Trilobatin in preparation of medicine for treating acute kidney injury and application of trilobatin

Abstract

The invention belongs to the field of new application of medicines, and particularly relates to trilobatin in preparation of a medicine for treating acute kidney injury and application of the trilobatin. It is found for the first time that trilobatin inhibits acute kidney injury and HK-2 cell ferroptosis by up-regulating Nrf2 / GPX4 in a glutathione pathway, and can improve acute kidney injury caused by cis-platinum. A new candidate drug is provided for clinical treatment of acute kidney injury.

Description

Technical Field

[0001] The present invention belongs to the field of new drug applications, and more specifically, relates to the use of trilobatin in the preparation of drugs for treating acute kidney injury and the use thereof. Background Art

[0002] Acute kidney injury (AKI) is a common critical illness with complex pathophysiological mechanisms and currently lacks effective drugs. Cisplatin is a common and effective anti-tumor drug. It is one of the main causes of clinical acute kidney injury. Its frequent use or overdose can cause nephrotoxicity. Nephrotoxicity mainly affects the kidneys through proximal tubular uptake. When cisplatin causes acute kidney injury, it can lead to oxidative stress, inflammation, endoplasmic reticulum stress, cell necrosis, apoptosis, and vascular damage. Recent studies have shown that ferroptosis, a newly discovered regulated cell death mode characterized by the lethal accumulation of iron and reactive oxygen-dependent lipid hydroperoxides, also plays a key role in acute kidney injury and has been shown to be involved in the progression of acute kidney injury.

[0003] Trilobatin is a glycosylated dihydrochalcone extracted from the leaves of Camellia oleifera. It has low cytotoxicity and good solubility. Currently, the research on trilobatin mainly focuses on hypoglycemic, anti-inflammatory, anti-cancer and antioxidant effects. In order to further expand the application scope of trilobatin, further research is needed on other pharmaceutical uses of trilobatin. Summary of the invention

[0004] The purpose of the present invention is to provide an application of trilobatin in the preparation of a medicine for treating acute kidney injury, so as to solve the above technical problems.

[0005] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0006] Application of trilobatin in the preparation of medicines for treating acute kidney injury.

[0007] Trilobatin is a sweetener derived from Lithocarpus polystachyus Rehd, CAS No.: 4192-90-9. Trilobatin is an HIV-1 inhibitor that targets the Gp41 envelope protein. It has a neuroprotective effect. Trilobatin is also an SGLT1 / 2 inhibitor that can selectively induce proliferation of human hepatoblastoma cells. The present invention first discovered that trilobatin can inhibit acute kidney injury and HK-2 cell ferroptosis by upregulating Nrf2 / GPX4 in the glutathione pathway, providing a new idea for the treatment of acute kidney injury and expanding the pharmaceutical use of trilobatin.

[0008] Furthermore, the acute kidney injury includes acute kidney injury caused by cisplatin.

[0009] Furthermore, the trilobatin is used in the preparation of a drug for treating acute kidney injury, wherein the trilobatin is the only active ingredient in the drug.

[0010] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0011] Furthermore, the auxiliary materials include any one or more of non-toxic fillers, stabilizers, diluents, and adjuvants.

[0012] Furthermore, the diluent is any one of water and physiological saline.

[0013] Based on the same inventive concept, the present invention also provides a drug for treating acute kidney injury, wherein the drug is prepared by mixing the trilobatin and excipients, and the content of trilobatin in the drug is 0.1wt% to 99wt%.

[0014] Furthermore, the dosage form of the drug is a solid dosage form or a solution dosage form, the solid dosage form includes granules, tablets, capsules, pills, and pellets, and the solution dosage form includes oral liquid preparations, intragastric lavages, and injection dosage forms.

[0015] Furthermore, the solution dosage form is a solution consisting of water and the trilobatin, or a solution consisting of physiological saline and the trilobatin.

