CN112274634A

CN112274634A - Application of phospholipid polyethylene glycol modified subminiature cerium oxide nanoenzyme in preparation of medicine for treating acute kidney injury

Info

Publication number: CN112274634A
Application number: CN201910666997.7A
Authority: CN
Inventors: 凌代舜; 翁勤洁; 李方园; 方春燕; 孙恒; 王金成; 谢安; 夏凡; 邱月萍
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2019-07-23
Filing date: 2019-07-23
Publication date: 2021-01-29

Abstract

The invention discloses an application of phospholipid polyethylene glycol modified subminiature cerium oxide nanoenzyme in preparation of a medicine for treating acute kidney injury, wherein the acute kidney injury is acute kidney injury induced by cisplatin when the cisplatin is used for treating solid cancer. The phospholipid polyethylene glycol modified subminiature cerium oxide nanoenzyme provided by the invention can generate a plurality of antioxidant mimic enzyme activities, can exert high-efficiency catalase activities in a physiological environment, effectively reduces the ROS level by acting on an Nrf2/Keap1 channel, inhibits apoptosis, plays an important role in protecting and treating acute kidney injury caused by cisplatin, and develops a more potential nanomaterial for realizing low-toxicity antitumor application of the cisplatin in clinic.

Description

Application of phospholipid polyethylene glycol modified subminiature cerium oxide nanoenzyme in preparation of medicine for treating acute kidney injury

Technical Field

The invention relates to application of a nano biological material, in particular to application of phospholipid polyethylene glycol modified subminiature cerium oxide nanoenzyme in preparation of a medicine for treating acute kidney injury.

Background

Drug-induced kidney injury refers to kidney injury caused by the drug itself or its metabolites after administration of one or more drugs. Drug-induced kidney injury can manifest as any type of acute or chronic kidney disease currently known. Acute Kidney Injury (AKI) is a clinical syndrome in which sudden decline in renal function, which may be accompanied by oliguria or anuresis, rapidly declines or loses renal function in a short period of time due to pathological changes such as glomeruli, tubules, renal interstitium, or blood vessels, and is manifested by symptoms such as imbalance of electrolytes in the body, failure to eliminate accumulation of metabolites, and hypomagnesemia. The main etiology of the medicine is ischemia, nephropathy is caused by continuous accumulation of toxic substances in kidney, and the pathological characteristics of the medicine are mainly characterized by renal tubular injury.

Cisplatin (CP) is a common first-line and highly effective anticancer drug in clinical practice, and is used for treating various solid cancers including ovarian cancer, testicular cancer, lung cancer, head and neck cancer, etc. In addition, cisplatin has been used in combination with other drugs, such as docetaxel, to enhance antitumor efficacy. Clinical investigations have shown that cisplatin has a greatly limited clinical application due to its dose-dependent acute nephrotoxic effects. Acute renal failure, characterized by a sharp rise in serum creatinine and urea nitrogen levels in the body, occurs following cisplatin overdose. Clinical cancer patients have the probability of acute kidney injury between 20 and 35 percent after cisplatin injection, and a few patients can have fulminant or severe renal failure with threat life, which is the key point for clinical monitoring and prevention of drug nephrotoxicity. There is no drug effective against cisplatin kidney injury in clinical and clinical trials, so the development of novel targeted drugs is of great significance in the treatment of cisplatin-induced kidney injury.

Cisplatin causes kidney damage through a variety of mechanisms, including inhibition of protein synthesis and mitochondrial damage. In addition, cisplatin causes apoptosis through the death receptor pathway, endoplasmic reticulum stress. In addition, cisplatin also activates cdk2 and p21 expression simultaneously, as well as DNA damage.

