A prefabricated nanozyme and its preparation method and application

Abstract

The present invention relates to the field of biomedicine technology, and discloses a prefabricated nanozyme, a preparation method thereof, and an application thereof. The prefabricated nanozyme comprises metal nanoparticles and a hydrogen sulfide releaser loaded on the metal nanoparticles; preferably, the metal nanoparticles are platinum nanoparticles, and the hydrogen sulfide releaser is sodium sulfide. The method for preparing the prefabricated nanozyme is: adding the metal nanoparticles and the hydrogen sulfide releaser to water, stirring overnight, then dialyzing to remove the free hydrogen sulfide releaser, and freeze-drying to obtain the prefabricated nanozyme. The prefabricated nanozyme of the present invention has good anti-inflammatory and antioxidant effects, and can be used to prepare drugs for alleviating or / and treating renal injury.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine and relates to a prefabricated nanozyme and a preparation method and application thereof. Background Art

[0002] Reactive oxygen species (ROS) are highly active oxygen-containing substances in organisms that participate in various physiological and pathological processes, mainly including superoxide anion free radicals (O2 ·- ), singlet oxygen, hydrogen peroxide (H2O2), and highly reactive hydroxyl radicals (·OH). Under normal physiological conditions, the concentration of ROS in cells is stable, and its production and clearance are in a dynamic equilibrium. Moderate levels of ROS participate in a variety of signal pathways that respond to changes in external conditions, and play an active role in physiological processes such as signal transduction, immune response, and cell function regulation. However, excessive production of ROS can disrupt the redox homeostasis in the human body, thereby causing oxidative stress in normal cells. This continuous oxidative stress can cause irreversible oxidative damage to lipids, proteins, and DNA, and even lead to a variety of physiological diseases such as inflammation and cancer.

[0003] Acute kidney injury (AKI) is a clinical syndrome caused by a rapid decline in renal function in a short period of time. AKI has become an increasingly serious health problem with high morbidity and mortality in countries around the world. It is a risk factor for chronic kidney disease (CKD) and end-stage renal disease (ESRD). Ischemia reperfusion injury (IRI) is an important cause of clinical AKI. During ischemia-reperfusion, a series of biochemical and molecular changes are triggered, resulting in a large amount of ROS, causing oxidative stress, inflammation and apoptosis, leading to damage to organs such as the kidney. Therefore, ROS is considered to be one of the key targets for the treatment of AKI. It can effectively remove excess ROS in the kidney, regulate the renal microenvironment, and prevent or reduce excessive oxidative damage. Some broad-spectrum ROS-scavenging antioxidants, such as n-acetylcysteine ​​and acetyl-l-carnitine, are considered to be the best candidate drugs for the treatment of AKI. However, due to the poor bioavailability of these antioxidants and their limited ability to scavenge ROS, their widespread clinical application is limited.

[0004] Under normal physiological conditions, O2 is produced during cell metabolism. ·-, ·OH, H2O2 and other ROS. This balance can be maintained by antioxidants such as CAT, ascorbic acid and glutathione. However, when the body is in a pathological state, excessive ROS are not cleared. With the development of nanotechnology, Yan et al. found in 2007 that Fe3O4NPs have enzyme-like activity. Nanozymes have been increasingly used in the field of biology / nanomedicine due to their unique nanosize, low production cost, easy modification and high stability. These properties enable nanoparticles to successfully mimic cellular antioxidant enzymes (such as CAT, GPx) and become new candidates for the treatment of diseases caused by oxidative stress. Mugesh et al. reported that V2O5 nanowires (Vn) mimic glutathione peroxides and exhibited excellent ROS scavenging ability in kidney, neuron, prostate and cervical cells. The ability of Vn to fully restore redox balance without interfering with cellular antioxidant defenses provides important cellular protection against harmful oxidative damage to biomolecules. However, due to the unique structure of the glomerular capillary wall, only ultra-small nanoparticles with a hydrodynamic diameter (HDs) less than 5.5 nm can pass through the kidneys and be rapidly excreted in the kidneys. Therefore, it is of great research value to design nanozymes of reasonable size for effective uptake, accumulation and clearance in the kidneys to treat and prevent ROS-related diseases.

[0005] Hydrogen sulfide (H2S) is the simplest sulfur-containing molecule, also known as sulfonamide, and is currently considered to be the third gas transmitter in the mammalian system, alongside nitric oxide (NO) and carbon monoxide (CO). Studies have shown that H2S can play an antioxidant role by stimulating ATP production and scavenging free radicals, thereby protecting protein function from oxidation due to excessive sulfidation. Liu et al. found that H2S can protect the kidney from IRI injury and reduce inflammation by inhibiting the activation of the Nod2 signaling pathway and the type A macrophage scavenger receptor signaling, upregulating endoplasmic reticulum stress-induced autophagy. However, nanozymes face a limitation in their large-scale application in the treatment of acute kidney injury, namely, the unique physiological structure of the kidney makes it impossible to effectively target and deliver them to the kidney. Therefore, the development of enzyme-mimicking nanomaterials (nanozymes) with good reactive oxygen species (ROS) and reactive nitrogen species (RNS) scavenging ability and biocompatibility, so that they can effectively enter the site of renal injury and play an antioxidant role, is a promising approach to treat AKI. Summary of the invention

[0006] In view of the problems and shortcomings in the prior art, the purpose of the present invention is to provide a prefabricated nanozyme and a preparation method and application thereof.

[0007] To achieve the purpose of the invention, the technical solution adopted by the present invention is as follows:

[0008] A first aspect of the present invention provides a prefabricated nanozyme, wherein the prefabricated nanozyme comprises metal nanoparticles and a hydrogen sulfide releasing agent loaded on the metal nanoparticles.

[0009] According to the above-mentioned prefabricated nanozyme, preferably, the metal nanoparticles are platinum nanoparticles (Ptzyme), and the hydrogen sulfide releaser is sodium sulfide or manganese sulfide. More preferably, the hydrogen sulfide releaser is sodium sulfide; most preferably, the sodium sulfide is nano sodium sulfide. According to the above-mentioned prefabricated nanozyme, preferably, the particle size of the prefabricated nanozyme is 4 to 100 nm.

[0010] The second aspect of the present invention provides a use of the preformed nanozyme described in the first aspect in the preparation of a drug for alleviating and / or treating kidney damage.

[0011] According to the above application, preferably, the renal injury is acute renal injury.

[0012] According to the above application, preferably, the acute kidney injury is acute kidney injury caused by oxidative stress or renal ischemia-reperfusion.

[0013] The third aspect of the present invention provides the use of the prefabricated nanozyme described in the first aspect in the preparation of a drug for alleviating and / or treating oxidative damage.

[0014] The fourth aspect of the present invention provides a method for preparing the preformed nanozyme described in the first aspect above, the preparation method comprising: adding metal nanoparticles and a hydrogen sulfide releaser into water, stirring overnight, then dialyzing to remove the free hydrogen sulfide releaser, and freeze-drying to obtain the preformed nanozyme.

[0015] According to the above preparation method, preferably, the mass ratio of the metal nanoparticles to the hydrogen sulfide releasing agent is 2:1 to 100:1. More preferably, the mass ratio of the metal nanoparticles to the hydrogen sulfide releasing agent is 10:1.

[0016] According to the above preparation method, preferably, the metal nanoparticles are platinum nanoparticles (Ptzyme), and the hydrogen sulfide releaser is sodium sulfide. More preferably, the sodium sulfide is nano sodium sulfide.

[0017] According to the above-mentioned preparation method, preferably, the preparation method of the platinum nanoparticles is: dissolving polyvinyl pyrrolidone (PVP) in a solvent to obtain a polyvinyl pyrrolidone solution, adding an H2PtCl6 aqueous solution to the polyvinyl pyrrolidone solution, stirring and refluxing at 60°C to 90°C for 2h to 24h, removing the solvent and PVP in the reaction product after the reaction is completed, and freeze-drying to obtain platinum nanoparticles (Ptzyme).

[0018] According to the above preparation method, preferably, the concentration of the H2PtCl6 aqueous solution is 1 to 60.0 mM, the solvent is methanol, and the mass ratio of PVP to H2PtCl6 is 2:1 to 10:1. In a fifth aspect, the present invention provides a drug for alleviating or / and treating renal injury, the drug comprising the preformed nanozyme described in the first aspect and a pharmaceutically acceptable excipient.

