Manganese dioxide nano-enzyme for treating inflammatory bowel disease as well as preparation method and application of manganese dioxide nano-enzyme

Abstract

The invention relates to the field of biomedical materials, and particularly discloses a manganese dioxide nano-enzyme for treating inflammatory bowel disease as well as a preparation method and application of the manganese dioxide nano-enzyme. The preparation method of the manganese dioxide nano-enzyme comprises the following steps: uniformly mixing an organic solution containing manganese nitrate hexahydrate with a montmorillonite aqueous solution to obtain a mixed reaction solution; the pH value of the mixed reaction solution is adjusted to be under the alkaline condition through alkali liquor, a synthetic reaction is carried out, then centrifugation and washing are carried out, the montmorillonite-loaded manganese dioxide nano-enzyme is obtained, and the manganese dioxide nano-enzyme is of a lamellar structure with the length being 20-400 nm. The manganese dioxide nano enzyme has excellent activity similar to superoxide dismutase, catalase and glutathione peroxidase; meanwhile, the compound also has the capability of removing various free radicals such as DPPH and ABTS, can be used for treating the inflammatory bowel disease, and has a wide application prospect.

Description

Technical Field

[0001] The present application relates to the field of biomedical materials, and more specifically, to a manganese dioxide nanozyme for treating inflammatory bowel disease and a preparation method and application thereof. Background Art

[0002] Inflammatory bowel disease (IBD), mainly including Crohn's disease (CD) and ulcerative colitis (UC), is a group of chronic, nonspecific, recurrent inflammatory diseases characterized by intestinal inflammation and mucosal damage, and may even cause colorectal cancer. The current effective treatment strategy for IBD is to remove reactive oxygen species and inhibit intestinal inflammatory reactions to achieve the purpose of relieving enteritis. At present, the drugs used in the clinical treatment of IBD mainly include anti-inflammatory drugs, immunomodulators, monoclonal antibodies, and antibiotics. However, these drugs have many adverse reactions, such as weakened efficacy after long-term use, antibiotic resistance, autoimmune diseases, electrolyte disorders, etc. Therefore, it is of great significance to develop new treatment methods for the inflammatory sites of inflammatory bowel disease.

[0003] Compared with biological enzymes, nanozymes have the advantages of simple preparation, stable structure, multiple types of enzyme activity, high and controllable catalytic activity, etc. They are widely used in biology, medicine and other fields. Constructing nanomaterials with multiple types of enzyme activity for the treatment of inflammatory bowel disease is an effective method.

[0004] Manganese dioxide nanomaterials have three types of antioxidant enzyme activities, including superoxide dismutase-like, catalase-like and glutathione peroxidase-like activities. Because manganese dioxide nanomaterials have multiple antioxidant enzyme activities, they can efficiently remove reactive oxygen and multiple free radicals, making them an effective method for treating inflammatory diseases. Chinese patent CN117285045A provides a clay mineral biomaterial and its preparation method and application, which uses montmorillonite as a carrier and grows nanometer-scale manganese dioxide particles in situ on the montmorillonite carrier through a hydrothermal reaction to obtain a biomaterial. Although the biomaterial also shows certain anti-superoxide dismutase and catalase-like activities, the activity is weak and it is difficult to apply to the treatment of inflammatory bowel disease. Summary of the invention

[0005] In order to enhance the enzyme-like activity of manganese dioxide biomaterials, to make their superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPX)-like activities higher, and to enhance their ability to scavenge various free radicals such as ABTS and DPPH, the present application provides a manganese dioxide nanozyme for the treatment of inflammatory bowel disease, and a preparation method and application thereof.

[0006] This application adopts the following technical solutions:

[0007] In a first aspect, the present application provides a method for preparing a manganese dioxide nanozyme, comprising:

[0008] The organic solution containing manganese nitrate hexahydrate and the montmorillonite aqueous solution are uniformly mixed to obtain a mixed reaction solution;

[0009] The pH of the mixed reaction solution is adjusted to alkaline conditions using alkaline solution, a synthesis reaction is carried out, and then centrifugation and washing are performed to obtain montmorillonite-supported manganese dioxide nanozyme, wherein the manganese dioxide nanozyme has a lamellar structure with a length of 20-400 nm.

