Application of dehydrodiisoeugenol in treating acute kidney injury by inhibiting ferroptosis

Abstract

The invention provides application of dehydrodiisoeugenol (DHE) to treatment of acute kidney injury by inhibiting ferroptosis, and belongs to the technical field of biological medicine. It is found that DHE can effectively inhibit cell ferroptosis, application of DHE as a ferroptosis inhibitor and application of DHE in preparation of drugs for treating acute kidney injury are provided, and DHE can inhibit renal tubular cell ferroptosis, recover the anti-oxidation defense function and relieve lipid peroxidation and iron deposition. It is proved that DHE is a natural small-molecule inhibitor of ACSL4, ACSL4-mediated ferroptosis can be effectively blocked, powerful protection is provided for cisplatin-induced acute kidney injury, and the DHE becomes a promising treatment candidate drug for cisplatin treatment of patients.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the use of dehydrodiisoeugenol in the treatment of acute kidney injury by inhibiting ferroptosis. Background Technology

[0002] Acute kidney injury (AKI) is a common and potentially fatal syndrome characterized by a rapid decline in renal function, leading to significant short-term and long-term morbidity and mortality. Cisplatin, a platinum-based chemotherapy drug widely used to treat various solid tumors, is nephrotoxic, with approximately one-third of patients experiencing dose-limited AKI. This not only reduces treatment efficacy but also poses a serious challenge to clinical prognosis. In proximal renal tubular epithelial cells, persistent cisplatin accumulation induces DNA damage, mitochondrial dysfunction, oxidative stress, and a strong inflammatory response, ultimately leading to cell death and renal impairment. Despite significant efforts in optimizing fluid resuscitation regimens, adjusting dosages, and developing nephroprotective agents, a reliable and clinically effective strategy for preventing cisplatin-induced AKI remains lacking.

[0003] Current evidence suggests that ferroptosis, a form of regulated cell death driven by iron-dependent lipid peroxidation, is a key mechanism in cisplatin-induced nephrotoxicity. Ferroptosis is characterized by glutathione depletion, GPX4 inactivation, and disruption of the cysteine / glutamate transport system X. c- Inhibition and accumulation of phospholipid peroxides. Unlike apoptosis, necrosis, and necroptosis, ferroptosis has unique mechanisms and morphological characteristics. In kidney diseases (especially acute kidney injury), the combined effects of excessive iron accumulation, impaired antioxidant defense, and lipid metabolism disorders exacerbate ferroptosis in renal tubular epithelial cells. Studies have confirmed that ferroptosis inhibitors and gene regulation of ferroptosis-related pathways can alleviate renal tubular damage, protect renal function, and reduce inflammatory responses, strongly demonstrating that ferroptosis plays a crucial role in cisplatin-induced AKI.

[0004] The core event in ferroptosis is the peroxidation of polyunsaturated fatty acids (PUFAs), a process regulated by member 4 of the long-chain acyl-CoA synthase family (ACSL4). ACSL4 catalyzes the activation of arachidonic acid and adrenaline to produce PUFA-acyl-CoA, which are key substrates for lipid peroxidation. ACSL4 expression is positively correlated with ferroptosis sensitivity in various cell types, and its gene or pharmacological inhibition can alleviate ferroptosis damage in multiple disease models, establishing it as a promising therapeutic target. 1 In the kidneys, ACSL4 is significantly upregulated in an experimental acute kidney injury (AKI) model, and its inhibition or knockout can alleviate renal tubular damage, reduce lipid peroxidation, and improve renal function. These findings highlight that ACSL4-mediated lipid remodeling is a key determinant of renal tubular ferroptosis, making ACSL4 a potential drug target for the prevention and treatment of cisplatin-induced AKI.

[0005] Dehydrodiisoeugenol (DHE) is a novel lignan, initially isolated from nutmeg and later discovered in various medicinal plants. It is renowned for its diverse pharmacological activities, including antioxidant, anti-inflammatory, anti-chemotoxicity, antibacterial, and antitumor effects. Studies have confirmed that DHE possesses potent free radical scavenging and cell-protective properties, making it a potential therapeutic agent. However, the therapeutic effects of DHE on AKI, especially cisplatin-induced AKI, are currently unexplored. Summary of the Invention

[0006] This invention provides the use of dehydrodiisoeugenol (DHE) in treating acute kidney injury by inhibiting ferroptosis. DHE provides significant renal protection against AKI by targeting ACSL4 and inhibiting cellular ferroptosis.

