Application of aloe-emodin in preparation of medicine for treating cisplatin-induced acute kidney injury

Abstract

The invention belongs to the technical field of medicines and disease treatment, and particularly relates to application of aloe-emodin in preparation of a medicine for treating cisplatin-induced acute kidney injury. Research results of the invention show that aloe-emodin significantly alleviates the oxidative stress of cis-platinum induced HK-2 cells by promoting the activity of Nrf2 and the expression of a downstream antioxidant gene GPX4. The invention provides an application of aloe-emodin in preparation of a medicine for treating cisplatin-induced acute kidney injury, and provides a novel method for treating platinum-induced acute kidney injury. Based on the purpose of fully exploring the potential of the aloe-emodin in the aspect of treating the cisplatin-induced acute kidney injury, the invention discovers the application potential of the aloe-emodin in preparation of the medicine for treating the cisplatin-induced acute kidney injury, and develops a new pharmaceutical application of the aloe-emodin.

Description

Technical Field

[0001] The invention belongs to the technical field of medicine and disease treatment, and particularly relates to application of aloe-emodin in preparing medicine for treating cisplatin-induced acute kidney injury. Background Art

[0002] Acute kidney injury (AKI) is a potentially life-threatening clinical syndrome characterized by a sharp rise in serum creatinine levels, a significant decrease in urine output, or both, leading to renal insufficiency or damage. AKI can be caused by a variety of factors, which may originate from sepsis, ischemia / reperfusion injury, immune system abnormalities, or exposure to nephrotoxic drugs. Among the various factors that cause AKI, about 20% of AKI cases are related to the use of nephrotoxic drugs, especially certain anti-tumor drugs such as cisplatin. AKI is not only an independent clinical symptom, but is also closely related to the progression of chronic kidney disease and the occurrence of end-stage renal disease. Although symptomatic treatment, supportive treatment, and renal replacement therapy are currently used clinically to relieve AKI, the efficacy of these methods is limited, and there is still a lack of effective drugs and treatment strategies for preventing and repairing renal injury.

[0003] Cisplatin is a chemotherapy drug widely used in the treatment of various malignant tumors. Due to its renal toxicity such as acute kidney injury, its application in clinical treatment is greatly limited. Among patients treated with cisplatin, about 30% will experience AKI related to nephrotoxicity. Even if they receive fluid rehydration before using cisplatin, the patients still show varying degrees of renal damage. The mortality rate of AKI should not be underestimated. According to studies, the related mortality rate of AKI patients is 23%. In an analysis of more than 5 million discharged patients, the mortality rate of AKI patients within 90 days was as high as 32%, and the mortality rate within 1 year rose to 48%. At present, there are few effective methods to alleviate cisplatin-induced nephrotoxicity. There is no doubt that effective drugs that can prevent cisplatin-induced AKI (Cis-AKI) are urgently needed in clinical practice. Therefore, further studying the mechanism of AKI caused by cisplatin and developing effective therapeutic drugs are of great significance for improving patient prognosis and improving treatment effects.

[0004] Aloe-emodin is a natural anthraquinone derivative and an active ingredient in many Chinese herbal medicines such as Cassia seed, Rheum palmatum, Aloe vera and Polygonum cuspidatum. It has been widely used as a raw material for medicines and cosmetics. Recent studies have revealed a variety of pharmacological effects of aloe-emodin, including anticancer, antiviral, anti-inflammatory, antibacterial, antiparasitic, neuroprotective and hepatoprotective activities. These pharmacological properties provide scientific basis for the treatment of a variety of diseases, including influenza virus, inflammation, sepsis, Alzheimer's disease, glaucoma, malaria, liver fibrosis, psoriasis, type 2 diabetes, growth disorders and several cancers. In addition, aloe-emodin has been reported to have antioxidant and cardiovascular protective effects and can treat doxorubicin (DOX)-induced cardiotoxicity by activating the Nrf2 signaling pathway. Although these potential therapeutic effects of aloe-emodin have been widely studied, its potential in treating cisplatin-induced acute kidney injury needs to be fully explored. Summary of the invention

[0005] In order to fully explore the potential of aloe-emodin in treating cisplatin-induced acute kidney injury, the present invention uses a cisplatin-induced AKI model as a research object to explore the regulatory effect of aloe-emodin on AKI and ferroptosis of renal tubular epithelial cells and its potential mechanism, and provides an application of aloe-emodin in the preparation of a drug for treating cisplatin-induced acute kidney injury.

[0006] To achieve the above purpose, the present invention adopts the following technical solution.

[0007] Aloe-emodin has shown significant potential and effects in pharmacological studies, including cardioprotection, antioxidant and anti-inflammatory effects, and antitumor activity. However, the potential of aloe-emodin in the treatment of cisplatin-induced acute kidney injury remains to be explored.

[0008] The present invention provides an application of aloe-emodin in preparing a drug for treating cisplatin-induced acute kidney injury. Based on the purpose of fully exploring the potential of aloe-emodin in treating cisplatin-induced acute kidney injury, the present invention discovers the application potential of aloe-emodin in preparing a drug for treating cisplatin-induced acute kidney injury, and develops a new pharmaceutical application of aloe-emodin.

[0009] Preferably, the drug contains the aloe-emodin as an active ingredient and is supplemented with pharmaceutically acceptable excipients or carriers.

[0010] Preferably, the auxiliary materials include any one or more of mannitol, microcrystalline cellulose, lactose, corn starch, stearic acid, dextrin, powdered sugar, glycerol, physiological saline, DMSO and Tween.

[0011] Mannitol is non-hygroscopic, dries quickly, has good chemical stability, and has the characteristics of being refreshing and having good granulation properties, and is used to prepare tablets. The present invention uses the aloe-emodin as an active ingredient, selects mannitol as an auxiliary material, and uses a conventional tablet preparation process to prepare a tablet for treating cisplatin-induced acute kidney injury.

[0012] Microcrystalline cellulose is commonly used as an adsorbent, suspending agent, diluent, and disintegrant. Microcrystalline cellulose is widely used in pharmaceutical preparations, mainly as a diluent and binder in oral tablets and capsules, and can be used not only for wet granulation but also for dry direct tableting. It also has certain lubrication and disintegration effects. The present invention uses the aloe-emodin as an active ingredient, selects microcrystalline cellulose as an auxiliary material, and uses a conventional tablet preparation process to prepare a tablet for the treatment of cisplatin-induced acute renal injury.

