-coated zinc / cerium-based nanozymes on intracellular reactive oxygen species, 1 mL (1×10 5 ) HT-29 cells, incubate for 24 hours, remove the supernatant. Then the experiment was divided into the following groups:

[0064] (1) Control group (fresh culture medium): Fresh DMEM culture medium was directly added and incubated for 4 h, followed by replacement of fresh culture medium and incubation for 4 h.

[0065] (2) H2O2 group: 500 μM H2O2 solution prepared with DMEM was added and incubated for 4 h, followed by replacement with 500 μM H2O2 solution prepared with DMEM and incubated for 4 h.

[0066] (3) H2O2+20 μg / mL ZCSO group: A 500 μM H2O2 solution prepared with DMEM was added and incubated for 4 h, followed by replacement with a solution containing 500 μM H2O2 and 20 μg / mL ZCSO prepared with DMEM and incubated for 4 h.

[0067] (4) H2O2+40 μg / mL ZCSO group: A 500 μM H2O2 solution prepared with DMEM was added and incubated for 4 h, followed by replacement with a solution containing 500 μM H2O2 and 40 μg / mL ZCSO prepared with DMEM and incubated for 4 h.

[0068] After the incubation of each group, the supernatant was aspirated, the culture was gently washed twice with PBS, and then 1 mL of DMEM-prepared 10 μM fluorescent dye 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) solution was added to each culture dish. The cells were incubated at 37°C in the dark for 30 min, and the ROS fluorescence images of HT-29 cells were observed under a fluorescence microscope. Fig. 9 This is the cell active oxygen staining image of the selenium-coated zinc / cerium-based nanozyme obtained in this example. It can be seen from the figure that the H2O2+40μg / mL ZCSO group has no obvious green fluorescence, indicating that it has a good ability to remove intracellular active oxygen.

[0069] Example 5

[0070] Fig.10 The selenium-coated zinc / cerium-based nanozyme obtained in this example promotes the migration of mouse intestinal epithelial cells. The characterization method is as follows: 1×10 5 For NCM460 cells, the cell monolayer was gently scraped along a straight line with a p200 pipette. After incubation with the cells in H2O2 solutions containing different concentrations of ZCSO, the cells moved toward the center of the well, and the cell migration was assessed by measuring the gap distance. Fig.10It can be seen that the addition of ZCSO greatly promoted the growth of NCM460 cells in the H2O2 environment.

[0071] Example 6

[0072] To verify the ability of the selenium-coated zinc / cerium-based nanozymes obtained in this example to relieve colitis, the following test was performed: sodium alginate was dissolved in 0.15 mol / L NaCl to form a sodium alginate solution with a final concentration of 1.4% (wt / vol), which was then mixed with ZCSO to obtain sodium alginate solutions containing different concentrations of ZCSO for administration.

[0073] 1. In the colon prevention experiment, C57BL / 6 male mice were randomly divided into four groups (n=3 in each group): (1) healthy control group (drinking water); (2) DSS control group (i.e., ulcerative colitis model group, drinking water containing 3% DSS); (3) low-concentration ZCSO group (drinking water containing 3% DSS + 100 μL ZCSO sodium alginate solution, the ZCSO dosage based on the weight of the mouse was 10 mg / kg); (4) high-concentration ZCSO group (drinking water containing 3% DSS + 100 μL ZCSO sodium alginate solution, the ZCSO dosage based on the weight of the mouse was 20 mg / kg). Group (1) was fed with a normal diet and allowed to drink water freely; Groups (2) to (4) were fed with a 3% (w / v) DSS solution prepared with ultrapure water and allowed to drink DSS-containing water freely for 7 days under normal diet conditions; Groups (3) and (4) were given 100 μL of DSS solution by gavage on days 1, 3, and 5, respectively, and then given 50 μL of 70 mM CaCl2 aqueous solution by gavage. On day 9, the mice were euthanized, and the colon was removed. Fig.11 As shown, compared with the normal control group, the colon of the DSS group was significantly shortened to about 5.93 cm, while the colon length of the mice in the high concentration ZCSO group was close to 7 cm, which was not much different from the normal control group.

[0074] In order to confirm the inhibitory effect of ZCSO nanozyme on M1 inflammatory macrophages in the disease site, colon lymphocytes were extracted from mice in each treatment group. Fig.12 The results of flow cytometry analysis showed that a large number of M1 inflammatory macrophages were produced in the DSS-induced mice, and the proportion was as high as 15.87±2.67% compared with the healthy control group mice. However, after treatment with Hy+20mg / kgZCSO, the proportion dropped to 6.97±2.41%, indicating that ZCSO nanozymes can reduce the level of M1 macrophages in mice.

