Zirconium-doped cerium oxide nano-enzyme for preparing allergic rhinitis drops and preparation method of zirconium-doped cerium oxide nano-enzyme

Abstract

The invention relates to the technical field of rhinitis medicine preparation, in particular to zirconium-doped cerium oxide nano-enzyme for preparing allergic rhinitis drops and a preparation method of the zirconium-doped cerium oxide nano-enzyme. The preparation method comprises the following steps: step 1, weighing a cerium acetylacetonate (III) hydrate and a zirconium acetylacetonate (IV) hydrate, adding into oleylamine, magnetically stirring and uniformly mixing, and carrying out ultrasonic treatment on the mixture; step 2, putting the mixed solution in the step 1 into a drying oven, heating, preserving heat, and aging to obtain a dark brown colloidal solution; step 3, naturally cooling the colloidal solution to room temperature, adding acetone, centrifuging, discarding supernate, and repeating centrifugal washing for several times; and 4, carrying out vacuum drying on the precipitate obtained in the step 3, and fully grinding to obtain the zirconium-doped cerium oxide nano-enzyme. The prepared zirconium-doped cerium oxide nano-enzyme is applied to preparation of allergic rhinitis drops. The zirconium-doped cerium oxide nano enzyme reduces the inflammatory factor level of the AR mouse and inhibits degranulation of nasal mucosa MCs, so that the pathological state and allergic symptoms of the AR mouse are improved.

Description

Technical Field

[0001] The invention relates to the technical field of rhinitis drug preparation, and in particular to a zirconium-doped cerium oxide nanozyme for preparing allergic rhinitis drops and a preparation method thereof. Background Art

[0002] Allergic rhinitis (AR) is a disease that generally affects the health and economy of the global population, involving more than 500 million people. Allergic rhinitis refers to a non-infectious chronic inflammatory disease of the nasal mucosa that is mainly mediated by IgE and involves a variety of immune-active cells (such as T lymphocytes, B lymphocytes, mast cells, basophils, etc.) and cytokines after atopic individuals come into contact with allergens. About 10.0% to 40.40% of the world's population has clinical manifestations of AR.

[0003] At present, the treatment of AR mainly adopts the principle of "combining prevention and treatment, four-in-one", including environmental control, drug therapy, immunotherapy and health education. Environmental control mainly refers to avoiding or reducing contact with allergens and various irritants, which is an important part of the AR prevention and treatment strategy. Drug therapy includes antihistamines, glucocorticoids, leukotriene receptor antagonists and mast cell membrane stabilizers. Allergen-specific immunotherapy is mainly divided into subcutaneous immunotherapy and sublingual immunotherapy.

[0004] Although the above treatment methods are widely used in clinical practice, they all have many limitations. Nasal glucocorticoids are prone to relapse after discontinuation and have many side effects; antihistamines are inevitable for irritation of local mucosa and systemic side effects; for mast cell membrane stabilizers, patient compliance is limited by short-term preventive effects and requires frequent local application; long-term use of decongestant nasal drops increases the risk of drug-induced rhinopathy; biological drugs are expensive and complex in dosage and are not widely used worldwide; immunotherapy is time-consuming and difficult for patients to adhere to, and is prone to cause local or systemic immune responses. Therefore, it is of great significance to find new, safe and effective drugs for the treatment of AR.

[0005] Studies have shown that oxidative stress is a key factor in AR. The accumulation of excessive reactive oxygen species (ROS) and reactive nitrogen species (RNS) leads to oxidative damage, consumes a large number of antioxidant molecules and attacks antioxidant enzymes. When redox homeostasis is disturbed, oxidative stress will occur, which in turn causes increased lipid peroxidation and increased airway reactivity and secretions. Chemical inducer molecules are produced and vascular permeability is increased, leading to AR and asthma attacks. Therefore, clearing excess ROS and RNS and blocking inflammatory responses may be one of the effective ways to treat AR.