[0013] Lactose is used as a filler, glidant, disintegrant, lubricant and binder in solid preparations and as an excipient in lyophilized preparations.

[0014] Corn starch is used as a filler and binder in the preparation of tablets.

[0015] Stearic acid, dextrin, powdered sugar, glycerin, saline and Tween can all be used as excipients in the drug preparation process. Different excipients can be selected according to the actual application scenario.

[0016] Preferably, the carrier comprises any one or more of liposomes, nanoparticles, methylcellulose and chitosan.

[0017] Preferably, the medicine includes any one of capsules, tablets, injections, granules, solutions, patches and ointments.

[0018] Capsules are often used to mask the bad smell or taste of drugs, making them easier to swallow. They can also protect drugs from being destroyed by stomach acid until they reach the small intestine for release. Tablets are the most common oral dosage form, which are easy to carry and store, with accurate dosage, easy to take, and relatively low cost. Injections are suitable for situations where rapid onset of action is required or oral administration is not possible, such as first aid or intravenous injection. Granules are easy to dissolve in water and are suitable for children and patients with difficulty swallowing, and can mask the bad smell of drugs. Solutions are easy to absorb and are suitable for situations where rapid onset of action is required. They can also be used for external use. Patches are administered through the skin, which can avoid the first-pass effect of the liver, reduce gastrointestinal irritation, and are easy to administer. They are suitable for situations where sustained release of drugs is required. Ointments are suitable for external use and can act directly on the skin or mucous membranes to treat local diseases such as skin inflammation or injury.

[0019] According to the actual treatment purpose and treatment plan, the aloe-emodin can be prepared into different types of medicines to achieve the effect of treating cisplatin-induced acute kidney injury.

[0020] Preferably, the drug is a solution.

[0021] The solution is prepared by compounding the aloe-emodin with a solvent.

[0022] The solvent includes any one of water, ethanol, vegetable oil, DMSO, glycerol, physiological saline and Tween.

[0023] Solution drugs have a large dispersion, fast absorption, rapid action, and good bioavailability; the active ingredients are evenly dispersed and can be accurately measured and used, especially suitable for children and elderly patients; the dosage size is easy to adjust and control; it can reduce the local irritation of certain soluble drugs; and it can increase the stability and safety of certain drugs. Therefore, the dosage form of the above-mentioned drug provided by the present invention is a solution.

[0024] Preferably, the concentration of the aloe-emodin in the drug is 1 μM to 3 μM.

[0025] Preferably, when the solvent is water, glycerol and / or physiological saline, the drug is a drug for human use.

[0026] When the solvent is ethanol, vegetable oil, DMSO and / or Tween, the drug is a drug for animal use.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] 1. The present invention provides the use of aloe-emodin in the preparation of a drug for treating cisplatin-induced acute kidney injury, and provides a new method for treating platinum-induced acute kidney injury. Based on the purpose of fully exploring the potential of aloe-emodin in treating cisplatin-induced acute kidney injury, the present invention discovered the application potential of aloe-emodin in the preparation of a drug for treating cisplatin-induced acute kidney injury, and developed a new pharmaceutical use of aloe-emodin.

[0029] 2. Aloe-emodin (AE) has shown potential in alleviating acute injury induced by cisplatin (CDDP) in mice. The present invention uses the CCK8 experimental method to evaluate the cell death inhibitory effect of AE on human renal tubular epithelial cells (HK-2) and its protective ability against nephrotoxicity. Further through Western blot and qRT-PCR analysis, the present invention deeply explores the molecular mechanism (MOA) of AE activating nuclear factor erythroid 2-related factor 2 (Nrf2), as well as the protective effect of downstream antioxidant genes regulated by Nrf2. In addition, fluorescence imaging technology was used to detect changes in intracellular reactive oxygen species, and the results showed that AE significantly alleviated erastin-induced HK-2 cell oxidative stress by promoting the activity of Nrf2 and the expression of its downstream antioxidant gene GPX4.

[0030] 3. The present invention found that AE can effectively alleviate cisplatin-induced oxidative stress in HK-2 cells by activating Nrf2 and its downstream antioxidant protein GPX4.

[0031] Based on these findings, the present invention proposes that Nrf2 is a key molecular target for AE to treat AKI. AE exerts a significant renal protective effect by inhibiting Nrf2-mediated ferroptosis of renal tubular epithelial cells. These research results not only provide a new perspective for the treatment strategy of cisplatin-induced acute kidney injury, but also open up a new path for the development of Nrf2 inhibitors as effective drugs for the clinical treatment of cisplatin-induced AKI. These characteristics of AE make it a promising candidate drug, providing new ideas and methods for the future clinical treatment of cisplatin-induced AKI. The research of the present invention not only deepens the understanding of the mechanism of action of AE, but also provides an important scientific basis for the development of new treatment strategies and drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 It is the Venny diagram of the intersection of diseases and drug targets in the present invention.

[0033] Figure 2 The PPI protein interaction network derived from String software in the present invention was constructed.

[0034] Figure 3 The association diagram between proteins was visualized by Cytoscape 3.7.0 and Cytoscape 3.9.1 in the present invention.

[0035] Figure 4 A three-in-one graph of GO enrichment analysis for the intersection targets in the present invention.

[0036] Figure 5 This is a mulberry diagram of the KEGG enrichment analysis in the present invention.

[0037] Figure 6 The structural formula of AE in the present invention, the concentration of Erastin inducing ferroptosis in HK-2 cells, and the determination of AE on HK-2 cell viability: wherein, Figure 6 Figure A in the figure is the chemical structure formula of AE; Figure 6 Figure B shows the optimal induction concentration of Erastin on ferroptosis in HK-2 cells; Figure 6 Figure C shows the reversal effect of AE on Erastin-induced ferroptosis in HK-2 cells.

[0038] Figure 7 The reactive oxygen species (ROS), ferroptosis markers MDA, Fe2+ And GSH level determination: Among them, Figure 7 Figure A shows the level of reactive oxygen species (ROS) in HK-2 cells; Figure 7 Figure B shows the MDA content of HK-2 cells; Figure 7 Figure C of HK-2 cells Fe 2+ content; Figure 7 Figure D shows the GSH content in HK-2 cells.

