Preparation method of cerium tannate nano-enzyme and application of cerium tannate nano-enzyme in treatment of acute kidney injury

Abstract

The preparation method comprises the following steps: mixing a tannic acid solution and a tetravalent cerium salt solution, adjusting the pH value to 9-11, and reacting to obtain the cerium tannate nano-enzyme, the molar ratio of the tannic acid to the tetravalent cerium salt is (1-4): 1; the hydration dynamic diameter of the cerium tannate nano-enzyme is 20 to 80 nm. The TA-Ce nano-enzyme prepared by the preparation method disclosed by the invention is smaller in particle size, can keep stable and good dispersibility in a physiological environment, and has good biocompatibility, low hemolysis rate and small systemic toxicity; meanwhile, the compound has good kidney targeting property, can be effectively enriched into the injured kidney, eliminates various ROS (reactive oxygen species) and relieves oxidative stress by utilizing unique redox cycle between Ce < 3 + > and Ce < 4 + > states, and has a good effect in treating acute kidney injury induced by rhabdomyolysis and / or cis-platinum. The research lays a foundation for the metal polyphenol nano material in the treatment of acute kidney injury.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical engineering materials, and more specifically, relates to a preparation method of cerium tannate nanozyme and application thereof in treating acute kidney injury. Background Art

[0002] Acute kidney injury (AKI) is a group of clinical syndromes caused by ischemic injury, nephrotoxic substances, sepsis, etc., which is characterized by a sharp decline in renal function in a short period of time, and its morbidity and mortality rate have been steadily increasing year by year. Clinically, AKI is characterized by increased serum creatinine levels, decreased urine output, or both. The causes of AKI are diverse, including prerenal factors (such as hypovolemia and decreased renal perfusion), intrinsic renal damage (caused by diseases such as acute tubular necrosis, glomerulonephritis or interstitial nephritis), and postrenal obstruction (such as urinary tract obstruction). Clinical data show that 10%-15% of hospitalized patients will develop AKI, and the incidence of AKI in intensive care units exceeds 50%. Although a large number of studies have been devoted to the development of drugs to prevent and treat AKI, most candidate drugs are still in the experimental stage. Currently approved therapies do not synergistically or comprehensively improve the pathogenesis of AKI; therefore, the treatment of AKI in clinical practice mainly relies on supportive therapies, including hemodialysis, renal replacement therapy, etc.

[0003] Rhabdomyolysis-induced AKI (RM-AKI) and cisplatin-induced AKI (CP-AKI) are two common forms of acute kidney injury in clinical practice. When skeletal muscle is damaged, myocyte contents (such as myoglobin, potassium ions, and phosphates) are released into the blood. These substances, especially myoglobin, can form casts in the renal tubules, leading to tubular obstruction and tubular necrosis, and subsequently inducing RM-AKI. CP-AKI is caused by the chemotherapy drug cisplatin. Although cisplatin has a therapeutic effect on a variety of solid tumors, it can damage renal tubular epithelial cells and lead to CP-AKI. Studies have shown that the pathogenesis of AKI is closely related to oxidative stress, which is characterized by excessive reactive oxygen species (ROS), mitochondrial dysfunction, endoplasmic reticulum (ER) stress, inflammation, and apoptosis. Specifically, in AKI, the reduced form of the nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase complex can be activated by a variety of stimuli, including ischemia-reperfusion injury and proinflammatory cytokines. This activation converts NADPH to nicotinamide adenine dinucleotide phosphate (NADP +), and produce a large amount of ROS, leading to oxidative stress. Mitochondrial damage plays a vital role in AKI. Due to the high metabolic demand of the kidney, it is rich in mitochondria and is prone to mitochondrial dysfunction. Damaged mitochondria can usually be cleared through the mitophagy pathway. Autophagosomes can recognize and engulf dysfunctional mitochondria, and then fuse with lysosomes to degrade damaged mitochondria. If mitophagy is impaired, damaged mitochondria will accumulate, leading to increased leakage of the electron transport chain, excessive ROS generation, and reduced adenosine triphosphate (ATP) generation, ultimately increasing the risk of cell death. ER is essential for protein folding and modification and calcium ion storage. Factors such as protein overload, oxidative stress, calcium imbalance, and nutrient deficiency can lead to imbalance in protein folding and metabolism, resulting in ER stress. In AKI, oxidative stress can disrupt the normal folding of proteins in the ER, thereby activating the unfolded protein response (UPR). Although the UPR is an adaptive response designed to restore protein balance, persistent stress can lead to cellular dysfunction and apoptosis, while producing excessive ROS, which can cause damage and death of renal tubular epithelial cells. In addition, the ER is the main reservoir of intracellular calcium ions, and ER stress can promote the release of calcium ions into the cytoplasm, thereby disrupting calcium balance and activating calcium-dependent signaling pathways, ultimately leading to cell damage. In addition, excessive ROS can shift the macrophage phenotype to the M1 proinflammatory state, which is characterized by the secretion of proinflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6). This polarization can aggravate the inflammatory response and lead to renal tissue damage. In summary, oxidative stress, mitochondrial damage, ER stress, inflammation, and apoptosis have complex interactions in the development of AKI. ROS lead to mitochondrial dysfunction and ER stress by affecting mitophagy and UPR, respectively. Damaged mitochondria and persistent ER stress, in turn, generate large amounts of ROS, forming a vicious cycle. This vicious cycle activates downstream inflammatory and apoptotic pathways, further impairing renal function. Despite advances in the treatment of a specific pathological mechanism of AKI, the limited efficacy of traditional anti-inflammatory drugs (such as N-acetylcysteine ​​(NAC)) highlights the importance of adopting therapeutic strategies targeting multiple aspects of AKI pathophysiology.

[0004] Metal polyphenol nanonetworks are nanocomposites formed by the interaction of metal ions and polyphenol compounds through coordination bonds. In recent years, metal polyphenol nanonetworks have become a promising therapeutic strategy due to their multifunctional properties, especially in regulating oxidative stress and restoring cellular homeostasis. However, their application is limited by stability, long-term toxicity, and targeting efficiency. Tannic acid (TA), a natural polyphenol, has attracted much attention due to its powerful antioxidant and anti-inflammatory effects. The antioxidant properties of TA are attributed to its structural components, including carbon-carbon double bonds, phenolic hydroxyl groups, gallic acid molecules, and quinone structures. It has been reported that TA can reduce inflammatory responses and promote tissue repair by regulating macrophage polarization. In addition, TA also plays an important role in stabilizing nanoparticles, improving bioavailability, and drug efficacy. Cerium nanoparticles (Ce NPs) have strong antioxidant activity, which can be used in the presence of reduced Ce. 3+ and oxidized Ce 4+ CeNPs alternate between the two states and are therefore another promising candidate for the treatment of AKI. This unique redox cycle enables CeNPs to mimic the enzymatic activities of superoxide dismutase (SOD) and catalase (CAT), thereby effectively neutralizing ROS and protecting cells from oxidative damage. In addition to their antioxidant properties, Ce NPs also have anti-inflammatory, anti-apoptotic and cytoprotective effects.

[0005] The prior art "Myocardial-Targeting Tannic Cerium Nanocatalyst AttenuatesIschemia / Reperfusion Injury" discloses adding 100 μL of tannic acid (TA) solution (24 mM) and 600 μL of Ce(SO4)2 solution (24 mM) into 50 mL of deionized water and stirring, then adding NaOH solution (2 M) to adjust the solution pH to 8.0, and reacting for 3 hours to obtain a TA-Ce nanozyme with a hydrodynamic diameter of 95.43 nm±2.52 nm and a Zeta potential of 23.5 mV in PBS. The enzyme has the ability to target human cardiomyocytes and has antioxidant properties, and can be used to treat ischemic heart disease caused by ischemia / reperfusion, significantly reducing the area of ​​myocardial infarction and restoring heart function. However, the prepared TA-Ce nanozyme has insufficient kidney targeting ability, its toxicity to normal cells needs to be improved, and its safety needs to be improved. At the same time, there are currently no reports on the use of TA-Ce nanozymes to target the kidneys for the treatment of rhabdomyolysis and / or cisplatin-induced acute kidney injury. Summary of the invention

[0006] The purpose of the present invention is to overcome the above-mentioned defects and shortcomings in the prior art and provide a method for preparing cerium tannate nanozyme.

[0007] The second object of the present invention is to provide cerium tannate nanozyme obtained according to the above preparation method.

[0008] The third object of the present invention is to provide the use of the above-mentioned cerium tannate nanozyme in the preparation of a product targeting the kidney to treat acute kidney injury.

[0009] The fourth object of the present invention is to provide a drug comprising the above-mentioned cerium tannate nanozyme.