[0075] 2. In the delayed treatment experiment of colitis, C57BL / 6 male mice were randomly divided into five groups (n=5 in each group): (1) healthy control group (drinking water); (2) DSS control group (i.e., ulcerative colitis model group, drinking water containing 3% DSS); (3) Hy group (i.e., blank sodium alginate group, drinking water containing 3% DSS + 100 μL of sodium alginate solution); (4) low-concentration ZCSO group (drinking water containing 3% DSS + 100 μL of sodium alginate solution in ZCSO, the ZCSO dosage based on the weight of the mouse was 10 mg / kg); (5) high-concentration ZCSO group (drinking water containing 3% DSS + 100 μL of sodium alginate solution in ZCSO, the ZCSO dosage based on the weight of the mouse was 20 mg / kg). Group (1) was fed with a normal diet and allowed to drink water freely; Groups (2) to (5) were fed with a 3% (w / v) DSS solution prepared with ultrapure water and allowed to drink DSS-containing water freely for 7 days under normal diet conditions; mice in treatment groups (3) to (5) were given 100 μL of DSS by gavage on days 8, 10, and 12 according to different treatment methods, and then given 50 μL of 70 mM CaCl2 aqueous solution by gavage after administration. The mice were killed on day 14. Fig.13 As shown, the colon length of mice in the DSS group was significantly shortened to about 4.70 cm, and the colon length of mice in the high-concentration ZCSO group was close to 6.27 cm, which was not much different from that of the normal control group.

[0076] To further elucidate the ability of ZCSO nanozymes to alleviate oxidative stress and cellular ferroptosis, Western blot analysis was performed. Fig.14 As shown in the figure, after DSS administration, the expression levels of Nrf2 and GPx4 proteins in the Hy+ZCSO group were significantly higher than those in the other treatment groups. These results indicate that ZCSO nanozymes have a good therapeutic effect on DSS-induced ulcerative colitis by promoting the activation of the Nrf2 signaling pathway and the expression of GPx4 protein.

[0077] 3. In the Crohn's disease mouse model, C57BL / 6 male mice were randomly divided into 5 groups (n=7 in each group): (1) healthy control group (drinking water); (2) TNBS group (i.e., Crohn's disease model group, drinking water + 1% TNBS); (3) Hy group (i.e., blank sodium alginate group, drinking water + 1% TNBS + 100μL sodium alginate solution); (4) low concentration ZCSO group (drinking water + 1% TNBS + 10mg / kg ZCSO); (5) high concentration ZCSO group (drinking water + 1% TNBS + 20mg / kg ZCSO). Except for the control group, mice in other experimental groups were given 1% TNBS enema on the first day. Groups (3) to (5) were gavaged with 100μL on the 2nd, 3rd, and 4th days, and then gavaged with 50μL of 70mM CaCl2 aqueous solution. They were euthanized on the 5th day. Fig.15It can be seen that the colon length of the high-concentration ZCSO nanozyme group was close to 7.03 cm, which is similar to that of normal mice, indicating that ZCSO nanozyme can reduce colon damage in CD mice.

[0078] 4. Use Venn diagram ( Fig.16 ) analyzed the overlapping and unique strains in the intestinal flora of each group of mice in the delayed treatment experiment of colitis. It is worth noting that the number of overlapping strains between the DSS control group and the healthy control group was significantly lower than that between the high-concentration ZCSO group and the healthy control group, indicating that the microbial strains of the ZCSO group and the healthy control group are closer. Family-level heat maps and bar graphs ( Fig.17 ) shows the relative abundance of intestinal microbiota, showing that the composition of the ZCSO-treated group was very similar to that of the healthy control group, indicating that the composition of the intestinal flora tended to move closer to normal levels.

[0079] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing selenium-coated zinc / cerium-based nanozymes, characterized in that: First, zinc / cerium-based nanozyme powder is synthesized, and then selenium nanoparticles are further coated to obtain selenium-coated zinc / cerium-based nanozyme.

2. The method for preparing selenium-coated zinc / cerium-based nanozymes according to claim 1, characterized in that: The following steps are involved: Step 1, 100-200 mg CTAB, 4-6 mg ascorbic acid, 10-15 mg HMTA and 24-28 mg Zn(NO3)2 are mixed in 30-40 mL deionized water and stirred evenly; the resulting mixture is placed in an oil bath pot at 75-85°C and magnetically stirred for 9-11 hours; after the reaction is completed, centrifuge, wash with water, wash with alcohol, and dry to obtain zinc-based nanomaterial powder; Step 2, dissolving zinc-based nanomaterial powder and Ce(NO3)4 in deionized water and stirring until uniformly mixed; placing the resulting mixture in an oil bath at 75-85°C with magnetic stirring for 11-12 hours; after the reaction, centrifuging, washing with water, washing with alcohol, and drying to obtain zinc / cerium-based nanozyme powder; Step 3, dissolve 10 mg of zinc / cerium-based nanozyme powder in 20 mL of deionized water containing 20-30 mg of Na2SeO3, stir at room temperature for 0.5-1 h, slowly add 10 mL of deionized water containing 40-60 mg of ascorbic acid, and then continue stirring for 1-2 h; after the reaction is completed, centrifuge, wash with water, wash with alcohol, and freeze-dry to obtain selenium-coated zinc / cerium-based nanozyme powder.

3. A selenium-coated zinc / cerium-based nanozyme powder prepared by the preparation method according to any one of claims 1 to 2.

4. The selenium-coated zinc / cerium-based nanozyme powder according to claim 3, characterized in that The particle size of the selenium-coated zinc / cerium-based nanozyme is 140-180 nm.

5. An application of the selenium-coated zinc / cerium-based nanozyme powder according to claim 3 or 4, characterized in that: Used to prepare drugs for regulating the intestinal microenvironment to treat inflammatory bowel disease.

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