[0039] Figure 8 The expression levels of Nrf2 and GPX4 proteins and mRNAs after Erastin-induced ferroptosis in HK-2 cells, the activity level of HK-2 cells after adding Nrf2 inhibitor ML385, and the expression level of Nrf2 in immunofluorescence: wherein, Figure 8 Figure A shows the protein expression levels of Nrf2 and GPX4 after ferroptosis and pre-protection; Figure 8 Panel B in the figure is the quantification of Nrf2 protein expression; Figure 8 Figure C shows quantification of GPX4 protein expression; Figure 8 Panel D in the figure shows the Nrf2 mRNA expression level; Figure 8 Panel E in the figure shows the GPX4 mRNA expression level; Figure 8 Figure F shows the cell viability levels of each group after adding ML385; Figure 8 Figure G shows the expression level of Nrf2 in each group after adding ML385.

[0040] Fig. 9 The expression of GPX4 in immunofluorescence after adding Nrf2 inhibitor ML385, Erastin-induced ferroptosis in HK-2 cells, the expression of Nrf2 and GPX4 proteins after adding ML385, ferroptosis, reactive oxygen species (ROS) in HK-2 cells pre-protected by ML385 and AE, and ferroptosis marker Fe 2+ , MDA and GSH level determination: Among them, Fig. 9 Figure A in the figure shows the expression level of GPX4 in each group; Fig. 9 Figure B shows the expression levels of Nrf2 and GPX4 proteins after ferroptosis, addition of ML385, and pre-protection; Fig. 9 Figure C in the figure is the quantification of Nrf2 protein expression; Fig. 9 Figure D in the figure is the quantification of GPX4 protein expression; Fig. 9 Panel E in the figure shows the reactive oxygen species (ROS) levels after ferroptosis, addition of ML385, and pre-protection; Fig. 9 The F diagram in the figure shows the Fe 2+ content; Fig. 9 Figure G in the figure shows the MDA content of each group; Fig. 9 GSH content in each group in Figure H.

[0041] Fig.10 The present invention is the mouse serum urea nitrogen (BUN), creatinine (Scr) and mouse kidney tissue H&E staining: wherein, Fig.10 Figure A shows the determination of serum urea nitrogen (BUN) content in each group of mice; Fig.10 Figure B shows the determination of creatinine (Scr) content in the serum of mice in each group; Fig.10 Figure C shows the serum urea nitrogen (BUN) content of mice without modeling and only given AE and normal mice; Fig.10 Figure D shows the serum creatinine (Scr) content of mice without modeling and only given AE and normal mice; Fig.10 Figure E shows H&E staining of kidney tissues of mice in each group.

[0042] Fig.11 Fe 2+ , GSH, MDA and serum MDA level determination: Among them, Fig.11 Figure A shows Fe in mouse kidney tissue 2+ content; Fig.11 Figure B shows the GSH content in mouse kidney tissue; Fig.11 Figure C shows the MDA content in mouse kidney tissue; Fig.11 Figure D shows the MDA content in mouse serum.

[0043] Fig.12 The expression levels of Nrf2 and GPX4 proteins in the renal tissue of mice after ferroptosis and pre-protection in the present invention are as follows: Fig.12 Figure A shows the expression levels of Nrf2 and GPX4 proteins in mouse kidney tissue; Fig.12 Panel B in the figure is the quantification of Nrf2 protein expression; Fig.12 Figure C shows quantification of GPX4 protein expression; Fig.12 Panel D in the figure shows the Nrf2 mRNA expression level in tissues; Fig.12 Figure E shows the GPX4 mRNA expression level in tissues. DETAILED DESCRIPTION

[0044] The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0045] Reagents, culture media, and experimental animals

[0046] 1. Reagents

[0047] Aloe-emodin: CAS No.: 481-72-1, purity: 97%; Cisplatin: CAS No.: 15663-27-1, purity: 99%; Aloe-emodin and cisplatin were purchased from Shanghai Bid Pharmaceutical Technology Co., Ltd.

[0048] Erastin: CAS number: 571203-78-6, purity: 99%, provided by Shanghai Aladdin Biochemical Technology Co., Ltd.

[0049] Dimethyl sulfoxide (DMSO): CAS number: 67-68-5, purity: ≥99.7%, purchased from Shanghai MacLean Biochemical Technology Co., Ltd.

[0050] 2',7'-Dichlorofluorescein diacetate (DCFH-DA) was obtained from Sigma-Aldrich (St Louis, USA).

[0051] Bovine serum albumin (BSA) and JC-1 probe were from Beyotime Company, Nantong, China.

[0052] Serum creatinine and urea nitrogen test kits were purchased from Shanghai JiNing Industry Co., Ltd.

[0053] 2. Culture medium

[0054] MEM culture medium was purchased from Solarbio, Beijing, China.

[0055] 3. Experimental animals

[0056] All animal experiments in the examples were reviewed and approved by the Experimental Animal Ethics Committee of the Air Force Medical University (approval number: SYXK (Shaanxi) 2023-004). The C57BL / 6J male mice used in the experiment were between 7±1 weeks old and weighed approximately 21±1 g, and were provided by the Experimental Animal Center of the Air Force Medical University. All operations involved in the animal experiments were approved by the Experimental Animal Ethics Committee of Yan'an University to ensure that the experimental process met ethical standards and animal requirements.

[0057] Example 1: Network pharmacology pathway and target prediction

[0058] 1. Screening of drugs and disease targets

[0059] Screening of aloe-emodin targets: Search for aloe-emodin targets in the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP), and then convert the target name of the relevant targets into symbol name through the Uniport database (http: / / www.Uniprotorg). Search for ferroptosis and acute kidney injury disease targets in the GeneCards database (http: / / www.genecards.org), and use the score ≥1.0 as the screening condition to obtain the corresponding disease targets.

[0060] 2. Construction of the intersection of drugs and disease-related targets

[0061] A total of 219 candidate sites for aloe-emodin (AE), 492 ferroptosis target genes, and 5932 acute kidney injury target genes were obtained. Then, the Venny diagram (http: / / bioinformatics.psb.ugent.be / webtools / Venn / ) was used to intersect the collected drug action target set with the disease-related target set, and 62 common targets (common drug-disease targets) were obtained.

[0062] The collected drug target set and disease-related target set are intersected to draw a Venny diagram. Figure 1 shown.

[0063] 3. PPI network analysis in target protein interactions

[0064] To further investigate the treatment of acute renal injury by AE through ferroptosis, 62 common drug-disease targets were analyzed through protein-protein interaction (PPI) networks using the String database, and hidden free nodes (such as Figure 2 The PPI network contains 60 interaction nodes and 584 interrelated action relationships, with an average node degree of 7.83 and an average clustering coefficient of 0.32. The results are saved in TSV format.