[0010] Furthermore, the temperature of the synthesis reaction is 30-80°C, and the reaction time is 2-3h. Preferably, the temperature of the synthesis reaction is 40-65°C, more preferably 55-65°C.

[0011] Furthermore, in the mixed reaction liquid, the mass ratio of the manganese nitrate hexahydrate to the montmorillonite is 1:2.3-5, preferably 1:3-5, and more preferably 1:3.5-4.5.

[0012] Furthermore, the organic solvent in the organic solution containing manganese nitrate hexahydrate is any one of ethylene glycol, propylene glycol and butanediol, preferably ethylene glycol.

[0013] Preferably, the alkali solution is ammonia water or sodium hydroxide solution, and the alkaline environment refers to a pH value of the mixed reaction solution of 7 to 11, preferably 7 to 9. The purpose of adding the alkali solution is to adjust the pH of the mixed reaction solution and to act as a precipitant so that the lamellar manganese dioxide produced by the thermal decomposition of manganese nitrate hexahydrate can be precipitated on the surface of montmorillonite.

[0014] Furthermore, the preparation of the mixed reaction solution includes:

[0015] The organic solution containing manganese nitrate hexahydrate is added dropwise into the montmorillonite aqueous solution, and stirred in a water bath at 40-70° C. for 20-40 minutes.

[0016] In a second aspect, the present application provides a manganese dioxide nanozyme, which is prepared using the above-mentioned preparation method.

[0017] In a third aspect, the present application provides the use of the above-mentioned manganese dioxide nanozyme in the preparation of a drug for treating inflammatory bowel disease.

[0018] Furthermore, the inflammatory bowel disease is any one of Crohn's disease and ulcerative colitis.

[0019] In a fourth aspect, the present application provides a composite preparation for treating inflammatory bowel disease, which includes the above-mentioned manganese dioxide nanozyme and pharmaceutical excipients.

[0020] In summary, this application has the following beneficial effects:

[0021] 1. This application prepares manganese dioxide nanozyme by an in-situ growth method, the principle of which is: using montmorillonite as a growth matrix, depositing manganese dioxide on the surface of montmorillonite by coprecipitation, and preparing a manganese dioxide montmorillonite composite material, namely, manganese dioxide nanozyme; the preparation method has the advantages of simplicity, low synthesis cost, good dispersibility, good stability, etc., which is conducive to large-scale production and application. In the prior art, potassium permanganate and montmorillonite are used as raw materials and synthesized by hydrothermal method, which needs to be carried out under high temperature (usually 100-400°C) and high pressure (from 0.1 MPa to hundreds of MPa), which puts higher requirements on the reaction equipment and operating conditions. The high temperature and high pressure environment not only increases the equipment cost and operation difficulty, but also may bring safety hazards, limiting the wide application of this method in industrial production.

[0022] 2. Compared with the prior art process, the manganese dioxide nanozyme obtained in this application has higher reaction activity and better therapeutic effect for inflammatory bowel disease. This is mainly due to the fact that the manganese dioxide in the material obtained by this method exists in the form of nanosheets, rather than nanoparticles or nanospheres, thereby changing or expanding the active sites of the manganese dioxide nanozyme, effectively improving its superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPX) activities; at the same time, it also has the ability to remove various free radicals such as DPPH and ABTS. This enzyme-like activity can enable manganese dioxide nanozyme to treat inflammatory bowel disease by eliminating ROS, with high efficiency and low side effects.

[0023] 3. The manganese dioxide nanozyme obtained in this application has specific intestinal targeting. The manganese dioxide nanozyme is modified by negatively charged substances such as montmorillonite, and can be targeted and enriched in inflammatory sites with positive charges, prolonging the action time of the nanozyme in the intestine. At the same time, it can protect the intestinal mucosa, promote the repair of damaged mucosa, and regulate the homeostasis of intestinal flora; and it has good biosafety, which is conducive to clinical use. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of the synthesis of manganese dioxide nanozyme prepared in Example 1.

[0025] Figure 2 This is a transmission electron microscopy image of the manganese dioxide nanozyme prepared in Example 1.