[0007] This invention provides the use of dehydrodiisoeugenol in the preparation of inhibitors of ferroptosis.

[0008] The present invention also provides the use of dehydrodiisoeugenol in the preparation of medicaments for treating acute kidney injury.

[0009] In one embodiment of the present invention, the acute kidney injury includes acute kidney injury induced by cisplatin.

[0010] In one embodiment of the present invention, the therapeutic effects of the drug include at least one of the following: targeted inhibition of ACSL4, inhibition of renal tubular cell ferroptosis, restoration of renal tubular antioxidant defense function, reduction of lipid peroxidation, reduction of iron deposition, improvement of renal function, improvement of histological damage, reduction of renal tubular injury, and reduction of renal tubular inflammatory response.

[0011] The present invention also provides the use of ferroptosis inhibitors in the preparation of medicaments for treating acute kidney injury.

[0012] In one embodiment of the present invention, the cell ferroptosis inhibitor comprises dehydrodiisoeugenol.

[0013] The present invention also provides a medicament for treating acute kidney injury, the active ingredient of which includes dehydrodiisoeugenol, and also includes pharmaceutically acceptable excipients.

[0014] In one embodiment of the present invention, the acute kidney injury includes acute kidney injury induced by cisplatin.

[0015] In one embodiment of the present invention, the dosage form of the drug includes a solid preparation or a liquid preparation.

[0016] In one embodiment of the present invention, the mass percentage of dehydrodiisoeugenol in the drug is 0.01~99.9%.

[0017] Beneficial effects: This invention discovered that DHE has an effect on the IC50 of Erastin-induced ferroptosis. 50 At 2.18 μM and 3.58 μM in HK-2 and NRK-52E cells, respectively, and at 5 μM, DHE could rescue cells from ferroptosis and almost completely block RSL3-induced ferroptosis, demonstrating that DHE can effectively inhibit ferroptosis. This invention evaluated the renal protective effect and molecular targets of DHE in cisplatin-induced AKI through model experiments. The interaction mechanism between DHE and its target was elucidated through renal tubular cell phenotypic screening, cisplatin-treated mouse experiments, and detection of ferroptosis and inflammatory markers, combined with activity-based proteomic analysis and complementary biophysical assays. DHE can inhibit renal tubular cell ferroptosis, restore antioxidant defense function, and reduce lipid peroxidation and iron deposition. In a cisplatin-treated mouse model, DHE significantly improved renal function and histological damage indicators, reduced renal tubular injury markers and inflammatory response levels, and alleviated the degree of ferroptosis. Proteomics and binding analysis revealed that ACSL4 is a direct target of DHE, and its dimerization and polyunsaturated fatty acid metabolism are inhibited by DHE. In summary, this invention confirms that the DHE is a natural small molecule inhibitor of ACSL4, which can effectively block ACSL4-mediated ferroptosis and provide strong protection against cisplatin-induced acute kidney injury, making it a promising therapeutic candidate for cisplatin-treated patients. Attached Figure Description