[0010] The above-mentioned object of the present invention is achieved through the following technical solutions: The invention provides a method for preparing tannic acid cerium nanozyme. The preparation method comprises the following steps: mixing a tannic acid solution and a tetravalent cerium salt solution, adjusting the pH to 9-11, and reacting the mixture. The molar ratio of the tannic acid to the tetravalent cerium salt is 1-4:1. The hydration kinetic diameter of the tannic acid cerium nanozyme is 20-80 nm.

[0011] The present invention adopts a self-assembly method to prepare TA-Ce, and TA chelates with cerium ions to form a stable TA-Ce nanozyme. By screening the molar ratio of TA to tetravalent cerium salt and pH conditions, a TA-Ce nanozyme with a smaller hydration kinetic diameter and a significantly different Zeta potential is obtained. The TA-Ce nanozyme can maintain stability and good dispersibility in a physiological environment, has good biocompatibility, a low hemolysis rate (hemolysis rate <5% at high concentrations), and low systemic toxicity. Due to the small particle size and surface characteristics of the TA-Ce nanozyme, it has good kidney targeting and can be effectively enriched in damaged kidneys. At the same time, the TA-Ce nanozyme utilizes Ce 3+ and Ce 4+ It can stimulate the unique redox cycle between states and simulate the activity of natural antioxidant enzymes such as SOD and CAT, thereby removing a variety of ROS and alleviating oxidative stress.

[0012] Furthermore, the molar ratio of the tannic acid to the tetravalent cerium salt is 1.5-3.5:1.

[0013] Furthermore, the molar ratio of the tannic acid to the tetravalent cerium salt is 2.5-3.5:1.

[0014] Preferably, the molar ratio of the tannic acid to the tetravalent cerium salt is 3:1.

[0015] Furthermore, the pH is 9.5 to 10.5.

[0016] Preferably, the pH is 10.

[0017] Furthermore, the reaction time is 2 to 4 hours.

[0018] Preferably, the reaction time is 3 hours.

[0019] Furthermore, the hydration kinetic diameter of the cerium tannate nanozyme is 20 to 60 nm.

[0020] Preferably, the hydration kinetic diameter of the cerium tannate nanozyme is 20 to 30 nm.

[0021] Furthermore, the Zeta potential of the cerium tannate nanozyme is -20 to -40 mV.

[0022] Furthermore, the tetravalent cerium salt is cerium sulfate Ce(SO4)2.

[0023] As a preferred embodiment, the preparation method is to add TA solution (86 mM, 500 μL) and Ce(SO4)2 solution (18 mM, 800 μL) to deionized water (40 mL), adjust the pH value to 10 with NaOH solution (2 M), and react for 3 hours. At this time, the particle size of the TA-Ce nanozyme obtained is the smallest, the hydration kinetic diameter is 25.6 nm, and the Zeta potential is -30.4 mV.

[0024] The present invention also provides a cerium tannate nanozyme prepared by the above preparation method.

[0025] Oxidative stress, mitochondrial damage, endoplasmic reticulum stress, inflammation and apoptosis have complex interactions in the development of acute kidney injury (AKI). There is still a lack of safe and effective therapeutic drugs for the treatment of AKI. Some interventional drugs are often used clinically to alleviate oxidative damage and inflammatory responses. However, these drugs cannot comprehensively regulate the multiple pathological mechanisms of AKI, and thus it is difficult to quickly reverse renal tissue damage and restore renal function. The present invention found that tannic acid cerium nanozyme TA-Ce can mimic a variety of antioxidant enzymes through valence transfer, thereby showing a broad-spectrum antioxidant capacity. In vitro experiments showed that TA-Ce can remove excess ROS, restore the ATP level and mitochondrial membrane potential of cells, and thus have a protective effect on human renal cortical proximal tubule epithelial cells (Human kidney-2, HK-2). In addition, TA-Ce can also promote the transformation of macrophages from M1 phenotype to M2 phenotype, thereby inhibiting inflammatory response. In vivo experiments showed that compared with the same dose of N-acetylcysteine ​​(NAC), TA-Ce had a stronger therapeutic effect on rhabdomyolysis-induced AKI (RM-AKI) and cisplatin-induced AKI (CP-AKI). TA-Ce effectively reduced the levels of blood ureanitrogen (BUN), creatinine (CRE) and lactate dehydrogenase (LDH) in serum, indicating that it can effectively protect kidney function. TA-Ce can inhibit the cascade of oxidative stress, mitochondrial autophagy, endoplasmic reticulum stress, inflammation and apoptosis, thereby breaking the vicious cycle and effectively alleviating AKI. Mechanistic studies have shown that TA-Ce nanozymes can restore mitochondrial autophagy flux by inhibiting the P62-LC3 signaling pathway and alleviate endoplasmic reticulum stress by inhibiting the IRE1-XBP1s pathway. This in turn prevented the activation of the Bcl-2-Bax-Cyt-c-Cleaved Casp-3 apoptotic pathway and the NF-κB inflammatory pathway, ultimately improving RM-AKI.

[0026] Therefore, the present invention provides the use of the above-mentioned cerium tannate nanozyme in preparing a product for targeting the kidney to treat acute kidney injury.

[0027] Furthermore, the acute kidney injury is induced by rhabdomyolysis and / or cisplatin.

[0028] The present invention also provides a medicine comprising the cerium tannate nanozyme.

[0029] The TA-Ce nanozyme of the present invention can specifically bind to mitochondrial outer membrane proteins. This targeted delivery can enable the nanozyme to effectively remove mitochondrial ROS, reverse mitochondrial depolarization and restore mitochondrial membrane potential. TA-Ce nanozyme can also regulate macrophage polarization, transforming it from a pro-inflammatory M1 phenotype to an anti-inflammatory M2 phenotype. In vivo distribution studies have shown that due to the small particle size and surface properties of TA-Ce, it can be enriched in damaged kidneys. In RM-AKI and CP-AKI models, the efficacy of TA-Ce is better than that of the traditional anti-inflammatory drug NAC. Mechanism studies have shown that TA-Ce promotes mitochondrial autophagy and removes damaged mitochondria by normalizing the P62-LC3 signaling pathway; by inhibiting the IRE1-XBP1s signaling pathway, TA-Ce reduces endoplasmic reticulum stress and prevents the accumulation of misfolded proteins, thereby inhibiting downstream inflammation and apoptosis pathways. TA-Ce can simultaneously target oxidative stress, mitochondrial dysfunction, ER stress, inflammation and apoptosis, and can comprehensively regulate the complex pathophysiological process of AKI, and is expected to be applied to other oxidative stress-related diseases. Therefore, TA-Ce nanozymes can be used in the preparation of drugs. The present invention provides a new strategy for the treatment of AKI and lays a foundation for the development of metal polyphenol nanomaterials as a therapeutic strategy for clinical AKI.

[0030] Compared with the prior art, the present invention has the following beneficial effects: The present application provides a method for preparing tannic acid cerium nanozyme, which adopts a self-assembly method, and obtains tannic acid cerium nanozyme with a smaller hydration kinetic diameter and significantly different Zeta potential by screening the molar ratio of tannic acid to tetravalent cerium salt and pH conditions. The TA-Ce nanozyme obtained by the preparation method of the present invention can maintain stability and good dispersibility in a physiological environment, has good biocompatibility, low hemolysis rate (hemolysis rate <5% at high concentration), and low systemic toxicity, and significantly improved safety. The TA-Ce nanozyme prepared by the present invention has good kidney targeting, can be effectively enriched in damaged kidneys, and utilizes Ce 3+ and Ce 4+ The unique redox cycle between the two states can remove multiple ROS and relieve oxidative stress, which has a good therapeutic effect in the treatment of rhabdomyolysis and / or cisplatin-induced acute kidney injury. This study lays a foundation for the use of metal polyphenol nanomaterials in the treatment of acute kidney injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Preparation and characterization of TA-Ce. Figure 1 A is the effect of pH on the particle size of TA-Ce; B is the effect of the molar ratio of raw materials on the particle size of TA-Ce; C is the particle size distribution diagram and transmission electron microscope image of TA-Ce; D is the potential and Tyndall effect of TA-Ce.

[0032] Figure 2 is the relative hemolysis rate of TA-Ce.

[0033] Figure 3 The stability of TA-Ce in PBS for 7 days.

[0034] Figure 4 is the in vitro ROS scavenging ability of TA-Ce. Figure 4 A, B, C, D, and E in the figure are respectively the TA-Ce pairs of ·O 2- , ·OH, PTIO·, DPPH· and ABTS· + F is the amount of O2 produced by H2O2 catalyzed by different concentrations of TA-Ce within 10 min; G is the POD-like activity of TA-Ce.