In recent years, studies have demonstrated a central role for mitochondria and an enhancement of apoptosis in cisplatin-induced acute renal failure, highlighting therapeutic strategies against cisplatin-induced nephrotoxicity starting from mitochondrial oxidative stress. Mitochondrial dysfunction is evidenced by a decrease in membrane electrochemical potential and a significant decrease in mitochondrial calcium uptake. Depletion of the mitochondrial antioxidant defense system, such as decreased levels of GSH and NADPH, decreased GSH/GSSG ratios and increased levels of GSSG. In addition, cisplatin induces oxidative damage to mitochondrial lipids, including oxidation of phospholipids and mitochondrial proteins, such as significantly reduced thiol protein concentrations and increased carbonylation protein levels. At the same time, they interfere with the mitochondrial Electron Transport Chain (ETC), leading to electron leakage from the chain and ultimately to Reactive Oxygen Species (ROS) formation. Excessive metabolism of cisplatin produces excessive amounts of NAPQI to form depleted cellular Glutathione (GSH), adducted proteins include mitochondrial proteins, and induce mitochondrial oxidative stress and dysfunction. Mitochondrial proteins cause fragmentation of nuclear DNA and death of necrotic cells and subsequent inflammatory responses, including release of pro-inflammatory cytokines and activation of immune cells. In many documents in the year, cis-platinum is accumulated in renal tubular cells due to abundant expression of OCT2 on renal proximal cell membranes, so that intracellular ROS and inflammatory factors are generated in a large quantity, the death of the renal tubular cells and the subsequent acute renal injury are facilitated, and meanwhile, the expression of oxidative stress related proteins Nrf2, HO-1, NQO1 and the like is up-regulated.

Nanoenzymes are artificial enzymes based on nanomaterials and have been widely used in the field of medical research due to their unique physicochemical properties. The ultra-small cerium oxide nanoparticle enzyme has an oxygen cavity in the structure and trivalent and tetravalent cerium ions on the surface, has various adjustable enzyme activities, and is a nanoenzyme with wide application prospect. Cisplatin often causes the ROS content in the kidney part to rise in the tumor chemotherapy process, and serious acute kidney injury is caused.

Disclosure of Invention

The invention aims to provide application of phospholipid polyethylene glycol modified subminiature cerium oxide nanoenzyme in preparation of a medicine for treating acute kidney injury, which can play a role in protecting and treating acute kidney injury caused by cisplatin without influencing the original anti-tumor effect of the cisplatin.

The invention provides the following technical scheme:

an application of ultra-small cerium oxide nanoenzyme modified by phospholipid polyethylene glycol in preparing medicine for treating acute renal injury is disclosed.

Further, the acute kidney injury is acute kidney injury induced by cisplatin when used to treat solid cancer.

Further, the acute kidney injury is kidney injury renal function, renal tubular cell apoptosis or renal oxidative injury.

Further, the solid cancer is ovarian cancer, testicular cancer, lung cancer, or head and neck cancer.

Further, the solid cancer is ovarian cancer.

Furthermore, the size of the subminiature cerium oxide nanoenzyme is 1-10nm, and the size of the subminiature cerium oxide nanoenzyme modified by phospholipid polyethylene glycol is 10-30 nm.

The phospholipid polyethylene glycol modified subminiature cerium oxide nanoenzyme is nanoenzyme, and the enzymatic activity of the nanoenzyme can be changed along with the change of the external environment. At the kidney part, the subminiature cerium oxide nanoenzyme can efficiently remove ROS generated by cisplatin drugs, reduce apoptosis and realize the effect of treating acute kidney injury caused by cisplatin. And secondly, the phospholipid polyethylene glycol is non-toxic and non-irritant, has high biological safety, can prolong the systemic circulation time of the subminiature cerium oxide nanoenzyme, and improves the treatment efficiency.

The size of the subminiature cerium oxide nanoenzyme is 1-10nm, and the size of the subminiature cerium oxide nanoenzyme modified by phospholipid polyethylene glycol is 10-30 nm. The subminiature cerium oxide nanoenzyme has a large number of oxygen cavities in the structure of enzyme body, and the surface of the subminiature cerium oxide nanoenzyme is simultaneously provided with Ce under the physiological pH environment³⁺And Ce⁴⁺Can mutually and rapidly switch, and shows strong catalase activity. In the acidic pH environment of the tumor, Ce⁴⁺To receive H⁺Influence of (3), Ce cannot be realized⁴⁺To Ce³⁺The catalase activity of (1) is low. Therefore, the ultra-small cerium oxide nanoenzyme shows strong catalase activity in a normal physiological environment, and can efficiently remove ROS; under the acidic pH environment, the ultra-small cerium oxide nanoenzyme shows weak catalase activity, and does not influence the ROS content of cisplatin generated at the tumor part.