[0018] Figure 1 The figure shows the results of DHE inhibiting Erastin-induced ferroptosis. A: Flowchart of the experiment validating ferroptosis inhibitors; B: Screening results for compounds with cell viability exceeding 60% as ferroptosis inhibitors; C: Chemical structure of DHE; D: Cell images of HK-2 and NRK-52E cells treated with 10 μM Erastin and simultaneously treated with 5 μM DHE or 5 μM Fer-1; E: Measurement of the IC50 of DEH against Erastin in HK-2 and NRK-52E cells. 50 The values, and the inhibition rates of ferroptosis by 5 μM DHE and 5 μM Fer-1; Figure 2The figure shows the results of DHE inhibiting RSL3-induced ferroptosis. Figure A: Cell images of HK-2 and NRK-52E cells treated with 1 μM RSL3 and simultaneously treated with 5 μM DHE or 5 μM Fer-1; B: Inhibition rate of ferroptosis by 5 μM DHE and 5 μM Fer-1 in HK-2 cells; C: Inhibition rate of ferroptosis by 5 μM DHE and 5 μM Fer-1 in NRK-52E cells. Data are expressed as mean ± standard deviation (SD), n=3; statistical analysis was performed using a two-tailed t-test. ### Compared with the control group, P <0.001; compared with the erastin group, P <0.05, P <0.01, P <0.001; Figure 3 Figure 1 shows the results of DHE in improving renal dysfunction and pathological changes in cisplatin-induced acute kidney injury. A: Animal experiment flowchart; B: Kidney injury score; C: Serum BUN level; D: Serum Scr level; E: Relative mRNA expression of Kim-1 in kidney tissue; F: Relative mRNA expression of NGAL in kidney tissue; G: H&E and PAS staining of kidney sections (scale bar = 100 μm); HJ: IHC staining and quantification results of NGAL and Kim-1 (scale bar = 100 μm). Figure 4 Figure 1 shows the results of DHE in reducing renal macrophage infiltration and inflammatory response in cisplatin-induced acute kidney injury. In the figure, AB: IHC staining and quantitative results of F4 / 80 renal tissue positive area (scale bar = 100 μm); CD: IHC staining and quantitative results of CD68 renal tissue positive area (scale bar = 100 μm); EG: relative mRNA expression levels of renal inflammatory factors IL-1β, IL-6 and TNF-α. Figure 5 The figure shows the results of DHE inhibiting ferroptosis in cisplatin-induced acute kidney injury. In the figure, AC: the results of measuring the levels of GSH, SOD and MDA in kidney tissue; DE: the results of TUNEL staining and quantitative analysis of the proportion of positive cells in kidney tissue (scale bar = 100 μm); FH: the expression levels and quantitative analysis of GPX4 and SLC7A11 proteins in kidney tissue; IJ: the relative expression levels of GPX4 and SLC7A11 mRNA in kidney tissue; K: the content of ferrous ions in kidney tissue; LM: the expression level of the lipid peroxidation marker 4-HNE in kidney tissue. Figure 6The figure shows the results of DHE inhibiting erastin-induced ferroptosis in renal tubular cells. In the figure, AF represents the levels of GSH, SOD, and MDA in HK-2 and NRK-52E cells; GH represents the ferrous ion content in HK-2 and NRK-52E cells; IL represents the level and quantification of lipid peroxidation in HK-2 and NRK-52E cells as detected by BODIPY-C11 staining combined with flow cytometry; and MN represents the expression levels and quantification of GPX4 and SLC7A11 proteins in HK-2 and NRK-52E cells. Figure 7 The figure shows the effects of DHE on the levels of GPX4 and SLC7A11 mRNA, which are antiferroptosis genes. In the figure, A: GPX4 mRNA level in different groups of HK-2 cells; B: GPX4 mRNA level in different groups of HK-2 cells; C: GPX4 mRNA level in different groups of NEK-52E cells; D: SLC7A11 mRNA level in different groups of NRK-52E cells. Figure 8 The diagram shows the results of ABPP proteomics analysis revealing ACSL4 as a target protein of DHE anti-ferroptosis. Figure A: Overall flowchart of ABPP analysis for identifying the target protein using photosynthetic group modification; B: Flowchart of DHE probe synthesis; C: Fluorescence imaging after incubation of cells with different concentrations of DHE probe; D: Fluorescence imaging of protein labeled with 40 μM DHE probe and competitively inhibited by 80 μM DHE; E: Differential protein sequencing diagram identified by ABPP; F: Verification of the captured target using Western blotting with the DHE probe; competitive inhibition using 80 μM DHE. Figure 9The diagram shows the results of DHE directly targeting ACSL4 and binding to its Lys690 residue. A: Western blot results of ACSL4 in mouse kidney tissue; B: Quantitative analysis of ACSL4 / β-actin protein expression, showing that the CDDP model group significantly upregulated ACSL4 levels, while DHE treatment reversed this change; C: ELISA detection of AA levels in the supernatant