[0065] The data were imported into Cytoscape 3.7.0 software for further visualization analysis (e.g. Figure 3 The larger the node, the stronger the interaction (as shown in Figure 4 shown).

[0066] PPI network and TSV format files of target protein interactions. (a) PPI network showing the interactions between related genes.

[0067] Filled nodes represent known or predicted 3D structural proteins. Edges represent the association between proteins. Edges of different colors show different interactions. (b) Download TSV format files from the STRING database and import them into Cytoscape 3.7.0 and Cytoscape 3.9.1 for visualization. Nodes are core targets. The larger the area, the more important they are in the network. The thicker the lines, the greater the correlation.

[0068] The DAVID database was used to perform biological function and signal pathway enrichment analysis on potential therapeutic targets, and p<0.01 was screened. GO enrichment analysis and KEGG pathway analysis were performed on 36 aloe-emodin in the treatment of cisplatin-induced acute kidney injury by inhibiting ferroptosis. Bioinformatics methods were used to perform GO functional enrichment analysis on 62 cross-target genes, and the results are shown in Figure 5.

[0069] The results were exported from the DAVID database, the data were sorted, and the results of the visual analysis by Micro-information showed that:

[0070] The main GO biological processes (BPs) include: negative regulation of apoptosis, positive regulation of DNA template transcription, positive regulation of phosphatidylinositol 3-kinase and positive regulation of gene expression, etc. In terms of GO molecular functions, they mainly involve: same protein binding, enzyme binding, ubiquitin protein ligase binding. In terms of GO cellular components, they are mainly enriched in: extracellular matrix, endoplasmic reticulum cavity and axon. In terms of GO molecular functions, they mainly involve: neurotransmitter receptor activity, transcription factor binding and kinase binding. In terms of GO cellular components, they are mainly enriched in: membrane rafts, components of presynaptic membranes and receptor complexes.

[0071] Example 2: In vivo and in vitro drug effect evaluation

[0072] 1. Experimental Methods

[0073] 1. Cell culture and cell grouping experiments

[0074] At 37°C, 5% CO 2 Under the culture conditions of , human renal tubular epithelial cells (HK-2 cells) were cultured using MEM medium containing 10% by volume of fetal bovine serum (FBS), 50 U / mL penicillin (Gibco) and 50 μg / mL gentamicin (Gibco). HK-2 cells were purchased from Shanghai JiNing Industry Co., Ltd.

[0075] To construct a cell injury model, HK-2 cells were treated with 4.6 μM erastin for 24 h. Subsequently, HK-2 cells were pretreated with 1 μM and 5 μM aloe-emodin to evaluate their protective effects on HK-2 cells.

[0076] The cell experiment groups and treatment methods are as follows:

[0077] Normal control group: cells were cultured normally using the above culture medium without any special treatment.

[0078] Erastin treatment group (4.6 μM): cells were treated with 4.6 μM Erastin and normal culture medium, each accounting for 50% of the total volume, wherein the "normal culture medium" was the same as the culture medium in the "normal control group".

[0079] Erastin (4.6 μM) + DMSO (3 μM) treatment group: 4.6 μM Erastin and 3 μM DMSO, each accounting for 50% of the total volume, acted on the cells together.

[0080] Erastin (4.6 μM) + AE (3 μM) treatment group: cells were first pre-protected with 3 μM AE for 12 h, and then treated with 4.6 μM Erastin.

[0081] 2. Cell Viability Assay

[0082] The cell counting kit-8 (CCK-8 kit) was used to evaluate the viability of HK-2 cells. The specific method is as follows:

[0083] First, cultured HK-2 cells were plated at 5×10 3 The cells were seeded at a density of 100 cells / mL in 96-well plates and incubated at 37°C with 5% CO 2 24 hours. Next, HK-2 cells were pretreated with different concentrations of aloe-emodin for 24 hours, and then treated with 4.6 μM erastin for 24 hours. After treatment, 10 μL of CCK8 reagent was added to each well and incubated for another 40 minutes. Finally, the optical density of each well was measured at a wavelength of 450 nm using a microplate reader (Molec μLar Devices), and the results were expressed as a percentage of the control to reflect relative cell viability.

[0084] Among them, the CCK-8 kit was provided by Beyotime Biotechnology.

[0085] 3. Malondialdehyde (MDA) determination

[0086] Based on the reactivity of thiobarbituric acid (TBA), the malondialdehyde (MDA) assay kit (Solarbio) was used to determine the MDA content. The specific method is as follows:

[0087] 5 million HK-2 cells were collected from the above different experimental groups, and then 1 mL of the extract was added. The HK-2 cells were broken using an ultrasonic device (power 200 W, ultrasound 3s, interval 10s, repeated 30 times) to release the MDA in the cells.

[0088] After the HK-2 cells were broken, the samples were centrifuged at 8000g for 10 minutes at 4°C, the supernatant was taken, and the working solution provided by the MDA assay kit was added. Finally, the absorbance of each sample was measured at wavelengths of 532nm and 600nm using a spectrophotometer. The mouse serum samples were processed and measured in the same way. According to the formula provided by the kit, the MDA concentration was calculated and compared with the relative concentration of the control group, expressed as a percentage, to evaluate the changes in the oxidative stress level.

[0089] 4. Determination of glutathione (GSH) levels

[0090] Using a total glutathione kit (Beyotime kit), oxidized glutathione (GSSG) is converted to its reduced form (GSH) by the action of glutathione reductase. GSH then reacts with the chromogenic substrate DTNB to produce yellow TNB and GSSG.

[0091] First, the HK-2 cell pellet collected by centrifugation was mixed with 3 volumes of protein removal solution and thoroughly ground to collect the cells to obtain the sample to be tested. The sample to be tested was then quickly frozen under liquid nitrogen conditions and then quickly thawed in a 37°C water bath. The sample was allowed to stand at 4°C for 5 minutes, then centrifuged at 10,000 g for 10 minutes at 4°C, and the resulting supernatant was added to the working solution provided by the total glutathione kit to measure its absorbance at a wavelength of 412 nm.

[0092] For mouse kidney tissue, first quickly freeze it in liquid nitrogen and then grind it into powder to obtain tissue powder. Add 30 ml of Protein Remover S solution to every 10 mg of tissue powder and grind it thoroughly. Then add 70 μL of Protein Remover Reagent S solution and mix evenly using a glass homogenizer. The tissue sample was allowed to stand at 4°C for 10 minutes and centrifuged at 10,000 g for 10 minutes at 4°C. Finally, the supernatant was added to the working solution provided by the total glutathione kit, and its absorbance was measured at a wavelength of 412 nm. The relative concentration of glutathione is expressed as a percentage of the control group.

