Deubiquitinating enzyme medicine as well as preparation method and application thereof

Abstract

The invention discloses a deubiquitination enzyme drug. The deubiquitination enzyme drug comprises a deubiquitination enzyme, an antioxidant nano-enzyme and a carrier for loading the deubiquitination enzyme and the antioxidant nano-enzyme. The deubiquitination enzyme medicine contains the deubiquitination enzyme and the antioxidant nano-enzyme, and the antioxidant nano-enzyme removes redundant ROS at the colon lesion, so that the biological activity of the deubiquitination enzyme can still be maintained in an inflammation microenvironment, and inflammation of the lesion is relieved. The invention further discloses a preparation method of the deubiquitination enzyme medicine. The preparation method is simple in process and short in time consumption. The invention also discloses application of the deubiquitinating enzyme medicine in preparation of medicines for treating and / or diagnosing inflammatory bowel diseases. The deubiquitinating enzyme medicine can activate an inertial cavitation mechanism through O2 microbubbles in an oxidative stress microenvironment of the inflammatory bowel disease, so that a photoacoustic signal is specifically generated, and accurate diagnosis of pathological changes of the inflammatory bowel disease and accurate treatment guided by photoacoustic imaging are realized.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a deubiquitinating enzyme drug and a preparation method and application thereof Background Art

[0002] Inflammatory bowel disease is a chronic gastrointestinal disease that affects millions of people worldwide and is common in young and middle-aged patients. Inflammatory bowel disease can cause symptoms such as diarrhea, abdominal pain, fatigue, rectal bleeding, weight loss, depression, etc., which have a significant impact on the patient's quality of life. Long-term progression of the disease will increase the risk of colorectal cancer. Immunosuppressive and anti-inflammatory drugs are widely used clinically to treat inflammatory bowel disease, but these drugs generally target downstream inflammatory mediators such as TNF-α, IL-6, etc. Anti-inflammatory drugs require high doses and high frequency of administration, and are prone to serious side effects such as bone marrow suppression, hepatotoxicity, and drug resistance, and the overall treatment effect is not ideal.

[0003] Post-translational modification of proteins by deubiquitinases plays a crucial role in regulating the inflammatory pathway of inflammatory bowel disease. Studies have shown that ovarian tumor deubiquitinase (OTUD1) effectively inhibits inflammatory bowel disease-associated colon inflammation by cleaving K63 ubiquitin chains on receptor-interacting serine / threonine protein kinase (RIPK1), an upstream mediator of NF-κB inflammatory signaling. Studies have found that Cezanne inhibits NF-κB activation by cutting ubiquitin chains of upstream regulatory factors. However, excessive reactive oxygen species (ROS) are usually produced in the inflamed colon tissue of patients with inflammatory bowel disease, and ROS directly oxidize deubiquitinases, causing them to lose their activity. Therefore, the poor stability of deubiquitinases in inflammatory colon tissues has seriously hindered the clinical translation of deubiquitinases for the treatment of inflammatory bowel disease.

[0004] Nanozymes are nanomaterials with similar activities to natural enzymes. Most of them are heterogeneous catalysts, such as metal, metal oxide or carbon-based nanozymes. Compared with natural enzymes, nanozymes have better stability and economic value. Nanozymes have the advantages of small size and high surface area ratio, which can enhance the tissue permeability of nanozymes, but they also need suitable carriers to reduce toxicity. Nanozymes can improve oxidative stress, remove reactive oxygen species, and regulate the local immune system.

[0005] Publication No. CN116173071A discloses a method for preparing MnNiPCN@PVP nanozyme and its application. Sodium chloride, manganese chloride, nickel chloride, glucose and urea are dissolved in deionized water, and the mixed solution is heated and fired in an argon atmosphere, then washed with distilled water for multiple times to remove the sodium chloride template, and finally polyvinyl pyrrolidone is used to coat the MnNiPCN nanoparticles to obtain MnNiPCN@PVP nanozyme. However, the preparation of the nanozyme requires high-temperature heating, which is easily restricted in industrial production.