The subminiature cerium oxide nanoenzyme can show strong catalase activity in HK-2 cells, can effectively remove excessive ROS (Reactive oxygen species) caused by cisplatin, and further can effectively inhibit apoptosis of human renal cortex proximal tubular epithelial cells HK-2 caused by the cisplatin. The subminiature cerium oxide nanoenzyme reduces the ROS level in human renal cortex proximal tubular epithelial cells HK-2 through the target regulation of Nrf2/Keap1 pathway.

Specifically, the subminiature cerium oxide nanoenzyme can act on an ROS-related pathway, namely an Nrf2/Keap1 pathway, and can activate the expression of the signal pathway and target genes downstream of the signal pathway, so that the aim of reducing the ROS level is fulfilled.

Meanwhile, the subminiature cerium oxide nanoenzyme effectively inhibits apoptosis of cells at the kidney part. That is, the ultra-small cerium oxide nanoenzyme is used to reduce apoptosis in renal tissue caused by cisplatin.

The subminiature cerium oxide nanoenzyme does not interfere with the original treatment effect of cisplatin on ovarian cancer. The subminiature cerium oxide nanoenzyme shows weak catalase activity under tumor acidic conditions, and does not reduce ROS (reactive oxygen species) generated by cisplatin at tumor parts, so that the original anti-tumor effect of the cisplatin is not influenced.

The invention discovers the application of the cerium oxide nanoenzyme in the medicines for relieving acute kidney injury related diseases caused by the cis-platinum through two aspects of animal experiments and in vitro cell experiments, and develops a more potential nano material for realizing low-toxicity anti-tumor application of the cis-platinum clinically.

Compared with the prior art, the invention has the beneficial effects that:

(1) the phospholipid polyethylene glycol modified subminiature cerium oxide nanoenzyme has an excellent morphological structure, is uniform and controllable in particle size, can effectively enter the kidney and plays a role in treatment.

(2) The phospholipid polyethylene glycol modified subminiature cerium oxide nanoenzyme can obviously remove overhigh ROS in cells and inhibit apoptosis by acting on an Nrf2/Keap1 channel.

(3) Due to the controllability of the enzyme activity of the phospholipid polyethylene glycol modified subminiature cerium oxide nanoenzyme, the antitumor effect of the cisplatin medicine can not be interfered while acute kidney injury is protected.

Drawings

FIG. 1 is a Transmission Electron Microscope (TEM) image of ultra-small cerium oxide nanoenzymes prepared in example 1;

FIG. 2 is a graph showing a dynamic light scattering particle size distribution of the phospholipid polyethylene glycol-modified ultra-small cerium oxide nanoenzyme prepared in example 1;

FIG. 3 shows catalase activities of phospholipid polyethylene glycol-modified ultra-small cerium oxide nanoenzymes prepared in example 1 under different pH conditions;

FIG. 4 is a graph showing the results of quantitative analysis of cell viability of HK-2 cells induced by inhibition of cisplatin by phospholipid polyethylene glycol-modified subminiature cerium oxide nanoenzymes using Sulforhodamine B colorimetry (Sulforhodamine B, SRB);

FIG. 5 shows the inhibition of Serum urea nitrogen (BUN) and creatinine (Cre) in AKI mice by cerium oxide nanoenzyme, which was detected by a blood biochemical analyzer;

FIG. 6 shows Hematoxylin-eosin (H & E) staining, and detection of the protective effect of cerium oxide nanoenzyme on the kidney structure of AKI mice;

FIG. 7 is a Western blot used for detecting the inhibition effect of cerium oxide nanoenzyme on the expression of apoptosis proteins Cleavd-PARP and Cleaved-caspase 3;