[0035] Figure 5 Cell viability after HK-2 cells were incubated with different concentrations of TA-Ce for 24 hours.

[0036] Figure 6 Fluorescence images and fluorescence semi-quantitative results of nanoparticle uptake by HK-2 cells stimulated by H2O2 after incubation for different times.

[0037] Figure 7 Cell survival rate of HK-2 cells stimulated with (A) cisplatin or (B) H2O2 after treatment with different concentrations of TA-Ce.

[0038] Figure 8 Fluorescence images and fluorescence semi-quantitative results of AM / PI staining after HK-2 cells stimulated by H2O2 were treated with different concentrations of TA-Ce.

[0039] Fig. 9 The intracellular ATP content of HK-2 cells after treatment with H2O2.

[0040] Fig.10 For imaging and semi-quantitative analysis of intracellular ROS. Fig.10 A and B are the ROS fluorescence images and semi-quantitative results of HK-2 cells stimulated by H2O2 24 h after treatment; C and D are the ROS levels and semi-quantitative results of each group of cells detected by flow cytometry.

[0041] Fig.11 Confocal images of mitochondrial membrane potential of HK-2 cells after different treatments.

[0042] Fig.12 This is the effect of TA-Ce on the survival rate of RAW 264.7 cells.

[0043] Fig.13is the effect of TA-Ce on macrophage polarization. Fig.13 A in the figure is the Western blot analysis and semi-quantitative results of CD86 and CD206 on LPS-stimulated macrophages after being treated with different concentrations of TA-Ce; B is the immunostaining image and semi-quantitative results of iNOS; C is the immunostaining image and semi-quantitative results of Arg-1.

[0044] Fig.14 Schematic diagram of the establishment and treatment schedule of the RM-AKI mouse model.

[0045] Fig.15 Fluorescence images of major organs (heart, liver, spleen, lung, and kidney) of RM-AKI mice 6 h after intravenous injection of RhB-labeled TA-Ce and semi-quantitative analysis of fluorescence intensity.

[0046] Fig.16 Representative kidney photos of RM-AKI mice after treatment with each preparation.

[0047] Fig.17 is the kidney coefficient of RM-AKI mice under different treatments.

[0048] Fig.18 Serum BUN, CRE, and LDH levels in RM-AKI mice 24 h after different treatments.

[0049] Fig.19 H&E staining images of kidney sections in different treatment groups of RM-AKI.

[0050] Fig. 20 These are the DHE staining fluorescence images and fluorescence semi-quantitative results of renal tissues of mice in each group of RM-AKI.

[0051] Fig.21 Figure 2 shows the serum SOD level and the GSH level in renal tissue homogenate of RM-AKI mice 24 h after different treatments.

[0052] Fig. 22 These are the fluorescence images and fluorescence semi-quantitative results of TUNEL staining of kidney sections in different treatment groups of RM-AKI.

[0053] Fig.23 The levels of TNF-α, IL-6 and IL-1β in the serum of mice in different treatment groups of RM-AKI.

[0054] Fig.24 This is to analyze and detect the therapeutic mechanism of TA-Ce on RM-AKI mice. Fig.24The Western blot analysis and semi-quantitative results of AL in the cells were IRE1, p-IRE1, XBP1s, TRAF2, NF-κB, JNK, p-JNK, P62, LC3, Bcl-2, Bax, Cyt-c, Cleaved Casp-3, and KIM-1.

[0055] Fig.25 is the therapeutic effect of TA-Ce on CP-AKI mice. Fig.25 A is a schematic diagram of the establishment of the CP-AKI mouse model and the treatment schedule; B is the kidney coefficient of CP-AKI mice in different treatment groups; CE is the serum BUN, CRE and LDH levels of CP-AKI mice 24 hours after different treatments; F is the H&E staining image of kidney sections in different treatment groups.

[0056] Fig.26 is the therapeutic effect of TA-Ce on CP-AKI mice. Fig.26 A in the figure is the DHE staining fluorescence image and fluorescence semi-quantitative results of renal tissues of mice in each group of CP-AKI; BD are the serum SOD levels and MDA and GSH levels in renal tissue homogenates of CP-AKI mice 24 h after different treatments.

[0057] Fig. 27 is the therapeutic effect of TA-Ce on CP-AKI mice. Fig. 27 A in the figure is the TUNEL staining fluorescence images and fluorescence semi-quantitative results of kidney sections in different CP-AKI treatment groups; BC are the TNF-α and IL-6 levels in the serum of mice in different CP-AKI treatment groups.

[0058] Fig.28 Evaluation of the biocompatibility of TA-Ce in vivo. Fig.28 A shows the weight change trend of healthy mice after being treated with PBS or TA-Ce for 7 days; BH shows the blood biochemistry and whole blood analysis of healthy mice after different treatments; I shows the H&E staining images of the main organs (heart, liver, spleen, lung and kidney) of each group.

[0059] Fig.29 Schematic diagram of the action pathway of TA-Ce nanozyme. DETAILED DESCRIPTION

[0060] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0061] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0062] Example 1 Preparation and characterization of TA-Ce nanozyme 1. Experimental Methods 1. Preparation of TA-Ce nanozymes. TA-Ce was prepared by self-assembly method. TA solution (86 mM, 500 μL) and Ce(SO4)2 solution (18 mM, 800 μL) were added to deionized water (40 mL). The pH value was adjusted to 10 with NaOH solution (2 M) and reacted for 3 hours. TA-Ce was dispersed in phosphate buffered saline (PBS, 0.01 M, pH 7.4), lyophilized into powder and set aside.

[0063] 2. Characterization of TA-Ce nanozymes. The hydration kinetic diameter, polymer dispersity index (PDI) and Zeta potential of TA-Ce nanozymes were measured using a Malvern Zetasizer Nano Instrument (ZEN3600, UK). The morphology of TA-Ce nanozymes was observed using a transmission electron microscope (TEM) (Hitachi-HT7700, Japan).

[0064] 3. Blood compatibility test of TA-Ce nanozyme. Fresh mouse blood was stirred with a glass rod to remove fibrin and obtain defibrinated blood. Then, the defibrinated blood was mixed with PBS and centrifuged at 4000 rpm for 5 min to separate the red blood cells. The red blood cells were diluted with PBS to obtain a 2% (v / v) red blood cell suspension. Different concentrations of TA-Ce nanozyme solution (20-1000µg / mL) were thoroughly mixed with the red blood cell suspension. 1% Triton X-100 and PBS were used as positive (100% hemolysis) and negative (0% hemolysis) controls, respectively. Each group of samples was incubated at 37°C for 2 h and centrifuged at 4000 rpm for 5 min. The absorbance of each sample was measured at a wavelength of 560 nm using an enzyme reader (BiotekSYNERGY H1 Instruments, Inc.) to calculate the hemolysis rate. Hemolysis rate (%) = {(A 样品 -A PBS ) / (A TritonX-100 -A PBS )}×100%.

[0065] 4. Dispersibility and stability of TA-Ce nanozymes. In order to evaluate the long-term stability of TA-Ce, TA-Ce nanozymes were stored at 4°C for 7 days, and the hydration kinetic size and PDI of the nanoparticles were monitored by dynamic light scattering (DLS).

[0066] 5. In vitro characterization of ROS scavenging ability of TA-Ce nanozymes (1) O2 - To evaluate the SOD-like activity of TA-Ce, the samples were reacted with the reagents in the kit according to the manufacturer’s instructions (Solabo, China), and the absorbance was then measured using a microplate reader (Biotek SYNERGY H1Instruments, Inc.).

[0067] (2) ·OH scavenging activity. The deoxyribose test was used to evaluate the ability of TA-Ce to scavenge ·OH. Deionized water, deoxyribose, EDTA-Na2, and FeCl3 solutions were added to TA-Ce at different concentrations (1-1000 μg / mL) and mixed thoroughly. Then, ascorbic acid solution was added to start the reaction and incubated in a 50°C metal bath for 20 min. TBA / TCA mixture (v / v=3:5) was added and incubated in a 100°C metal bath for 15 min. The samples were transferred to a 96-well plate and the absorbance was measured at a wavelength of 532 nm.

[0068] (3) 1,1-Diphenyl-2-picrylhydrazyl (DPPH) scavenging experiment. DPPH was mixed with an equal volume of TA-Ce solution of different concentrations (1-200 μg / mL) and incubated at room temperature for 30 min. The absorbance at a wavelength of 517 nm was measured and the DPPH scavenging activity was calculated.