[0026] Figure 3 This is the multi-type enzyme activity diagram of manganese dioxide nanozymes and manganese dioxide nanospheres in Experiment 2, including superoxide dismutase activity (SOD), catalase activity (CAT), and glutathione peroxidase (GPX).

[0027] Figure 4This is the analysis diagram of manganese dioxide nanozyme removing DPPH and ABTS free radicals in experiment three.

[0028] Figure 5 This is the hemolysis experiment diagram of manganese dioxide nanozyme in Experiment 4.

[0029] Figure 6 This is a comparison chart of the colon length of mice after manganese dioxide nanozyme was used to treat colitis in Experiment 5.

[0030] Figure 7 This is the HE staining image of mouse colon sections after manganese dioxide nanozyme was used to treat mouse colitis in Experiment 5.

[0031] Figure 8 This is a map of intestinal microbial diversity after manganese dioxide nanozyme treated colitis in mice in experiment five. DETAILED DESCRIPTION

[0032] The embodiments of the present invention will be described in detail below in conjunction with examples. However, those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. The specific conditions not specified in the examples are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased commercially.

[0033] The specific embodiments of the present invention are described in detail below. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0034] Example

[0035] Example 1

[0036] This embodiment provides a manganese dioxide nanozyme, and its synthesis schematic diagram is as follows Figure 1 As shown, the preparation method comprises:

[0037] ① Dissolve 126 mg of manganese nitrate hexahydrate (Mn(NO3)2·6H2O) crystals in 10 mL of ethylene glycol to obtain a manganese nitrate hexahydrate ethylene glycol solution for later use.

[0038] ② Disperse the montmorillonite powder in water, stir vigorously for 24 hours, centrifuge repeatedly to remove impurities, and dry to obtain purified montmorillonite powder. Subsequently, dissolve 5g of purified montmorillonite powder in 100mL of water to prepare a uniformly dispersed montmorillonite suspension with a concentration of 50mg / mL for later use.

[0039] ③ Add 10mL of solution ① dropwise into 10mL of the suspension of ②. After the solution is evenly mixed, place it in a 60°C water bath and stir rapidly for 30 minutes. Then quickly add 2mL of concentrated ammonia water to make the pH value of the mixed reaction liquid above 7. Stir rapidly and continue the reaction for 3 hours. After the reaction is completed, centrifuge and wash with deionized water until the filtrate reaches neutrality. After freeze-drying, obtain manganese dioxide nanozyme.

[0040] Example 2

[0041] This embodiment provides a manganese dioxide nanozyme, and the preparation method thereof comprises:

[0042] ① Dissolve 126 mg of manganese nitrate hexahydrate (Mn(NO3)2·6H2O) crystals in 10 mL of ethylene glycol to obtain a manganese nitrate hexahydrate ethylene glycol solution for later use.

[0043] ②Using clinically used montmorillonite powder as raw material, dissolve 3g of purified montmorillonite powder in 100mL of water to prepare a uniformly dispersed montmorillonite suspension for later use.

[0044] ③ Add 10mL of solution ① dropwise into 10mL of the suspension of ②. After the solution is evenly mixed, place it in a 40°C water bath and stir rapidly for 40 minutes. Then quickly add 2mL of concentrated ammonia water to make the pH value of the mixed reaction liquid above 7. Stir rapidly and continue the reaction for 2 hours. After the reaction is completed, centrifuge and wash with deionized water until the filtrate reaches neutrality. After freeze-drying, obtain manganese dioxide nanozyme.

[0045] Example 3

[0046] This embodiment provides a manganese dioxide nanozyme, and the preparation method thereof comprises:

[0047] ① Dissolve 126 mg of manganese nitrate hexahydrate (Mn(NO3)2·6H2O) crystals in 10 mL of ethylene glycol to obtain a manganese nitrate hexahydrate ethylene glycol solution for later use.

[0048] ② The montmorillonite powder was dispersed in water, stirred vigorously for 24 hours, centrifuged repeatedly to remove impurities, and dried to obtain purified montmorillonite powder. Subsequently, 6.3 g of the purified montmorillonite powder was dissolved in 100 mL of water to prepare a uniformly dispersed montmorillonite suspension for later use.