[0019] Compared with the prior art, the present invention has the following positive and beneficial effects:

[0020] (1) The present invention prepares a prefabricated nanozyme (Pt 5.65 S), the prefabricated nanozyme is a Pt-based nanozyme that integrates the mimetic enzyme properties of catalase, superoxide dismutase and glutathione peroxidase, and can release loaded hydrogen sulfide (H2S), has the ability to antagonize renal oxidative stress, relieves renal damage by alleviating the degree of renal oxidative stress, and can protect renal cell damage mediated by ROS / RNS at extremely low doses.

[0021] (2) The prefabricated nanozyme (Pt 5.65 S) can reduce oxidative damage and inflammation by releasing hydrogen sulfide gas, promote the activation of the Nrf2 pathway, and further enhance the expression of antioxidant molecules and enzymes; moreover, the prefabricated nanozyme (Pt 5.65 S) can significantly alleviate renal pathological symptoms and has significant advantages in treating acute kidney injury and other oxidative damage-related diseases, providing new research directions and methods for the treatment of related diseases.

[0022] (3) Prefabricated nanozyme Pt ​​prepared by the present invention 5.65 S, under normal physiological conditions, when the kidneys are not damaged by oxidative stress, the preformed nanozyme Pt 5.65 S does not play a role and can be excreted from the kidneys normally; however, once the kidneys are damaged by oxidative stress, the pH value inside the kidneys will change. 5.65 S begins to release hydrogen sulfide gas, and the Pt-based nanozyme combines with the released hydrogen sulfide (H2S) gas to exert a synergistic antioxidant effect, specifically removing reactive oxygen in the kidneys, alleviating oxidative stress, and effectively treating kidney damage. Moreover, the present invention also verifies the prefabricated nanozyme Pt ​​through the study of acute kidney injury model mice. 5.65 The therapeutic effect of S in alleviating renal pathological symptoms provides a new method for the treatment of acute kidney injury and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Pt 5.65Schematic diagram of the preparation and characterization results of prefabricated nanozymes; A is Pt 5.65 S is a schematic diagram of the preparation process of prefabricated nanozymes; B and C are Pt 5.65 S is the TEM image of the prefabrication (scale bar = 5 nm), D is Pt 5.65 S is the SAED spectrum of prefabricated nanozyme; E is Pt 5.65 S is the EDS mapping spectrum of the prefabricated; F is the EDS mapping spectrum of Ptzyme and Pt 5.65 S is the Zeta potential detection diagram of prefabricated nanozyme; G is the detection diagram of Ptzyme and Pt 5.65 S is the FTIR spectrum of prefabricated nanozyme; H is Ptzyme and Pt 5.65 PXRD pattern of S preformed nanozyme; I and J are Pt 5.65 XPS spectra of S preformed; K is Pt at pH = 6.5 and pH = 7.4 5.65 The results of the detection of H2S released by S prefabricated nanozymes;

[0024] Figure 2 The results of the cell experiment verifying the active oxygen scavenging effect of Pt5.65S prefabricated nanozyme; A is the result of the cytotoxicity experiment; B is the result of the cell proliferation inhibition experiment; C is the result of the flow cytometry apoptosis detection; D is the result of the DCFH-DA active oxygen detection experiment; E is the result of Mito-tracker cell mitochondrial morphology staining;

[0025] Figure 3 These are the experimental results of the renal protection effect of the prefabricated nanozyme in vivo experiment on mice; wherein, A is a schematic diagram of the construction of the mouse model of acute kidney injury in mice; B is a graph of the Scr detection results; C is a graph of the BUN detection results; D is a graph of the SOD detection results; E is a graph of the MDA detection results; F is a graph of the GSH (GSSG) detection results; G is a graph of the H&E staining, Masson staining, and Tunel staining results; H is the result of ICP-MS detection of Pt ions in various organs of mice after 24 hours; I is the result of ICP-MS detection of Pt ions in various organs of mice after 48 hours; J is a graph of Western blot detection results; in the figure, IRI represents the IRI-AKI model group; Sham represents the sham operation group, i.e., the negative control group. DETAILED DESCRIPTION

[0026] The following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0027] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. When the terms "comprise" and / or "include" are used in this specification, it indicates that there are features, steps, operations, parts and / or combinations thereof.

[0028] The experimental methods in the following examples without specifying specific conditions all adopt conventional techniques in the art or follow the conditions recommended by the manufacturers; the reagents or instruments used without specifying the manufacturers are all conventional products that can be obtained commercially.

[0029] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0030] Example 1: Pt 5.65 Preparation and characterization of prefabricated nanozymes

[0031] 1. Pt 5.65 Preparation method of prefabricated nanozyme:

[0032] Pt 5.65 The preparation method of S prefabricated nanozyme is specifically as follows ( Figure 1 As shown in A), platinum nanoparticles (Ptzyme, 10 mg) and sodium sulfide were added to 10 mL of water, with the mass ratio of platinum nanoparticles to sodium sulfide being 10:1. The mixture was stirred overnight, and then dialyzed to remove free sodium sulfide. The mixture was freeze-dried to obtain Pt 5.65 S preformed nanozymes.

[0033] Among them, the preparation process of platinum nanoparticles (Ptzyme) is as follows: 133 mg PVP powder is dissolved in 180 mL methanol (180 mL), and then 6.0 mM H2PtCl6 aqueous solution (20.0 mL) is dropped, and after continuous stirring for a few minutes, the mixture is stirred and refluxed at 60°C to 90°C for 3 hours. After the reaction is completed, the reaction product is rotary evaporated to remove the solvent (methanol and water), and then washed to remove free PVP, and freeze-dried to obtain platinum nanoparticles (Ptzyme).

[0034] 2. Pt 5.65 Characterization of preformed nanozymes:

[0035] (1)Pt 5.65 Experimental methods for characterization of preformed nanozymes:

[0036] 1) Morphological characterization:

[0037] The prepared Pt 5.65The prefabricated nanozyme powder was prepared into a 1 mg / mL solution with deionized water and dropped onto a copper grid for sample preparation. The morphology of the nanoparticles was detected by transmission electron microscopy (TEM) with a data acquisition range of 5°-45° and a scanning speed of 15° / min.

[0038] 2) Crystal structure characterization:

[0039] The prepared Pt 5.65 The preformed nanozyme powder was detected by X-ray diffractometer (XRD).

[0040] 3) Ultraviolet spectrum detection:

[0041] Pt 5.65 S prefabricated nanozyme powder, Ptzyme, and Na2S were prepared into a 1 mg / mL solution using deionized water, added into a cuvette, and detected by a UV-visible spectrophotometer.

[0042] 4) Zeta potential determination:

[0043] Pt 5.65 After the S prefabricated nanozyme was configured into a 1 mg / mL solution, the Zeta potential changes of different nanoparticles were analyzed using a nanoparticle size potentiostat.

[0044] 5) Fourier Transform Infrared Detection (FITR):

[0045] Pt 5.65 The preformed nanozyme was prepared into a 1 mg / mL solution and placed in a NaCl plate. The spectrum was generated by passing infrared light through the sample and detector through an interferometer, which splits the beam into two paths. The difference between these paths generates a signal that is sent to a computer for analysis.

[0046] 6) X-ray photoelectron spectroscopy (XPS) detection:

[0047] X-ray diffractometer was used to analyze the Pt 5.65 S prefabricated nanozymes for detection and analysis of Pt 5.65 Surface chemical properties of S preformed nanozymes.

[0048] 7) Pt 5.65 Detection of H2S gas release from prefabricated nanozymes:

[0049] First, draw a standard curve for H2S gas release. Weigh an appropriate amount of Na2S crystals and prepare them into different concentrations with deionized water. Then use a hydrogen sulfide detection kit to detect the H2S gas content in the solution of different concentrations, and use a UV-visible spectrophotometer to measure the absorbance (OD value). Draw a standard curve with the H2S gas content as the X-axis and the OD value measured by the UV-visible spectrophotometer as the Y-axis. 5.65 The prefabricated nanozyme material was dispersed in a PBS solution with a pH of 6.8 and a concentration of 1 mg / mL, and gently shaken at 37 °C. Subsequently, 2 mL of Pt 5.65 The H2S content in the prefabricated nanozyme solution was measured using a hydrogen sulfide detection kit in a UV-visible spectrophotometer, and the corresponding H2S gas content was calculated from the previously drawn H2S gas release standard curve.