FIG. 8 is a flow cytometer detecting the inhibition effect of cerium oxide nanoenzyme on cisplatin-induced apoptosis by Annexin V/PI double staining;

FIG. 9 shows the inhibition of cisplatin-induced apoptosis by cerium oxide nanoenzyme detected by TUNEL staining;

wherein the solid white line represents the boundary between the kidney and the background and the dashed white line represents the boundary between the renal cortex and the medulla;

FIG. 10 is a graph showing the effect of cerium oxide nanoenzyme on changes in the amount of ROS in HK-2 cells, using a ROS detection kit;

FIG. 11 is a graph showing the results of in vitro investigation of oxidative stress-related proteins (Nrf2, Keap1 and DJ-1 and apoptosis-related proteins (Cleavd-PARP and Cleaved-caspase 3) responsible for cerium oxide-protected cisplatin-induced renal injury after knock-out of Nrf2 using the SiRNA technique and Western blot;

FIG. 12 is a graph showing the results of detecting the up-regulation of the antioxidant gene NQO1 by cerium oxide nanoenzyme to cisplatin using a real-time fluorescent quantitative PCR method;

FIG. 13 is a graph of the results of examining the Relative Tumor Volume (RTV) changes at different times in ovarian cancer tumor-bearing nude mice administered with cisplatin or (and) ceria nanoenzyme;

FIG. 14 is a graph of the results of administering cisplatin or (and) cerium oxide nanoenzyme to nude mice with ovarian cancer tumor for 10 days and examining the tumor weight;

FIG. 15 is a graph of the results of administering cisplatin or (and) cerium oxide nanoenzyme to nude mice with ovarian cancer tumor for 12 days and examining the survival rate of the nude mice.

Detailed Description

The invention is further described with reference to the following specific embodiments and the accompanying drawings. The characterization method and the steps adopted in the embodiment of the invention are as follows:

SRB method: after the tested substances and the like act on the cells for 24 hours, removing the culture solution, adding a 10% trichloroacetic acid (TCA) solution precooled at 4 ℃ into each hole to fix the cells, placing the cells in a refrigerator at 4 ℃ to fix the cells for more than 2 hours or overnight, washing each hole of the culture plate for more than 5 times by using tap water or deionized water (D3W) to remove redundant trichloroacetic acid, placing the culture plate in a 60 ℃ oven to dry for 30 minutes, adding an SRB solution (4mg/ml) prepared by 1% acetic acid into each hole, placing the culture plate at room temperature for 20min to 30min, removing the liquid in each hole, and washing the cells for more than 5 times by using 1% acetic acid, so as to clean the unbound SRB dye. Drying in 60 deg.C oven for 30min, adding 70-80 μ l 10mM Tris-base (Tris-hydroxymethyl aminomethane) solution with pH of 10.5 into each well, dissolving, shaking on plate shaker for 10min, and measuring absorbance OD value with enzyme-labeling instrument.

Hematoxylin-Eosin staining (Hematoxylin-Eosin staining): the kidney was obtained by transection, paraffin-embedded kidney, slice thickness 3 μm, and HE staining. Dewaxing a paraffin section for 15min by an environment-friendly transparent agent I and 12min by an environment-friendly transparent agent II; dehydrating the materials in 100%, 95%, 90%, 80% and other grades of ethanol for about 5 min; the step of distilling the water for 5 min; fourth, dyeing in hematoxylin for about 2.5min, and washing with tap water for about 15 min; fifthly, putting the slices into 0.5% hydrochloric acid ethanol for color separation, wherein the slices turn red and are lighter in color; sixthly, washing with water for 5min, and turning ammonia water to blue; dewatering with gradient, 70% ethanol for 2min, 80% ethanol for 2min, 90% ethanol for 2min, and 90% ethanol for 2 min; 95% ethanol for 2 min; in contrast with 0.5 eosin ethanol for 2 s; the self-sustaining effect is that the absolute ethyl alcohol I is 3min and the absolute ethyl alcohol II is 5 min; the sealing piece is transparent after the environment-friendly transparent agent is prepared.