of HK-2 cells treated with erastin, showing that DHE significantly inhibited erastin-induced AA production; D: Drug affinity-responsive target stability (DARTS) experiment in HK-2 cells, showing different... Under pronase digestion at certain concentrations, DHE treatment can protect ACSL4 from protease degradation; E: Cell thermal displacement assay (CETSA) shows that under gradient temperature increases of 37–72 °C, the ACSL4 protein band in the DHE-treated group is more stable than that in the DMSO group; F: Quantitative results from CETSA indicate that DHE significantly improves the thermal stability of ACSL4; G: Plasmon resonance imaging (SPRi) results show the binding curves of ACSL4 with different concentrations of DHE; H: SPRi binding analysis calculates the equilibrium dissociation constant KD of DHE and ACSL4 to be 8.6. μM; IJ: Schematic diagram of the three-dimensional docking conformation and two-dimensional interaction mode of DHE and ACSL4, showing that DHE interacts with multiple amino acid residues in the active pocket through hydrogen bonds and π forces; K: RMSD curve of the ACSL4–DHE complex over time in molecular dynamics simulation, indicating that the overall conformation of the complex is relatively stable during the simulation process; L: Analysis of the contribution of DHE to the binding of ACSL4 amino acid residues in molecular dynamics simulation, the results show that Lys690, Leu691 and Arg693 contribute the most to DHE binding and are key binding residues; M: DARTS experiment was performed in HK-2 cells overexpressing Flag-labeled WT or different mutants (Lys690A, Lys691A, Lys693A) ACSL4, the results showed that only the Lys690A mutation eliminated the protective effect of DHE on ACSL4; N: CETSA of Lys690A mutant showed that DHE no longer improves the thermal stability of ACSL4 under this mutant background. Figure 10 The results show that DHE probe and DHE have comparable inhibitory effects on ferroptosis. In the figure, A: In HK-2 cells, DHE probe and DHE have the same inhibitory effect on erastin-induced ferroptosis; B: In NRK-52E cells, DHE probe and DHE have the same inhibitory effect on erastin-induced ferroptosis. Figure 11The figures show the molecular dynamics simulation results. A: RMSF curve: The RMSF curve of the ACSL4 protein fluctuates within 1 nm, with no significant fluctuations. B: Radius of gyration (Rg) curve: The Rg curve of the ACSL4 protein-DHE complex fluctuates around 2.5 nm and remains stable throughout, indicating that the ACSL4 protein and DHE form a tight and stable complex. C: Hydrogen bond number fluctuation curve: The number of hydrogen bonds between ACSL4 and DHE remains stable between 1 and 3 throughout, reaching a maximum of 4. The hydrogen bond curve shows stable fluctuations, indicating a good hydrogen bond interaction between the ACSL4 protein and DHE, resulting in high complex stability. D: Solvent accessible surface area (SASA) curve: The SASA curve remains stable throughout, with no significant fluctuations, and the fluctuation range is within 300 nm. 2 ; Figure 12 The figures show the results of DHE antagonizing ACSL4-mediated ferroptosis by inhibiting ACSL4 dimerization and activating its antagonistic effect. Figure A: Representative immunoblot of ACSL4 protein expression after transfection with the ACSL4 plasmid in HK-2 cells; B: Quantitative analysis of the relative expression levels of ACSL4 / β-actin proteins; C: Detection of lipid peroxidation levels in HK-2 cells under different treatment groups using BODIPY 581 / 591 C11 staining combined with flow cytometry; D: Detection of HK-2 cell viability under different treatment conditions using the CCK-8 assay, showing that ACSL4 overexpression weakens the protective effect of DHE against Erastin-induced cell death; E: Quantitative analysis of lipid peroxidation levels based on BODIPY 581 / 591 C11, indicating that ACSL4 overexpression increases lipid peroxidation and counteracts the inhibitory effect of DHE; F: Detection of GPX4 and SLC7A11 protein expression in HK-2 cells under different treatments using Western blot; G: Detection of intracellular ferrous iron (Fe2+) in HK-2 cells under different treatment conditions using an iron ion detection kit. 2+ Relative content; H: Immunoblotting results of ACSL4 dimers and monomers in HK-2 cells, showing that DHE treatment reduces Erastin-induced ACSL4 dimerization levels; I: Immunoblotting results of ACSL4 dimers and monomers in mouse kidney tissue, showing that DHE treatment reduces ACSL4 dimerization in kidney tissue in the CDDP-induced model; Data are expressed as mean ± SD; Compared with the control group, P <0.05, P <0.01, P <0.001. Detailed Implementation