[0006] "Nanozymes" are nanomaterials with natural enzyme activity. Nanozymes have made significant research progress in biomedical fields such as medical diagnosis, targeted therapy and biosensors due to their advantages such as simple synthesis, good stability, recyclability, easy surface modification and adjustable catalytic activity. Cerium dioxide (CeO2NPs) nanozymes can remove a variety of ROS and relieve oxidative stress. In recent years, they have been used in the antioxidant treatment of diseases such as periodontitis, urolithiasis, orthopedic diseases, atherosclerosis, acute kidney injury and dry eye. The enzyme-mimicking activity of CeO2NPs mainly comes from Ce 3+ and Ce 4+ The redox cycle between 4+ ) to increase Ce 3+ / Ce4 + The ratio can be further improved

[0007] CeO2NPs have the ability to remove ROS and are used to treat sepsis, neuropathic pain, and renal injury. In addition to the ability to efficiently eliminate a variety of ROS, CeO2NPs also have phosphohydrolase activity, which can catalyze the hydrolysis of phosphate bonds, inhibit the degranulation-related phosphate signaling cascade in allergen-stimulated MCss, and prevent the release of inflammatory mediators, thereby achieving long-term prevention of skin allergic diseases. 4+ The doped CeO2NPs not only have significant SOD mimetic enzyme activity, CAT mimetic enzyme activity and ·OH antioxidant capacity, but also have phosphohydrolase mimetic activity. Summary of the invention

[0008] In order to comprehensively solve the above problems, the purpose of the present invention is to overcome the defects in the prior art and provide a zirconium-doped cerium oxide nanozyme for preparing allergic rhinitis drops and a preparation method thereof. The present invention prepares zirconium-doped cerium oxide nanozyme with good biocompatibility, uses rat basophilic leukemia cells (RBL-2H3) and AR mice as models, utilizes the phosphohydrolase activity and ROS removal ability of the nanomaterial, and regulates the phosphorylation signal cascade associated with MCs degranulation to inhibit MCs degranulation and subsequent pathological reactions, thereby improving the prevention and treatment efficacy of AR.

[0009] In order to achieve the above object, the first aspect of the present invention provides a method for preparing zirconium-doped cerium oxide nanozyme, comprising:

[0010] Step 1: Weigh cerium (III) acetylacetonate hydrate and zirconium (IV) acetylacetonate hydrate, add them to oleylamine, stir them magnetically, and then treat the mixture with ultrasound;

[0011] Step 2: placing the mixed solution of step 1 in an oven, heating and keeping warm, and aging to obtain a dark brown colloidal solution;

[0012] Step 3: After the colloidal solution is naturally cooled to room temperature, acetone is added, the supernatant is discarded after centrifugation, and the centrifugation washing is repeated several times;

[0013] Step 4: The precipitate from step 3 is vacuum dried and ground thoroughly to obtain zirconium-doped cerium oxide nanozyme.

[0014] Preferably, in step 1, the amount of cerium (III) acetylacetonate hydrate is 0.36 g, the amount of zirconium (IV) acetylacetonate hydrate is 0.14 g, and the amount of oleylamine is 15 mL.

[0015] Preferably, the ultrasonic temperature in step 1 is 20° C. and the ultrasonic time is 20 min.

[0016] Preferably, the insulation temperature of step 2 is 80° C. and the time is 24 hours.

[0017] Preferably, the acetone in step 3 is 100 mL.

[0018] The second aspect of the present invention provides a zirconium-doped cerium oxide nanozyme prepared by the above method.

[0019] A third aspect of the present invention provides an application of a zirconium-doped cerium oxide nanozyme for preparing allergic rhinitis drops.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] 1. The present invention provides strong evidence for the anti-inflammatory and anti-allergic effects of CZ NPs. CZNPs were synthesized by precipitation method, and mPEG(2000)-PE was modified on their surface to improve the water solubility and biocompatibility of CZ NPs. In addition, the nanoparticles showed good scavenging ability for a variety of ROS and RNS including O2-·, H2O2, ·OH and .DPPH, and had phosphohydrolase-like activity, and still maintained high multiple enzyme activities at lower concentrations, which will provide a strong basis for exploring the anti-inflammatory and anti-allergic effects of CZ NPs at the cell and activity levels.