[0069] (4) 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulphonate), ABTS + ) clearance experiment. Equal volumes of ABTS solution (7.4 mM) and K2S2O8 solution (2.6 mM) were thoroughly mixed and incubated at room temperature for 18 h. Different concentrations (1-400 μg / mL) of TA-Ce were added to the ABTS working solution and incubated at room temperature for 5 min. The absorbance at 405 nm was measured and the ABTS· + Scavenging activity.

[0070] (5) 1-lambda1-Oxidanyl-4,4,5,5-tetramethyl-3-oxido-2-phenylimidazol-3-ium (PTIO·) scavenging experiment. Nanoparticles of different concentrations (1-1000 μg / mL) were mixed with equal volumes of PTIO· solution. The absorbance at 557 nm was measured and the scavenging activity of PTIO· was calculated.

[0071] (6) H2O2 scavenging ability. To determine the CAT-like activity of TA-Ce, 30% H2O2 (1.2 mL) was added to PBS (8.4 mL), and then nanoparticles of different concentrations (25-400 μg / mL, 200 μL) were added. A dissolved oxygen electrode (Strater 400D, Ohaus, China) was immersed in the solution to monitor the dissolved oxygen concentration in real time. The measurement was performed once every minute for 10 minutes.

[0072] (7) POD-like activity. Nanoparticles at different concentrations (2-200 μg / mL), 30% H2O2, and 3,3',5,5'-tetramethylbenzidine (TMB, 25 mM) were added to acetic acid / sodium acetate buffer (0.1 M, pH 4.5). The reaction was monitored by measuring the absorbance of oxidized TMB at a wavelength of 652 nm.

[0073] 2. Experimental Results 1. Preparation and characterization of TA-Ce nanozymes As a kind of polyphenol, TA can chelate with metal ions through ligand interaction to form metal polyphenol nanoparticles. Figure 1 As shown in Figure 1, TA-Ce was synthesized by a one-pot method. The molar ratio of the raw materials and the pH value of the solution are the key factors affecting the particle size of TA-Ce. The study showed that when the pH was 10 and the molar ratio of TA:Ce was 3:1, the particle size of TA-Ce was the smallest ( Figure 1 A and B). The hydration kinetic diameter, PDI and Zeta potential of TA-Ce dispersed in PBS were determined by DLS. The hydration kinetic diameter was 25.6 nm and the PDI was 0.208 ( Figure 1 C), Zeta potential is -30.4 mV ( Figure 1 D). At the same time, the morphological characteristics of TA-Ce nanozymes are as follows Figure 1 C, the Tyndall effect is Figure 1 As shown in D.

[0074] 2. The results of in vitro hemolysis experiment of TA-Ce nanozyme are as follows Figure 2As shown, even at a high concentration of 1000 μg / mL, the hemolysis rate of TA-Ce was still less than 5%, indicating that the nanoparticles had good blood compatibility.

[0075] 3. Dispersibility and stability of TA-Ce nanozymes Figure 3 As shown, the particle size and PDI of TA-Ce remained almost unchanged at 4 °C for 7 days, indicating that the nanoparticles were stable and well dispersed.

[0076] 4. ROS scavenging ability of TA-Ce nanozymes The pathological process of AKI is closely related to the excess of reactive oxygen and reactive nitrogen. Therefore, scavenging free radicals is essential for protecting the kidneys from oxidative damage. TA is a polyphenolic compound found in a variety of plants that has the functions of scavenging ROS, chelating metal ions, and inhibiting lipid peroxidation. 3+ and Ce 4+ The cerium nanoparticles can switch between oxidation states and exhibit strong antioxidant properties. This redox cycle enables the cerium nanoparticles to mimic the activity of antioxidant enzymes such as SOD and CAT, thereby effectively scavenging superoxide radicals and H2O2. TA-Ce prepared by assembling TA with cerium can scavenge a variety of free radicals.

[0077] like Figure 4 As shown in the figure, TA-Ce exhibits significant scavenging activity, and its scavenging activity is closely related to its concentration. At a concentration of 20 μg / mL, TA-Ce can scavenge about 91% of ·O2 - ( Figure 4 A), showing SOD-like activity. At a concentration of 200 μg / mL, TA-Ce removed more than 37% of ·OH ( Figure 4 B). In addition, TA-Ce also has a strong scavenging effect on oxygen free radical PTIO·. At a concentration of 200 μg / mL, the scavenging rate reaches 81% ( Figure 4 C). To further confirm the antioxidant properties of TA-Ce, its scavenging ability against nitrogen free radicals was evaluated, such as Figure 4 As shown in D, TA-Ce was able to remove about 40% of DPPH· at a low concentration (10 μg / mL). In addition, the removal of ABTS· by TA-Ce was also evaluated. + At a concentration of 50 μg / mL, TA-Ce almost removed 100% of ABTS· + ( Figure 4E). H2O2 solution was incubated with various concentrations of TA-Ce, and the ability of TA-Ce to decompose H2O2 (CAT-like activity) was evaluated by monitoring the concentration of dissolved O2 in the solution. The results showed that TA-Ce could effectively catalyze the decomposition of H2O2. It is worth noting that with the increase of TA-Ce concentration (0 μg / mL to 400 μg / mL), the production of O2 increased from 10 mg / mL to 38.5 mg / mL ( Figure 4 F). The POD-like activity of TA-Ce in catalyzing the conversion of H2O2 into cytotoxic free radicals (such as ·OH) was evaluated by colorimetric reaction. The results showed that the POD-like activity of TA-Ce was low ( Figure 4 G). In conclusion, TA-Ce has a good affinity for a variety of free radicals (including ·OH, PTIO·, DPPH· and ABTS· + ) have good scavenging ability. TA-Ce catalyzes the cascade reaction of SOD and CAT to convert ·O2 - It can also convert H2O2 into non-toxic molecules, thus achieving antioxidant activity. In addition, due to its low POD-like activity, its effect in decomposing H2O2 into highly cytotoxic free radicals (·OH) is minimal.

[0078] Example 2 Cellular level study of TA-Ce nanozymes 1. Experimental Methods 1. Determination of the effect of TA-Ce nanozymes on cell viability HK-2 cells were cultured in MEMα medium containing 10% (v / v) fetal bovine serum (FBS) and 1% (v / v) penicillin-streptomycin (100 IU / mL) at 37°C and 5% CO2. HK-2 cells were plated at 1×10 4 The cells were seeded at a density of 100 μg / mL in a 96-well plate. Then different concentrations (10-600 μg / mL) of TA-Ce were added and incubated with the cells for 24 h.

[0079] 2. Effect of TA-Ce nanozymes on HK-2 cells stimulated by H2O2 (1) Cellular uptake. To prepare rhodamine B (RhB)-labeled TA-Ce, RhB solution (1 mg / mL, 1 mL) was added dropwise to TA-Ce dispersion (1 mg / mL, 1 mL) and stirred overnight. RhB-labeled TA-Ce was collected by centrifugation and washed three times with water to remove excess RhB.

[0080] RhB-labeled TA-Ce was used for cellular uptake experiments. RhB-labeled TA-Ce was incubated with H2O2 (400 μM)-stimulated HK-2 cells for 6 h or 12 h, respectively. After 4',6-diamidino-2-phenylindole (DAPI) staining and PBS washing, the fluorescence intensity in the cells was observed using a fluorescence microscope (CYT7UMW, Agilent, USA).

[0081] (2) Cell protection. 4 HK-2 cells were seeded in 96-well plates at a density of 10 μM. To evaluate the protective effect of nanoparticles against cisplatin- or H2O2-induced cell damage, cisplatin (10 μM) or H2O2-stimulated HK-2 cells were treated with different concentrations of TA-Ce for 24 h. Cell viability was then assessed using the MTT assay.

[0082] (3) Anti-apoptotic effect. The anti-apoptotic effect of TA-Ce was evaluated using the Calcein-AM / PI kit (C2015M, Beyotime Biotech, China). HK-2 cells stimulated with H2O2 were treated with different concentrations of TA-Ce (50, 100, or 200 μg / mL) for 24 h. The culture medium was discarded, the cells were washed three times with PBS, and then observed under an inverted fluorescence microscope (CYT7UMW, Agilent, USA). The percentage of PI-positive cells was quantified using ImageJ software.

[0083] (4) Determination of adenosine triphosphate (ATP) content. HK-2 cells were plated at 1×10 5 The density of 100 μg / mL was inoculated in a 12-well plate. After the HK-2 cells stimulated with H2O2 were treated with different concentrations of TA-Ce (50, 100 or 200 μg / mL) for 24 h, the intracellular ATP content was determined according to the instructions of the ATP detection kit (S0027, Beyotime Biotech, China).