[0019] This invention provides the use of dehydrodiisoeugenol in the preparation of inhibitors of ferroptosis.

[0020] The DHE described in this invention has Figure 1 The structure shown in C is lignin isolated from nutmeg. Analysis revealed that DHE has an IC50 effect on Erastin-induced ferroptosis. 50 At 2.18 μM and 3.58 μM in HK-2 cells and NRK-52E cells, respectively, the DHE almost completely blocked RSL3-induced ferroptosis, demonstrating that the DHE can act as an inhibitor of ferroptosis.

[0021] The present invention also provides the use of dehydrodiisoeugenol in the preparation of medicaments for treating acute kidney injury.

[0022] In this embodiment of the invention, an acute kidney injury model was induced using cisplatin, and the model was treated with DHE. DHE treatment significantly alleviated cisplatin-induced acute kidney injury. The specific therapeutic effects include at least one of the following: targeted inhibition of ACSL4, inhibition of renal tubular cell ferroptosis, restoration of renal tubular antioxidant defense function, reduction of lipid peroxidation, reduction of iron deposition, improvement of renal function, improvement of histological damage, reduction of renal tubular injury, and reduction of renal tubular inflammatory response.

[0023] The present invention also provides the use of ferroptosis inhibitors in the preparation of medicaments for treating acute kidney injury.

[0024] The cell ferroptosis inhibitors described in this invention include DHE.

[0025] The present invention also provides a medicament for treating acute kidney injury, the active ingredient of which includes dehydrodiisoeugenol, and also includes pharmaceutically acceptable excipients.

[0026] The acute kidney injury described in this invention includes cisplatin-induced acute kidney injury, and the dosage form of the drug includes solid or liquid formulations. The drug of this invention contains 0.01~99.9% dehydrodiisoeugenol by mass.

[0027] To further illustrate the present invention, the use of dehydrodiisoeugenol provided by the present invention in treating acute kidney injury by inhibiting ferroptosis is described in detail below with reference to embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0028] Unless otherwise specified, the materials and methods used in the embodiments of this invention are all conventional commercially available materials and methods in the art.

[0029] In this embodiment of the invention, the experimental content involved is as follows: 1. Cell Culture HK-2 and NEK-52E cells were purchased from the American Type Culture Collection (ATCC). HK-2 and NRK-52E cells were cultured in DMEM / F12 medium (Gibco, USA) containing 10% fetal bovine serum (Sigma-Aldrich, Germany) and 1% penicillin-streptomycin (100 U / mL, Gibco, USA). IPSCs were provided by AIMINGMED. Cells were seeded in 6-well plates coated with reduced-growth factor Matrigel (354277; Corning Life Sciences, USA) at a concentration of 0.013 mg / cm³. 2 The culture medium was mTeSR (85875; Stemcell Technologies, Canada). Cells were cultured in a 37°C, 5% CO2 incubator.

[0030] 2. Reagents and plasmids Dehydrodiisoeugenol (2680-81-1), Erastin (T1765), RSL3 (T3646), cisplatin (CDDP, T1564), and Ferrostatin-1 (Fer-1, T6500) were purchased from Targetmol (Shanghai, China). Dimethyl sulfoxide (DMSO, D2650) was purchased from Sigma-Aldrich (Germany). Primary antibody types and sources are shown in Table 1. Secondary antibodies were purchased from Cell Signaling Technology (USA). Flag-ACSL4, Flag-ACSL4-LysS690A, Flag-ACSL4-Leu691A, and Flag-ACSL4-Arg693A plasmids were provided by Genechem Co., Ltd.

[0031] Table 1. Types and sources of antibodies

[0032] 3. Animals and Processing Male C57BL / 6J mice (weighing 22–24 g) were purchased from GemPharmatech Ltd. (Beijing, China) and housed in an SPF environment at 22±2℃ and 50–60% relative humidity, using a 12-hour light-dark cycle. Standard feed and water were provided for free access. After a one-week acclimatization period, cisplatin (CDDP; 20 mg / kg) was administered intraperitoneally to induce kidney injury. Mice were randomly assigned to six groups (n=6 per group): sham-operated group, model group, CDDP+L-DHE group (10 mg / kg), CDDP+M-DHE group (20 mg / kg), CDDP+H-DHE group (40 mg / kg), and CDDP+Fer-1 group (2 mg / kg). DHE was administered intraperitoneally 6 hours before cisplatin injection, and subsequent dosing regimens were as per protocol. Figure 2 As shown in Figure A. The sham-operated group received an equal volume of physiological saline. Mice were sacrificed 72 h after cisplatin administration, and serum and kidney tissue were collected for subsequent analysis. The same administration regimen was used in the mouse DHE toxicity experiment. All animal operations followed the guidelines of the China Laboratory Animal Management Committee and were approved by the Animal Ethics Committee of the Cancer Hospital of the Chinese Academy of Medical Sciences (Approval No.: NCC2025A626).