[0006] Publication No. CN118105522A discloses a synthesis method and application of a targeted nano-delivery system with biocatalytic activity, firstly, a metal platinum nanozyme with antioxidant activity and a metal organic framework (MOF) with catalytic activity are synthesized separately, MOF and dopamine induce polymerization through chelation competition, dopamine replaces the imidazole skeleton to obtain hollow dopamine particles, and finally mixed with a nano-carrier to obtain a targeted nano-delivery system with biocatalytic activity. This preparation method is time-consuming and complex. Summary of the invention

[0007] In order to solve the above technical problems, the present invention provides a deubiquitinase drug, which contains a deubiquitinase and an antioxidant nanozyme, which can remove excess ROS at the colon lesions, so that the deubiquitinase can still maintain its biological activity in the inflammatory microenvironment, thereby reducing inflammation at the lesion site.

[0008] A deubiquitinase drug comprises a deubiquitinase, an antioxidant nanozyme and a carrier loaded with the deubiquitinase and the antioxidant nanozyme.

[0009] Deubiquitinase is an important regulator of inflammatory bowel disease and plays an important role in regulating protein post-translational modification. However, in the oxidative stress microenvironment of colonic tissue in patients with inflammatory bowel disease, deubiquitinase will be oxidized by ROS and lose its activity. Antioxidant nanozymes can mimic the properties of catalase and superoxide dismutase, and can achieve the removal of highly reactive oxygen free radicals in the lesion site, thereby reducing inflammation in the lesion site.

[0010] The deubiquitinase drug of the present invention contains both a deubiquitinase and an antioxidant nanozyme, and the two produce a synergistic effect. The antioxidant nanozyme can remove excess ROS in colon lesions, and at the same time, it can also enable the deubiquitinase to maintain its biological activity in the inflammatory microenvironment, and the two can synergistically exert an anti-inflammatory effect in the inflammatory microenvironment.

[0011] Preferably, the mass ratio of the deubiquitinating enzyme and the antioxidant nanozyme in the deubiquitinating enzyme drug to the mass ratio of the carrier is 1:1-6.

[0012] Preferably, the mass ratio of the deubiquitinating enzyme to the antioxidant nanozyme is 1:1-6 μg / mg.

[0013] Preferably, the deubiquitinating enzyme is UBPs or OTU.

[0014] Deubiquitinases are important regulators of inflammatory bowel disease, and their post-translational modification of proteins plays a key role in inflammatory bowel disease. Ubiquitin-specific proteases (UBPs) and ovarian tumor-associated proteases (OTUs) are both cysteine ​​protease-type deubiquitinases, which mediate the deubiquitination of upstream inflammatory regulatory factors and regulate downstream inflammatory mediators (such as TNF-α, IL-6, IL-8, etc.), thereby improving inflammatory bowel disease.

[0015] Further preferably, the OTU is OTUD1 or Cezanne.

[0016] OTUD1 of the OTU family can bind to receptor-interacting serine / threonine protein kinase (RIPK1) and remove the K63 polyubiquitination modification of lysine 627, thereby hindering RIPK1's activation of the NF-κB signaling pathway, thereby inhibiting the production of proinflammatory cytokines (including TNF-α, IL-6, and IL-1β, etc.) in intestinal immune cells and the occurrence of enteritis. Cezanne inhibits the activation of NF-κB by cutting off the ubiquitin chain of upstream regulatory factors.

[0017] Preferably, the antioxidant nanozyme is at least one of platinum nanozyme, iridium nanozyme and ruthenium nanozyme.

[0018] Platinum nanozymes, iridium nanozymes, and ruthenium nanozymes all have the properties of mimicking catalase and superoxide dismutase, and can effectively remove ROS and regulate the microenvironment of the inflammatory site.

[0019] Preferably, the deubiquitinating enzyme is OTU, and the antioxidant nanozyme is platinum nanozyme.

[0020] In the best case, OTU inhibits inflammation-related signal transduction by cutting off the ubiquitin chain of upstream regulatory factors, thereby reducing inflammation in the lesion site. Platinum nanozymes maintain the activity of OTU by clearing ROS in the lesion site, and the two work synergistically to exert anti-inflammatory effects.