[0050] (2)Pt 5.65 Characterization experimental results of S prefabricated nanozymes:

[0051] Pt 5.65 The characterization experimental results of S preformed nanozymes are shown in Figure 1 As shown in B~K.

[0052] Figure 1 Wherein B and C are Pt prepared by the present invention 5.65 TEM image of S prefabricated nanozyme. Figure 1 As shown in B and C, the Pt prepared by the present invention 5.65 The S preformed nanozyme is a uniform spherical nanoparticle with an average particle size of 4 nm, which meets the glomerular filtration threshold (~6 nm).

[0053] Figure 1 D is Pt 5.65 Selected area electron diffraction (SAED) pattern of S preformed nanozyme, which showed that the nanozyme complex was amorphous.

[0054] Figure 1 E is Pt 5.65 EDS mapping spectrum of S prefabricated nanozyme, where E(1) is the morphology of the prefabricated nanozyme; E(2) is the composite image of the electronic signal after the prefabricated nanozyme is synthesized; E(3) is the C signal image of the prefabricated nanozyme; E(4) is the Pt signal image of the prefabricated nanozyme; E(5) is the S signal image of the prefabricated nanozyme. The results of Figure E show that Pt 5.65 The presence of Pt and S signals in the S-preformed nanozyme provides solid evidence for the successful loading of Na2S on Ptzyme.

[0055] Figure 1 F is Pt 5.65Zeta potential detection results of S prefabricated nanozymes. It can be seen from the figure that the Na2S reaction reduces the zeta potential of Ptzymes.

[0056] Figure 1 G and H are Pt 5.65 FTIR spectrum and PXRD spectrum of S prefabricated nanozyme, by Figure 1 As shown in G and H, Na2S successfully reacts with Ptzymes; moreover, in contrast to Ptzyme, Pt 5.65 New peaks appeared in the PXRD and FTIR spectra of S preformed nanozymes, confirming that S 2- Effectively incorporated into Pt 5.65 S preformed nanozyme.

[0057] Figure 1 I and J are Pt 5.65 XPS spectrum of S preformed nanozyme, Figure 1 Pt can be clearly seen in I and J 5.65 The visible absorption peaks of the main elements (Pt and S) of the S-preformed nanozymes; moreover, S2p 1 / 2 and S2p 3 / 2 The binding energies of the Pt 4f and Pt 4f are 168.28 eV and 162.18 eV, respectively, confirming the chemical state of the S element. In addition, the high-resolution XPS spectrum of the Pt orbital shows that the Pt 4f 7 / 2 The corresponding binding energies are 72.6 (PtII) and 71.2 (Pt0) eV. According to the Pt 4f 7 / 2 The mass fractions of Pt and PtS were calculated from the peak areas, and the proportions of Pt and PtS were approximately 17.7% and 82.3%, respectively.

[0058] Figure 1 When K is pH=6.5 and pH=7.4, Pt 5.65 The detection result of H2S released by S prefabricated nanozyme, Figure 1 As can be seen from Figure 5, slight H2S release was observed in the acidic microenvironment (pH = 6.8), indicating that H2S is continuously produced in the inflammatory environment.

[0059] The above characterization experimental results show that the present invention successfully synthesized Pt 5.65 S preformed nanozymes.

[0060] Example 2: Cell experiment verification of Pt 5.65 S-prepared nanozyme active oxygen scavenging effect

[0061] The cell line used in the cell experiment of this example is HEK293 cells (human embryonic renal tubular epithelial cells), which were purchased from the Institute of Cell Biology, Chinese Academy of Sciences, and stored in a liquid nitrogen tank in the laboratory on the 4th floor of the Postdoctoral Building, School of Medical Sciences, Zhengzhou University. Cell culture conditions: complete medium (containing 10% fetal bovine serum FBS, 90% DMEM medium and 1% double anti-penicillin / streptomycin), 37°C, 5% CO2 cell culture incubator.

[0062] 1. Cytotoxicity assay:

[0063] CCK-8 method was used to detect Ptzyme, Na2S, Pt 5.65 The toxicity and damage of S preformed nanozymes on HEK293 cells. The specific experimental method is:

[0064] Three experimental groups were set up: (1) Ptzyme group; (2) Na2S group; (3) Pt 5.65 S pre-made nanozyme group. HEK 293 cells were digested and centrifuged, and 8×10 4 Resuspend the cells in complete medium at a concentration of 100 μL / ml, add 100 μL of the resuspended cell suspension to each well of the 96-well plate, and culture in a cell culture incubator for 24 h. 5.65 After diluting the pre-made original drug of S, carefully aspirate the original culture medium in each well of the 96-well plate, and then add 100 μL of culture medium containing different concentrations of drugs to each well in sequence, and place them in a cell culture incubator for incubation for 12 hours. Add 10 μL of CCK8 reagent to each well, gently shake the 96-well plate horizontally to mix it, and place it in a cell culture incubator for incubation for 2-3 hours. Use an enzyme reader to detect the absorbance (OD value) of the cells in each well of the 96-well plate at a wavelength of 450nm. The above experiment was set up with a blank control group and a negative control group, and 6 replicate wells were set up for the control group and the experimental group. The cell survival rate was calculated according to the following formula: Cell survival rate (%) = (absorbance of the experimental group - absorbance of the blank control group) / (absorbance of the negative control group - absorbance of the blank control group) × 100%. The experimental results are as follows Figure 2 As shown in A.

[0065] The experimental results are as follows Figure 2 As shown in A.

[0066] Depend on Figure 2 From A, we can see that Pt 5.65 The OD value of the cell survival rate of the S-preformed nanozyme was almost the same as that of the Control group, proving that Pt 5.65 S preformed nanozymes have no cytotoxicity.

[0067] The CCK-8 assay is a rapid, highly sensitive, non-radioactive assay for detecting cell activity based on WST-8 and widely used in cell proliferation and cytotoxicity. The CCK-8 solution can be directly added to the cell sample without the need to pre-mix various components.

[0068] In the presence of an electron coupling agent, WST-8 can be reduced by some dehydrogenases in the mitochondria to generate orange-yellow formazan. The more and faster the cell proliferation, the darker the color; the greater the cytotoxicity, the lighter the color. For the same cells, the depth of color (the amount of formazan generated) is linearly related to the number of cells. Therefore, we first performed a CCK8 toxicity test on the synthesized nanozymes, and compared the Ptzyme group; the Na2S group; the Pt 5.65 The CCK8 detection and cell activity calculation of three groups with different concentrations of S prefabricated nanozyme group showed that when the concentration of nanozyme was added to 200μg, the activity was basically the same as that of the group without nanozyme, that is, the activity rate of the group with a concentration of 0μg / ml. This proves that the synthesized nanozyme itself is non-toxic to kidney cells, has biosafety, and can be used in subsequent cell protection experiments.

[0069] 2. Cell proliferation inhibition experiment:

[0070] In order to verify the Pt 5.65 The antioxidant effect of S preformed nanozymes in vitro was evaluated by CCK-8 method. 5.65 S preformed nanozymes, Ptzyme, and Na2S protected HEK293 cells from proliferation inhibition under hydrogen peroxide (H2O2) injury conditions, allowing for screening of appropriate drug concentrations for subsequent cell experiments.

[0071] The specific experimental method is:

[0072] Five experimental groups were set up: (1) H2O2 damage group; (2) Ptzyme group; (3) Na2S group; (4) Pt 5.65 S pre-made nanozyme group. HEK293 cells were digested and centrifuged at 8×10 4The cells were resuspended in complete medium at a concentration of 100 μg / ml, and 100 μL of the resuspended cell suspension was added to each well of the 96-well plate, and the cells were cultured in a cell culture incubator for 24 hours. The pre-prepared original drugs of Ptzyme, Na2S, and Pt5.65S were diluted to seven concentration gradients of 2, 5, 8, 11, 14, 17, and 20 μg / mL using complete medium. After carefully removing the original medium in each well of the 96-well plate, 100 μL of complete medium without drugs was added to the Control group, and 50 μL of complete medium was added to the H2O2 injury group, followed by 50 μL of 800 μM H2O2 diluted with complete medium, so that the final concentration of the H2O2 injury group was 400 μM. 50 μL of complete medium containing the corresponding drug concentration was added to the remaining nanozyme treatment groups, and the 96-well plate was placed in a cell culture incubator for 30 minutes. After that, 50 μL of 800 μM H2O2 diluted with complete medium was added to each well in sequence, and the cells were cultured in a cell culture incubator for 12 hours. Add 10 μL of CCK-8 reagent to each well, gently shake the 96-well plate horizontally to mix, and place it in a cell culture incubator for 2-3 hours. Use an ELISA reader to detect the absorbance (OD value) of cells in each well of the 96-well plate at a wavelength of 450 nm.