[0049] ③ Add 10mL of solution ① dropwise into 10mL of the suspension of ②. After the solution is evenly mixed, place it in a 50°C water bath and stir rapidly for 20 minutes. Then quickly add 2mL of concentrated ammonia water to make the pH value of the mixed reaction liquid above 7. Stir rapidly and continue the reaction for 2.5 hours. After the reaction is completed, centrifuge and wash with deionized water until the filtrate reaches neutrality. After freeze-drying, obtain manganese dioxide nanozyme.

[0050] Example 4

[0051] This embodiment provides a manganese dioxide nanozyme, and the preparation method thereof comprises:

[0052] ① Dissolve 126 mg of manganese nitrate hexahydrate (Mn(NO3)2·6H2O) crystals in 10 mL of ethylene glycol to obtain a manganese nitrate hexahydrate ethylene glycol solution for later use.

[0053] ② Disperse the montmorillonite powder in water, stir vigorously for 24 hours, centrifuge repeatedly to remove impurities, and dry to obtain purified montmorillonite powder. Subsequently, dissolve 5g of purified montmorillonite powder in 100mL of water to prepare a uniformly dispersed montmorillonite suspension with a concentration of 50mg / mL for later use.

[0054] ③ Add 10mL of solution ① dropwise into 10mL of the suspension of ②. After the solution is evenly mixed, place it in a 60°C water bath and stir rapidly for 30 minutes. Then quickly add 2mL of concentrated ammonia water to make the pH value of the mixed reaction liquid above 7. Stir rapidly and continue the reaction for 3 hours. After the reaction is completed, centrifuge and wash with deionized water until the filtrate reaches neutrality. After freeze-drying, obtain manganese dioxide nanozyme.

[0055] Example 5

[0056] This embodiment provides a manganese dioxide nanozyme, and the preparation method thereof comprises:

[0057] ① Dissolve 126 mg of manganese nitrate hexahydrate (Mn(NO3)2·6H2O) crystals in 10 mL of ethylene glycol to obtain a manganese nitrate hexahydrate ethylene glycol solution for later use.

[0058] ② Disperse the montmorillonite powder in water, stir vigorously for 24 hours, centrifuge repeatedly to remove impurities, and dry to obtain purified montmorillonite powder. Subsequently, dissolve 5g of purified montmorillonite powder in 100mL of water to prepare a uniformly dispersed montmorillonite suspension with a concentration of 50mg / mL for later use.

[0059] ③ Add 10mL of solution ① dropwise into 10mL of suspension ②. After the solution is evenly mixed, place it in a 30°C water bath and stir rapidly for 30 minutes. Then quickly add 2mL of concentrated ammonia water to make the pH value of the mixed reaction liquid above 7. Stir rapidly and continue the reaction for 3 hours. After the reaction is completed, centrifuge and wash with deionized water until the filtrate reaches neutrality. After freeze-drying, obtain manganese dioxide nanozyme.

[0060] Example 6

[0061] This embodiment provides a manganese dioxide nanozyme, and the preparation method thereof comprises:

[0062] ① Dissolve 126 mg of manganese nitrate hexahydrate (Mn(NO3)2·6H2O) crystals in 10 mL of propylene glycol to obtain a manganese nitrate hexahydrate propylene glycol solution for later use.

[0063] ② Disperse the montmorillonite powder in water, stir vigorously for 24 hours, centrifuge repeatedly to remove impurities, and dry to obtain purified montmorillonite powder. Subsequently, dissolve 5g of purified montmorillonite powder in 100mL of water to prepare a uniformly dispersed montmorillonite suspension with a concentration of 50mg / mL for later use.

[0064] ③ Add 10mL of solution ① dropwise into 10mL of the suspension of ②. After the solution is evenly mixed, place it in a 30°C water bath and stir rapidly for 30 minutes. Then quickly add concentrated ammonia water to make the pH value of the mixed reaction liquid between 7 and 9. Stir rapidly and continue the reaction for 3 hours. After the reaction is completed, centrifuge and wash with deionized water until the filtrate reaches neutrality. After freeze-drying, obtain manganese dioxide nanozyme.