Western blot: immunoblot analysis of proteins in mouse tissue lysates was performed by homogenizing frozen tissues in lysis buffer containing 50mM Tris-HCl, 150mM sodium chloride, 2mM EDTA, 2mM Ethylene Glycol Tetraacetic Acid (EGTA), 1% Triton-X100. And protease inhibitor (pH 7.4), followed by centrifugation (10000 rpm, 4 ℃) for 30 minutes to obtain a supernatant, and a BCA protein assay kit (Yeasen Biotech, Hong Kong, China) was used. Proteins were separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes (Millipore, Massachusetts, USA) and then detected with the following primary antibodies: anti-Nrf 2 (1: 1000, ab62352, Abcam, Cambridge, UK), anti-Keap1 (1: 1000, ab119403) anti-DJ-1 (1: 1000, sc-55572, Santa Cruz, Dallas, Texas, USA), anti-Cleaved-caspase 3 (1: 1000, 9661L, Cell Signaling Technology) and anti-GAPDH (1: 1000, sc-1615).

Annexin V/PI double staining: the cells and cell supernatants were collected in 15ml centrifuge tubes and centrifuged (1.8X 10)³rpm, 5min), discard the supernatant, add 1ml of 4 ℃ 1 XPBS, resuspend, centrifuge and discard the supernatant. Add l × Binding Buffer to resuspend cells to l × l0⁶cells/ml. Carefully transferred to a 1.5ml EP tube and 5ul annexin V-FITC and 5ul PI were added. Cells were mixed well and incubated at room temperature for 15min in the dark. 400. mu. l l Xbinding Buffer was added to each tube and the machine was set up for 1h internal flow assay.

TUNEL (TdT-msidated dUTP Nick-End Labeling) staining: after the section is embedded in paraffin, apoptotic cells are detected by a TUNEL method, and the specific operation is carried out according to the instruction. The TUNEL method for detecting apoptosis comprises the following steps: 1) slicing, dewaxing conventionally, and washing with xylene for 5min each time for 2 times; gradient ethanol immersion washing, 3 minutes each time, 1 time in total; 2) rinsing with PBS for 2 times, and treating the tissue with a protease K working solution for 15-30 minutes; 3) rinsing with PBS for 2 times, adding prepared TUNEL reaction mixture (treating group is mixed with 50 μ L of TdT +450 μ L of fluorescein-labeled dUTP solution, negative control group is added with only 50 μ L of fluorescein-labeled dUTP solution, positive control group is added with 100 μ L of DNase I, reacting for 10min, and the subsequent steps are the same as the treating group; 4) add 50. mu.L TUNEL reaction mix (negative control group add only 50. mu.L fluorescein labeled dUTP solution) on the specimen with cover slip and react for 1 hour at 37 ℃ in dark and wet box; 5) PBS rinsing 3 times, adding 50. mu.L coverter-POD on the specimen, adding a cover glass, and reacting for 30 minutes at 37 ℃ in a dark and wet box; 6) rinsing with PBS for 3 times, adding 50 mu L of DAB substrate, and reacting for 10 minutes; 7) rinsing with PBS 3 times, counterstaining with hematoxylin, dehydrating by conventional method, clearing with xylene, and sealing with neutral gum. TUNEL staining was observed under a Leica optical microscope, 3 fields were selected for each section at 400-fold field, the number of TUNEL positive cells and the number of normal cells were counted, and the apoptosis rate was calculated.

And (3) detecting the ROS kit: HK-2 cells were confocal plated in 6-well plates (2X 10 cells per well)⁵Individual cells). Treating the cells with cerium oxide nanoenzyme for 24 hr, collecting cells by trypsinization, and centrifuging (1.8 × 10)³rpm, 5min), the supernatant was discarded. Using serum-free 1640 culture solution 1: ROS fluorescent probe (2, 7-dichlorodihydrofluorescein diacetate, DCFH-DA) was diluted at 1000 to a final concentration of 10. mu.M. After the cells are collected, the cells are resuspended in diluted DCFH-DA, incubated in a cell incubator at 37 ℃ for 20min, and inverted and mixed evenly every 5min to ensure that the probe is fully contacted with the cells. Cells were washed three times with serum-free medium to remove DCFH-DA that did not enter the cells. Only Rosup was added to the positive control wells as a positive control. ROS content was detected by flow cytometry.