[0093] 5. Fe 2+ Level determination

[0094] The ferrous iron content test kit (mLbio kit) can be used to determine the ferrous iron (ferrous iron: Fe 2+ ) level. Ferrous iron can react with the specific components in the ferrous iron content detection kit to form a purple-red compound, which has a characteristic absorption peak at a wavelength of 562nm. This feature can be used to calculate the ferrous iron content by measuring absorbance.

[0095] First, HK-2 cells from different experimental groups were collected, with about 5 million cells in each group. The cells were mixed with 1 mL of the extract, and the cells were broken by ultrasound in an ice bath (power was 200 W, each ultrasonic treatment was 3 s, interval was 10 s, and repeated 30 times). After that, the mixture was centrifuged at 12000r for 10 min at 4°C, the supernatant was collected and the working solution provided by the ferrous iron content detection kit was added, and finally the absorbance of the sample at a wavelength of 562nm was measured to evaluate the ferrous iron content.

[0096] For mouse kidney tissue samples, weigh about 0.1 g of tissue, add 1 mL of extract solution, and homogenize in an ice bath. Then, centrifuge at 12000r for 5 min at 4°C, take the supernatant, and add the working solution provided by the ferrous iron content detection kit. Finally, measure the absorbance of the sample at a wavelength of 562 nm. 2+ The relative concentrations were expressed as percentages compared with the control group to facilitate comparison and analysis.

[0097] 6. Protein extraction and Western blot:

[0098] The cultured HK-2 cells were removed from the cell culture incubator, the original culture medium was first aspirated, and then the cells were rinsed three times with ice PBS to remove the residual culture medium. Total protein was then extracted from mouse kidney tissue and human HK-2 cells using ripa lysis buffer (Beyotime, China) and PMSF. The cell lysate was centrifuged at 12,000r for 20 min at 4°C, the supernatant was collected, and the corresponding loading buffer was added to prepare the sample, which was then stored at -20°C for subsequent use. PBS was purchased from Solarbio, Beijing, China, with a pH of 7.3 and a concentration of 0.01M.

[0099] When performing protein electrophoresis analysis, equal amounts of protein samples were separated by SDS-PAGE on a 10% bis-tris gel. The separated protein bands were transferred to a polyvinylidene fluoride (PVDF) membrane with a pore size of 0.22 μM. The membrane was blocked with a 5% by mass skim milk powder solution for 1.5 hours to reduce nonspecific binding. After blocking, the membrane was washed three times with 1× TBST buffer for 10 minutes each to remove unbound blocking agents. The membrane was then incubated with two corresponding specific primary antibodies at 4°C overnight to specifically bind to the target protein. Among them, overnight refers to a time of ≥12h.

[0100] On the second day, the primary antibody was recovered and washed again with 1×TBST buffer three times for 10 minutes each time to remove unbound primary antibody. Then, the corresponding secondary antibody was incubated at room temperature for 1.5 hours to detect the binding of the primary antibody. After the secondary antibody incubation was completed, it was washed three times with 1×TBST for 10 minutes each time to remove unbound secondary antibody.

[0101] Finally, an enhanced chemiluminescence system (Biosharp) was used to detect the signal of the target protein. After detection, the signal was quantified using ImageJ software (NIH, Bethesda, MD, USA) to evaluate the protein expression level.

[0102] 7. RNA extraction and quantitative real-time PCR (QRT-PCR)

[0103] First, total RNA was extracted from mouse kidney tissue and HK-2 cells using Trizol reagent, and the extracted RNA was properly stored at -80°C to ensure its stability and integrity. Ⅱ1st StrandcDNA Synthesis Kit (YEASEN, China) was used to reverse transcribe RNA into cDNA, and the cDNA was stored at −80°C for subsequent qPCR analysis.

[0104] To detect the mRNA expression level of specific genes, we used qPCR SYBR Green Master Mix (Yeasen, China) and simpliAmp PCR detection system (Applied Biosystems, USA) were used for qPCR analysis.

[0105] The PCR amplification procedure used a two-step method: first, a 2-minute pre-denaturation treatment at 95°C; followed by 40 cycles, each cycle including a 10-second denaturation step at 95°C and a 30-second annealing / extension step at 60°C.

[0106] The primers used were designed and synthesized by Wuhan Jinkairui Bioengineering Co., Ltd., and the sequence information is shown in Table 1.

[0107] The PCR amplification reaction system was 1 μL of DNA template, 0.5 μL each of forward primer and reverse primer (10 μM), 10 μL of 2×RealStarFast SYBR qPCR Mix, and 8 μL of Sterile Water. The total reaction system was 20 μL.

[0108] After PCR amplification is completed, the specificity of the PCR product is further verified by melting curve analysis to ensure the accuracy of amplification. ΔΔCT , by comparing the time point at which the fluorescence signal of the sample at the threshold cycle (Ct) is first significantly higher than the background, the difference in mRNA expression levels between different samples is calculated. This method can accurately evaluate the relative changes in gene expression and provide reliable data support for subsequent biological analysis.

[0109] Table 1 Primer information

[0110]

[0111]

[0112] 8. Immunofluorescence (IMF) detection

[0113] Culture to 1×10 4 HK-2 cells of equal density were seeded in 24-well plates, and 500 μL of MEM medium containing FBS, Gibco and Gibco was added to each well. The plates were then placed at 37°C with 5% CO 2 The cells were cultured in an environment for 24 hours. After the cells adhered to the wall, they were washed three times with PBS, fixed with 4% paraformaldehyde at room temperature for 20 minutes, and then incubated with 0.2% Triton×100 by volume at 4°C for 10 minutes. After that, they were blocked with 5% BSA at room temperature for 1 hour. After blocking, the primary antibody was added and incubated overnight at 4°C. The next day, the primary antibody was recovered, the corresponding fluorescent secondary antibody was added, and incubated at 37°C in the dark for 1 hour. After incubation, the anti-fluorescence quenching sealing agent containing DAPI was used for sealing. Finally, the fluorescence signal was detected and displayed under a confocal microscope.