[0033] 4. Cell viability detection Cell viability was determined using the Cell Count Kit-8 (CCK-8) method.

[0034] 5. Western blot Cells and tissues were lysed in RIPA buffer, and total protein concentration was determined. Protein samples were mixed with loading buffer and denatured at 98°C for 10 min. Proteins were then separated by SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, IPVH00010). The membrane was blocked with 5% skim milk powder and incubated overnight at 4°C with primary antibody, followed by three washes with Tris-buffered saline (TBST) containing Tween-20. The membrane was then incubated with the corresponding secondary antibody at room temperature for 1 h, washed three times with TBST, and signal detection was performed.

[0035] 6. Immunohistochemical (IHC) staining Formalin-fixed paraffin-embedded tissue sections were treated using a standard immunohistochemical staining protocol. The specific steps were as follows: sections were dewaxed with xylene and rehydrated using a gradient of ethanol to distilled water. Endogenous peroxidase activity was blocked using 3% hydrogen peroxide, followed by heat-induced antigen retrieval in citrate buffer. Immunohistochemical staining was then performed using a horseradish peroxidase (HRP) detection system. After blocking non-specific binding sites, sections were incubated with a moderately diluted primary antibody, followed by incubation with the corresponding secondary antibody and color development. The staining results were semi-quantitatively evaluated using an immunoreaction scoring method.

[0036] 7. Quantitative Real-Time PCR Total RNA was extracted from tissues or cells using an RNA extraction kit (AG21024, Accurate Biology). Complementary DNA (cDNA) was synthesized using a reverse transcription kit (AG11615, Accurate Biology). Quantitative PCR was performed using a SYBR Green ProTaq HS qPCR kit (AG11762, Accurate Biology). GAPDH was used as an internal control gene. (The last sentence appears to be incomplete and requires further context.) -ΔΔCt The relative RNA expression level was calculated using this method.

[0037] Table 2 Primer information used in quantitative real-time PCR

[0038] 8. Detection of lipid peroxidation Lipid peroxidation levels were quantitatively determined using the oxidation-sensitive dye BODIPY 581 / 591 C11 (Thermo Fisher Scientific). After specified treatment, the culture medium was replaced with fresh complete medium supplemented with 10 μM BODIPY 581 / 591 C11, and cells were incubated at 37°C for 1 h. Subsequently, cells were washed with PBS, collected, and the fluorescence intensity of BODIPY 581 / 591 C11 was analyzed by flow cytometry as an indicator of lipid peroxidation.

[0039] 9. Labeling and Protein Targeting Experiments HK-2 cells were seeded in 10 cm culture dishes and divided into three groups: an untreated control group, a DHE probe-labeled group, and a DHE pretreatment competition group. In the competition experiment, cells were first pre-incubated with 80 μM DHE for 3 h, and then a DHE probe containing 0 or 40 μM was added. Figure 8Freshly cultured cells (B) were labeled after incubation at 37°C for 4 h. Cells were then irradiated with 365 nm UV light for 10 min, washed twice with PBS, lysed, and proteins extracted. The supernatant was adjusted to a final concentration of 1 mM TCEP, 0.1 mM TBTA, 1 mM CuSO4, and 10 mM TAMRA-N3, and click reaction was performed at room temperature. Proteins were precipitated with cold methanol, separated by SDS-PAGE, analyzed by in-gel fluorescence imaging, and stained with Coomassie brilliant blue. To identify the target protein, biotin-N3 (10 mM) was used instead of TAMRA-N3 (10 mM) in the click reaction. After cold methanol precipitation, proteins were incubated overnight at 4°C on NeutrAvidin magnetic beads (88816; Thermo Fisher Scientific). The bead-protein complex was then collected for liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis (ABPP based proteomic analysis). For Western blot analysis following precipitation, the precipitation procedure is as described above. The captured proteins are eluted with protein loading buffer and detected by Western blot analysis.