[0021] Preferably, the carrier is at least one of a metal organic framework, a liposome, a hydrogel, a micelle, and a polymer vesicle.

[0022] The present invention also provides a method for preparing the deubiquitinating enzyme drug, which has simple process and short time consumption.

[0023] A method for preparing a deubiquitinating enzyme drug comprises the following steps:

[0024] (1) adding sodium borohydride to a mixed solution of a metal salt and a capping agent to react, and precipitating with a poor solvent to obtain an antioxidant nanozyme;

[0025] (2) The deubiquitinating enzyme and the antioxidant nanozyme are co-loaded on a carrier to obtain a deubiquitinating enzyme drug.

[0026] Preferably, in step (1), the metal salt is at least one of chloroplatinic acid, iridium chloride and ruthenium chloride.

[0027] Preferably, in step (1), the capping agent is at least one of polyvinyl pyrrolidone, glutathione, polyethylene glycol and triethanolamine.

[0028] Preferably, in step (1), the poor solvent is at least one of anhydrous ether, dimethyl sulfoxide, N,N-dimethylformamide and acetone.

[0029] Preferably, in step (1), the mass ratio of sodium borohydride to metal salt is 1:6-15.

[0030] Preferably, in step (1), the particle size of the antioxidant nanozyme is 1-20 nm.

[0031] The present invention also provides the use of the above-mentioned deubiquitinase drug as a drug for the preparation of a drug for the treatment and / or diagnosis of inflammatory bowel disease. The deubiquitinase drug combines the anti-inflammatory ability of the deubiquitinase and the ROS scavenging ability of the antioxidant nanozyme to reduce inflammation in the lesion. At the same time, the deubiquitinase drug can reduce the inflammatory response of the inflammatory bowel disease microenvironment through O 2 Microbubbles activate the inertial cavitation mechanism, thereby specifically generating photoacoustic signals, enabling accurate diagnosis of inflammatory bowel disease lesions and precise treatment under the guidance of photoacoustic imaging.

[0032] Compared with the prior art, the beneficial effects achieved by the present invention are:

[0033] (1) The deubiquitinating drug of the present invention combines an antioxidant nanozyme for scavenging ROS with a deubiquitinating enzyme for inhibiting inflammation-related pathways, and utilizes the ROS scavenging ability of the antioxidant nanozyme to maintain the activity of the deubiquitinating enzyme and reduce inflammation at the lesion site.

[0034] (2) Deubiquitinase drugs can inhibit the oxidative stress microenvironment of inflammatory bowel disease by 2 Microbubbles activate the inertial cavitation mechanism, thereby specifically generating photoacoustic signals, enabling accurate diagnosis of inflammatory bowel disease lesions and precise treatment under the guidance of photoacoustic imaging.

[0035] (3) The preparation method of the present invention is simple in process and takes a short time. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1These are transmission electron microscope photos of the platinum nanozyme and the deubiquitinating enzyme drug prepared in Example 1, wherein A is the image of the platinum nanozyme and B is the image of the deubiquitinating enzyme drug.

[0037] Figure 2 This is a transmission electron microscope photograph of the deubiquitinase drug prepared in Example 2.

[0038] Figure 3 These are transmission electron microscope photos of the platinum nanozyme and the deubiquitinating enzyme drug prepared in Example 3, wherein A is the image of the platinum nanozyme and B is the image of the deubiquitinating enzyme drug.

[0039] Figure 4 The figure below is a test result of the catalase-like activity of the platinum nanozyme prepared in Example 1, wherein the left figure is the test result of the scavenging ability of the platinum nanozyme of different concentrations on hydrogen peroxide, and the right figure is the test result of the effect of the platinum nanozyme of different concentrations catalyzing hydrogen peroxide to produce oxygen.

[0040] Figure 5 This is a graph showing the test results of the superoxide dismutase activity of the platinum nanozyme prepared in Example 1.