RNA extraction and RT-PCR. RNA was extracted from treated cells using Trizol reagent and absorbance values were measured at 260/280nm to determine sample purity and concentration RNA was reverse transcribed into cDNA according to the reagent instructions. Primers specific to NQO1mRNA were designed using Primer5.0 software, with GAPDH as an internal control, and synthesized by Shanghai Co., Ltd. Quantitative RT-PCR was performed using SYBR Green Supermix (Bio-Rad, Hercules, California, USA) and 20. mu.L of the reaction mixture. The PCR reactions were performed on a Quant Studio 6Flex real-time PCR system (Applied Biosystems, Carlsbad, California, USA) with the following program: step 1, the temperature of 95 ℃ lasts for 3 minutes to activate Taq polymerase; step 2, denaturing the DNA at 95 ℃ for 3 seconds; step 3, annealing/extension at 60 ℃ for 31 seconds, and steps 2-3 for 39 cycles. Relative mRNA levels were quantified by the 2- Δ Δ Ct method and all data were normalized to GAPDH (internal control).

Example 1: synthesis of phospholipid polyethylene glycol modified subminiature cerium oxide nanoenzyme

(1) Synthesizing the ultra-small cerium oxide nanoenzyme:

adding 0.4g of cerium acetate hydrate and 3.2g of oleylamine into 15ml of dimethylbenzene, raising the temperature to 90 ℃ at the rate of 2 ℃ per minute, and stirring for 4 hours to form a complex; injecting 1ml of deionized water into an inert gas protection reaction system, aging for three hours, precipitating with anhydrous ether, and centrifuging to obtain the ultra-small cerium oxide nanoenzyme.

The result is shown in figure 1, and the subminiature cerium oxide nanoenzyme is characterized by the shape by a transmission electron microscope, and the particle size is about 1-10 nm.

(2) Synthesis of phospholipid polyethylene glycol modified subminiature cerium oxide nanoenzyme:

0.01g of polyethylene glycol phosphate and 1ml of subminiature cerium oxide nanoenzyme are added into 5ml of chloroform, and ultrasound treatment is carried out in a water bath ultrasound pot for 3 min. And (3) carrying out rotary evaporation on the mixed solution at 50 ℃ for 2 hours, and adding 1ml of deionized water for hydration to obtain the phospholipid polyethylene glycol modified subminiature cerium oxide nanoenzyme.

The result is shown in fig. 2, the phospholipid polyethylene glycol modified ultra-small cerium oxide nanoenzyme is characterized in morphology, and the particle size is about 10-30 nm.

As a result, as shown in FIG. 3, the ultra-small cerium oxide nanoenzyme has a strong hydrogen peroxide activity under normal physiological pH conditions and a weak hydrogen peroxide activity under acidic conditions.

Example 2: influence of cerium oxide nanoenzyme on HK-2 injury caused by cisplatin

Preparation of the medicament: the phospholipid polyethylene glycol modified ultra-small cerium oxide nanoenzyme prepared in the embodiment 1 is dispersed into an aqueous solution to obtain the final ultra-small cerium oxide nanoenzyme with the concentration of 8.3 mg/ml.

Establishing a cell model: the cell culture solution containing cisplatin was added to HK-2 cells, and the drug was redispersed with the cell culture solution to give a concentration of 200 mM.

Group setting:

a. control group: normal cells were given fresh medium.

b. Model group: only the cell culture broth containing cisplatin was added to HK-2 cells to a final concentration of 10. mu.M.

c. Treatment groups: the cell culture solution containing 200mM cisplatin and different concentration gradients of cerium oxide was added to HK-2 cells to give final concentrations of cerium oxide of 0.78. mu.M, 1.56. mu.M, 3.13. mu.M, 6.25. mu.M, 12.5. mu.M, 25. mu.M, and 50. mu.M, respectively.