[0084] (5) Cell antioxidant assay. HK-2 cells were cultured at 1×10 6The cells were seeded at a density of 100 μg / mL in 6-well plates. After the cells were treated with different concentrations of TA-Ce for 24 h, the fluorescent probe 2′,7′-dichlorofluorescein diacetate (DCFH-DA) was added and incubated with the cells at 37°C for 30 min. Fluorescence images were collected using an inverted fluorescence microscope (CYT7UMW, Agilent, USA), and ROS levels were quantified using a flow cytometer (CYT7UMW, Agilent, USA). Fluorescence images were semi-quantitatively analyzed by ImageJ software.

[0085] (6) Mitochondrial membrane potential (MMP) detection. HK-2 cells were plated at 1×10 4 The cells were seeded at a density of 100 μg / mL in a confocal culture dish and treated with different methods after the cells were fully adhered. The cells were incubated with the fluorescent probe in the JC-1 staining kit (C2003S, Bio-Tech, China) at 37°C for 20 min and then rinsed twice with PBS. The changes of MMP were observed under a confocal microscope (Nikon, Japan).

[0086] 4. Effect of TA-Ce on macrophage polarization RAW 264.7 cells were cultured in DMEM medium containing 10% (v / v) FBS and 1% (v / v) penicillin-streptomycin (100 IU / mL) at 37°C and 5% CO2. To evaluate the cytotoxicity of TA-Ce on RAW 264.7 cells, cells were plated at 1×10 4 The cells were seeded at a density of 1.54 μg / mL in a 96-well plate. Different concentrations of TA-Ce were added and incubated with the cells for 24 h.

[0087] (1) Western blot analysis. 6RAW264.7 cells were seeded into 6-well plates at a density of 100 μg / mL. Cells were stimulated with lipopolysaccharide (LPS, 100 ng / mL) and treated with different concentrations of TA-Ce for 24 h. Cell lysates were collected and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins in the lysates were separated using 10% SDS-PAGE and then transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% skim milk and Tris-buffered saline with tween (TBST) at room temperature for 2 h, then incubated with primary antibodies including CD86 (1:1000), CD206 (1:5000), and GAPDH (1:20000) at 4°C overnight, followed by incubation with horseradish peroxidase-conjugated secondary antibodies and detected by enhanced chemiluminescence. Images were acquired using a multifunctional gel imaging system (ChemiDoc™ Imaging System, Bio-Rad, USA), and protein bands were quantified using ImageJ software.

[0088] (2) Immunofluorescence staining. RAW 264.7 cells were fixed with 4% paraformaldehyde for 10 min and blocked with 5% bovine serum albumin for 1 h at 37°C. Subsequently, cells were incubated with primary antibodies against inducible nitric oxide synthase (iNOS) (1:500) and arginase-1 (Arg-1) (1:500) overnight. Cells were stained with DAPI for 15 min, and images were captured using a fluorescence microscope (CYT7UMW, Agilent, USA). Fluorescence images were semi-quantitatively analyzed using ImageJ software.

[0089] 2. Experimental Results 1. The potential nephrotoxicity of nanoparticles is a major problem, which greatly limits their application in the treatment of AKI. Therefore, the toxicity of nanoparticles to HK-2 cells was evaluated. Figure 5 As shown, when the concentration of TA-Ce was lower than 400 μg / mL, the survival rate of HK-2 cells remained above 95%, indicating its good safety.

[0090] 2. Effect of TA-Ce nanozymes on HK-2 cells stimulated by H2O2 (1) The uptake effect of TA-Ce by HK-2 cells Figure 6As shown in the figure, by comparing the cellular uptake at two time points (6 h and 12 h), there was no significant difference in the uptake of nanoparticles by healthy HK-2 cells. In contrast, the uptake of TA-Ce by cells stimulated with H2O2 was significantly higher than that of healthy cells at both time points.

[0091] (2) Tubular injury in AKI occurs earlier than glomerular injury, and damage to tubular epithelial cells triggers a series of events that aggravate renal dysfunction and accelerate disease progression. CP (Cisplatin) is a chemotherapeutic drug widely used to treat a variety of solid tumors, but its nephrotoxicity can adversely affect clinical outcomes. Previous studies have shown that a single high-dose of CP can induce tubular cell damage. In order to evaluate the protective effect of TA-Ce against AKI, 10 μM CP was used to induce HK-2 cell damage, and the survival rate of HK-2 cells dropped to approximately 22%. The results are shown in Figure 2. Figure 7 As shown in A, TA-Ce can increase the viability of HK-2 cells in a concentration-dependent manner. When the concentration of TA-Ce reached 200 μg / mL, the cell viability increased to 72%. These results indicate that TA-Ce has a significant protective effect against CP-induced HK-2 cell injury.

[0092] Excessive production of ROS can lead to persistent renal oxidative stress, resulting in renal damage in patients with AKI. To investigate whether the renal protective effect of TA-Ce is related to the reduction of ROS levels, HK-2 cells stimulated with H2O2 were used for the study. Figure 7 As shown in Figure B, TA-Ce can increase the cell survival rate from 19% to 98% in a dose-dependent manner. These results indicate that TA-Ce has a significant protective effect against HK-2 cell injury stimulated by H2O2.

[0093] (3) AM / PI double staining experiment Figure 8 As shown, the increase of ROS can lead to cell death, which can be confirmed by the increase in the number of dead cells (PI-positive cells) in the model group. TA-Ce treatment can reduce the number of dead cells, indicating that TA-Ce can protect H2O2-stimulated cells from ROS-induced damage.

[0094] (4) Compared with healthy cells, H2O2 treatment induced pathological damage, resulting in reduced ATP production. Fig. 9 As shown, the addition of TA-Ce significantly increased the intracellular ATP level, thereby restoring cell vitality and function.

[0095] (5) DCFH-DA is a ROS probe that is widely used for imaging and semi-quantitative analysis of intracellular ROS. Fig.10As shown in A and B, the intracellular ROS level in the negative control group was low, showing a weak signal under the fluorescence microscope. After stimulation with H2O2, the fluorescence intensity of the model group increased significantly, indicating that the intracellular ROS level increased. Treatment of HK-2 cells stimulated with H2O2 with different concentrations of TA-Ce significantly reduced the intracellular ROS level. In addition, as the concentration of TA-Ce increased, the intracellular ROS level gradually decreased and approached the level of the control group. The flow cytometry results further confirmed the ROS scavenging ability of TA-Ce ( Fig.10 C and D).

[0096] (6) MMP is an important indicator of mitochondrial function. Therefore, the JC-1 fluorescent probe was used to evaluate changes in MMP. When MMP is high, JC-1 will aggregate in the mitochondrial matrix and emit red fluorescence. Fig.11 As shown in the figure, the MMP level in HK-2 cells was significantly reduced after H2O2 stimulation. After TA-Ce treatment, MMP was restored, indicating that TA-Ce has a protective effect on mitochondrial function. This may be due to the ability of TA-Ce to target mitochondria by binding to mitochondrial outer membrane proteins, voltage-dependent anion channels, and outer membrane transporters, thereby effectively clearing excess reactive oxygen species in mitochondria and reversing mitochondrial depolarization. In conclusion, TA-Ce exhibits excellent antioxidant activity, indicating its potential application value in the treatment of AKI.

[0097] 3. Effect of TA-Ce on macrophage polarization Since oxidative stress plays a key role in macrophage polarization in inflammatory-related diseases, and macrophages are crucial in the occurrence and development of AKI, the effect of TA-Ce on macrophage polarization was evaluated. Macrophages exhibit different phenotypes depending on the microenvironment and activation state. M1 macrophages, also known as proinflammatory macrophages, can secrete a variety of proinflammatory cytokines, including TNF-α, IL-1β, IL-6, and nitric oxide (NO), which are mainly involved in the initiation and expansion of inflammation. In contrast, M2 macrophages exhibit an anti-inflammatory phenotype and can secrete anti-inflammatory cytokines such as IL-10 to promote immune regulation and tissue remodeling. First, the MTT method was used to evaluate the cytotoxicity of TA-Ce on macrophages. The results are shown in Figure 2. Fig.12 As shown, when the TA-Ce concentration was lower than 200 μg / mL, the viability of RAW 264.7 cells remained above 86%.