[0041] Figure 6 The deubiquitinase drug prepared in Example 1 was used at different concentrations H 2 O 2 Standard curve of the photoacoustic signal after processing.

[0042] Figure 7 These are confocal laser scanning microscopy (CLSM) images of the drugs prepared in Example 1 and Comparative Examples 1-2 clearing intracellular ROS.

[0043] Figure 8 The effects of the drugs prepared in Example 1 and Comparative Examples 1-2 on the mRNA expression levels of IL-1β (left figure), IL-6 (middle figure) and TNF-α (right figure) in RAW264.7 cells induced by LPS.

[0044] Fig. 9 This is a photoacoustic imaging intensity diagram of the deubiquitinase drug prepared in Example 1 used in healthy or inflammatory bowel disease mice.

[0045] Fig.10 The effects of the drugs prepared in Example 1 and Comparative Examples 1-2 on the disease activity index (left figure) and body weight (right figure) of inflammatory bowel disease model mice.

[0046] Fig.11 The effects of the drugs prepared in Example 1 and Comparative Examples 1-2 on the colon length of inflammatory bowel disease model mice. DETAILED DESCRIPTION

[0047] The present invention will be further described in detail below in conjunction with the examples, but the embodiments of the present invention are not limited to the following examples.

[0048] The raw materials used in the present invention are all commercially available.

[0049] Example 1

[0050] (1) Preparation of antioxidant nanozymes:

[0051] According to the weight ratio, 1 part of chloroplatinic acid and 20 parts of polyvinyl pyrrolidone (58000Da) were dissolved in 200 parts of deionized water and stirred at room temperature for 15 minutes. 0.15 parts of sodium borohydride were added to the above system, stirred at room temperature for 12 hours, and then precipitated with acetone and centrifuged to obtain platinum nanozyme. The transmission electron microscopy image of platinum nanozyme is shown in FIG. Figure 1 As shown in A.

[0052] (2) Preparation of deubiquitinating enzyme drugs:

[0053] According to the volume fraction, 1 part of OTUD1 (50 μg / mL) and 1 part of platinum nanozyme (300 mg / mL) were added to 18 parts of imidazole solution (280 mg / mL), stirred at room temperature for 15 minutes to fully disperse, and then 2 parts of zinc nitrate solution (90 mg / mL) were added dropwise, and then allowed to stand at 4°C for 12 hours. Subsequently, the deubiquitinase drug was obtained by centrifugation (8000 rpm, 20 min) and washing several times with deionized water. The transmission electron microscopy image of the deubiquitinase drug is shown in FIG. Figure 1 As shown in B.

[0054] Example 2

[0055] (1) Preparation of antioxidant nanozymes:

[0056] According to the weight ratio, 1 part of chloroplatinic acid and 20 parts of polyvinyl pyrrolidone (58000Da) were dissolved in 200 parts of deionized water and stirred at room temperature for 15 minutes. 0.15 parts of sodium borohydride were added to the above system, stirred at room temperature for 12 hours, and then precipitated with acetone and centrifuged to obtain platinum nanozyme.

[0057] (2) Preparation of deubiquitinating enzyme drugs:

[0058] According to the volume fraction, 1 part of OTUD1 (50 μg / mL) and 1 part of platinum nanozyme (60 mg / mL) were added to 18 parts of imidazole solution (280 mg / mL), stirred at room temperature for 15 minutes to fully disperse, and then 2 parts of zinc nitrate solution (90 mg / mL) were added dropwise, and then allowed to stand at 4°C for 12 hours. Subsequently, the deubiquitinase drug was obtained by centrifugation (8000 rpm, 20 min) and washing several times with deionized water. The transmission electron microscopy image of the deubiquitinase drug is shown in FIG. Figure 2 shown.

[0059] Example 3

[0060] (1) Preparation of antioxidant nanozymes:

[0061] According to the weight ratio, 1 part of chloroplatinic acid and 20 parts of polyvinyl pyrrolidone (58000Da) were dissolved in 200 parts of deionized water and stirred at room temperature for 15 minutes. 0.08 parts of sodium borohydride were added to the above system, stirred at room temperature for 12 hours, and then precipitated with acetone and centrifuged to obtain platinum nanozyme. The transmission electron microscopy image of platinum nanozyme is shown in FIG. Figure 3 As shown in A.