After 24 hours of action, the cell activity was quantified by SRB method, and the results of cerium oxide inhibition of cisplatin-induced tubular cell HK-2 death are shown in FIG. 4, where cisplatin has a significant effect of promoting tubular cell HK-2 death, and the cell activity was only 38.4%. The renal tubular cell death is inhibited after the administration of cerium oxide, the cell activity exceeds 78.5 percent, and the effect of obviously inhibiting the renal tubular cell death induced by cisplatin is proved.

Example 3: effect of cerium oxide nanoenzyme on cisplatin-induced acute renal injury

Taking 20-22g male ICR mice, and adopting cisplatin drug induction to establish an AKI model. Firstly, the mice are randomly divided into 4 groups, namely a blank control group, a cis-platinum model group and a cerium oxide 0.5m/kg administration group, and a cis-platinum model group and a cerium oxide 1.5m/kg administration group, wherein each group comprises 6 mice. 15mg/kg cis-platinum is injected into the abdominal cavity, and 0.5mg/kg or 1.5mg/kg cerium oxide nano enzyme is injected into the tail vein at the same time. And (3) taking the medicine for dissection on the third day, taking blood from eyeballs, collecting serum, dissecting, perfusing the heart, and taking the kidney of the mouse for paraffin embedding, HE staining and TUNEL staining.

As shown in fig. 5 and 6, cisplatin induced a significant increase in serum urea nitrogen and creatinine and increased lesions such as tubular necrosis and shedding, and administration of cerium oxide reduced the levels of urea nitrogen and creatinine and improved kidney structure, and was dose-dependent. The results are shown in fig. 9, cisplatin induced an increase in tunee-positive apoptotic cells, and administration of ceria nanoenzyme significantly reduced the apoptotic cell proportion.

Example 4: effect of cerium oxide nanoenzyme on cisplatin-induced tubular cell HK-2 apoptosis in vitro

HK-2 cells (human normal tubular cell line) were applied in vitro and cells were incubated for 24h with 5. mu.M cisplatin and 50. mu.M cerium oxide nanoenzyme. And detecting the ratio of the late apoptotic cells by using Western blot to detect the cleared-PARP and the cleared-caspase 3 and using Annexin V/PI double staining flow cytometry.

Results as shown in fig. 7 and 8, cisplatin induced significant increase in clear-PARP and clear-caspase 3 protein and significant increase in the proportion of late apoptotic cells, and administration of cerium oxide reduced the levels of clear-PARP and clear-caspase 3 protein and the proportion of positive apoptotic cells, consistent with animal results.

Example 5: influence of cerium oxide nanoenzyme on cisplatin-induced renal tubular cell HK-2 oxidative damage and Nrf2/Keap1 signal pathway in vitro

HK-2 cells (human normal tubular cell line) were used in vitro, while cells were incubated for 24h with 5. mu.M cisplatin and 50. mu.M cerium oxide nanoenzyme, while a blank control (Vehicle) and a single cerium oxide nanoenzyme group were also set. Detecting the level of ROS in the HK-2 cells by using an ROS kit; detecting Nrf2, Keap1, DJ-1, cleaned-PARP and cleaned-caspase 3 by using Western blot; the mRNA level of NQO1 was detected using a real-time fluorescent quantitative PCR instrument.

The results are shown in FIG. 10, where cisplatin induced a significant increase in the intracellular ROS level of HK-2, and administration of cerium oxide was able to reduce excessive ROS levels.

The results are shown in fig. 11 and fig. 12, cisplatin-induced DJ-1 reduction, and cleared-PARP and cleared-caspase 3 proteins were significantly increased, and after Nrf2 knockout, cerium oxide administration failed to reverse the trend of change, while the mRNA level of the target gene NQO1 downstream of Nrf2 remained low and failed to exert protective effect well. It can be seen that Nrf2 is a key protein in protecting kidney injury by ceria nanoenzymes.