[0114] 9. Reactive oxygen species (ROS) detection

[0115] The ROS detection experiment was performed using a reactive oxygen species (ROS) fluorescence detection kit (Elabscience). DCFH-DA (2,7-dichlorofluorescein diacetate) is a fluorescent probe that can freely pass through the cell membrane and does not emit fluorescence itself. Once inside the cell, DCFH-DA will be hydrolyzed by intracellular esterases and converted into DCFH (dichlorofluorescein). Under the action of reactive oxygen species, DCFH is further oxidized to produce a strong green fluorescent substance DCF (dichlorofluorescein), which cannot pass through the cell membrane. The fluorescence intensity of DCF reaches a peak at an excitation wavelength of about 502nm, and also shows a maximum fluorescence intensity at an emission wavelength of about 530nm, and its fluorescence intensity is proportional to the level of reactive oxygen in the cell. The experimental operation was carried out strictly in accordance with the instructions of the kit, and the fluorescence signal was finally detected by fluorescence microscopy.

[0116] 10. Acute kidney injury and drug treatment

[0117] In this embodiment, we selected 30 male C57BL / 6J mice, each weighing about 22 g. All mice were randomly divided into seven groups: a normal control group, a normal saline control group, a cisplatin treatment group, a cisplatin + aloe-emodin high-dose treatment group, a cisplatin + aloe-emodin low-dose treatment group, and an aloe-emodin high- and low-dose single treatment group.

[0118] The mice in the normal control group were raised under standard feeding conditions and did not receive any treatment, serving as a negative control. The mice in the other groups were given the corresponding drugs by intraperitoneal injection.

[0119] Among them, in the cisplatin + aloe-emodin high-dose treatment group, mice were intraperitoneally injected with aloe-emodin once a day in the morning and evening six days before cisplatin administration as a pre-protective measure. The dose of aloe-emodin injected was 40 mg / kg, and aloe-emodin was a solution of 0.352 g of aloe-emodin in 8 mL of normal saline. On the seventh day, 15 mg / kg of cisplatin was intraperitoneally injected to induce AKI.

[0120] In the cisplatin + aloe-emodin low-dose treatment group, mice were intraperitoneally injected with aloe-emodin (doses of 10 mg / kg and 40 mg / kg, respectively) once a day six days before cisplatin administration as a pre-protective measure. The dose of aloe-emodin injected was 10 mg / kg, and aloe-emodin was a solution of 0.088 g aloe-emodin in 8 mL of normal saline. On the seventh day, 15 mg / kg cisplatin was intraperitoneally injected to induce AKI.

[0121] Mice were treated with aloe-emodin alone at the same dose as that in the cisplatin + aloe-emodin high- and low-dose groups as a positive control.

[0122] Mice in the normal saline control group were intraperitoneally injected with 0.2 mL of 0.9% saline in the morning and evening every day.

[0123] Mice in the cisplatin-treated group were housed normally for the first six days without any treatment, and were injected with 15 mg / kg cisplatin on the seventh day to induce AKI.

[0124] Twelve hours after the model was established, all mice were killed, blood was collected through orbital bleeding, and kidney tissue samples were taken for subsequent biochemical and pathological analyses.

[0125] 11. Serum creatinine measurement

[0126] Creatinine (CRE) is a product produced during muscle metabolism and is mainly excreted from the body through glomerular filtration. In a healthy state, the level of creatinine in the body usually remains relatively stable. The concentration of creatinine in the blood is an important indicator for assessing the glomerular filtration rate (GFR).

[0127] In the experiment in this embodiment, we use creatinase to specifically catalyze the conversion of creatinine into creatine, and then creatine generates hydrogen peroxide under the continuous action of creatinase and sarcosine oxidase. Hydrogen peroxide reacts with a specific color developer to produce a purple colored substance, which has a maximum absorption peak at 546 nanometers (nm). By measuring this absorption peak, we can calculate the content of creatinine in the sample. The specific experimental operation steps are as follows:

[0128] Blood samples from each group of mice were collected by orbital bleeding. The blood samples were centrifuged at 12,000 rpm for 10 minutes at room temperature to separate the serum. Subsequently, the separated serum samples were added to a 96-well plate and mixed evenly with reagent 1. After incubation at 37°C for 5 minutes, the absorbance value was read at a wavelength of 546 nm using an ELISA reader and recorded as A. 1 Then add reagent 2 and mix again, and continue to incubate at 37°C for 5 minutes. Read the absorbance at 546nm again and record it as A 2 , calculate the change in absorbance (△A=A 2 -A 1 ). Finally, according to the calculation formula provided by the blood creatinine test kit, the change in absorbance is converted into the content of creatinine in serum, in micromoles per liter (μmol / L).

[0129] 12. Serum urea nitrogen determination

[0130] Urea is the final product of the metabolism of nitrogenous compounds in organisms. It can be decomposed and converted into ammonia under the action of urease. Urea nitrogen (BUN) in the blood is one of the key indicators for evaluating renal function. The content of urea nitrogen can reflect the kidney's ability to remove nitrogenous waste.

[0131] In the experiment of this embodiment, we use the diacetyl monoxime method to determine the urea nitrogen content in the sample. The specific steps are as follows:

[0132] The urea nitrogen in the animal serum sample is heated together with diacetyl monoxime and thiamine urea in a ferric chloride monophosphate solution. This process will cause the urea nitrogen to be converted into a red diazine compound. The color intensity of this red diazine compound is proportional to the content of urea nitrogen in the sample. The specific experimental operation steps are as follows:

[0133] First, blood samples from each group of mice were collected by orbital bleeding. The obtained blood samples were centrifuged at 12,000 revolutions per minute (rpm) at room temperature for 10 minutes to separate the serum. Subsequently, the separated serum samples were added to a 96-well plate and mixed evenly with reagents 1 and 2. The mixed samples were heated in a boiling water bath for 10 minutes to promote the chemical reaction. After heating, the samples were allowed to cool naturally. After cooling, the absorbance of the samples was measured at a wavelength of 540 nanometers (nm) using an ELISA reader, recorded as A. 测定 At the same time, the absorbance value of the blank control group was measured and recorded as A 空白 Then calculate the change in absorbance (△A = A 测定 -A 空白 ). Finally, according to the calculation formula of serum urea nitrogen, the change in absorbance value is converted into the content of urea nitrogen in serum, in milligrams per milliliter (mg / ml).

[0134] 13. Histology

[0135] The removed kidney tissue was first fixed in a 4% paraformaldehyde solution for 48 hours to maintain its structural integrity. Subsequently, the tissue samples were dehydrated with gradient alcohol, a step that helps remove water from the tissue in preparation for subsequent paraffin embedding. After paraffin embedding, the tissue was cut into 4 micron (μm) thick sections, which are thick enough to provide clear tissue structure.