[0073] The experimental results are as follows Figure 2 As shown in B. Figure 2 As shown in Figure B, after HEK293 cells were treated with 400 μM H2O2, cell viability decreased significantly, indicating that H2O2 can significantly reduce cell activity; Pt 5.65 The OD value of cell survival rate in the S-prefabricated nanozyme group was significantly improved compared with the H2O2 group, and the Pt 5.65 The OD value of the S prefabricated nanozyme group was higher than that of the Ptzyme group and the Na2S group, which proved that the Pt 5.65 S preformed nanozymes have excellent antioxidant capacity.

[0074] 3. DCFH-DA fluorescence detection of active oxygen scavenging:

[0075] The Bio-Tian active oxygen detection kit (Cat. No. S0033S) was used to detect Ptzyme, Na2S, Pt 5.65 The ability of S preform to remove ROS under H2O2 damage conditions. The specific experimental method is:

[0076] Five experimental groups were set up: (1) Control group; (2) H2O2 injury group; (3) Ptzyme group; (4) Na2S group; (5) Pt 5.65 S pre-made nanozyme group. HEK 293 cells were digested and centrifuged at 1.5×10 5Resuspend the cells in complete medium at a concentration of cells / mL, add 2 mL of the resuspended cell suspension to each well of the six-well plate, and culture in a cell culture incubator for 24 h. 5.65 The original drug of S prefabricated nanozyme was diluted to 20 μg / mL, the original culture medium in each well of the six-well plate was discarded, and after washing once with 1 mL of PBS, 2 mL of complete culture medium without drugs was added to the control group, 1 mL of complete culture medium was added to the H2O2 injury group, and 1 mL of complete culture medium containing the corresponding drug concentration was added to each of the other groups. The six-well plate was placed in a cell culture incubator and incubated for 30 minutes. Except for the Control group, 1 mL of 800 μM H2O2 diluted with complete culture medium was added to the other four wells. After the six-well plate was placed in a cell culture incubator for 12 hours, the culture medium in each well of the six-well plate was aspirated, the serum-free culture medium was washed once, 1 mL of DCFH-DA probe (10 μM) was added to each well, and it was placed in a cell culture incubator for 20 minutes, washed three times with serum-free culture medium, and 2 mL of serum-free culture medium was added. The staining results were observed using a laser confocal microscope.

[0077] The Reactive Oxygen Species Assay Kit (also known as ROS Assay Kit) is a kit for detecting reactive oxygen species using the fluorescent probe DCFH-DA. DCFH-DA itself has no fluorescence and can freely pass through the cell membrane. After entering the cell, it can be hydrolyzed by the esterase in the cell to produce DCFH. However, DCFH cannot penetrate the cell membrane, making it easy for the probe to be loaded into the cell. The reactive oxygen species in the cell can oxidize the non-fluorescent DCFH to produce fluorescent DCF (green). By detecting the fluorescence of DCF, the level of reactive oxygen species in the cell can be known. That is, the more green fluorescence in the cell, the higher the level of reactive oxygen species in the cell. The changes in the level of green fluorescence in the cell can be used to compare the changes in the level of reactive oxygen species in the cell.

[0078] The experimental results are as follows Figure 2 As shown in D.

[0079] Depend on Figure 2 As shown in Figure D, the green fluorescence intensity of the H2O2 group is much higher than that of the Control group, indicating the generation of a large amount of reactive oxygen species. 5.65 The green fluorescence intensity of the S-prefabricated nanozyme group was significantly lower than that of the H2O2 group, which indicated that the Pt 5.65 In addition, compared with the Ptzyme group and the Na2S group, the Pt 5.65 The fluorescence brightness of the S preformed nanozyme group was also low. This indicates that the Pt 5.65The scavenging effect of S preformed nanozyme on active oxygen is higher than that of the other two, indicating that it has a stronger ability to scavenging active oxygen. 5.65 The high efficiency of S-preformed nanozymes in scavenging reactive oxygen species lays an experimental foundation for its application in the treatment of diseases such as oxidative damage.

[0080] 4. Cell apoptosis detection:

[0081] In this experiment, the AnnexinV-FITC / PI cell apoptosis detection kit (manufactured by Biyuntian, catalog number C1062S) was used to detect Ptzyme, Na2S, Pt 5.65 The anti-apoptotic ability of S-preformed nanozymes in HEK 293 cells under H2O2 injury conditions was analyzed by flow cytometry. The specific experimental methods are as follows:

[0082] Five experimental groups were set up: (1) Control; (2) H2O2 injury group; (3) Ptzyme group; (4) Na2S group; (5) Pt 5.65 S pre-made nanozyme group. HEK 293 cells were digested and centrifuged at 1.5×10 5 Resuspend the cells in complete medium at a concentration of cells / mL, add 2 mL of the resuspended cell suspension to each well of the six-well plate, and culture in a cell culture incubator for 24 h. 5.65 The original drug of S preformed nanozyme was diluted to 20 μg / mL, and the original culture medium in each well of the six-well plate was discarded. After washing once with 1 mL of PBS, 2 mL of complete culture medium without drugs was added to the Control group, 1 mL of complete culture medium was added to the H2O2 injury group, and 1 mL of complete culture medium containing the corresponding drug concentration was added to each of the other groups. The six-well plate was placed in a cell culture incubator and incubated for 30 minutes. After that, 1 mL of 800 μM H2O2 diluted with complete culture medium was added to the other four wells except the Control group. After culturing the six-well plate in a cell culture incubator for 12 hours, remove the culture medium, wash once with PBS, add 1 mL of trypsin to each well for 1 minute, add 1 ml of complete culture medium to terminate digestion, collect the original culture medium, washing solution, and cell suspension, centrifuge at 1000 rpm for 5 minutes, carefully remove the supernatant, wash twice with PBS again, resuspend each group of cells with 195 μL Annexin V-FITC binding solution, transfer the cell suspension to a flow tube, add 5 μL Annexin V-FITC and 10 μL PI staining solution to each group of tubes, blow evenly, incubate at room temperature in the dark for 10 minutes, and detect using a flow cytometer. The experimental results are as follows: Figure 2 As shown in C.

[0083] In normal cells, phosphatidylserine (PS) is only distributed on the inner side of the lipid bilayer of the cell membrane, while in the early stage of apoptosis, the phosphatidylserine (PS) in the cell membrane flips from the inner side of the lipid membrane to the outer side. Annexin V is a Ca2+-dependent phospholipid binding protein with a molecular weight of 35-36kDa, which has a high affinity with phosphatidylserine. Therefore, Annexin V is recognized as one of the sensitive indicators for detecting early cell apoptosis. Annexin V is labeled with green fluorescence (FITC), and the labeled Annexin V is used as a probe to detect the occurrence of cell apoptosis using flow cytometry. Propidium iodide (PI) is a nucleic acid dye that cannot penetrate the intact cell membrane of normal cells or early apoptotic cells, but for cells in the middle and late stages of apoptosis and necrotic cells, PI can penetrate the cell membrane and dye the cell nucleus red. Therefore, by matching Annexin V with PI, cells in different stages of apoptosis can be distinguished.

[0084] Therefore, by Figure 2 In C, the apoptosis rate of cells in the H2O2 injury group was 26.03%, and the cell survival rate was 34.18%; 5.65 After treatment with S preformed nanozyme, the cell survival rate was 52.96%. The comparison of cell survival rates proved that the preformed nanozyme prepared by the present invention has the ability to protect cells from apoptosis caused by oxidative stress damage.