[0016] Beneficial effects:

[0017] The present invention found through research that trilobatin inhibits acute kidney injury in mice and ferroptosis of HK-2 cells by upregulating Nrf2 / GPX4 in the glutathione pathway. And trilobatin cannot further protect HK-2 cells from ferroptosis using the specific Nrf2 inhibitor ML385. Experimental data show that trilobatin needs to rely on Nrf2 to prevent ferroptosis in acute kidney injury. This provides a new candidate drug for the clinical treatment of acute kidney injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Figure 1 is a graph of serum urea nitrogen (BUN) and blood creatinine (Scr) in mice treated with trilobatin, where: A is a graph of serum urea nitrogen levels in the trilobatin treatment group. B is a graph of blood creatinine levels in the trilobatin treatment group.

[0019] Figure 2 This is a cisplatin-induced acute kidney injury model in mice and its treatment plan.

[0020] Figure 3 The graphs are the serum urea nitrogen and serum creatinine of mice treated with cisplatin, where: A is the serum urea nitrogen level graph of the cisplatin-treated group. B is the serum creatinine level graph of the cisplatin-treated group.

[0021] Figure 4 Representative images of HE-stained mouse kidney sections, where CDDP represents cisplatin and TLB represents trilobatin.

[0022] Figure 5 The figure shows the detection of physiological indicators of mouse tissues, where A is the content of glutathione (GSH) in tissues. B is the content of Fe 2+ C is the content of malondialdehyde (MDA) in tissues. D is the content of malondialdehyde in tissues and serum.

[0023] Figure 6 This is the half-inhibitory concentration curve of Erastin on HK-2 cells.

[0024] Figure 7 This is the half-maximal effect concentration of trilobatin on HK-2 cells induced by Erastin.

[0025] Figure 8 Representative images of DCFH-DA staining.

[0026] Fig. 9 It is a cell physiological index detection diagram, where: A is the percentage of intracellular glutathione. B is the intracellular Fe 2+ A is a graph showing the content of MDA in cells. C is a graph showing the content of MDA in cells. D is a graph showing the cell specificity.

[0027] Fig.10 It is a bioinformatics prediction analysis diagram, where: A is a Venny diagram of the number of overlapping genes between acute kidney injury, trilobatin and ferroptosis. B is a protein-protein interaction network diagram between acute kidney injury, ferroptosis and trilobatin.

[0028] Fig.11 Gene ontology (GO) analysis of the cross-talk between acute kidney injury, trilobatin and ferroptosis using the DAVID bioinformatics resource.

[0029] Fig.12 Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of the cross-talk genes between acute kidney injury, trilobatin and ferroptosis for DAVID bioinformatics resources.

[0030] Fig.13 The figures are immunoblot images of mouse kidney tissue proteins, where A is the immunoblot image of Nrf2 and GPX4 proteins, B is the analysis image of Nrf2 protein expression, and C is the analysis image of GPX4 protein expression.

[0031] Fig.14 The following are gene expression diagrams of mouse kidney tissue, where A is the Nrf2 gene expression diagram and B is the GPX4 gene expression diagram.

[0032] Fig.15The following are the immunoblot images of cell proteins, where A is the immunoblot image of cell Nrf2 and GPX4 proteins. B is the analysis image of cell Nrf2 protein expression. C is the analysis image of cell GPX4 protein expression.

[0033] Fig.16 : Cell gene expression diagram, where: A is the cell Nrf2 gene expression diagram. B is the cell GPX4 gene expression diagram.

[0034] Fig.17 The figure is a graph showing the effect of ML385, where A is the half-inhibitory concentration curve of ML385 on HK-2 cells and B is the cell viability value graph.

[0035] Fig.18 The following is a diagram of the physiological level of cells under the action of trilobatin, where A is the percentage of intracellular glutathione. B is the percentage of intracellular Fe 2+ A is a graph showing the content of malondialdehyde in cells. C is a graph showing the content of malondialdehyde in cells. D is a representative graph showing DCF-DA staining.

[0036] Fig.19 The following are the immunoblot images of cell proteins under the action of trilobatin, where A is the immunoblot image of cell Nrf2 and GPX4 proteins. B is the analysis image of cell Nrf2 protein expression. C is the analysis image of cell GPX4 protein expression.

[0037] Fig. 20 The following are analysis diagrams of inflammatory factor expression, where A is the tissue TNF-α gene expression diagram, B is the tissue IL-6 gene expression diagram, and C is the tissue IL-1β gene expression diagram. DETAILED DESCRIPTION

[0038] 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.