[0098] Previous studies have shown that TA can inhibit LPS-induced macrophage polarization toward the M1 phenotype. In this study, the effect of TA-Ce on macrophage polarization was systematically evaluated. CD86 and CD206 were used as markers of the M1 and M2 phenotypes, respectively, and their expression levels were evaluated by Western blotting. Fig.13 As shown in A, with the increase of TA-Ce concentration, the expression of CD86 decreased significantly, while the expression of CD206 increased significantly. The effect of nanoparticles on macrophage phenotypic transformation was further verified by immunofluorescence staining. It is reported that iNOS is highly expressed in M1 macrophages and is a common marker for identifying the M1 phenotype. LPS treatment can significantly increase the expression of iNOS in macrophages. Fig.13 As shown in B, the fluorescence intensity of the LPS-treated model group was higher than that of the healthy control group. However, treatment with different concentrations of TA-Ce significantly reduced the fluorescence intensity of iNOS in M1 macrophages. In addition, immunofluorescence staining of Arg-1, a marker of M2 macrophages, was performed. Fig.13 As shown in Figure C, compared with the model group, the mean fluorescence intensity of Arg-1 in macrophages was significantly increased after treatment with different concentrations of TA-Ce. These results indicate that TA-Ce can effectively prevent macrophages from polarizing to the M1 phenotype and promote their transformation to the M2 phenotype, thereby improving the inflammatory microenvironment of AKI.

[0099] Example 3 Establishment of mouse AKI model and in vivo therapeutic effect of TA-Ce 1. Experimental Methods 1. Establishment of mouse AKI model Institute of Cancer Research (ICR) mice (6–8 weeks, 20–25 g, female) were provided by Guangzhou Mindray Biotechnology Co., Ltd. (Guangzhou, China). All animal experiments were in accordance with the Guide for the Care and Use of Laboratory Animals and experimental protocols approved by the Institutional Animal Care and Use Committee of Sun Yat-sen University.

[0100] Rhabdomyolysis-induced acute kidney injury (RM-AKI) mouse model. Schematic diagram of the establishment and treatment schedule of the RM-AKI mouse model is shown in the figure. Fig.14 As shown, 50% glycerol (10 mL / kg) was injected intramuscularly into the hind limbs of ICR mice. In the treatment groups, TA-Ce (15 mg / kg) or an equivalent dose of N-acetylcysteine ​​(NAC) was injected intravenously 2 h after glycerol injection. 24 h later, the mice were euthanized, and blood and kidney samples were collected.

[0101] Cisplatin-induced acute kidney injury (CP-AKI) mouse model. Schematic diagram of the establishment and treatment schedule of the CP-AKI mouse model is shown in the figure. Fig.25 As shown in A, ICR mice were injected intraperitoneally with cisplatin (18 mg / kg). The treatment groups were injected intravenously with TA-Ce (10 mg / kg) or an equal dose of NAC 2 h and 26 h after cisplatin injection, respectively. Three days after cisplatin injection, mice were euthanized. Blood samples and kidney tissues were collected for analysis.

[0102] 2. Biodistribution of TA-Ce in RM-AKI mice In the RM-AKI mouse model, the biodistribution of nanoparticles was studied by intravenous injection of RhB-labeled TA-Ce (15 mg / kg). Six hours after injection, the mice were euthanized, and the main organs were collected and imaged using a small animal in vivo imaging system (PerkinElmer IVIS Lumina Series III, USA).

[0103] 3. In vivo therapeutic effect of TA-Ce In the RM-AKI and CP-AKI mouse models, mice were randomly divided into four groups. Group 1: healthy mice injected with PBS; Group 2: AKI mice injected with PBS; Group 3: AKI mice injected with NAC; Group 4: AKI mice injected with TA-Ce. RM-AKI mice were euthanized 24 h after intravenous injection, and CP-AKI mice were euthanized three days after cisplatin injection. Blood and kidney samples were collected for subsequent experimental analysis.

[0104] (1) Kidney coefficient. Mice were weighed before euthanasia, and kidneys were collected, washed, and weighed. The kidney coefficient was calculated using the following formula: kidney coefficient (%) = kidney weight (mg) / body weight (g) × 100%.

[0105] (2) Renal function test. After euthanasia, blood samples were collected from mice. Serum was obtained by centrifugation at 4000 rpm and 4°C for 10 min. Renal function was assessed using a BUN kit (BC1535, Solebao, China), a CRE kit (C011-2-1, Nanjing Jiancheng, China), and a LDH kit (A020-2-2, Nanjing Jiancheng, China) according to the manufacturer's instructions.

[0106] (3) Hematoxylin-eosin (H&E) staining. After euthanasia of mice, kidneys were collected and fixed with 4% paraformaldehyde. Samples were embedded in paraffin, sections were prepared and H&E staining was performed. Images were acquired using a microscope (CYT7UMW, Agilent, USA).

[0107] (4) Evaluation of in vivo antioxidant properties. The SOD level in the serum of each group was analyzed using a commercial kit (BC165, Solebao, China) according to the manufacturer's instructions. Kidney tissue homogenate was prepared, and the GSH level was determined using a Glutathione (GSH) kit (BC1175, Solebao, China). The MDA level was determined using a Malondialdehyde (MDA) detection kit (BC0025, Solebao, China). To evaluate the generation of ROS, the kidney tissue was cryosectioned, and 5 μm thick frozen kidney tissue sections were prepared, stained with 10 μM dihydroethidium (DHE) for 30 min, washed three times with PBS, and counterstained with DAPI for 20 min. The sections were mounted with anti-fluorescence attenuation medium. Fluorescence images were acquired using an inverted fluorescence microscope (CYT7UMW, Agilent, USA).

[0108] (5) Anti-apoptotic effect in vivo. Apoptotic cells were stained using the Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) detection kit (C1088, Bio-Tech, China) according to the manufacturer's instructions. After washing three times with PBS, the cell nuclei were counterstained with DAPI. Fluorescence images were acquired using an inverted fluorescence microscope (CYT7UMW, Agilent, USA).

[0109] (6) In vivo anti-inflammatory properties. The levels of TNF-α, IL-6, and IL-1β in serum were measured using enzyme-linked immunosorbent assay kits (Elabscience, ELISA, China) according to the manufacturer's instructions.

[0110] 4. The therapeutic mechanism of TA-Ce on RM-AKI mice (1) Western blot detection. Kidney tissue homogenates were prepared and subjected to immunoblotting to evaluate the levels of key proteins involved in mitochondrial autophagy, endoplasmic reticulum stress, inflammation, and apoptosis. Proteins in the lysate were separated by 10% SDS-PAGE and transferred to a PVDF membrane. The membrane was blocked with 5% skim milk plus TBST at room temperature for 2 h. Primary antibodies were added and incubated overnight at 4°C, including CD86 (1:1000), CD206 (1:5000), anti-IRE1 (1:1000), p-IRE1 (1:1000), TRAF2 (1:1000), JNK (1:1000), p-JNK (1:1000), NF-κB (1:1000), XBP1s (1:2000), P62 (1:5000), LC3 (1:1000), KIM-1 (1:1000), Bcl-2 (1:2000), Bax (1:6000), Cyt-c (1:1000), Casp-3 (1:500), β-actin (1:5000), and GAPDH (1:20000). Then, the samples were incubated with secondary antibodies conjugated to horseradish peroxidase and detected by enhanced chemiluminescence. Images were acquired using a multifunctional gel imaging system (ChemiDoc™ Imaging System, Bio-Rad, USA). The intensity of protein bands was semi-quantitatively analyzed using ImageJ software.

[0111] 5. In vivo biocompatibility test. TA-Ce (15 mg / kg) was injected into the tail vein of mice once every other day for a total of three times. PBS was used as a control. The body weight of mice was monitored daily and a body weight curve was drawn. After seven days, the mice were euthanized, and serum and whole blood samples were collected to evaluate relevant biochemical indices. Major organs, including heart, liver, spleen, lung, and kidney, were collected, sliced, and stained with H&E.

[0112] 2. Experimental Results 1. Biodistribution of TA-Ce in mice. Fig.15 As shown in the figure, 6 h after tail vein injection of RhB-labeled TA-Ce, nanoparticles were observed to accumulate in the kidneys of AKI mice, about twice as much as in healthy mice. During AKI, the permeability of the glomerular filtration barrier increases due to structural damage, inflammation, and loss of capsule cells. The increase in permeability allows nanoparticles to pass through the barrier and accumulate in the kidneys. In addition, damaged renal cells show a stronger endocytic capacity, further promoting the uptake of nanoparticles.

[0113] 2. Use the commonly used anti-inflammatory drug NAC as a control to compare the therapeutic effects of TA-Ce and NAC on AKI at the same dose. Fig.16Macroscopic photos showed that the kidneys of healthy mice were pink, while the kidneys of RM-AKI mice were swollen, khaki and lackluster, indicating kidney damage. Notably, the kidneys of RM-AKI mice improved significantly after TA-Ce treatment. At a dose of 15 mg / kg, the kidneys of the TA-Ce-treated group showed no obvious signs of swelling and remained pink like the kidneys of the healthy group, while the results of the NAC-treated group were comparable to those of the model group.