[0022] 2. The present invention provides CZ NPs that exhibit good biocompatibility, anti-oxidative stress ability and anti-allergic properties in in vitro experiments. According to the results of in vitro cell viability experiments, CZ NPs have little toxic effect on RBL-2H3 cells when the concentration is ≤30μg mL-1, and can maintain cell proliferation activity at a high level. In the oxidative stress model, the cell viability after treatment with CZ NPs was significantly enhanced, and the oxidative stress induced by H2O2 was effectively reduced. Further experiments have shown that the levels of ROS and RNS in RBL-2H3 cells were significantly increased by adding Rousp and H2O, respectively, and then the levels of ROS and RNS in RBL-2H3 cells were significantly reduced by adding CZ NPs, indicating that CZ NPs can protect cells from in vitro damage induced by oxidative stress.

[0023] 3. After the mast cell degranulation model was constructed by inducing RBL-2H3 cells with Anti-DNP-IgE and DNP-HSA, the levels of ROS and RNS in RBL-2H3 cells increased significantly. CZ NPs reduced the levels of intracellular ROS and RNS in a concentration-dependent manner, demonstrating its ability to resist oxidative stress. In addition, CZ NPs effectively inhibited the degranulation process of MCs and reduced the release of allergic mediators such as β-HEX and histamine.

[0024] 4. The present invention provides for the first time evidence that CZ NPs are effective in the treatment and prevention of AR, providing new ideas and methods for clinical treatment. The present invention successfully established an OVA-induced AR mouse model and verified that it conforms to the pathophysiological characteristics of AR. The results showed that CZ NPs exhibited significant anti-allergic inflammatory effects in the AR mouse model. CZ NPs intervention can significantly alleviate nasal symptoms caused by AR, including symptoms such as scratching the nose, sneezing, and sparse hair around the nose, and improve the structure of the nasal mucosa and reduce the inflammatory response. At the same time, the intervention of CZ NPs can also reduce the degranulation of MCs in the nasal mucosa of mice, thereby reducing the degree of inflammation of the nasal mucosa and improving the allergic symptoms of AR mice. In addition, by reducing the levels of inflammatory factors in the serum of AR mice, such as OVA-sIgE, IL-4, IL-13 and TNF-α, and increasing the content of IFN-γ, CZ NPs effectively reduced the inflammatory response. At the same time, CZ NPs exhibited good biosafety.

[0025] 5. The zirconium-doped cerium oxide nanozyme prepared by the present invention has efficient phosphatase-like activity and the ability to remove ROS and RNS; the zirconium-doped cerium oxide nanozyme can reduce the level of excessive ROS and RNS in cells, inhibit the release of β-HEX and histamine, and thus inhibit the degranulation of MCs; the zirconium-doped cerium oxide nanozyme reduces the level of inflammatory factors in the serum of AR mice and inhibits the degranulation of MCs in the nasal mucosa, thereby improving the pathological state and allergic symptoms of AR mice. Zirconium-doped cerium oxide nanozyme can improve the nasal allergy symptoms of OVA-induced AR mouse models, and may become a potential drug for the prevention and treatment of AR, providing new ideas and methods for clinical treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The accompanying drawings are used to provide further understanding of the present invention and constitute a part of the specification. They are used to explain the present invention together with the embodiments of the present invention and do not constitute a limitation of the present invention.

[0027] In the attached picture:

[0028] Figure 1 CZ NPs have SOD-like activity. (A) UV-visible absorption spectra of O2·- scavenging by CZ NPs at different concentrations; (B) Inhibition rates of CZ NPs solutions at different concentrations.

[0029] Figure 2 CZ NPs have the ability to scavenge ·OH. (A) UV-visible absorption spectra of CZ NPs at different concentrations scavenging ·OH; (B) The ability of CZ NPs at different concentrations to scavenge ·OH.

[0030] Figure 3 CZ NPs have the ability to scavenge DPPH·. (A) UV-visible absorption spectra of CZ NPs at different concentrations scavenging DPPH·; (B) scavenging rates of CZ NPs at different concentrations on DPPH·.

[0031] Figure 4 Evaluation of the protective effect of CZ NPs on RBL-2H3 cells co-incubated with H2O2.

[0032] Figure 5 CZ NPs can effectively remove ROS at the cellular level.