[0062] (2) Preparation of deubiquitinating enzyme drugs:

[0063] According to the volume fraction, 1 part of OTUD1 (50 μg / mL) and 1 part of platinum nanozyme (60 mg / mL) were added to 18 parts of imidazole solution (280 mg / mL), stirred at room temperature for 15 minutes to fully disperse, and then 2 parts of zinc nitrate solution (90 mg / mL) were added dropwise, and then allowed to stand at 4°C for 12 hours. Subsequently, the deubiquitinase drug was obtained by centrifugation (8000 rpm, 20 min) and washing several times with deionized water. The transmission electron microscopy image of the deubiquitinase drug is shown in FIG. Figure 3 As shown in B.

[0064] Example 4

[0065] (1) Preparation of antioxidant nanozymes:

[0066] According to the weight ratio, 1 part of chloroplatinic acid and 20 parts of polyvinyl pyrrolidone (58000Da) were dissolved in 200 parts of deionized water and stirred at room temperature for 15 minutes. 0.08 parts of sodium borohydride were added to the above system, stirred at room temperature for 12 hours, and then precipitated with acetone and centrifuged to obtain platinum nanozyme.

[0067] (2) Preparation of deubiquitinating enzyme drugs:

[0068] According to the volume fraction, 1 part of OTUD1 (50 μg / mL) and 1 part of platinum nanozyme (300 mg / mL) were added to 18 parts of imidazole solution (280 mg / mL), stirred at room temperature for 15 minutes to fully disperse, and then 2 parts of zinc nitrate solution (90 mg / mL) were added dropwise, and then allowed to stand at 4°C for 12 hours. Subsequently, the deubiquitinase drug was obtained by centrifugation (8000 rpm, 20 min) with deionized water and washed several times.

[0069] Comparative Example 1

[0070] (1) Preparation of antioxidant nanozymes:

[0071] According to the weight ratio, 1 part of chloroplatinic acid and 20 parts of polyvinyl pyrrolidone (58000Da) were dissolved in 200 parts of deionized water and stirred at room temperature for 15 minutes. 0.15 parts of sodium borohydride were added to the above system, stirred at room temperature for 12 hours, and then precipitated with acetone and centrifuged to obtain platinum nanozyme.

[0072] (2) Preparation of platinum-loaded nanozyme drugs:

[0073] According to the volume fraction, platinum nanozyme (300 mg / mL) was added to 18 parts of imidazole solution (280 mg / mL), stirred at room temperature for 15 minutes to fully disperse it, and then 2 parts of zinc nitrate solution (90 mg / mL) were added dropwise, and then allowed to stand at 4°C for 12 hours. Subsequently, it was washed several times by centrifugation (8000 rpm, 20 min) with deionized water to obtain only the platinum nanozyme drug loaded.

[0074] Comparative Example 2

[0075] (1) Preparation of antioxidant nanozymes:

[0076] According to the weight ratio, 1 part of chloroplatinic acid and 20 parts of polyvinyl pyrrolidone (58000Da) were dissolved in 200 parts of deionized water and stirred at room temperature for 15 minutes. 0.15 parts of sodium borohydride were added to the above system, stirred at room temperature for 12 hours, and then precipitated with acetone and centrifuged to obtain platinum nanozyme.

[0077] (2) Preparation of only loaded deubiquitinase drug:

[0078] According to the volume fraction, 1 part of OTUD1 (50 μg / mL) was added to 18 parts of imidazole solution (280 mg / mL), stirred at room temperature for 15 minutes to fully disperse it, and then 2 parts of zinc nitrate solution (90 mg / mL) were added dropwise, and then allowed to stand at 4°C for 12 hours. Subsequently, the deubiquitinase drug was obtained by washing with deionized water by centrifugation (8000 rpm, 20 min) for multiple times.