[0114] (1) In RM-AKI, the kidneys swell due to inflammation, cell proliferation, and congestion, resulting in an increase in the renal coefficient. Fig.17 As shown, the renal coefficient of the TA-Ce-treated group was significantly lower than that of the model group and the NAC group, close to the level of healthy mice, indicating that TA-Ce can reduce renal swelling and effectively alleviate RM-AKI.

[0115] (2) In addition, serum levels of BUN and CRE, two key indicators of renal function, were also measured. Fig.18 As shown in the figure, after 24 h of treatment, the BUN and CRE levels in the model group were about 12 and 10 times those in the healthy group, respectively. In contrast, the BUN and CRE levels in the TA-Ce-treated group were significantly lower than those in the model group and the NAC group, close to the levels in healthy mice, further confirming the powerful therapeutic effect of TA-Ce on RM-AKI. During RM-AKI, the release of intracellular LDH into the blood indicates that cell membrane damage leads to irreversible cell death. To evaluate the cytoprotective effect of TA-Ce, serum LDH levels were measured. Compared with the model group and the NAC-treated group, the LDH levels in RM-AKI mice treated with TA-Ce were significantly reduced, close to normal levels, indicating that TA-Ce can effectively protect the renal cell membrane of RM-AKI mice from damage, thereby inhibiting the release of LDH.

[0116] (3) In kidney disease, denatured proteins will precipitate in the renal tubules to form cast structures, which can be used as a diagnostic marker for kidney pathological changes. Fig.19 As shown in the figure, the kidneys of both the model group and the NAC group had severe histological damage, including tubular casting and intratubular obstruction, tubular lumen dilation, and cell loss. In contrast, TA-Ce treatment significantly alleviated the histological damage in RM-AKI mice, indicating the protective potential of TA-Ce against renal injury.

[0117] (4) ROS generated during the reoxidation of ischemic tissues can cause cell damage and trigger a series of harmful cellular reactions. In order to further evaluate the ability of TA-Ce to remove ROS in vivo, DHE staining was performed on renal tissues. Fig. 20As shown, the red fluorescence of the model group was significantly enhanced, indicating that the level of ROS increased during RM-AKI. Compared with the model group and NAC group, treatment with TA-Ce significantly reduced the level of ROS in RM-AKI mice, indicating the excellent antioxidant capacity of TA-Ce. Therefore, clearing excess ROS in ischemic tissues is an effective strategy to alleviate RM-AKI.

[0118] SOD is a key enzyme in the antioxidant defense system, which protects cells and tissues from ROS damage by inhibiting the free radical chain reaction that triggers lipid peroxidation. GSH is the main antioxidant in cells, which can directly scavenge H2O2 and hydroxyl radicals, and plays an important role in neutralizing ROS and maintaining redox balance. During RM-AKI, both SOD activity and GSH levels decrease, thereby impairing the mitochondrial antioxidant system and making cells more susceptible to oxidative damage. Lipid peroxidation caused by oxidative stress releases catabolic byproducts such as MDA, which are both markers of renal damage and indirect indicators of ROS levels. Fig.21 As shown in Figure 3, serum SOD levels and renal tissue GSH levels in the model group were significantly decreased, indicating that RM-AKI significantly inhibited SOD activity and GSH levels. However, TA-Ce could effectively restore SOD activity and GSH levels to close to normal values.

[0119] (5) Apoptosis of renal tubular cells plays a key role in the pathogenesis of RM-AKI, as extensive apoptosis leads to the loss of nephrons and ultimately accelerates the progression of the disease. Therefore, the anti-apoptotic effect of TA-Ce was further evaluated using TUNEL staining. Fig. 22 As shown, quantitative analysis showed that TA-Ce treatment significantly reduced the proportion of TUNEL-positive cells in the renal tissue of RM-AKI mice compared with the model group and NAC group.

[0120] (6) During the pathological progression of RM-AKI, infiltrating immune cells can mediate inflammatory damage and repair, among which M1 macrophages can aggravate damage by releasing proinflammatory cytokines. Therefore, ELISA kits were used to evaluate the levels of various inflammatory cytokines in serum. The results are shown in Figure 2. Fig.23 As shown in the results, the levels of proinflammatory cytokines (i.e., TNF-α, IL-6, and IL-1β) in the serum of RM-AKI mice were increased, while the levels of each proinflammatory cytokine in the TA-Ce treatment group were significantly reduced, indicating that TA-Ce can alleviate RM-AKI by inhibiting the production of proinflammatory cytokines. In general, TA-Ce can improve the renal function and alleviate renal injury in RM-AKI mice by reducing oxidative stress, apoptosis, and inflammatory response.

[0121] 3. AKI is usually caused by renal ischemia-reperfusion injury or nephrotoxicity, manifested as a rapid loss of renal function. The endoplasmic reticulum (ER) is an important organelle involved in protein synthesis, folding, and post-translational modification. Under cellular stress conditions such as ischemia-reperfusion injury, the accumulation of unfolded or misfolded proteins in the ER lumen activates the unfolded protein response (UPR), a cellular mechanism designed to restore protein balance. However, if ER stress persists for too long or is excessive, the UPR triggers apoptotic pathways, leading to cell death. It has been reported that ER stress plays an important role in the progression of various AKI models. For example, in ischemia-reperfusion injury, activation of the UPR can lead to upregulation of C / EBP homologous protein (CHOP) and glucose-regulated protein 78 (GRP78), thereby promoting apoptosis and inflammation in the post-ischemic kidney. Similarly, in CP-AKI, activation of the UPR leads to increased levels of CHOP and GRP78, further promoting ER stress and apoptosis. Mitochondrial function is crucial to the pathophysiology of kidney-related diseases, as the metabolic demands of the kidney are high and the content of mitochondria in the kidney is second only to that of the heart. Damaged mitochondria promote excessive ROS production, which in turn further impairs mitochondrial function and increases the risk of cell death. Mitophagy is a selective form of autophagy that can eliminate damaged or dysfunctional mitochondria and plays a key role in mitochondrial quality control. This process involves the recognition and engulfment of damaged mitochondria by autophagosomes, followed by fusion with lysosomes for degradation. Selective elimination of damaged mitochondria is essential for cell survival and helps prevent the release of pro-apoptotic factors such as cytochrome c (Cyt-c). In AKI, the efficiency of mitophagy is impaired, resulting in the accumulation of damaged mitochondria and aggravation of AKI.To explore the molecular mechanism of TA-Ce in the treatment of RM-AKI, the proteins involved in ER stress (phosphorylated inositol-requiringenzyme 1, p-IRE1), spliced ​​X-box binding protein 1 (XBP1s), mitochondrial autophagy (sequestosome 1, P62), microtubule-associated protein 1 light chain 3 II (LC3 II), apoptosis (phosphorylated c-Jun N-terminal kinase, p-JNK), B-cell lymphoma 2 (Bcl-2), Bcl-2 associated X The changes in the levels of key proteins in the inflammatory cytokine pathway (inflammatory cytokines, such as Bax), Cyt-c, caspase-3 (Casp-3), and inflammation (tumor necrosis factor receptor-associated factor 2 (TRAF2), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB)).

[0122] IRE1 is a transmembrane protein that functions as an ER stress sensor. When misfolded or incompletely folded proteins accumulate in the ER lumen, IRE1 undergoes dimerization and autophosphorylation, leading to its activation. Activated IRE1 has endonuclease activity that promotes unconventional splicing of XBP1 mRNA. This splicing removes an intron to produce the activated XBP1s (spliced ​​XBP1) transcription factor. This process is critical for the UPR and initiates a program signaling cascade in response to ER stress. Fig.24As shown in A and B, IRE1 was significantly phosphorylated in the kidneys of AKI mice, increasing 2.6-fold compared with the normal group, further inducing 2.3-fold upregulation of XBP1s. TA-Ce treatment reduced the levels of p-IRE1 and XBP1s to near normal levels, while NAC treatment did not reverse IRE1 phosphorylation and XBP1s activation. In addition, during ER stress, IRE1 formed a complex with IRE1α and TRAF2, thereby recruiting κB kinase inhibitors, leading to upregulation of NF-κB-related inflammatory genes and exacerbating the inflammatory response. Compared with the model group, the levels of TRAF2 and NF-κB decreased by approximately 60% and 64%, respectively, after TA-Ce treatment ( Fig.24 C and D). These results indicate that TA-Ce can effectively inhibit the cascade of ER stress and inflammation. Under persistent ER stress, p-IRE1 interacts with TRAF2, leading to phosphorylation of the downstream protein c-Jun N-terminal Kinase (JNK), which in turn activates apoptosis-related protein kinases. Phosphorylated JNK (p-JNK) induces apoptosis by inhibiting the anti-apoptotic protein Bcl-2 and activating the pro-apoptotic protein Bax. Compared with the normal group, the expression of p-JNK protein in the model group increased by approximately 1.6 times, while the p-JNK level was significantly reduced after TA-Ce treatment and returned to near normal levels ( Fig.24 E). These results indicate that TA-Ce can inhibit the expression of key proteins in the downstream UPR pathway, alleviate ER stress, and block subsequent inflammatory and apoptotic signaling pathways.