[0039] Example 1

[0040] 1. Experimental Materials

[0041] 1. Trilobatin with a purity of ≥98% was provided by Shanghai McLean Biochemical Technology Co., Ltd. (Shanghai, China). Dimethyl sulfoxide was provided by Shanghai Myrel Chemical Technology Co., Ltd. (Shanghai, China). Cisplatin was provided by Shanghai Bid Pharmaceutical Technology Co., Ltd. (Shanghai, China). Erastin with a purity of 99.96% and ML385 with a purity of 99.96% were purchased from MCE. Anti-TNF-α and IL-6 antibodies were provided by Abcam (Cambridge, USA); Nrf2, GPX4, Cy3 fluorescent goat anti-rabbit IgG, β-actin, and enzyme-labeled goat anti-rabbit IgG were purchased from Proteintech (Chicago, USA). In addition, serum creatinine and blood urea nitrogen detection kits were from milbio (Shanghai, China); glutathione kits and protein BCA quantification kits were purchased from Beyotime Biotechnology Institute (Jiangsu, China). Reverse transcription kits and real-time fluorescence PCR kits were provided by GenStar (Beijing, China). All other reagents were purchased from SolarBio unless otherwise specified.

[0042] 2. The experimental animals were SPF grade male C57 mice. The mice were kept in an environment with a temperature of 20-24℃ alternating between day and night for 12 hours, and had free access to sufficient feed and water.

[0043] 2. Experimental Methods

[0044] 1. Establishment of acute renal injury model: After acclimation for 1 week, the mice were randomly divided into 5 groups, with 6-8 mice in each group, namely: (1) normal group; (2) cisplatin group: 0.9% saline was intraperitoneally injected for 6 consecutive days. At the end of the 6th day, a single intraperitoneal injection of cisplatin was given at a concentration of 15 mg / kg. (3) Cisplatin + trilobatin concentration 10 mg / kg group: trilobatin was intraperitoneally injected for 6 consecutive days, and cisplatin was injected intraperitoneally at the end of the 6th day; (5) Cisplatin + trilobatin concentration 40 mg / kg group: trilobatin was intraperitoneally injected for 6 consecutive days, and cisplatin was injected intraperitoneally at the end of the 6th day. On the 7th day, blood was collected from the mouse orbit and perfused, and then serum was prepared, and blood urea nitrogen and blood creatinine were measured to evaluate renal function. The kidneys were removed in time for further analysis.

[0045] 2. Cell culture and grouping Cell experiments: HK-2 cells were cultured in MEM medium containing 10% fetal bovine serum, 50 μg / mL penicillin and 50 μg / mL streptomycin. The culture was placed in an incubator at 37°C with 5% carbon dioxide. The cell experiment groups were: control group, Erastin concentration 4.6 μM group, Erastin concentration 4.6 μM + DMSO concentration 3 μM group, Erastin concentration 4.6 μM + trilobatin concentration 1 μM group, Erastin concentration 4.6 μM + trilobatin concentration 3 μM group, Erastin concentration 4.6 μM + ML385 concentration 2.73 μM group, Erastin concentration 4.6 μM + ML385 concentration 2.73 μM + trilobatin concentration 3 μM group. After 24 hours of culture, the cells were collected for immunoblot analysis, real-time quantitative polymerase chain reaction, and other experimental methods for detection of cell activity, ferroptosis, oxidative stress and inflammatory response.