[0079] In vitro test:

[0080] The platinum nanozymes and deubiquitinating drugs prepared in the examples were subjected to in vitro experiments, specifically:

[0081] (1) Using transmission electron microscopy, the morphology of the platinum nanozymes and deubiquitinase drugs prepared in Examples 1-3 was characterized:

[0082] The results are as follows Figure 1 As shown in A and 3A, the platinum nanozymes prepared in Examples 1-3 are spherical or quasi-spherical under a transmission electron microscope, with relatively uniform size and good dispersibility. Figure 1As shown in Figures 2B, 2 and 3B, the deubiquitinating enzyme drugs prepared in Examples 1-3 are relatively uniform in morphology and size under a transmission electron microscope.

[0083] (2) Characterization of the catalase activity of the platinum nanozyme prepared in Example 1:

[0084] The results are as follows Figure 4 As shown, the ability of platinum nanozymes of different concentrations to scavenge hydrogen peroxide and catalyze the production of oxygen from hydrogen peroxide were tested respectively, indicating that the prepared platinum nanozymes have good catalase-like activity and can catalyze the decomposition of hydrogen peroxide.

[0085] (3) Characterization of the superoxidase-like activity of the platinum nanozyme prepared in Example 1:

[0086] The results are as follows Figure 5 As shown, the scavenging ability of platinum nanozymes of different concentrations on superoxide anions was tested, indicating that the prepared platinum nanozymes can effectively scavenge superoxide anions.

[0087] (4) In vitro photoacoustic imaging evaluation: In order to investigate H 2 O 2 The photoacoustic signal standard curve of the deubiquitinase drug after stimulation was obtained. 12.5 μg / mL of the deubiquitinase drug prepared in Example 1 was mixed with different concentrations of H 2 O 2 (0, 100, 200, 400, 600, 800, 1000 μM) and incubated for 5 min. The photoacoustic signal intensity was measured at 800 nm using MSOT inVision 256 photoacoustic imaging instrument.

[0088] Figure 6 For different concentrations of H 2 O 2 The standard curve of the photoacoustic signal after treatment shows that the deubiquitinating enzyme drug has the ability to pass through O 2 Microbubbles activate inertial cavitation mechanism to achieve toxicity to overexpressed H 2 O 2 Performance of photoacoustic imaging of inflammatory colonic tissue.

[0089] (5) Intracellular ROS scavenging test: In order to investigate the activity of nanomedicines with different compositions in scavenging intracellular ROS, the intracellular ROS level was analyzed using a DCFH-DA fluorescent probe. RAW264.7 cells were inoculated in confocal dishes and cultured to a suitable density. After discarding the old culture medium, the experimental group dishes were added with culture medium containing 50 μg / mL of nanomedicines of different compositions (only platinum nanozyme drugs loaded in comparative example 1, only deubiquitinase drugs loaded in comparative example 2, and deubiquitinase drugs prepared in Example 1), and the blank control group and the positive control group dishes were added with an equal volume of new culture medium. Subsequently, lipopolysaccharide (LPS) was added to each dish of the positive control group and the experimental group, so that the final concentration of LPS was 500 ng / mL. After all the above cells were incubated under hypoxic conditions for 3 hours, the culture medium was discarded, and 1 mL of DCFH-DA solution (10 μM) was added to each well and incubated at a constant temperature and in the dark for 30 minutes. After rinsing with PBS three times to remove excess dye, the cells were fixed with 4% paraformaldehyde for 15 minutes, rinsed with PBS three times, and stained with DAPI for 30 minutes. After rinsing with PBS three times again, the cell fluorescence distribution was observed with a confocal laser scanning microscope (CLSM).

[0090] Figure 7 The cell fluorescence distribution images after drug treatment prepared in Example 1 and Comparative Examples 1-2 indicate that both the drug loaded with only platinum nanozyme and the deubiquitinase drug have good activity in removing intracellular reactive oxygen species, and the drug loaded with only deubiquitinase has very low activity in removing intracellular reactive oxygen species.