Example 6: cerium oxide nanoenzyme does not interfere with the effect of cisplatin in the treatment of ovarian cancer

(1) Preparing a model: ES-2 cells (1X 10)⁷cells/mouse) were inoculated into the left axilla of BALB/c nude mice, tumor mass was grown to 2cm (diameter) about 20 days, the tumor was aseptically exfoliated to 2mm × 2mm × 2mm, and the tumor mass was transplanted subcutaneously into the left axilla of 24 nude mice with a trocar. The whole inoculation process needs to be completed within 1 hour. Growing again for 16-20 days until 2cm (diameter) is aseptically stripped to 1mm³And transplanting and inoculating the nude mice again.

(2) Grouping and administration: when the tumor mass in the nude mouse body grows to 75-100mm³The animals in each group were divided into groups, and the average tumor volume of the animals in each group was close to that of the control groupPlatinum group, cerium oxide group, cisplatin + cerium oxide group, each group consisting of 6 nude mice. The dose of cisplatin is 3mg/kg, and the cisplatin is administered 2 times a week by intraperitoneal injection for only one week. The dosage of cerium oxide is 1.5mg/kg, and the cerium oxide is injected into a nude mouse body by tail vein and is injected with cisplatin at the same time. The control group was given an equal volume of physiological saline simultaneously. The long diameter and the short diameter of the tumor of the nude mice are measured every other day and recorded in time.

(3) Data acquisition: the nude mice were sacrificed 10 days after the administration, tumor masses of each group of nude mice were detached, and the tumor masses were weighed.

Calculating the tumor volume V, the relative tumor volume RTV and the like, and performing statistical detection.

The calculation formula is as follows: a.V ═ 1/2 × D²Where D and D represent the major and minor diameters of the tumor, respectively, the major and minor diameters must be measured perpendicularly. Rtv ═ V_t/V₀In which V is₀Is the tumor volume, V, measured in groups_tFor the tumor volume at each measurement.

The results are shown in fig. 13, fig. 14 and fig. 15, cisplatin has significant effect on ovarian cancer, while the relative tumor volume RTV and tumor weight are not affected by the cerium oxide nanoenzyme. Meanwhile, cisplatin can cause death of mice due to over-high dosage when used for treating cancer, and the cerium oxide nanoenzyme can remarkably improve the survival rate of the mice from 0% to 75%.

The above embodiments are described in detail to explain the technical solutions and advantages of the present invention, and it should be understood that the above embodiments are only specific examples of the present invention and are not intended to limit the present invention, and any modifications, additions, equivalents, etc. made within the scope of the principles of the present invention should be included in the scope of the present invention.

Claims

1. An application of ultra-small cerium oxide nanoenzyme modified by phospholipid polyethylene glycol in preparing medicine for treating acute renal injury is disclosed.

2. The use of subminiature cerium oxide nanoenzyme according to claim 1, in the preparation of a medicament for the treatment of acute kidney injury, wherein the acute kidney injury is that induced by cisplatin when used to treat solid cancer.

3. The use of subminiature cerium oxide nanoenzyme according to claim 2, in the manufacture of a medicament for the treatment of acute kidney injury, wherein the acute kidney injury is renal function injury, tubular apoptosis, or renal oxidative injury.

4. The use of the ultra-small cerium oxide nanoenzyme according to claim 1, in the preparation of a medicament for the treatment of acute kidney injury, wherein the solid cancer is ovarian cancer, testicular cancer, lung cancer or head and neck cancer.

5. The use of subminiature cerium oxide nanoenzyme according to claim 1, in the preparation of a medicament for the treatment of acute kidney injury, wherein the solid cancer is ovarian cancer.

6. The use of subminiature cerium oxide nanoenzymes according to any one of claims 1-5 in the manufacture of a medicament for the treatment of acute kidney injury, wherein the subminiature cerium oxide nanoenzyme has a size of 1-10nm and a phospholipid polyethylene glycol modified size of 10-30 nm.