[0136] The sections are heated in a slide oven for about 20 minutes to fix the sections and enhance the staining effect. After that, they are dewaxed and hydrated using xylene and graded alcohols. This process helps remove the paraffin in the sections and rehydrates the tissue in preparation for staining. Next, the sections are stained with hematoxylin and eosin (H&E). Hematoxylin is a basic dye that makes the cell nucleus appear blue; eosin is an acidic dye that makes the cytoplasm appear red. This staining method can clearly show the morphological structure of cells and tissues. The stained sections are dehydrated and transparentized through graded alcohol and xylene to remove excess water and staining agents, and finally sealed with neutral gum to protect the sections and increase their transparency for easy observation.

[0137] Kidney sections were observed under a microscope, and the application of microscope software optimized the imaging process, improving the quality of images and the accuracy of analysis. At a magnification of 10×10, detailed tissue damage analysis was performed on kidney tissue sections to evaluate the pathological changes of the kidney.

[0138] 14. Statistical analysis

[0139] All experiments were performed at least 3 times, and the results were expressed as mean ± SD. The t-test was used to evaluate the statistical difference between the two groups, and p < 0.05 and p < 0.01 were considered statistically significant.

[0140] 2. Results

[0141] 1. AE can alleviate HK-2 cell damage induced by erastin

[0142] The chemical structure of AE is Figure 6 As shown in Figure A.

[0143] The results of cell viability experiments showed that AE at concentrations of 1 μM and 3 μM could effectively reverse the decrease in cell viability induced by erastin in a dose-dependent manner ( Figure 6 Figure B and Figure 6 Figure C in the figure).

[0144] 2. AE effectively protects HK-2 cells from damage by reducing oxidative stress response

[0145] It is well known that iron-induced oxidative stress is closely related to ferroptosis, which plays an important role in cisplatin-induced acute kidney injury. To evaluate the effect of AE on ferroptosis-related oxidative stress markers, we performed a series of experimental tests.

[0146] Through DCFH-DA (a reactive oxygen species (ROS) fluorescent probe) staining experiments, we observed that AE significantly inhibited the accumulation of intracellular ROS associated with erastin-induced ferroptosis ( Figure 7 Figure A in Figure 1).

[0147] In addition, we measured intracellular malondialdehyde (MDA) and free iron ions (Fe 2+ ) levels, and it was found that AE could significantly reduce the levels of MDA and Fe in cells treated with erastin. 2+ level( Figure 7 Figure B and Figure 7 Figure C in the figure).

[0148] We further tested the level of glutathione (GSH), a key indicator of intracellular redox balance, and the results showed that AE could increase the total GSH level and GSH / GSSG ratio in cells ( Figure 7 This indicates that AE enhanced the antioxidant capacity of cells.

[0149] In summary, the above experimental results consistently prove that AE exhibits significant antioxidant stress effects during erastin-induced ferroptosis of HK-2 cells, thereby providing protection for the cells.

[0150] 3. AE inhibits ferroptosis by activating the Nrf2 signaling pathway and the expression of downstream signals

[0151] As a key antioxidant transcription factor, Nrf2 plays a central role in regulating a series of downstream genes involved in xenobiotic detoxification and antioxidant responses. It can effectively reduce the accumulation of lipid peroxides during ferroptosis. In addition, Nrf2 is also involved in regulating the expression of a variety of genes related to iron metabolism. GPX4, as a key downstream target of the Nrf2 signaling pathway, plays a vital role in inhibiting ferroptosis by catalyzing the reduction of lipid peroxides by GSH. The transcriptional activity of GPX4 is directly regulated by Nrf2. Based on these reports, we hypothesized that AE may alleviate erastin-induced ferroptosis in HK-2 cells by activating Nrf2 and affecting its downstream signaling pathways.

[0152] Western blot and qRT-PCR experimental results showed that erastin treatment reduced the expression levels of Nrf2 and GPX4 proteins and mRNA in HK-2 cells, while cells pre-treated with AE showed a significant increase in the levels of Nrf2 and GPX4 proteins and mRNA, which was significantly different from the erastin-treated group ( Figure 8 Figure A in Figure 8 Figure B in Figure 8 Figure C in Figure 8 Figure D in Figure 8 Immunofluorescence detection further confirmed this result ( Figure 8 The G graph in Fig. 9 Figure A in Figure 1).

[0153] To verify the mechanism of Nrf2 in the protective effect of AE, we used the Nrf2 inhibitor ML385 to inhibit the expression of Nrf2 in HK-2 cells. Cell viability assay showed that the cell viability decreased after the addition of ML385 ( Figure 8(F) Western blot analysis showed that Nrf2 and GPX4 protein levels were significantly decreased in cells treated with ML385 compared with untreated HK-2 cells, indicating that ML385 inhibited the protective effect of AE ( Fig. 9 Figure B in Fig. 9 Figure C in Fig. 9 Figure D in the figure).

[0154] We also measured the level of ROS, and the results showed that compared with the normal group and the erastin-treated group, the addition of ML385 increased the accumulation of reactive oxygen species, and the AE pretreatment group significantly reduced the level of intracellular reactive oxygen species ( Fig. 9 Figure E in the figure).

[0155] In addition, we measured the levels of Fe2+, MDA, and GSH in HK-2 cells after adding ML385, which was consistent with the above results ( Fig. 9 Figure F in Fig. 9 The G graph in Fig. 9 These findings support the involvement of Nrf2 in the protective effect of AE on erastin-induced ferroptosis in HK-2 cells, indicating that AE activated the Nrf2 signaling pathway and enhanced the expression of GPX4, thereby exerting its antioxidant and anti-ferroptosis effects in cells.

[0156] 4. AE can alleviate cisplatin-induced AKI in mice

[0157] To investigate whether AE has a protective effect in vivo, we used a cisplatin-induced AKI mouse model for in-depth study. Through biochemical analysis, we found that AE significantly alleviated the sharp increase in serum urea nitrogen (BUN) and serum creatinine (Scr) levels induced by cisplatin ( Fig.10 Figure A in Fig.10 The improvement of these indicators indicates that AE has potential efficacy in alleviating renal damage, while AE itself has no effect on BUN and Scr ( Fig.10 Figure C in Fig.10 Figure D in the figure).