[0046] 3. Network pharmacology prediction: The chemical structure of trilobatin was searched using the PubChem platform (https: / / pubchem.ncbi.nlm.nih.gov) and saved as an SDF format file. It was then imported into the PharmMapper Server platform (https: / / lilab-ecust.cn / pharmmapper / index.html) to predict its targets, setting the human protein target as the pharmacophore map and the number of targets returned to 300. The results were then converted by searching and selecting the species "Homo sapiens" in the Uniprot database (https: / / www.uniprot.org / ) to obtain trilobatin targets and potential target genes. GeneCards database (https: / / www.genecards.org / ) and FerrDb database (http: / / www.zhounan.org / ferrdb / ) were used to obtain targets related to acute kidney injury and ferroptosis, and the names of these targets were standardized using the UniProt database. The Venny online tool was used to analyze ferroptosis-related genes and common target genes between trilobatin and acute kidney injury (https: / / bioinfogp.cnb.csic.es / tools / venny / index.html), and 33 common target genes were obtained between the three. The common target genes were entered into the STRING database to construct the PPI network, and these data were analyzed using Cytoscape (version 3.9.1) to construct a visualized compound-target network. GO is an important bioinformatics tool for annotating genes and analyzing the biological processes (BP), cellular components (CC), and molecular functions (MF) of these genes. KEGG is a database for systematic analysis of gene function, which links genomic information with high-order functional information and assigns functional significance to genes and genomes at the molecular and higher levels. The shared genes were functionally annotated and pathway enrichment analyzed using the Database for Annotation, Visualization, and Integrated Discovery (DAVID, version 6.8; http: / / david.ncifcrf.gov).

[0047] 3. Sample Testing

[0048] 1. Serum creatinine and blood urea nitrogen blood sample analysis: Serum was centrifuged at 12000 rpm for 15 minutes at 4°C. The supernatant was transferred to a new centrifuge tube, and the sample was added according to the instructions of the serum creatinine and blood urea nitrogen test kit, and then the test was analyzed under the microplate reader.

[0049] 2. Cell viability assay: Cell counting kit was used to detect cell viability. HK-2 cells were plated at 3×10 3Cells were seeded in 96-well plates at a density of 100 μL / well culture medium overnight, and then cultured for 24 h with Erastin, trilobatin or ML385 in the experimental protocol, either alone or in combination. The original culture medium was then replaced with 100 μL of fresh culture medium containing 10 μL CCK-8 solution, incubated at 37°C for 1 h, and the absorbance at 450 nm was recorded on a microplate reader. The relative cell survival rate was expressed as a percentage of the control.

[0050] 3. Cytotoxicity test: The toxicity of trilobatin to HK-2 cells was detected using a cell counting kit. 3 The cells were inoculated into a 96-well plate at a density of 1.5 and cultured overnight in 100 μL / well culture medium. Then, the cells were incubated with trilobatin for 12 h at a certain concentration gradient. The original culture medium was replaced with 100 μL of fresh culture medium containing 10 μL CCK-8 solution and incubated at 37°C for 1 h. Finally, the absorbance at 450 nm was recorded on a microplate reader, and the relative cell survival rate was expressed as a percentage of the control.

[0051] 4. Determination of malondialdehyde, glutathione, and iron content: Malondialdehyde, glutathione, and iron ion detection kits were used to detect lipid peroxidation in renal tissue and HK-2 cells according to the manufacturer's instructions.

[0052] 5. Reactive oxygen species (ROS) measurement: The generation of reactive oxygen species was observed using the 2',7'-dichlorofluorescein diacetate (DCF-DA) cell reactive oxygen species kit under dark conditions. HK-2 cells were plated at 1×10 4 The cells were seeded into 24-well plates at the same density and cultured overnight in normal culture medium to adhere to the plate. After the treatment of different groups of drugs, the cells were stained with DCF-DA for 45 min, and then the level of reactive oxygen species was detected using a fluorescence microscope (Leica, Germany) (magnification × 100).

[0053] 6. Western blotting: Equal amounts of tissues or cells after disruption were lysed with pre-cooled cell lysis buffer at 4°C for 40 min. The supernatant was collected after centrifugation at 12000rpm, 4°C for 10 min. The protein concentration was determined using the BCA protein assay kit. The samples were heated at 100°C for 10 min to denature. Equal amounts of protein were separated using 10% SDS-PAGE and then transferred to PVDF membranes. They were blocked with 10% skim milk for 1 h and incubated with primary antibodies at 4°C overnight. The membranes were then washed 3 times with TBST, incubated with rabbit HRP-conjugated secondary antibodies at room temperature for 2 h, and finally washed 3 times with TBST and displayed with ECL chemiluminescent substrate. The band intensity was quantified using Image J software and normalized to the band intensity of β-actin.