[0085] 5. MitoTracker staining:

[0086] MitoTracker kit (Biyuntian, Cat. No. C1032) was used to detect Ptzyme, Na2S, Pt 5.65 The ability of S preform to remove ROS under H2O2 damage conditions. The specific experimental method is:

[0087] Five experimental groups were set up: (1) Control group; (2) H2O2 injury group; (3) Ptzyme group; (4) Na2S group; (5) Pt 5.65 S pre-made nanozyme group. HEK 293 cells were digested and centrifuged at 1.5×10 5 Resuspend the cells in complete medium at a concentration of cells / mL, add 2 mL of the resuspended cell suspension to each well of the six-well plate, and culture in a cell culture incubator for 24 h. 5.65The original drug dilution of S prefabricated nanozyme was 20.μg / mL. The original culture medium in each well of the six-well plate was discarded, and after washing once with 1mL PBS, 2mL of complete culture medium without drugs was added to the Control group, 1mL of complete culture medium was added to the H2O2 damage group, and 1mL of complete culture medium containing the corresponding drug concentration was added to each of the other groups. The six-well plate was placed in a cell culture incubator and incubated for 30 minutes. Except for the Control group, 1mL of 800μM H2O2 diluted with complete culture medium was added to the other four wells. After the six-well plate was placed in a cell culture incubator and cultured for 12 hours, the culture medium in each well of the six-well plate was aspirated, the serum-free culture medium was washed once, 1mL of MitoTracker (200nM) was added to each well, and the plate was placed in a cell culture incubator and incubated for 20 minutes. The serum-free culture medium was washed three times, and 2mL of serum-free culture medium was added. The staining results were observed using a laser confocal microscope. The experimental results are shown in the figure. Figure 2 As shown in E.

[0088] Mito-Tracker Red CMXRos (mitochondrial red fluorescent probe), also known as MitoTracker RedCMXRos, is a cell-permeable X-rosamine derivative (Chloromethyl-X-rosamine, CMXRos for short), which can specifically label biologically active mitochondria in cells and detect mitochondrial membrane potential. This probe is an oxidized red fluorescent dye that can be passively transported through the cell membrane by simply incubating with cells, and specifically label biologically active mitochondria with the help of the weakly thiol-reactive chloromethyl functional group contained in this probe. The weakly thiol-reactive chloromethyl contained in this probe can react with the thiol group of the protein in the mitochondria and covalently link. The probe fluorescent labeling can be used to reflect the changes in mitochondrial morphology, and the function of mitochondria can be evaluated by observing the morphological changes of mitochondria.

[0089] Depend on Figure 2 From E, we can see that Pt 5.65 The mitochondrial morphology of the S preformed enzyme protection group was intact, proving the protective effect of preformed nanozymes on mitochondria.

[0090] Under normal circumstances, mitochondria present a complete filamentous structure. When damaged by oxidative stress, the mitochondrial morphology becomes blurred or even destroyed. Compared with the Ptzyme group and the Na2S group, the Pt 5.65 The protective effect of the S-preformed nanozyme group on mitochondrial morphological damage caused by oxidative stress induced by hydrogen peroxide was more significant. 5.65In the S preformed nanozyme group, the morphology of mitochondria was significantly improved, while in the other two groups, although the morphology of mitochondria changed slightly, the rupture of mitochondria was still relatively serious.

[0091] It is obvious from this comparison that Pt 5.65 S-prepared nanozymes have excellent mitochondrial protection and anti-oxidative stress effects, which provides strong experimental evidence for their application in the treatment of diseases such as oxidative damage.

[0092] Example 3: Acute kidney injury mouse model (IRI-AKI mouse model) verifies the protective effect of Pt5.65S preformed nanozyme on kidney injury

[0093] 1. Construction of acute kidney injury mouse model (IRI-AKI mouse model):

[0094] The method for constructing an acute kidney injury mouse model (IRI-AKI mouse model) is as follows (eg Figure 3 (As shown in A): 5-6 week old male C57 mice (purchased from Beijing Weitong Lihua and raised in the Experimental Animal Center of Zhengzhou University) were selected and fed for 7 days before surgery. On the 7th day, the bilateral renal arteries were clamped for 25 minutes with micro arterial clamps to block blood flow, causing acute kidney injury caused by renal ischemia-reperfusion, and an IRI mouse animal model was established. Various samples were taken on the 8th day for related experiments). The sham operation group mice, namely the Sham group mice, were subjected to the same surgical method, laparotomy and exposure time, but the bilateral renal arteries were not clamped, and no treatment was performed. They were used as negative controls to eliminate the errors caused by the surgical method.

[0095] After 24 hours, IRI-AKI model mice and sham-operated mice (Sham) were killed to obtain kidney tissue and serum samples. Renal function of IRI-AKI mouse model was evaluated by measuring serum creatinine (Scr) and urea nitrogen (BUN) levels; oxidative stress was determined by measuring antioxidant SOD activity, GSH and MDA levels.

[0096] 2. Creatinine (Scr) and urea nitrogen (BUN) detection:

[0097] The main function of the kidney is to produce urine through glomerular filtration and tubular reabsorption and secretion, secrete metabolic waste and maintain the body's metabolic balance of water, electrolytes, acid and base. When the kidneys suffer from oxidative stress due to various nephrotoxic substances and acute kidney injury occurs, the filtration function of the renal tubules decreases and metabolic waste is retained in the body. Serum creatinine (CRE) and blood urea nitrogen (BUN) are the main nitrogen-containing end products of renal metabolism. When the renal tissue is damaged and the filtration capacity decreases, they accumulate in the blood. Therefore, CRE and BUN are widely used in clinical renal function testing and are the gold standard for diagnosing acute kidney injury.

[0098] (1) Experimental methods:

[0099] The IRI-AKI mouse model was established according to the above animal model construction method. The mice were divided into 5 groups, 5 mice in each group. The specific groups of animal experiments are as follows: (1) Sham group (negative control group); (2) IRI-AKI model group (positive control group); (3) Ptzyme treatment group; (4) Na2S treatment group; (5) Pt 5.65 S prefabricated nanozyme treatment group. Among them, the mice in the sham group were not injected with any fluid; the mice in the IRI-AKI model group were treated according to the above-mentioned IRI-AKI model construction method; the mice in the Ptzyme treatment group were injected with Ptzyme (injection dose of 15 mg / Kg body weight) through the tail vein 30 minutes before the renal artery clamping operation for pre-protection against ischemia-reperfusion injury; the mice in the Na2S treatment group were also injected with Na2S (injection dose of 15 mg / Kg body weight) through the tail vein 30 minutes before the renal artery clamping operation; 5.65 The mice in the S-preformed nanozyme treatment group were injected with Pt 5.65 S preformed nanozyme (injection dose is 15 mg / Kg body weight).

[0100] After 24 hours, the eyeballs of mice in each group were removed and blood was collected. The whole blood was placed in a 1.5 mL sterile EP tube, and then placed at 4 ° C for 1-2 hours. After centrifugation at 2000g and 4 ° C for 15 minutes, the upper serum was removed and placed in a new EP tube, which was placed in a negative 80 ° C refrigerator for subsequent processing. The Scr value in the serum was detected using a creatinine (Scr) assay kit (Nanjing Jiancheng, item number C011-2-1). First, the sample to be tested, the standard in the kit, and 6 μL of double distilled water were added to each well of the 96-well plate, and then 180 μL of enzyme solution A was added to each detection well. After incubation at 37 ° C for 5 minutes, the absorbance value A1 of each detection well was detected at 546 nm using a full-wavelength microplate reader. Subsequently, 60 μL of enzyme solution B was added to each detection well, and after incubation at 37 ° C for 5 minutes, the absorbance value A2 of each detection well was detected at 546 nm. Finally, the CRE value of the serum sample to be tested was calculated using the formula. The specific formula is as follows: ΔA=A2-K*A1, K=0.756 Creatinine content (μmol / L)=(ΔA 测定 -ΔA 空白 ) / (ΔA 标准 -ΔA 空白 )*C 标准 , C 标准:Standard concentration, 442μmol / L. The BUN value in serum was detected using the urea nitrogen (BUN) detection kit (Nanjing Jiancheng, item number C013-2-1). First, the enzyme buffer and BUN standard application solution were prepared. The enzyme buffer was prepared as follows: the enzyme stock solution: enzyme diluent = 3:1000 was prepared as buffered enzyme solution before use, and it was prepared immediately before use; 10mmol / LBUN standard application solution was prepared: before use, 1 powder was added with 1mL double distilled water to prepare a 100mmol / L standard stock solution, and the 100mmol / L standard stock solution was diluted with double distilled water at 1:9 (i.e., 10 times dilution) to prepare a 10mmol / LBUN standard application solution. The sample to be tested and 20μL of the 10mmol / LBUN standard application solution and double distilled water in the kit were added to a 10mL EP tube, and then 250μL of buffered enzyme solution was added to each tube, mixed and placed in a 37℃ water bath for 10min. Subsequently, 1 mL of phenol colorimetric agent and 1 mL of alkaline sodium hypochlorite were added to each tube in turn, mixed thoroughly, and placed in a 37°C water bath for 10 min. The absorbance (OD) value of each tube was measured using an ultraviolet spectrophotometer at a wavelength of 640 nm, a light path of 1 cm, and double distilled water at zero. Finally, the BUN value in serum was calculated using the following formula: BUN concentration (mmol / L) = (ΔA 测定 -ΔA 空白 ) / (ΔA 标准 -ΔA 空白 )*C 标准 , C 标准 : Standard concentration, 10mmol / L (280.1mg / L).