[0033] Figure 6 CZ NPs cleared RNS well at the cellular level.

[0034] Figure 7 H&E staining of nasal mucosal tissues of mice in each group (400 times).

[0035] Figure 8 Toluidine blue staining (400 times) of the nasal mucosal tissue of mice in each group.

[0036] Fig. 9 Figure 2 shows the levels of OVA-sIgE and cytokines in the serum of mice in each group.

[0037] Fig.10 TEM characterization of CZ NPs (A) low magnification TEM; (B) HRTEM; (C) HAADF-STEM; (DF) corresponding element distribution diagram.

[0038] Fig.11 XRD and XPS characterization of CZ NPs (A) XRD spectrum; (B) Ce 3d core energy level spectrum; (C) Zr 3d core energy level spectrum.

[0039] Fig.12 ICP-MS characterization of CZ NPs. (A) Standard curve of Ce; (B) Standard curve of Zr.

[0040] Fig.13 Cell viability of RBL-2H3 cells after treatment with different concentrations of CZ NPs for 24 h.

[0041] Fig.14 The present invention is a flow chart of the preparation method of zirconium-doped cerium oxide nanozyme. DETAILED DESCRIPTION

[0042] The following combination Figure 1-Figure 14 It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0043] Embodiment 1:

[0044] A method for preparing zirconium-doped cerium oxide nanozyme, comprising:

[0045] Step 1: Weigh 0.36 g of cerium (III) acetylacetonate hydrate and 0.14 g of zirconium (IV) acetylacetonate hydrate and add them into 15 mL of oleylamine. After magnetic stirring, the mixture is ultrasonically treated at 20° C. for 20 min.

[0046] Step 2: placing the mixed solution of step 1 in an oven, heating from room temperature to 80° C., and aging at 80° C. for 24 h to obtain a dark brown colloidal solution;

[0047] Step 3: After the colloidal solution is naturally cooled to room temperature, 100 mL of acetone is added, centrifuged at 125,000 rpm for 10 min, the supernatant is discarded, and the centrifugation washing is repeated 3 times;

[0048] Step 4: The precipitate from step 3 is dried under vacuum at 40°C, and then ground thoroughly to obtain zirconium-doped cerium oxide nanozyme.

[0049] Step 5: Weigh 50 mg of zirconium CZ solid powder and add it to 5 mL of chloroform, and ultrasonicate for 14 h to completely disperse it. Take 100 mg of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000](ammonium salt) (mPEG(2000)PE) and dissolve it in 5 mL of chloroform. Mix 5 mL of CZ NPs sample chloroform dispersion (10 mg mL-1) with 10 mL of mPEG(2000)PE (10 mg mL-1) chloroform solution, let it stand for 10 min, and then remove the chloroform with a rotary evaporator (50 ° C, 28 rmin-1). The sample is dried in a vacuum oven (70 ° C) for 2 h to ensure that the chloroform is completely removed. Then, 5 ml of deionized water was added to the sample to obtain a transparent colloidal suspension, which was filtered using a 0.45 μm filter membrane to remove large particles, and then centrifuged using an ultracentrifuge (200,000 × g, 30 min), and the supernatant was discarded to remove excess mPEG (2000) -PE. Finally, the sample was dispersed in deionized water to obtain a CZ NPs solution.

[0050] Embodiment 2:

[0051] The zirconium-doped cerium oxide nanozyme prepared in Example 1 is used to prepare allergic rhinitis drops to achieve the treatment of allergic rhinitis.

[0052] Experimental and characterization:

[0053] 1. Characterization of Zr-doped Cerium Oxide Nanozymes

[0054] The morphology, structure and chemical state of the samples were comprehensively characterized by transmission microscopy (TEM), high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM), energy dispersive X-ray spectroscopy (EDX-mapping), particle size analysis, X-ray diffraction pattern (XRD), and X-ray photoelectron spectroscopy (XPS). Fig.10 , 11 ). The element content (such as Fig.12 ). Evaluate its phosphohydrolase activity and scavenging efficiency against various ROS and RNS (such as Figure 5 , 6 ).

[0055] ROS include hydrogen peroxide (H2O2), superoxide anion (O2·-), hydroxyl radical ·OH, and RNS is nitric oxide radical (NO·).