[0123] Mitochondria autophagy is a key quality control mechanism that clears damaged mitochondria and regulates ROS production. However, when mitochondria are damaged, mitophagy is impaired, leading to the accumulation of LC3 II and P62. The degradation of LC3 II and P62 is a key indicator of smooth autophagic flux. Fig.24 As shown in F and G, both P62 and LC3 II in the model group were upregulated by approximately 3 times. Compared with the model group, TA-Ce treatment reduced the expression of P62 and LC3 II by 48.9% and 62.3%, respectively, thereby restoring the autophagic flux to normal. As mentioned above, p-JNK can activate cell apoptosis. In addition, dysfunction of the P62-LC3 II signaling pathway can also promote cell apoptosis. Specifically, the upregulation of P62 and LC3 II can lead to mitochondrial autophagy disorders, resulting in the accumulation of damaged mitochondria and increased mitochondrial ROS generation. This process exacerbates oxidative stress and ultimately leads to apoptosis of renal tubular cells. Therefore, the changes in the levels of downstream apoptosis-related proteins (i.e., Bcl-2, Bax, Cyt-c, and Caspase-3) after endoplasmic reticulum stress and mitochondrial autophagy were studied. Fig.24As shown in H and I, after AKI mice were treated with TA-Ce, the Bcl-2 level increased by 85.2%, while the Bax level decreased by 49.4%. The release of Cyt-c plays a vital role in the apoptosis signaling pathway. In the case of mitochondrial dysfunction or increased outer membrane permeability, Cyt-c is released into the cytoplasm, forms a complex with apoptotic protease activating factor-1 (Apaf-1) and caspase-9, and finally cleaves and activates Casp-3. In the pathological condition of AKI, the decrease in Bcl-2 levels and the increase in Bax expression promote the release of Cyt-c, thereby activating Casp-3 and leading to cell apoptosis. Fig.24 As shown in J and K, the Cyt-c level in the model group increased by approximately 1.4 times, while the cleaved Casp-3 level increased by approximately 1.5 times, indicating enhanced apoptosis during the progression of AKI. After TA-Ce treatment, the Cyt-c level decreased by 25% and the cleaved Casp-3 level decreased by 42%. In contrast, NAC treatment failed to reduce the Cyt-c level. These results suggest that TA-Ce can inhibit apoptosis in AKI by restoring mitophagy flux, clearing damaged mitochondria, and regulating downstream apoptosis-related factors.

[0124] Kidney injury molecule-1 (KIM-1) is a transmembrane glycoprotein that is considered a key biomarker for AKI. KIM-1 is not detectable in healthy kidneys, but its expression is rapidly upregulated after injury. KIM-1 is localized to the apical membrane of proximal tubular cells, especially in areas most affected by ischemia or toxic injury. Therefore, the level of KIM-1 in the kidney after TA-Ce treatment was investigated. In the RM-AKI model, the expression of KIM-1 was significantly increased, approximately twice that of the control group, indicating severe kidney damage. Compared with the model group, TA-Ce treatment reduced KIM-1 levels by 61.7%, close to normal levels. In contrast, NAC treatment only reduced KIM-1 levels by 20.7% ( Fig.24 L). These results suggest that TA-Ce can effectively improve RM-AKI, and its effect is superior to that of NAC at the same dose.

[0125] 4. The therapeutic effect of TA-Ce on CP-AKI mice CP is a widely used chemotherapeutic drug that can also induce AKI. CP promotes oxidative stress by generating ROS in mitochondria. Excessive ROS can damage cellular components, including lipids, proteins, and DNA, leading to mitochondrial dysfunction and cell death, which in turn causes renal damage. To evaluate the therapeutic effect of TA-Ce on CP-AKI, an AKI model was established by intraperitoneal injection of CP. Mice in the NAC group received the same dose of NAC at the same time point to compare the therapeutic effects ( Fig.25 A).

[0126] (1) Similar to its effect on RM-AKI, TA-Ce significantly reduced the renal coefficient and serum BUN, CRE, and LDH levels of CP-AKI mice, bringing them close to healthy levels ( Fig.25 BE). In addition, H&E staining showed that TA-Ce could restore the renal tubular structure and repair the renal tubular damage caused by protein deposition ( Fig.25 F). These findings suggest that TA-Ce can effectively reverse CP-induced renal dysfunction in vivo, and its therapeutic effect is superior to that of NAC at the same dose.

[0127] (2) DHE staining results showed that the red fluorescence of the kidneys in the model group and NAC group was significantly higher than that in the control group, indicating that the level of ROS was elevated. After treatment with TA-Ce, the red fluorescence intensity returned to normal levels ( Fig.26 A). SOD activity, MDA and GSH levels are important indicators of oxidative stress severity and ROS-induced damage. Compared with the model group and NAC group, the TA-Ce treatment group had higher SOD and GSH levels and lower MDA level ( Fig.26 B, C, and D). These changes indicated that TA-Ce alleviated renal injury in CP-AKI mice by reducing oxidative stress.

[0128] (3) TUNEL staining showed that the number of apoptotic cells in the renal tissue of CP-AKI mice increased significantly. After treatment with TA-Ce, the fluorescence intensity decreased significantly, indicating that TA-Ce has a strong anti-apoptotic effect ( Fig. 27 A). TA-Ce can also significantly reduce the levels of pro-inflammatory cytokines (i.e., TNF-α and IL-6) in the serum of CP-AKI mice ( Fig. 27 B and C). These findings suggest that TA-Ce can alleviate oxidative stress, apoptosis, and inflammatory responses, thereby improving renal function in CP-AKI mice.

[0129] 5. In vivo biocompatibility like Fig.28 As shown in AH, the body weight of mice in the TA-Ce group did not decrease significantly, and there were no significant changes in liver and kidney function and hematological indicators. Fig.28As shown in Figure I, the tissue characteristics of the TA-Ce-treated group were similar to those of the control group, and no adverse reactions were observed. These findings indicate that TA-Ce has good biosafety and has the potential for clinical application.

[0130] The schematic diagram of the action pathway of TA-Ce nanozyme of the present invention is as follows Fig.29 As shown, the TA-Ce nanozyme prepared by the present invention has good kidney targeting and can be effectively enriched in the damaged kidney. 3+ and Ce 4+ It can clear a variety of ROS through a unique redox cycle between states, inhibit the cascade reaction of oxidative stress, mitochondrial autophagy, endoplasmic reticulum stress, inflammation and apoptosis, thereby breaking the vicious cycle and playing a good therapeutic effect in the treatment of rhabdomyolysis and / or cisplatin-induced acute kidney injury.

Claims

1. A method for preparing cerium tannate nanozyme, characterized in that: The preparation method comprises the following steps: mixing a tannic acid solution and a tetravalent cerium salt solution, adjusting the pH to 9-11, and reacting to obtain the nanozyme; the molar ratio of the tannic acid to the tetravalent cerium salt is 1-4:1; and the hydration kinetic diameter of the tannic acid cerium nanozyme is 20-80 nm.

2. The preparation method according to claim 1, characterized in that: The molar ratio of the tannic acid to the tetravalent cerium salt is 1.5-3.5:

1.

3. The preparation method according to claim 1, characterized in that: The pH is 9.5 to 10.

5.

4. The preparation method according to claim 1, characterized in that: The reaction time is 2 to 4 hours.

5. The preparation method according to claim 1, characterized in that: The hydration kinetic diameter of the cerium tannate nanozyme is 20 to 60 nm.

6. The preparation method according to claim 1, characterized in that: The zeta potential of the cerium tannate nanozyme is -20 to -40 mV.

7. Cerium tannate nanozyme prepared by the preparation method according to any one of claims 1 to 6.

8. Use of the cerium tannate nanozyme according to claim 7 in the preparation of a product for targeting the kidney to treat acute kidney injury.

9. The use according to claim 8, characterized in that: The acute kidney injury is induced by rhabdomyolysis and / or cisplatin.

10. A drug, characterized in that Comprising the cerium tannate nanozyme as described in claim 7.

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