[0101] (2) Experimental results:

[0102] The test results of serum creatinine (Scr) and urea nitrogen (BUN) levels of mice in each experimental group are as follows Figure 3 As shown in B and C.

[0103] like Figure 3 As shown in B and C, the CRE and BUN values ​​of mice in the IRI-AKI group were significantly higher than those in the Sham group (P<0.0001), confirming that the renal ischemia-reperfusion model induced by surgery was successfully established. 5.65 The CRE and BUN values ​​of mice in the S pretreatment group were significantly lower than those in the IRI-AKI group (P<0.0001), and were even almost the same as those in the Sham group, indicating that Pt 5.65S preformation has an excellent therapeutic effect on IRI-AKI and has a strong ability to resist oxidative stress; the CRE and BUN values ​​of mice in the Ptzyme group decreased slightly (P<0.05), proving that the release of H2S gas by preformed nanozymes in the kidneys has a slight anti-inflammatory effect; the CRE and BUN values ​​of mice in the Na2S group decreased slightly (P<0.0001), proving that it can resist oxidation to a certain extent. Therefore, the experimental results show that Pt 5.65 S preformulation has good antioxidant capacity and can treat AKI caused by oxidative stress.

[0104] 3. SOD, MDA, GSH (GSSG) detection:

[0105] The IRI-AKI mouse model was established according to the above animal model construction method. The mice were divided into 5 groups, 5 mice in each group. The specific groups of animal experiments are as follows: (1) Sham group (negative control group); (2) IRI-AKI model group (positive control group); (3) Ptzyme treatment group; (4) Na2S treatment group; (5) Pt 5.65 S prefabricated nanozyme treatment group. Among them, the mice in the sham group were not injected with any fluid; the mice in the IRI-AKI model group were treated according to the above-mentioned IRI-AKI model construction method; the mice in the Ptzyme treatment group were injected with Ptzyme through the tail vein 30 minutes before surgery (injection dose was 15 mg / Kg body weight); the mice in the Na2S treatment group were injected with Na2S through the tail vein 30 minutes before surgery (injection dose was 15 mg / Kg body weight); Pt 5.65 The mice in the S-preformed nanozyme treatment group were injected with Pt 5.65 S preformed nanozyme (injection dose is 15 mg / Kg body weight).

[0106] Kidney tissues were collected from mice in each group, and the tissues were broken according to the instructions of the SOD detection kit (Biyuntian, item number S0101S), MDA detection kit (Biyuntian, item number S0131S), and GSH / GSSG detection kit (Biyuntian, item number S0053), and the indicators of each group were detected under an ELISA instrument. The test results are shown in Figure 3 As shown in D, E, and F.

[0107] Superoxide dismutase (SOD) can catalyze the dismutation of superoxide anions to generate hydrogen peroxide (H2O2) and oxygen (O2), and is an important antioxidant enzyme in the body. WST-8 can react with superoxide anions (O2.-) catalyzed by xanthine oxidase (XO) to produce water-soluble formazan dye. Since SOD can catalyze the dismutation of superoxide anions, this reaction step can be inhibited by SOD. Therefore, the activity of SOD is negatively correlated with the amount of formazan dye generated, so the enzyme activity of SOD can be calculated by colorimetric analysis of WST-8 products. The detection of SOD activity in kidney tissue can reflect the degree of kidney oxidative stress to a certain extent. As an important antioxidant enzyme in the human body, SOD enzyme plays an important role in alleviating the generation of oxidative stress in the human body. Therefore, we detected the SOD enzyme activity in the kidneys of mice in different groups, and compared the effect of nanozymes in antagonizing oxidative stress and protecting the kidneys through the changes in SOD enzyme activity. SOD test results are as follows Figure 3 As shown in D. Figure 3 As shown in Figure 3, the renal SOD (superoxide dismutase) value decreased in the mice in the IRI-AKI group, indicating that the kidneys were damaged. In the group of mice treated with Pt5.65S preformed nanozyme, the SOD value increased significantly compared with the Ptzyme group and the Na2S group, indicating that Pt 5.65 It is worth mentioning that compared with the Ptzyme group and the Na2S group, the Pt 5.65 The SOD value of the S-preformed nanozyme group was higher, which further proved its significant advantages and effects in kidney protection. 5.65 S-preformed nanozymes have significant potential in the treatment of ischemia-reperfusion injury-induced acute kidney injury (IRI-AKI), and their effects on restoring renal function and protecting the kidneys from oxidative damage deserve further in-depth research and application.

[0108] Malondialdehyde (MDA) is a natural product of lipid oxidation in organisms. Lipid oxidation occurs when animal or plant cells are under oxidative stress. After oxidation, some fatty acids gradually decompose into a series of complex compounds, including MDA. At this time, the level of lipid oxidation can be detected by detecting the level of MDA, so the determination of MDA is widely used as an indicator of lipid oxidation. Therefore, we observed the level of lipid peroxidation in the kidneys of each group of mice by detecting the level of MDA in the kidneys, and evaluated the protective effect of prefabricated nanozymes on renal oxidative stress. The MDA test results are as follows: Figure 3As shown in E. Figure 3 As shown in Figure E, in the IRI-AKI group, the MDA value of the mouse kidney increased significantly, indicating the occurrence of renal lipid oxidative damage. This further verifies the successful construction of the model. In contrast, the MDA value of the mouse kidney in the Pt5.65S preformed nanozyme treatment group was significantly reduced, indicating the protective effect of the Pt5.65S preformed nanozyme on renal oxidative stress. Compared with the Ptzyme group and the Na2S group, the MDA value of the Pt5.65S preformed nanozyme group was lower, indicating that the Pt5.65S preformed nanozyme has more significant effects and advantages in renal antioxidant effects.

[0109] In summary, the MDA test results further verified the protective effect of Pt5.65S preformed nanozymes on renal oxidative stress. These experimental evidences provide strong support for the application of Pt5.65S preformed nanozymes in the treatment of acute kidney injury (AKI).

[0110] Glutathione is a small peptide composed of three amino acid residues. Its full name is glutamyl-cysteinyl-glycine, and its English name is glutamyl-cysteinyl-glycine, abbreviated as glutathione. Since the sulfhydryl group (SH) on cysteine ​​is the active group of glutathione, it is often abbreviated as G-SH or GSH. Glutathione includes two forms: reduced glutathione (commonly known as GSH) and oxidized glutathione (oxidized glutathionedisulfide). Since oxidized glutathione is formed by the dehydrogenation of two GSH groups through sulfhydryl groups, it is often abbreviated as GSSG or GSSG. Reduced glutathione is the main source of sulfhydryl groups in most living cells. It plays an important role in maintaining the proper redox state of sulfhydryl groups in proteins and is a key antioxidant in animal cells. Usually 90-95% of the total glutathione is reduced glutathione. By detecting the changes in the ratio of the two, we can evaluate the degree of kidney damage and oxidative stress, so we tested the kidney tissue samples of each group of mice. Figure 3 As shown in F. Figure 3 As shown in Figure 5, the renal GSH / GSSG value of mice in the IRI-AKI group decreased, indicating that the kidneys were damaged by lipid oxidation. 5.65 In the group of mice treated with S-preformed nanozymes, the GSH / GSSG value increased significantly, indicating that Pt 5.65 S preformed nanozymes have a protective effect on the kidneys and can reduce oxidative damage to the kidneys. It is worth noting that compared with the Ptzyme group and the Na2S group, the Pt 5.65The GSH / GSSG value of the S-preformed nanozyme group was higher, which further proved that the Pt 5.65 S-preformed nanozymes have more significant effects and advantages in kidney antioxidant function. These results emphasize that Pt 5.65 The important role of S-preformed nanozymes in the treatment of ischemia-reperfusion injury-induced acute kidney injury (IRI-AKI) helps maintain the redox balance of the kidney and protect the kidney from oxidative stress damage. This provides important support for its potential application in kidney protection and treatment.