[0040] 10. Biochemical and ELISA detection Serum creatinine (Scr) and blood urea nitrogen (BUN) levels were determined using commercial kits (Nanjing Jiancheng, Nanjing, China). Superoxide dismutase (SOD) activity, malondialdehyde (MDA), and glutathione (GSH) levels in kidney homogenate and cell lysates were measured using SOD kits (Bristol-Myers Squibb, Shanghai, China), MDA kits (Solarbio, Shanghai, China), and GSH kits (Solarbio, Shanghai, China), respectively. Intracellular iron content was quantified using an iron assay kit (Iron Assay Kit–Colorimetric, I291, Dojindo). Arachidonic acid (AA) concentration in culture supernatant was determined using an ELISA kit (ZK-1104, Zhenke Biotechnology) according to the manufacturer's instructions.

[0041] 11. Hematoxylin-eosin (H&E) staining After fixation, the kidneys were embedded in paraffin and sectioned for histological evaluation. Sections were stained with hematoxylin and eosin (H&E) according to standard procedures. Renal tubular injury was assessed semi-quantitatively using a 0-4 grading system: Grade 0: no injury; Grade 0.5: <10% tubular involvement; Grade 1: 10-25%; Grade 2: 26-50%; Grade 3: 51-75%; Grade 4: 76-100% tubular involvement. At least six fields of view of the renal cortex were randomly selected from each specimen for observation under a light microscope.

[0042] 12. Surface Plasmon Resonance (SPR) Analysis Surface plasmon resonance (SPR) experiments were performed using a CM5 sensor chip via standard amine coupling chemistry. Immediately before ligand immobilization, an activation solution containing 400 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 100 mM N-hydroxysuccinimide (NHS) was prepared and continuously injected into the chip surface at a flow rate of 10 μL / min for 420 s. ACSL4 was diluted to a concentration of 20 μg / mL with immobilization buffer and injected into the sample flow cell (Fc2) at a flow rate of 10 μL / min, achieving an immobilization level of approximately 12,600 response units (RU). The control flow cell (Fc1) was kept blank as a reference surface. Residual active esters were blocked by injecting 1 M ethanolamine hydrochloride (flow rate 10 μL / min, 420 s). During analyte injection, DHE was diluted with running (analyte) buffer to eight concentration gradients from 0.31 to 20 μM. Samples of all concentrations were injected into Fc1 and Fc2 at a flow rate of 20 μL / min using a multi-cycle kinetic mode: a 100 s binding phase followed by a 180 s dissociation phase (both performed in flow buffer). Analytes were injected in ascending order of concentration, and the sensor surface was regenerated according to the instrument procedure before the next injection after each cycle.

[0043] 13. Drug Affinity Response Target Stability (DARTS) Cell pellets were lysed in a suitable lysis buffer to extract total protein. The resulting cell lysates were clarified by centrifugation at 12,000 × g for 10 min at 4 °C, and the supernatant was collected. The clarified lysates were incubated with dimethyl sulfoxide (DMSO) or DHE at 4 °C for 2 h, respectively. Subsequently, a protease (T13827, TargetMol) was added to the lysates, and digestion continued for 2 h at room temperature. At the end of the reaction, a mixture of protease inhibitors and SDS-PAGE sample buffer were added, and the samples were analyzed by Western blot.

[0044] 14. Cell thermal denaturation analysis (CETSA) After cell harvesting, the cell pellet was washed once with ice-cold PBS and then lysed in NP-40 lysis buffer containing a phosphatase inhibitor at 4°C for 30 min. The lysis buffer was clarified by centrifugation at 12,000 g for 10 min (4°C), and the supernatant was collected. The clarified lysis buffer was incubated with dimethyl sulfoxide (DMSO) or DHE at 4°C for 2 h. After incubation, aliquots of samples from each treatment group were heated at a specified temperature for 5 min, briefly cooled in an ice bath, and then centrifuged again at 4°C for 10 min. Soluble fractions were subsequently analyzed by Western blotting.

[0045] 15. Molecular Modeling and Docking The experiment was conducted according to the method described in the reference (Trott O, Olson AJ. AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J Comput Chem. 2010 Jan 30;31(2):455-61. doi: 10.1002 / jcc.21334. PMID: 19499576; PMCID: PMC3041641.).