[0065] Example 7

[0066] This embodiment provides a manganese dioxide nanozyme, and the preparation method thereof comprises:

[0067] ① Dissolve 126 mg of manganese nitrate hexahydrate (Mn(NO3)2·6H2O) crystals in 10 mL of ethylene glycol to obtain a manganese nitrate hexahydrate ethylene glycol solution for later use.

[0068] ② Disperse the montmorillonite powder in water, stir vigorously for 24 hours, centrifuge repeatedly to remove impurities, and dry to obtain purified montmorillonite powder. Subsequently, dissolve 5g of purified montmorillonite powder in 100mL of water to prepare a uniformly dispersed montmorillonite suspension with a concentration of 50mg / mL for later use.

[0069] ③ Add 10mL of solution ① dropwise into 10mL of the suspension of ②. After the solution is evenly mixed, place it in a 60°C water bath and stir rapidly for 30 minutes. Then quickly add 2mL of sodium hydroxide solution (1.5M) to make the pH value of the mixed reaction liquid above 7. Stir rapidly and continue the reaction for 3 hours. After the reaction is completed, centrifuge and wash with deionized water until the filtrate reaches neutrality. After freeze-drying, obtain manganese dioxide nanozyme.

[0070] Comparative Example 1

[0071] This comparative example provides a manganese dioxide nanosphere, and the synthesis method thereof comprises:

[0072] 35 mL of H2O was added to 1.45 g of KMnO4 and 500 mg of medical grade montmorillonite to form a reaction solution, which was transferred to a reactor and subjected to a hydrothermal reaction at 160°C for 48 hours to generate manganese dioxide on the montmorillonite. The resulting precipitate was washed with deionized water and freeze-dried to obtain the final product, montmorillonite / manganese dioxide (i.e., manganese dioxide nanospheres).

[0073] Performance testing

[0074] 1. Structural Characterization

[0075] The manganese dioxide nanozyme prepared in Example 1 was characterized by transmission electron microscopy, and the results were as follows: Figure 2 As shown in the figure, the manganese dioxide nanozyme has good dispersion performance and structural stability, presenting a uniformly dispersed sheet structure (length of 20-400nm), and the manganese dioxide nanozyme for the treatment of inflammatory bowel disease was successfully prepared.

[0076] 2. Determination of enzyme activity:

[0077] 1. Determination method:

[0078] The manganese dioxide nanozyme obtained in Example 1 and the manganese dioxide nanospheres obtained in Comparative Example 1 were used as experimental objects to measure their enzyme-like activities:

[0079] (1) Detection of superoxide dismutase (SOD) activity: The superoxide dismutase activity is determined by the NBT photoreduction method. The blue formazan, the photoreduction product of NBT, has a maximum absorption at 560 nm. Add 0.00015 g riboflavin, 0.0387 g methionine and 0.00123 g NBT to a test tube containing 20 mL phosphate buffer, mix thoroughly and store in the dark. Take 900 μL and add it to a 1.5 mL EP tube, then add 100 μL (1 mg / mL) of manganese dioxide nanosheets or manganese dioxide nanospheres. After rapid mixing, irradiate under an incandescent lamp for 5 minutes and detect the UV-vis spectrum at 400-800 nm.

[0080] (2) Catalase (CAT) activity detection: The model reaction of catalase is used to evaluate the activity of CAT enzymes; when nanozymes coexist with H2O2, they can rapidly catalyze H2O2 to produce O2, so the CAT activity of the material can be determined by monitoring the change in the absorbance of H2O2 at 240nm. The specific method is: prepare 900μ of PBS (0.1M, pH=7.4) solution containing 20mM hydrogen peroxide and add it to the EP tube, then add 100μL (1mg / mL) of manganese dioxide nanosheets or manganese dioxide nanospheres, mix well and place at room temperature for 15 minutes, take the supernatant and detect the UV-vis spectrum at 230-300nm.