[0111] 4. H&E staining:

[0112] In order to further determine the therapeutic effect of Pt5.65S on renal organ damage, we performed H&E staining on the kidneys of mice in different drug treatment groups.

[0113] (1) Experimental methods:

[0114] The IRI-AKI mouse model was established according to the above animal model construction method. The mice were divided into 5 groups, 5 mice in each group. The specific groups of animal experiments are as follows: (1) Sham group (negative control group); (2) IRI-AKI model group (positive control group); (3) Ptzyme treatment group; (4) Na2S treatment group; (5) Pt 5.65 S prefabricated nanozyme treatment group. Among them, the mice in the sham group were not injected with any fluid; the mice in the IRI-AKI model group were treated according to the above-mentioned IRI-AKI model construction method; the mice in the Ptzyme treatment group were injected with Ptzyme through the tail vein 30 minutes before surgery (injection dose was 15 mg / Kg body weight); the mice in the Na2S treatment group were injected with Na2S through the tail vein 30 minutes before surgery (injection dose was 15 mg / Kg body weight); Pt 5.65 The mice in the S-preformed nanozyme treatment group were injected with Pt 5.65 S preformed nanozyme (injection dose is 15 mg / Kg body weight).

[0115] After 24 hours, mice in each group were killed by dislocation, and their kidneys were removed by dissection, fixed with paraformaldehyde (4% PBS), embedded in paraffin, and then sliced ​​and stained with hematoxylin-eosin. Finally, images were captured using an upright microscope.

[0116] (2) Experimental results:

[0117] The cell nucleus contains acidic nucleic acids, which have a strong affinity with alkaline dyes (hematoxylin), while the cytoplasm contains alkaline substances with a strong affinity with acidic dyes (eosin). Therefore, after the tissue section is stained with HE, the cell nucleus is stained blue-purple by hematoxylin, while the cytoplasm, muscle fibers, and collagen fibers appear red to varying degrees. The experimental results are as follows Figure 3 As shown in G. Figure 3 As shown in Figure G, the mice in the IRI-AKI group clearly showed severe cellular vacuoles, diffuse or multifocal cell collapse and detachment, and rupture of the tubular basement membrane. In addition, the H&E staining results of the IRI-AKI group showed that the nuclear staining deepened, there was inflammatory cell infiltration, and the cells were arranged in a very disordered manner. From this, it can be judged that the IRI-AKI mouse model was successfully constructed. The renal tubular cells of the mice in the sham group were neatly arranged, without obvious tubular damage. At the same time, according to the experimental results of the Pt5.65S prefabricated nanozyme group and the IRI-AKI group, the following situations were observed: 1. The degree of renal cavitation was reduced: This shows that the Pt5.65S prefabricated nanozyme can reduce vacuoles in renal tissue, suggesting a protective effect on renal cells. 2. Tubular basement membrane repair: Studies have shown that in the Pt5.65S prefabricated nanozyme group, the tubular basement membrane was repaired, which means that the Pt5.65S prefabricated nanozyme has a positive effect on the protection and repair of tubular cells. 3. Reduction of diffuse inflammatory foci: It was observed that the distribution range of inflammatory foci was reduced in the Pt5.65S preformed nanozyme group, indicating that Pt5.65S preformed nanozyme can alleviate the inflammatory response.

[0118] In summary, the experiment proved that Pt5.65S preformed nanozyme has excellent antioxidant activity, can protect renal tubular cells, prevent cell necrosis caused by oxidative stress, and effectively treat acute kidney injury (AKI). In contrast, the pathological changes in the Ptzyme group and the Na2S group were less severe, which also confirmed the effectiveness of preformed nanozyme in kidney protection. These results provide strong experimental evidence for the application of Pt5.65S preformed nanozyme in the treatment of kidney diseases.

[0119] 5. Masson staining:

[0120] Masson staining is a histological staining method that stains tissue keratin and muscle fibers red, collagen and bone blue or green, cytoplasm light red or pink, and cell nuclei dark brown or black. 5.65 In order to investigate the therapeutic effect of S on renal organ fibrosis, we performed Masson staining on the kidneys of mice in different drug treatment groups.

[0121] (1) Experimental methods:

[0122] The IRI-AKI mouse model was established according to the above animal model construction method. The mice were divided into 5 groups, 5 mice in each group. The specific groups of animal experiments are as follows: (1) Sham group (negative control group); (2) IRI-AKI model group (positive control group); (3) Ptzyme treatment group; (4) Na2S treatment group; (5) Pt5.65 S prefabricated nanozyme treatment group. Among them, the mice in the sham group were not injected with any fluid; the mice in the IRI-AKI model group were treated according to the above-mentioned IRI-AKI model construction method; the mice in the Ptzyme treatment group were injected with Ptzyme through the tail vein 30 minutes before surgery (injection dose was 15 mg / Kg body weight); the mice in the Na2S treatment group were injected with Na2S through the tail vein 30 minutes before surgery (injection dose was 15 mg / Kg body weight); Pt 5.65 The mice in the S-preformed nanozyme treatment group were injected with Pt 5.65 S preformed nanozyme (injection dose is 15 mg / Kg body weight).

[0123] After 24 hours, mice in each group were killed by dislocation, and their kidney tissues were removed by dissection, fixed with paraformaldehyde (4% PBS), embedded in paraffin, and then sectioned and stained with Masson Ponceau acid fuchsin dye. Finally, images were captured using an inverted microscope.

[0124] (2) Experimental results:

[0125] The experimental results are as follows Figure 3 As shown in G. Figure 3 As shown in Figure G, renal fibrosis can be clearly seen in the IRI-AKI group, and the degree of blue staining deepens, proving that the kidney can be used to judge that the IRI-AKI mouse model was successfully constructed. At the same time, Masson staining of the prefabricated nanozyme group showed a lighter degree of blue, suggesting a lower degree of renal interstitial fibrosis. This shows that the Pt5.65S prefabricated nanozyme has a significant effect in inhibiting renal interstitial fibrosis. In contrast, Masson staining in the IRI-AKI group showed a darker degree of blue, indicating a higher level of renal interstitial fibrosis, which further confirmed the renal damage caused by acute kidney injury. In the Masson staining results of the Ptzyme group and the Na2S group, a darker degree of blue was shown relative to the prefabricated nanozyme group, suggesting that the degree of renal interstitial fibrosis was relatively high.

[0126] In summary, the experiments demonstrated that Pt5.65S preformed nanozymes have a significant effect in inhibiting renal interstitial fibrosis. In contrast, the IRI-AKI group had a higher level of renal interstitial fibrosis, and the preformed nanozyme group performed well in this regard. These results provide strong experimental evidence for the application of Pt5.65S preformed nanozymes in the treatment of acute kidney injury.

[0127] 6. Tunel dyeing:

[0128] In order to further determine Pt 5.65In order to investigate the therapeutic effect of S on kidney organs, we performed Tunel staining on the kidneys of mice in different drug treatment groups.

[0129] (1) Experimental methods:

[0130] The IRI-AKI mouse model was established according to the above animal model construction method. The mice were divided into 5 groups, 5 mice in each group. The specific grouping of animal experiments is as follows: (1) Sham group (negative control group); (2) IRI-AKI model group (positive control group); (3) Ptzyme treatment group; (4) Na2S treatment group; (5) Pt 5.65 S prefabricated nanozyme treatment group. Among them, the mice in the sham group were not injected with any fluid; the mice in the IRI-AKI model group were treated according to the above-mentioned IRI-AKI model construction method; the mice in the Ptzyme treatment group were injected with Ptzyme through the tail vein 30 minutes before surgery (injection dose was 15 mg / Kg body weight); the mice in the Na2S treatment group were injected with Na2S through the tail vein 30 minutes before surgery (injection dose was 15 mg / Kg body weight); Pt 5.65 The mice in the S-preformed nanozyme treatment group were injected with Pt 5.65 S preformed nanozyme (injection dose is 15 mg / Kg body weight).

[0131] After 24 hours, mice in each group were killed by dislocation, and their kidney tissues were removed by dissection, fixed with paraformaldehyde (4% PBS), embedded in paraffin, and then sliced ​​and stained with Tunel staining solution. Finally, images were captured using an inverted microscope.