[0056] result:

[0057] TEM observation showed that zirconium-doped ceria nanozymes were spherical nanoparticles with uniform morphology and size (e.g. Fig.10 ).

[0058] Dynamic light scattering (DLS) showed that its hydration dynamic particle size was mainly distributed in the range of 10-40 nm, the most probable particle size was 15.90 nm, and the Zeta potential was -13.57 mV.

[0059] XRD and XPS analysis showed that Ce in Zr-doped ceria nanozymes 3+ and Ce 4+ The existence of Fig.11 ).

[0060] Experiments have shown that it has antioxidant enzyme and phosphohydrolase activities such as superoxide dismutase (SOD) and catalase (CAT), and can effectively remove reactive oxygen species (ROS) and reactive nitrogen species (RNS).

[0061] 2. In vitro experimental study on the anti-allergic effect of zirconium-doped cerium oxide nanozymes

[0062] The mast cell (MCs) degranulation model was established by inducing RBL-2H3 cells with anti-dinitrophenol (anti-DNP) IgE monoclonal antibody and human serum albumin (HSA) (Anti-DNP-HSA). The cytoprotective toxicity, antioxidant and anti-allergic effects of zirconium-doped ceria nanozymes and their effects on degranulation were evaluated by CCK-8 method, ROS and RNS fluorescent probes, colorimetric method and enzyme-linked immunosorbent assay (ELISA).

[0063] result:

[0064] Zirconium-doped cerium oxide nanozymes had no significant toxicity to RBL-2H3 cells at a concentration of 0-30 μg mL-1 (e.g. Fig.13 ).

[0065] Laser confocal microscopy results showed that zirconium-doped cerium oxide nanozymes could reduce the levels of ROS and RNS in cells after stimulation with H2O2 or Anti-DNP-HSA.

[0066] The results of the degranulation experiment showed that the release rates of β-HEX in RBL-2H3 cells under the action of different concentrations of zirconium-doped cerium oxide nanozymes (10, 20, 30 μg mL-1) were 88.48±7.09%,

[0067] 74.86±3.56% and 49.86±7.31%, and the histamine release rates were 80.90±6.08%,

[0068] 70.29±0.92% and 47.21±3.01%.

[0069] It was confirmed that zirconium-doped cerium oxide nanozymes have the ability to inhibit MCs degranulation. Compared with the blank group, there was no significant difference in the cell β-HEX release rate in the 10μg mL-1 zirconium-doped cerium oxide nanozyme treatment group (P>0.05), and the cell β-HEX release rate in the 20 and 30μg mL-1 zirconium-doped cerium oxide nanozyme treatment groups was significantly reduced (P<0.01 and P<0.0001, respectively). Compared with the control group, the cell histamine release rate in the 10, 20 and 30μg mL-1 zirconium-doped cerium oxide nanozyme treatment groups was significantly reduced (P<0.0001).

[0070] 3. In vivo experimental study of zirconium-doped cerium oxide nanozyme nasal drops in the treatment of OVA-induced AR mice

[0071] The mice were randomly divided into 3 groups: blank group Con, model group AR and drug-treated group (2 mg kg -1 )CZ+AR.

[0072] The OVA-induced AR model in mice was used to evaluate the effects of zirconium-doped ceria nanozymes (2 mg kg -1 ) The therapeutic effect of nasal drops was evaluated. After treatment, biological behaviors were observed, AR symptom scores were recorded, tissue samples were collected and analyzed, key inflammatory factor levels were detected by ELISA, and histological changes of nasal mucosa and the degree of MCs degranulation were observed by H&E and toluidine blue staining to evaluate the therapeutic potential of zirconium-doped ceria nanozyme.

[0073] result:

[0074] Pathological changes of nasal mucosa of mice in each group

[0075] H&E staining Fig. 9 As shown, compared with the Con group, the H&E staining results showed that the nasal mucosa of the AR group mice was significantly swollen, the goblet cells increased, filled with mucus, focal cilia loss, and a large number of lymphocytes and eosinophils infiltrated under the mucosa; after the intervention of zirconium-doped ceria nanozyme, the swelling of the nasal mucosa of the mice in the zirconium-doped ceria nanozyme + AR group was reduced, the proliferation of submucosal glands was reduced, and the infiltration of lymphocytes and eosinophils was reduced.