[0054] 7. RNA extraction and real-time fluorescence quantitative polymerase chain reaction: Add 1 ml of pre-cooled Trizl reagent to an equal amount of kidney tissue or cells, and disrupt the cells and tissues. Then centrifuge at 12000 rpm and 4°C for 5 minutes. Transfer the supernatant to a new EP tube and incubate on ice for 5 minutes, then add 200 μL of chloroform. Take the supernatant and centrifuge it at 12000 rpm for 30 minutes, mix it with an equal amount of isopropanol, place it at -20°C for 40 minutes, centrifuge it at 12000 rpm for 30 minutes, and discard the supernatant. After adding 1 ml of 75% ethanol, the supernatant was centrifuged at 7500 rpm for 20 minutes. Discard the supernatant and air-dry the precipitate. Use GenStar reverse transcription polymerase chain reaction to detect the mRNA levels of Nrf2, GPX4, IL-1β, TNF-α, IL-6, etc. Ct(2 -ΔΔCt )-related gene expression and β-actin gene expression were normalized. The primer sequences are shown in the following table.

[0055] Table 1 Primer sequences

[0056]

[0057] 8. Immunofluorescence staining: According to the instructions of the kit, HK-2 cells were cultured on a cell slide at 1×10 4 The cells were seeded into 24-well plates at a high density and cultured overnight in normal culture medium to adhere to the wall. Then they were fixed with 4% paraformaldehyde for 10 min at 37°C, blocked with 10% bovine serum albumin, incubated with rabbit anti-Nrf2 and rabbit anti-GPX4 at 4°C overnight, and then incubated with fluorescent secondary antibodies. The cell nuclei were stained with DAPI for 5 min. Then they were observed with a fluorescence microscope (Leica, Germany) (magnification × 100).

[0058] 9. Histological staining: After the kidney was taken, it was cut along the coronal plane, fixed with 4% paraformaldehyde for 48 hours, embedded in paraffin after gradient dehydration, and sliced ​​into 4μM slices. These slices were dewaxed with xylene and then hydrated in gradient concentrations of alcohol. HE staining was used for histological observation. Staining was performed according to the instructions of the hematoxylin-eosin staining kit, then dehydrated with gradient alcohol, transparent with xylene, and finally sealed with a neutral adhesive. The staining images were observed with a fluorescence microscope (Leica, Germany) (magnification × 100) and electron microscope observation.

[0059] 4. Experimental Data Analysis

[0060] Statistical analysis: All data are expressed as mean ± standard deviation. One-way ANOVA was used for analysis, and GraphPad Prism software was used for T test and graphical representation of experimental data. P value < 0.05, the difference was statistically significant. Mean ± SD, n = 3, ns-not significant. *, #, p < 0.05; **, ##, p < 0.01, ***, ###, p < 0.001, ****, ####, p < 0.0001.

[0061] V. Experimental Results

[0062] 1. Protective effect of trilobatin on cisplatin-induced acute kidney injury

[0063] According to previous reports, renal tubular cell ferroptosis plays an important role in the pathogenesis of acute kidney injury. Based on the effective inhibitory effect of trilobatin on ferroptosis, the possible role of trilobatin in acute kidney injury was explored. Figure 1 As shown in A and B, trilobatin itself had no effect on the blood urea nitrogen and blood creatinine levels of normal mice. Figure 2 As shown. Figure 3 As shown in A and B, compared with the control group, the levels of serum creatinine and blood urea nitrogen in mice were significantly increased after cisplatin injection; however, the levels of serum creatinine and serum urea nitrogen were reversed after trilobatin treatment (P<0.05). HE staining of renal tissue further showed the protective effect of trilobatin on renal injury. Figure 4 As shown in the results, cisplatin-induced morphological damage to the renal tubules of mice included necrosis, loss of brush edges, cast formation, tubular degeneration, and severe cavitation. These damages were inhibited by trilobatin treatment, and the results showed that trilobatin could significantly reduce histological damage. Next, the effects of trilobatin on lipid peroxidation and ferroptosis in cisplatin-induced acute kidney injury were evaluated in vivo. Compared with the control group, Figure 5 As shown in A, cisplatin significantly increased the Fe 2+ ,like Figure 5 As shown in Figure B, cisplatin significantly reduced the percentage of glutathione in the kidneys of mice. Figure 5 As shown in C and D, cisplatin significantly increased the content of malondialdehyde in the kidney and serum of mice, and trilobatin significantly reversed the changes in these contents in the kidney of mice (P<0.05). The results showed that trilobatin can treat acute kidney injury by inhibiting ferroptosis of renal tubular epithelial cells.