[0132] (2) Experimental results:

[0133] The principle of TUNEL staining is that dUTP labeled with fluorescein can be linked to the 3-OH end of the broken DNA in apoptotic cells under the action of deoxyribonucleotide terminal transferase (TdTEnzyme), and specifically bind to the fluorescein antibody linked to horseradish peroxidase (HRP, horse-radish peroxidase), which reacts with the HRP substrate diaminobenzidine (DAB) to produce a strong color reaction (dark brown), specifically and accurately locating apoptotic cells, so apoptotic cells can be observed under an optical microscope; since normal or proliferating cells have almost no DNA breaks, no 3-OH is formed and they can rarely be stained. In this experiment, the type of cell death and differentiation stage were determined by the two-color method through the in situ detection of apoptosis in kidney tissue at the single-cell level. The experimental results were shown by the staining results of kidney tissue sections, that is, the brown part is the apoptotic part of kidney cells, and the darker the kidney is stained brown, the more apoptotic cells there are.

[0134] The experimental results are as follows Figure 3 As shown in G. Figure 3 As shown in Figure G, the renal tissue staining of the IRI-AKI group showed obvious brown areas, indicating the apoptosis of renal tubular cells. The depth of the brown part is positively correlated with the degree of cell apoptosis, which also proves that the model was successfully constructed. In contrast, the renal tissue staining of mice in the Sham group showed a light color, indicating a relatively low level of cell apoptosis. Comparing the experimental results of the Pt5.65S prefabricated nanozyme group and the IRI-AKI group, it can be clearly seen that the renal tissue staining color of the Pt5.65S prefabricated group is lighter, indicating a lower level of cell apoptosis. This further verifies the superiority of the Pt5.65S prefabricated nanozyme in resisting renal tubular cell apoptosis. Compared with the Ptzyme group and the Na2S group, the tubular cell staining color of the Pt5.65S prefabricated nanozyme group was significantly lighter, indicating its superior ability to resist renal tubular cell apoptosis.

[0135] In summary, the experiments demonstrated that Pt5.65S preformed nanozymes have a significant effect in inhibiting renal tubular cell apoptosis. These results provide strong experimental evidence for the application of Pt5.65S preformed nanozymes in the treatment of acute kidney injury (AKI).

[0136] 7. Pt 5.65 Biodistribution study of S preform in mouse tissues:

[0137] (1) Experimental method: Pt 5.65 The heart, liver, spleen, lung and kidney organ tissues of mice in the S-prefabricated nanozyme treatment group were analyzed using an ICP-MS instrument to detect the platinum ion content of the organs 24 and 48 hours after treatment.

[0138] ICP-MS can detect the accumulation of metal ions in organ tissues. By comparing the content of pt ions in various organs of mice, it can be demonstrated whether the prefabricated nanozymes are targeted and delivered to the kidneys.

[0139] (2) Experimental results:

[0140] Test results such as Figure 3 As shown in H and I. Over time, the Pt ion content in various organs changed. The results showed that even after 48 hours, the Pt ion content in the kidney was still higher than that in other organs. This result shows that the preformed nanozyme has a significant kidney-targeted enrichment ability. This experiment demonstrates the superiority of preformed nanozymes in treatment, especially its targeted therapeutic ability in the kidney.

[0141] 8. Western blot analysis:

[0142] Western blot was used to detect the expression levels of NGAL, NRF2, GPX4 and TNF-α proteins in the kidney tissues of each group of mouse models.

[0143] (1) Experimental methods:

[0144] The IRI-AKI mouse model was established according to the above animal model construction method. The mice were divided into 5 groups, 5 mice in each group. The specific groups of animal experiments are as follows: (1) Sham group (negative control group); (2) IRI-AKI model group (positive control group); (3) Ptzyme treatment group; (4) Na2S treatment group; (5) Pt 5.65 S prefabricated nanozyme treatment group. Among them, the mice in the sham group were not injected with any fluid; the mice in the IRI-AKI model group were treated according to the above-mentioned IRI-AKI model construction method; the mice in the Ptzyme treatment group were injected with Ptzyme through the tail vein 30 minutes before surgery (injection dose was 15 mg / Kg body weight); the mice in the Na2S treatment group were injected with Na2S through the tail vein 30 minutes before surgery (injection dose was 15 mg / Kg body weight); Pt 5.65 The mice in the S-preformed nanozyme treatment group were injected with Pt 5.65 S preformed nanozyme (injection dose is 15 mg / Kg body weight).

[0145] After 24 hours, mice in each group were killed by dislocation, and the kidney tissue of mice was removed by dissection. 20 mg of kidney tissue was taken from each group of mice, and an appropriate amount of tissue protein lysis solution was added. It was ground in a liquid nitrogen grinder until it was ground into a homogenate, poured into an EP tube, centrifuged at 4°C 12000rpm for 15 minutes, and the supernatant was collected. After quantitatively detecting the total cell protein concentration by BCA method (Thermo Kit), an appropriate amount of protein sample was mixed with 5× sodium dodecyl sulfate (SDS) loading buffer, boiled in a 100°C water bath for 10 minutes, and stored at -20°C. Prepare SDS-PAGE gel electrophoresis with 12% separation gel, then take 10μg of protein in equal amounts for SDS-PAGE gel electrophoresis, 80V constant voltage electrophoresis sample from the concentrated gel to the separation gel, and then 120V constant voltage electrophoresis until bromophenol blue reaches the bottom of the separation gel. After transferring the band to the PVDF membrane, cut the position of the target protein according to the instructions of the maker. Remove the PVDF membrane and block it with 5% TBST skim milk at room temperature for 60 minutes. After blocking, remove the PVDF membrane, put it into the corresponding primary antibody working solution, and place it on a shaker at 4°C overnight. Wash the membrane with TBST solution for 15 minutes each time, and wash it 4 times. Transfer to the secondary antibody working solution and incubate at room temperature for 60 minutes. Wash the membrane with TBST solution for 15 minutes each time, and wash it 4 times. Add an appropriate amount of developer in the darkroom to color the protein bands on the PVDF membrane, expose it to X-ray film, and develop it.

[0146] (2) Experimental results:

[0147] The experimental results are as follows Figure 3 As shown in J.

[0148] Depend on Figure 3 It can be seen from J that Pt 5.65 The protein expression of NGAL and TNF-A in the S preformed nanozyme group was reduced, which proved the renal protective effect of the preformed nanozyme; Pt 5.65 The expression of nrf2 protein in the S-prefabricated nanozyme group increased, proving that Pt 5.65 S-preformed nanozymes can induce renal demethylation through the nrf2 promoter sequence, promote nrf2 expression, and thus protect the kidneys; Pt 5.65 The expression of S-preformed nanozyme gpx4 increased, proving that Pt 5.65 S-preformed nanozymes can treat kidney damage by alleviating renal ferroptosis.

[0149] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A prefabricated nanozyme, characterized in that: The invention comprises metal nanoparticles and a hydrogen sulfide releasing agent loaded on the metal nanoparticles; the metal nanoparticles are platinum nanoparticles, and the hydrogen sulfide releasing agent is sodium sulfide; the particle size of the prefabricated nanoenzyme is 4 to 100 nm, and the preparation method of the platinum nanoparticles is as follows: polyvinyl pyrrolidone is dissolved in a solvent to obtain a polyvinyl pyrrolidone solution, an H2PtCl6 aqueous solution is added to the polyvinyl pyrrolidone solution, and the mixture is stirred and refluxed at 60°C to 90°C for 2h to 24h for reaction, and after the reaction is completed, the solvent and PVP in the reaction product are removed, and the reaction product is freeze-dried to obtain platinum nanoparticles.

2. Use of the preformed nanozyme according to claim 1 in the preparation of a drug for alleviating and / or treating renal injury, wherein the renal injury is acute renal injury, and the acute renal injury is acute renal injury caused by oxidative stress or renal ischemia-reperfusion.

3. The method for preparing the prefabricated nanozyme according to claim 1, characterized in that: The metal nanoparticles and the hydrogen sulfide releaser are added into water, stirred overnight, then dialyzed to remove the free hydrogen sulfide releaser, and freeze-dried to obtain the prefabricated nanozyme.

4. The preparation method according to claim 3, characterized in that: The mass ratio of the metal nanoparticles to the hydrogen sulfide releasing agent is 2:1 to 100:

1.

5. A drug for alleviating and / or treating renal injury, characterized in that: The drug comprises the preformed nanozyme according to claim 1 and pharmaceutically acceptable excipients.

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