[0076] Toluidine blue staining of nasal mucosa of mice in each group:

[0077] Toluidine blue staining results Fig.10 As shown, Fig.10 For TEM characterization of CZ NPs, Fig.10(A) Low-magnification TEM; (B) HRTEM; (C) HAADF-STEM; (DF) Corresponding element distribution diagram Compared with the Con group, there were more MCs infiltration and degranulation (purple-red metachromatic granules) in the nasal mucosa of the AR group; while after the intranasal intervention of zirconium-doped cerium oxide nanozyme (2 mg kg-1), there were a small number of mast cells and degranulation scattered in the nasal mucosa of mice.

[0078] The contents of OVA-sIgE, IL-4, IL-13, IFN-γ and TNF-α in the serum of mice in each group:

[0079] like Fig.11 As shown, the ELISA results showed that the levels of OVA-sIgE, IL-4, IL-13 and TNF-α in the serum of mice in the AR group were significantly increased (P<0.01, P<0.0001, P<0.01 and P<0.01, respectively), and the content of IFN-γ was significantly decreased (P<0.0001).

[0080] After intranasal intervention with zirconium-doped cerium oxide nanozyme (2 mg kg-1), the levels of OVA-sIgE, IL-4, IL-13, and TNF-α in the serum of mice were significantly lower than those in the AR group (P<0.05, P<0.0001, P<0.05, and P<0.05, respectively), and the content of IFN-γ was significantly reduced (P<0.001).

[0081] The experimental results showed that zirconium-doped cerium oxide nanozymes can effectively improve the inflammatory response and allergic reaction of AR mice.

[0082] 4. Determination of SOD-like activity of zirconium-doped cerium oxide nanozymes

[0083] Since the reduced Ce 3+ It can effectively remove O2·-, and the SOD simulation activity of zirconium-doped cerium oxide nanozyme was determined by using a SOD kit.

[0084] result:

[0085] like Figure 1 As shown, Figure 1 (A) shows that the mixed solution has an obvious absorption peak at 450nm. As the concentration of CZ NPs increases, the absorbance of the system gradually decreases and the color of the solution gradually becomes lighter. Figure 1 (B) shows that with the increase of CZ NPs concentration, the inhibition rate gradually increases. When the concentration of CZ NPs reaches 0.32 mg mL-1, the inhibition rate reaches more than 20%. The experimental results show that zirconium-doped cerium oxide nanozyme has SOD mimetic enzyme activity, the specific activity of the enzyme is 49.88±10.18 U mg-1, and it can catalyze O2·- to generate O2 and H2O2.

[0086] 5. Determination of OH scavenging ability of zirconium-doped ceria nanozymes

[0087] The ability of zirconium-doped ceria nanozymes to scavenge ·OH was evaluated by a hydroxyl radical test kit.

[0088] result:

[0089] like Figure 2 As shown, Figure 2 (A) shows that as the concentration of CZ NPs increases, the absorbance of the system gradually decreases. Figure 2 (B) shows that as the concentration of CZ NPs increases, the inhibition rate gradually increases. When the concentration of CZ NPs reaches 0.40 mg mL-1, the inhibition rate reaches more than 50%. The experimental results show that zirconium-doped cerium oxide nanozymes have the ability to scavenge ·OH. When the concentration of zirconium-doped cerium oxide nanozymes reaches 0.40 mg mL-1, the inhibition rate reaches more than 50%. The ability to inhibit hydroxyl radicals is 65.21±7.94 U mg -1 .

[0090] 6. Determination of RNS elimination ability of zirconium-doped cerium oxide nanozymes

[0091] The RNS scavenging ability of zirconium-doped cerium oxide nanozymes was evaluated by DPPH reagent.