[0064] 2. Trilobatin inhibits ferroptosis of renal tubular cells

[0065] Erastin is a classic inducer of ferroptosis. Studies on the mechanism of ferroptosis induced by it have shown that ferroptosis is a unique form of regulated cell death. Erastin can induce ferroptosis in HK-2 cells. Figure 6 As shown in Figure 2, after treating HK-2 cells with Erastin for 24 hours, cell viability decreased in a dose-dependent manner. Under the stimulation of 4.6 μM Erastin, the cell survival rate was close to 50%. Therefore, 4.6 μM Erastin was used to induce ferroptosis in HK-2 cells to evaluate the protective effect of trilobatin on HK-2 cells. Figure 7 As shown, the concentration of 100nM-1uM trilobatin had no effect on the viability of HK-2 cells, but the cell viability began to decrease at around 3uM. Based on the effect of trilobatin on the viability of HK-2 cells, the optimal trilobatin treatment concentration was selected for subsequent experiments. The results showed that trilobatin dose-dependently attenuated Erastin-induced HK-2 cell ferroptosis, and the use of trilobatin at concentrations of 1μM and 3μM most significantly increased cell viability.

[0066] 3. Trilobatin inhibits the generation of reactive oxygen species and lipid peroxidation

[0067] When cells undergo ferroptosis, a large amount of reactive oxygen species is produced. Free iron interacts with reactive oxygen species to form lipid free radicals, which react rapidly with oxygen to induce lipid peroxidation. First, fluorescent probes were used to study intracellular reactive oxygen species. The results showed that, Figure 8 As shown in the results, Erastin induced the generation of reactive oxygen species in HK-2 cells, while trilobatin reversed the level of reactive oxygen species. In addition, to further confirm that the inhibition of ferroptosis by trilobatin was related to lipid peroxidation, the levels of cellular glutathione, malondialdehyde and Fe 2+ The results show that Fig. 9 As shown in A, glutathione was significantly downregulated after Erastin treatment. Fig. 9 B and C show upregulation of MDA and Fe 2+ , and these trends were reversed by trilobatin treatment. In addition, it is worth noting that trilobatin could not rescue cell viability in H2O2-induced apoptosis and cisplatin-induced pyroptosis of HK-2 cells. These results indicate that trilobatin can selectively inhibit ferroptosis of HK-2 cells.

[0068] 4. Prediction of Nrf2 as a key indicator for the treatment of acute kidney injury with trilobatin

[0069] like Fig.10 , Fig.11 , Fig.12As shown, in order to further explain the relationship between trifoliate glycosides, ferroptosis and acute kidney injury, their common target genes were analyzed. According to the Cytoscape software degree algorithm to screen common target genes, Nrf2 and other genes ranked high, revealing that these genes may be the main hub genes of trifoliate glycosides, which can achieve anti-acute kidney injury effects by regulating ferroptosis.

[0070] 5. Trilobatin inhibits acute kidney injury by regulating the Nrf2 / GPX4 pathway

[0071] To evaluate whether Nrf2 is the pharmacological target of trilobatin, in vivo experiments were first performed. Western blot analysis showed that Fig.13 As shown in Figure 2, Nrf2 and GPX4 protein levels were significantly downregulated after cisplatin treatment but recovered after trilobatin treatment. Fig.14 As shown in the figure, the mRNA levels of Nrf2 and GPX4 in the kidney tissue of mice in the cisplatin group were reduced, and trilobatin treatment significantly increased the transcription levels of Nrf2 and GPX4. The experimental results suggest that the mechanism by which trilobatin alleviates acute kidney injury is related to Nrf2.