[0158] Furthermore, we observed the effect of AE on cisplatin-induced renal injury by H&E pathological staining. The results of H&E staining showed that in the AKI model group, the renal tubular epithelial cells showed pathological changes such as turbidity and swelling, vacuolar degeneration, disordered cell arrangement, and protein cast formation, accompanied by the dissolution of renal tubular cells. In contrast, the pathological changes of renal tissue in the AE-treated group were significantly alleviated, while there were no significant changes in renal tissue in the AE- and saline-treated groups ( Fig.10 Figure E in the figure).

[0159] The above results indicate that AE can significantly improve cisplatin-induced renal pathological damage and has no obvious toxic effect on renal tissue, which provides a strong experimental basis for the application of AE in the treatment of AKI.

[0160] 5. AE inhibits lipid peroxidation and ferroptosis in cisplatin-induced AKI mice

[0161] To further explore whether AE can regulate the ferroptosis process in renal tubular epithelial cells of AKI mice, we investigated the iron ion (Fe 2+ ) and glutathione (GSH) levels were detected. The experimental results revealed that AE significantly reduced the accumulation of Fe2+ in the kidneys of mice induced by cisplatin, and at the same time, AE also effectively reduced the consumption of glutathione ( Fig.11 Figure A in Fig.11 This finding further confirms the potential role of AE in alleviating renal ferroptosis.

[0162] In addition, we also analyzed the ferroptosis-related metabolite MDA. The results showed that AE could significantly reduce the accumulation of MDA in the kidney tissue and serum of AKI mice ( Fig.11 Figure C in Fig.11 Figure 2 (D). The decreased level of MDA, an important marker of lipid peroxidation, further confirmed the role of AE in inhibiting lipid peroxidation and ferroptosis in cisplatin-induced AKI mice.

[0163] These results indicate that AE has a significant protective effect on the cisplatin-induced AKI mouse model by regulating iron metabolism and antioxidant response.

[0164] 6. AE regulates ferroptosis in cisplatin-induced AKI mice by activating the Nrf2 signaling pathway and downstream GPX4 expression

[0165] To further verify whether AE regulates ferroptosis in cisplatin-induced AKI mice by activating the Nrf2 signaling pathway, we used Western blot and qRT-PCR techniques to analyze the expression levels of Nrf2 and -GPX4, a downstream target of the Nrf2 signaling pathway.

[0166] Western blot and qRT-PCR experimental results showed that the expression levels of Nrf2 and GPX4 proteins and mRNA were significantly reduced in the cisplatin-induced AKI mouse model. However, AE treatment significantly restored the expression levels of Nrf2, GPX4 proteins and mRNA ( Fig.12 Figure A in Fig.12 Figure B in Fig.12 Figure C in Fig.12 Figure D in Fig.12 This result indicates that AE can counteract cisplatin-induced ferroptosis by activating the Nrf2 signaling pathway. This not only confirms the role of GPX4 as a downstream target of Nrf2 in AKI, but also further supports the hypothesis that AE enhances GPX4 expression by activating the Nrf2 signaling pathway, thereby playing a protective role in the cisplatin-induced AKI mouse model. Through this mechanism, AE can enhance the antioxidant defense capacity of the kidney and alleviate ferroptosis-related damage, which provides strong support.

[0167] These findings confirm the potential mechanism of action of AE in regulating ferroptosis and provide strong molecular mechanistic evidence for AE as a potential therapeutic drug for cisplatin-induced AKI. By activating the Nrf2 pathway, AE can enhance the antioxidant capacity and iron metabolism regulation of mouse kidneys, thereby alleviating cisplatin-induced AKI and ferroptosis.

[0168] The above experimental results show that AE can inhibit ferroptosis and alleviate cisplatin-induced acute kidney injury by activating the Nrf2 signaling pathway. AE, as a new type of Nrf2 ferroptosis inhibitor, has clinical potential for the treatment of cisplatin-induced acute kidney injury.

[0169] Compared with existing studies, the present invention's research deeply reveals the potential of AE as a new type of ferroptosis inhibitor. The present invention found that AE can effectively alleviate cisplatin-induced oxidative stress in HK-2 cells by activating Nrf2 and its downstream antioxidant protein GPX4. In addition, AE also finely regulates the occurrence of ferroptosis by regulating intracellular iron metabolism and reducing lipid peroxidation.

[0170] Based on these findings, the present invention proposes that Nrf2 is a key molecular target for AE in the treatment of AKI. AE exerts a significant renal protective effect by inhibiting Nrf2-mediated ferroptosis of renal tubular epithelial cells. These research results not only provide a new perspective for the treatment strategy of cisplatin-induced acute kidney injury, but also open up a new path for the development of Nrf2 inhibitors as effective drugs for the clinical treatment of cisplatin-induced AKI.

[0171] These characteristics of AE make it a promising candidate drug, providing new ideas and methods for the future clinical treatment of cisplatin-induced AKI. The research of the present invention not only deepens the understanding of the mechanism of action of AE, but also provides an important scientific basis for the development of new treatment strategies and drugs.

[0172] It should be noted that when the present invention involves a numerical range, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes a preferred embodiment.

[0173] Although preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they understand the basic inventive concepts, and all such changes and modifications fall within the scope of the present invention.

Claims

1. Application of aloe-emodin in the preparation of drugs for treating cisplatin-induced acute kidney injury.

2. The use according to claim 1, characterized in that: The medicine uses the aloe-emodin as an active ingredient and is supplemented with pharmaceutically acceptable excipients or carriers.

3. The use according to claim 2, characterized in that: The auxiliary materials include any one or more of mannitol, microcrystalline cellulose, lactose, corn starch, stearic acid, dextrin, powdered sugar, glycerol, physiological saline, DMSO and Tween.

4. The use according to claim 2, characterized in that: The carrier includes any one or more of liposomes, nanoparticles, methylcellulose and chitosan.

5. The use according to claim 2, characterized in that: The medicine includes any one of capsules, tablets, injections, granules, solutions, patches and ointments.

6. The use according to claim 5, characterized in that: The drug is a solution; The solution is prepared by compounding the aloe-emodin with a solvent; The solvent includes any one of water, ethanol, vegetable oil, DMSO, glycerol, physiological saline and Tween.

7. The use according to claim 6, characterized in that: The concentration of aloe-emodin in the medicine is 1 μM to 3 μM.

8. The use according to claim 6, characterized in that: When the solvent is water, glycerol and / or physiological saline, the drug is a drug for human use; When the solvent is ethanol, vegetable oil, DMSO and / or Tween, the drug is a drug for animal use.

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