[0091] (6) Evaluation of intracellular anti-inflammatory effect: In order to investigate the in vitro anti-inflammatory activity of nanomedicines with different compositions, the expression of intracellular inflammatory factor mRNA was detected by RT-qPCR technology. RAW264.7 cells were inoculated in a 6-well plate and cultured to an appropriate density. After discarding the old culture medium, the experimental wells were added with culture medium containing 50 μg / mL of nanomedicines of different compositions (only platinum nanozyme drugs loaded in comparative example 1, only deubiquitinase drugs loaded in comparative example 2, and deubiquitinase drugs prepared in Example 1). An equal volume of new culture medium was added to the blank control wells and the positive control wells, and then lipopolysaccharide (LPS) was added to each well of the positive control group and the experimental group to a final concentration of LPS of 500 ng / mL. All the above cells were incubated under hypoxic conditions for 3 hours. The culture medium was discarded, rinsed twice with pre-cooled PBS, 1 mL of Trizol reagent was added to each well to extract total RNA, and then a total of 1 μg of RNA samples was reverse transcribed into cDNA. Finally, the expression of each pro-inflammatory cytokine mRNA was detected by RT-qPCR system, and 2 -ΔΔCT Method for quantification.

[0092] Figure 8The effect of the drug treatment prepared in Example 1 and Comparative Examples 1-2 on the expression level of the mRNA of related inflammatory factors. Compared with the blank group, after stimulating RAW264.7 cells with LPS alone, the expression levels of IL-1β, IL-6, and TNF-α mRNA increased significantly. When compared with the LPS group, only the antioxidant nanozyme drug loaded, only the deubiquitinase drug loaded, and the deubiquitinase drug loaded with both can inhibit the expression of IL-1β, IL-6, and TNF-α mRNA levels in RAW264.7 cells, and the inhibitory effect of the deubiquitinase drug is better than that of Comparative Examples 1-2.

[0093] Application Example 1: The diagnostic and therapeutic effects of the deubiquitinating enzyme drug prepared in Example 1 were evaluated, specifically:

[0094] (1) In vivo photoacoustic diagnostic evaluation of deubiquitinating enzyme drugs

[0095] The deubiquitinase drug prepared in Example 1 was dispersed in physiological saline to obtain a deubiquitinase drug solution. The C57BL / 6 experimental mice used were first adaptively cultured for one week before the experiment began. Then, a mouse colitis model was induced by continuously drinking 2.5% dextran sodium sulfate (DSS) solution for subsequent experiments. In order to perform photoacoustic imaging of the colon, the deubiquitinase drug solution (10 mg / kg) was administered rectally to healthy mice and colitis mice, respectively. After 6 hours, the MSOT inVision 256 photoacoustic imaging instrument was used to perform photoacoustic imaging of the colon of healthy mice (healthy group), colitis mice (model group), healthy mice treated with deubiquitinase drug solution (healthy administration group) and deubiquitinase drug solution treated colitis mice (treatment group).

[0096] The results are as follows Fig. 9 As shown, the photoacoustic signal intensity in the colon of the treatment group was significantly higher than that of the other groups, indicating that the prepared deubiquitinase drug can be used for in vivo photoacoustic imaging.

[0097] (2) Evaluation of the therapeutic effects of deubiquitinating enzyme drugs

[0098] The deubiquitinase drug prepared in Example 1 was dispersed in physiological saline to obtain a deubiquitinase drug solution. The C57BL / 6 experimental mice used were first adaptively cultured for one week before the experiment began. Fifteen 8-week-old mice were randomly divided into three groups, each with 5 mice, including a control group, a 2.5% dextran sulfate sodium (DSS)-induced model group, and a deubiquitinase drug solution (10 mg / kg) treatment group. The different treatments of the three groups of mice are as follows:

[0099] Mice in the control group drank water during the experiment;

[0100] Model group mice: drank 2.5% dextran sulfate sodium (DSS) for 7 consecutive days;

[0101] The mice in the platinum nanozyme drug-treated group prepared in Comparative Example 1 were fed with 2.5% dextran sulfate sodium (DSS) for 7 consecutive days, and during the modeling period, the platinum nanozyme drug solution (10 mg / kg) was administered rectally on days 1, 3, 5, and 7;