[0081] (3) Glutathione peroxidase (GPx) activity detection: The 5,5'-dithio-bis-(2-nitrobenzoic acid) (DTNB) method was used to determine the glutathione peroxidase activity. The enzyme activity can catalyze H2O2 to oxidize GSH to produce GSSG. GSH and DTNB react to produce yellow 2-nitro-5-mercaptobenzoic acid, which has a characteristic absorption peak at 412nm. The glutathione peroxidase activity can be evaluated by the rate of decrease in absorbance. The specific method is: 200μL GSH and 100μL (1mg / mL) manganese dioxide nanosheets or manganese dioxide nanospheres are added to 500μL PBS solution and incubated for 30min, and then 200μL DTNB is added to the mixture. After 2min, the UV-vis spectrum at 350-600nm is detected.

[0082] 2. Measurement results

[0083] The results are as follows Figure 3 As shown, in the tests of superoxide dismutase-like activity, catalase-like activity, and glutathione peroxidase-like activity, when manganese dioxide nanozyme or manganese dioxide nanospheres were added, the ultraviolet absorbance values ​​of the three were significantly reduced, and the manganese dioxide nanozyme in the present application exhibited stronger enzyme-like activity.

[0084] 3. Free radical scavenging experiment:

[0085] 1. Experimental methods:

[0086] Scavenging of DPPH free radicals: Prepare different concentrations of manganese dioxide nanozymes (provided in Example 1), mix the manganese dioxide nanozyme solution with the DPPH ethanol solution (5mM) evenly, place it in a 37°C incubator to avoid light for 30 minutes, and measure the ultraviolet absorbance of the solution at 517nm. The free radical scavenging activity is evaluated by the percentage decrease in the absorbance of the sample at 517nm. Scavenging of ABTS free radicals: Mix 7.4mM ABTS solution with 2.6mM potassium persulfate (K2S2O8) at room temperature in the dark for 12h to prepare ABTS + Then, different concentrations of manganese dioxide nanozymes were prepared and the manganese dioxide nanozyme solution was mixed with ABTS + The solution was mixed evenly and reacted in the dark for 30 minutes. The UV absorbance of the solution at 734 nm was measured, and the free radical scavenging activity was evaluated by the percentage decrease of the absorbance of the sample at 734 nm.

[0087] Scavenging of ABTS free radicals: 7.4mM ABTS solution was mixed with 2.6mM potassium persulfate (K2S2O8) at room temperature in the dark for 12h to prepare ABTS+. Then, manganese dioxide nanozymes of different concentrations (provided in Example 1) were prepared, and the manganese dioxide nanozyme solution was evenly mixed with the ABTS+ solution, and reacted in the dark for 30min. The ultraviolet absorbance of the solution at 734nm was measured, and the free radical scavenging activity was evaluated by the percentage decrease in the absorbance of the sample at 734nm. Figure 4 The results showed that manganese dioxide nanozyme exhibited good ABTS scavenging activity. When manganese dioxide nanozyme was added, the UV absorbance value decreased significantly. The absorbance of the solution at 734 nm was recorded to evaluate the ability of manganese dioxide to scavenge ABTS free radicals.

[0088] 2. Experimental results:

[0089] like Figure 4 The results showed that manganese dioxide nanozyme exhibited good DPPH scavenging activity and ABTS scavenging activity. When manganese dioxide nanozyme was added, the UV absorbance value was significantly reduced.

[0090] 4. Manganese dioxide nanozyme blood compatibility test:

[0091] Manganese dioxide nanozymes (provided in Example 1) with concentrations of 25 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL, 400 μg / mL, and 800 μg / mL were prepared respectively, and the above-mentioned manganese dioxide nanozyme solutions (0.5 mL) of different concentrations were mixed with 0.2 mL of blood (0.5 mL of fresh blood was added to 4.5 mL of normal saline, centrifuged and washed 5 to 8 times, the centrifugal speed was 3000 rpm, the centrifugal time was 10 min, and the blood supernatant was clear and transparent, the supernatant was discarded, and the volume was fixed to 5 mL with normal saline) and 0.6 mL of normal saline, incubated at 37 ° C for 4 h, and then centrifuged at 3500 rpm for 10 min. The absorbance value of the supernatant at OD 492nm was measured to calculate the hemolysis rate.

[0092] The results are as follows Figure 5 As shown in the figure, it can be seen that the hemolysis rate of manganese dioxide nanozymes is less than 5%, indicating that manganese dioxide nanozymes have good biocompatibility.