[0092] result:

[0093] like Figure 3 As shown, Figure 3 (A) shows that as the concentration of CZ NPs increases, the absorbance of the mixed solution at 517 nm gradually decreases. The results show that CZ NPs have the ability to scavenge RNS. Figure 3 (B) shows that as the concentration of CZ NPs increases, the clearance rate gradually increases. When the concentration of CZ NPs reaches 0.6 mg mL-1, the inhibition rate reaches more than 80%. The results show that zirconium-doped cerium oxide nanozymes have the ability to clear RNS. The RNS clearing capacity of zirconium-doped cerium oxide nanozymes is 12.97±7.16 Uμg -1 .

[0094] 7. Evaluation results of the protective effect of zirconium-doped cerium oxide nanozymes on RBL-2H3 cells co-incubated with H2O2:

[0095] like Figure 4 As shown, compared with the H2O2 group, the cell viability was enhanced after treatment with zirconium-doped ceria nanozyme, and when the concentration of zirconium-doped ceria nanozyme reached 30 μg mL-, the cell viability was restored to more than 80%, revealing the cytoprotective effect of zirconium-doped ceria nanozyme against H2O2-induced oxidative stress.

[0096] 8. Evaluation of the ability of zirconium-doped cerium oxide nanozymes to remove intracellular ROS and RNS

[0097] Fluorescence images taken using a confocal microscope, such as Figure 5 As shown in the figure, compared with the blank group, RBL-2H3 cells showed strong green fluorescence under Rousp stimulation, indicating that the level of ROS in the cells was significantly enhanced and the cells were in a state of oxidative stress. The fluorescence intensity of cells treated with zirconium-doped cerium oxide nanozymes was significantly weakened under the stimulation of Rousp, indicating that zirconium-doped cerium oxide nanozymes can effectively remove ROS in RBL-2H3 cells.

[0098] like Figure 6 As shown in the figure, compared with the blank group, RBL-2H3 cells showed strong green fluorescence under the stimulation of 200μΜH2O2, indicating that the level of RNS in the cells was significantly enhanced and the cells were in a state of oxidative stress. After the cells were treated with zirconium-doped cerium oxide nanozymes, the fluorescence intensity of the cells was significantly weakened under the stimulation of 200μΜH2O2, indicating that zirconium-doped cerium oxide nanozymes can effectively remove RNS in RBL-2H3 cells.

[0099] Therefore, CZ NPs reduced the intracellular ROS and RNS levels in a concentration-dependent manner, demonstrating its ability to resist oxidative stress. CZ NPs effectively inhibited the degranulation process of MCs and reduced the release of allergic mediators such as β-HEX and histamine.

[0100] The above shows and describes the basic principles, main features and advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A method for preparing zirconium-doped cerium oxide nanozyme, characterized in that: include: Step 1: Weigh cerium (III) acetylacetonate hydrate and zirconium (IV) acetylacetonate hydrate, add them to oleylamine, stir them magnetically, and then treat the mixture with ultrasound; Step 2: placing the mixed solution of step 1 in an oven, heating and keeping warm, and aging to obtain a dark brown colloidal solution; Step 3: After the colloidal solution is naturally cooled to room temperature, acetone is added, the supernatant is discarded after centrifugation, and the centrifugation washing is repeated several times; Step 4: The precipitate from step 3 is vacuum dried and ground thoroughly to obtain zirconium-doped cerium oxide nanozyme.

2. The method for preparing a zirconium-doped cerium oxide nanozyme according to claim 1, characterized in that: In step 1, 0.36 g of cerium (III) acetylacetonate hydrate, 0.14 g of zirconium (IV) acetylacetonate hydrate, and 15 mL of oleylamine were used.

3. The method for preparing a zirconium-doped cerium oxide nanozyme according to claim 2, characterized in that: The ultrasonic temperature of step 1 is 20° C. and the ultrasonic time is 20 min.

4. The method for preparing a zirconium-doped cerium oxide nanozyme according to claim 3, characterized in that: The insulation temperature of step 2 is 80°C and the time is 24h.

5. The method for preparing zirconium-doped cerium oxide nanozyme according to claim 4, characterized in that: The acetone in step 3 was 100 mL.

6. The zirconium-doped cerium oxide nanozyme prepared by the method according to any one of claims 1 to 5.

7. The use of the zirconium-doped cerium oxide nanozyme according to claim 6, characterized in that: Use for preparing allergic rhinitis drops.

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