[0102] The mice in the deubiquitinase-only drug-treated group prepared in Comparative Example 2 were fed with 2.5% dextran sulfate sodium (DSS) for 7 consecutive days, and during the modeling period, deubiquitinase-only drug solution (10 mg / kg) was administered rectally on days 1, 3, 5, and 7;

[0103] The mice in the deubiquitinase drug treatment group prepared in Example 1 were fed with 2.5% dextran sulfate sodium (DSS) for 7 consecutive days. During the modeling period, deubiquitinase drug solution (10 mg / kg) was administered rectally on days 1, 3, 5 and 7.

[0104] The weight changes, feces hardness and blood content of the mice were recorded every day. After the model was established, the mice were killed by cervical dislocation under deep anesthesia, and the colon tissue was dissected to measure the length of the colon in each group.

[0105] The results are as follows Fig.10 As shown in the figure, compared with the model group mice, the deubiquitinase drug significantly improved the weight loss and disease activity index of mice caused by DSS-induced colitis, and was better than the control group of drugs loaded with only antioxidant nanozymes or only deubiquitinase. Fig.11 As shown, compared with the model group mice, deubiquitinase drugs improved the shortening of colon length in mice caused by DSS-induced colitis.

[0106] The embodiments described above provide a detailed description of the technical solutions of the present invention. It should be understood that the above are only specific examples of the present invention and are not intended to limit the present invention. Any modifications, supplements or similar substitutions made within the scope of the principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A deubiquitinating enzyme drug, characterized in that: It includes deubiquitinating enzymes, antioxidant nanozymes, and carriers loaded with deubiquitinating enzymes and antioxidant nanozymes.

2. The deubiquitinating enzyme drug according to claim 1, characterized in that The mass ratio of the deubiquitinating enzyme and the antioxidant nanozyme in the deubiquitinating enzyme drug to the mass ratio of the carrier is 1:1-6.

3. The deubiquitinating enzyme drug according to claim 1 or 2, characterized in that The mass ratio of the deubiquitinating enzyme and the antioxidant nanozyme is 1:1-6 μg / mg.

4. The deubiquitinating enzyme drug according to claim 1, characterized in that The deubiquitinating enzyme is UBPs or OTU; the antioxidant nanozyme is at least one of platinum nanozyme, iridium nanozyme, and ruthenium nanozyme; and the carrier is at least one of metal organic framework, liposome, hydrogel, micelle, and polymer vesicle.

5. The deubiquitinating enzyme drug according to claim 4, characterized in that The deubiquitinating enzyme is OTU, and the antioxidant nanozyme is platinum nanozyme.

6. The method for preparing the deubiquitinating enzyme drug according to any one of claims 1 to 5, comprising the following steps: (1) adding sodium borohydride to a mixed solution of a metal salt and a capping agent to react, and precipitating with a poor solvent to obtain an antioxidant nanozyme; (2) The deubiquitinating enzyme and the antioxidant nanozyme are co-loaded on a carrier to obtain a deubiquitinating enzyme drug.

7. The method for preparing a deubiquitinating enzyme drug according to claim 6, characterized in that: In step (1), the metal salt is at least one of chloroplatinic acid, iridium chloride and ruthenium chloride; the capping agent is at least one of polyvinyl pyrrolidone, glutathione, polyethylene glycol and triethanolamine; and the poor solvent is at least one of anhydrous ether, dimethyl sulfoxide, N,N-dimethylformamide and acetone.

8. The method for preparing a deubiquitinating enzyme drug according to claim 6, characterized in that: In step (1), the mass ratio of sodium borohydride to metal salt is 1:6-15.

9. The method for preparing a deubiquitinating enzyme drug according to claim 6, characterized in that: In step (1), the particle size of the antioxidant nanozyme is 1-20 nm.

10. Use of the deubiquitinating enzyme drug according to any one of claims 1 to 5 as a drug for the treatment and / or diagnosis of inflammatory bowel disease.

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