[0093] 5. Animal trials for the treatment of inflammatory bowel disease:

[0094] Taking DSS-induced ulcerative colitis as an example; male C57BL / 6 mice aged 6 to 8 weeks and weighing 18 to 20 g (purchased from Chengdu Dashuo Experimental Animal Co., Ltd.) were selected to construct a DSS-induced ulcerative colitis model, and divided into a control group, a model group and a manganese dioxide nanozyme group, with 5 mice in each group, for a total of 15 mice; except for the mice in the control group who were given sterile water as free drinking water, the mice in other groups were given 2.5% DSS solution instead of sterile water for 7 days for modeling.

[0095] The mice in the manganese dioxide nanozyme group were given a daily dose of 20 mg / kg (provided in Example 1), and the mice in the control group and the model group were enema with the same volume of sterile water. After 12 days of feeding, samples were collected, the colon tissue of the mice was dissected, the colon length of each group of mice was recorded, and the colon tissue was embedded and sliced, and hematoxylin eosin staining (HE) was performed to observe the intestinal treatment effect, and the 16sRNA of the intestinal contents was measured to evaluate the changes in microbial diversity.

[0096] from Figure 6 It can be seen that the colon length of mice in the manganese dioxide nanozyme group was about 6.5 cm, which was closest to the colon length of mice in the normal group.

[0097] according to Figure 7 The results showed that HE staining showed severe abnormal inflammatory response and intestinal mucosal barrier damage in the colon of model mice. After manganese dioxide nanozyme treatment, the colon mucosal barrier of mice was restored and the morphology was intact.

[0098] according to Figure 8The results showed that DSS-induced ulcerative colitis significantly changes the structure and composition of intestinal flora. Manganese dioxide nanozymes can change and restore the composition of intestinal flora after treatment, increase beneficial flora, reduce harmful flora, and alleviate intestinal flora imbalance. The results show that manganese dioxide nanozymes can effectively treat and relieve ulcerative colitis by removing reactive oxygen species in intestinal inflammatory sites, relieving intestinal inflammation, and maintaining intestinal microbial homeostasis.

[0099] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed, but such modifications are protected by the patent law as long as they are within the scope of the claims of the present application.

Claims

1. A method for preparing a manganese dioxide nanozyme, characterized in that: It includes: The organic solution containing manganese nitrate hexahydrate and the montmorillonite aqueous solution are uniformly mixed to obtain a mixed reaction solution; The pH of the mixed reaction solution is adjusted to alkaline conditions using alkaline solution, a synthesis reaction is carried out, and then centrifugation and washing are performed to obtain montmorillonite-supported manganese dioxide nanozyme, wherein the manganese dioxide nanozyme has a lamellar structure with a length of 20-400 nm.

2. The method for preparing the manganese dioxide nanozyme according to claim 1, characterized in that: The temperature of the synthesis reaction is 30-80° C., and the reaction time is 2-3 hours.

3. The method for preparing the manganese dioxide nanozyme according to claim 1, characterized in that: In the mixed reaction liquid, the mass ratio of the manganese nitrate hexahydrate to the montmorillonite is 1:2.3-5.

4. The method for preparing the manganese dioxide nanozyme according to claim 1, characterized in that: The organic solvent in the organic solution containing manganese nitrate hexahydrate is any one of ethylene glycol, propylene glycol and butanediol.

5. The method for preparing the manganese dioxide nanozyme according to claim 1, characterized in that: The preparation of the mixed reaction solution comprises: The organic solution containing manganese nitrate hexahydrate is added dropwise into the montmorillonite aqueous solution, and stirred in a water bath at 40-70° C. for 20-40 minutes.

6. A manganese dioxide nanozyme, characterized in that It is prepared by the preparation method according to any one of claims 1 to 5.

7. Use of the manganese dioxide nanozyme as claimed in claim 6 in the preparation of a drug for treating inflammatory bowel disease.

8. The use according to claim 7, characterized in that: The inflammatory bowel disease is any one of Crohn's disease and ulcerative colitis.

9. A composite preparation for treating inflammatory bowel disease, characterized in that: It comprises the manganese dioxide nanozyme as claimed in claim 6, and pharmaceutical excipients.

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