[0072] 6. Trilobatin inhibits ferroptosis of HK-2 cells by regulating the Nrf2 / GPX4 pathway

[0073] In vitro experiments were then performed to explore the antioxidant defense of the cells. Western blot analysis showed that Fig.15 As shown, Nrf2 and GPX4 protein levels were significantly downregulated after erastin treatment, but recovered after trilobatin treatment. Fig.16 As shown in the figure, the mRNA levels of Nrf2 and GPX4 in HK-2 cells treated with Erastin decreased, and the transcription levels of Nrf2 and GPX4 were significantly increased by trilobatin treatment, suggesting that the mechanism by which trilobatin alleviates Erastin-induced ferroptosis in HK-2 cells is related to Nrf2.

[0074] 7. Inhibition of Nrf2 can eliminate the effects of trilobatin on ROS generation, lipid peroxidation and ferroptosis

[0075] Considering the role of Nrf2 in preventing lipid peroxidation and ferroptosis, the possibility that Nrf2 mediates the protective effect of trilobatin against ferroptosis was investigated. Fig.17 As shown in Figure B, consistent with the above results, trilobatin effectively increased the cell survival rate of HK-2 cells induced by erastin. However, in the ML385 treatment of Nrf2-inhibited cells, Fig.17 As shown in Figure A, the protective effect of trilobatin on erastin-induced ferroptosis in HK-2 cells was weakened. Fig.18The effects of trilobatin on glutathione and Fe 2+ The protective effect of consumption is almost eliminated. Fig.18 The protective effect of trilobatin on the formation of malondialdehyde was almost eliminated, as shown in C. Fig.18 As shown in D in Figure 1, the protective effect of trilobatin on the generation of reactive oxygen species was almost abolished. Next, the protein expression of key factors regulating cellular iron homeostasis and ferroptosis was detected. Fig.19 As shown in Figure 2, the regulatory effect of trilobatin on Nrf2 and GPX4 was almost abolished in Nrf2-inhibited cells. Western blot analysis showed that the expression of Nrf2 and Gpx4 proteins increased in the trilobatin group compared with the erastin group. These changes were reversed in Nrf2-inhibited cells. This suggests that the Nrf2 signaling pathway plays an important role in the protective effect of trilobatin on ferroptosis.

[0076] 8. Trilobatin has anti-inflammatory effects in the body

[0077] In acute kidney injury, inflammation of the proximal tubules has been shown to be essential. In vivo experiments, the expression of three proinflammatory cytokines in renal tissue increased after cisplatin stimulation, such as Fig. 20 As shown, administration of trilobatin had a significant inhibitory effect on the expression of TNF-a, IL-1b and IL-6 mRNA in renal tissue.

[0078] It should be noted that when the claims of the present invention involve numerical ranges, 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.

[0079] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0080] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. Application of trilobatin in the preparation of drugs for the treatment of acute kidney injury.

2. The use of trilobatin according to claim 1 in the preparation of a medicament for treating acute kidney injury, characterized in that: In the medicine, trilobatin is the only effective ingredient.

3. The use of trilobatin according to claim 1 in the preparation of a medicament for treating acute kidney injury, characterized in that: The drug also includes pharmaceutically acceptable excipients.

4. The use of trilobatin according to claim 3 in the preparation of a medicament for treating acute kidney injury, characterized in that: The auxiliary materials include any one or more of non-toxic fillers, stabilizers, diluents and adjuvants.

5. The use of trilobatin according to claim 4 in the preparation of a medicament for treating acute kidney injury, characterized in that: The diluent is any one of water and physiological saline.

6. A drug for treating acute kidney injury, characterized in that: The drug is prepared by mixing the trilobatin described in claim 1 with auxiliary materials, and the content of trilobatin in the drug is 0.1wt% to 99wt%.

7. The drug according to claim 6, characterized in that The dosage form of the drug is a solid dosage form or a solution dosage form. The solid dosage form includes granules, tablets, capsules, pills, and drip pills. The solution dosage form includes oral liquid preparations, intragastric lavages, and injection dosage forms.

8. The drug according to claim 7, characterized in that The solution dosage form is a solution consisting of water and the trilobatin, or a solution consisting of physiological saline and the trilobatin.

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