[0046] 16. Molecular Dynamics Simulation The experiment was conducted according to the method described in the reference (Genheden S, Ryde U. The MM / PBSA and MM / GBSA methods to estimate ligand-binding affinities. Expert Opin Drug Discov. 2015 May;10(5):449-61. doi: 10.1517 / 17460441.2015.1032936. Epub 2015 Apr 2. PMID: 25835573;PMCID: PMC4487606.).

[0047] 17. Statistical Analysis All experiments were independently repeated at least three times, and quantitative data are expressed as mean ± standard deviation (SD). Differences between two groups were assessed using a two-tailed Student's t-test. When more than three groups were involved, a two-way ANOVA combined with Tukey's post-hoc test was used. A p-value <0.05 was considered statistically significant.

[0048] Example 1: DHE is a natural small molecule that inhibits ferroptosis. The DHE described in this invention is a lignin isolated from nutmeg, possessing various activities, including antitumor, antioxidant, and anti-inflammatory effects. Figure 1 Experiments using the procedure described in section A found that DHE has an inhibitory effect on ferroptosis, and based on... Figure 1 The results from DHE indicated that it was an inhibitor of ferroptosis. DHE had an IC50 effect on Erastin-induced ferroptosis. 50In HK-2 and NRK-52E cells, concentrations of 2.18 μM and 3.58 μM, respectively, showed that a concentration of 5 μM significantly rescued cells from ferroptosis, consistent with the effect of ferrostatin-1 (Fer-1). Figure 1 D and E). Similarly, DHE almost completely blocked RSL3-induced ferroptosis ( Figure 2 (AB). Therefore, DHE can effectively inhibit ferroptosis.

[0049] Example 2: DHE improves renal dysfunction and pathological changes in cisplatin-induced acute kidney injury. To investigate the protective effect of DHE on the kidneys, this invention... Figure 3 Figure A shows a model of cisplatin-induced acute kidney injury. Figure 3 The BF model group showed significant renal function impairment, with significantly elevated serum BUN and Scr levels, and an increasing trend in the mRNA levels of Kim-1 and NGAL (two markers of renal tubular injury). Furthermore, cisplatin-induced acute kidney injury also caused severe pathological changes; H&E and PAS staining indicated significant tubular damage, including reduced brush borders and tubular dilation. Figure 3 (G).

[0050] To verify the role of ferroptosis in acute kidney injury, this invention evaluated the effect of the ferroptosis inhibitor Fer-1. Clearly, Fer-1 treatment alleviated renal function impairment and pathological damage in mice with kidney injury. Similar to the results observed with Fer-1, DHE treatment significantly alleviated cisplatin-induced acute kidney injury. It alleviated the cisplatin-induced elevation of serum BUN and Scr levels and reduced the cisplatin-induced pathological changes (…). Figure 3 (BD and G). Further, IHC was used to detect Kim-1 and NGAL protein expression. The results showed that cisplatin-induced Kim-1 and NGAL showed an increasing trend, which was significantly eliminated by DHE treatment. Figure 3 (H). The results were consistent with mRNA expression ( Figure 3 (EF). In summary, DHE can effectively prevent renal insufficiency and pathological damage caused by cisplatin-induced acute kidney injury.

[0051] Example 3: DHE reduces renal macrophage infiltration and inflammatory response in cisplatin-induced acute kidney injury. Since tubulointerstitial inflammation is a common pathological feature of acute kidney injury (AKI), this invention investigated the effect of dihydroepiandrosterone (DHE) on the renal inflammatory state in cisplatin-induced acute kidney injury. The area of ​​F4 / 80 and CD68 positive cells in the renal tissue of the cisplatin model group was significantly increased, suggesting a significantly enhanced infiltration of renal interstitial macrophages. Figure 4(AD). Compared with the model group, DHE treatment significantly reduced the positive area of ​​F4 / 80 and CD68, and its effect in inhibiting macrophage infiltration was similar to that of the ferroptosis inhibitor Fer-1. Meanwhile, qPCR results showed that the mRNA levels of inflammatory factors IL-1β, IL-6, and TNF-α in the renal tissue of the model group were significantly increased, while the expression of these inflammatory factors was significantly downregulated after DHE intervention. The Fer-1 group also showed a similar trend. Figure 4 (EG). The above results indicate that DHE can reduce macrophage infiltration and inhibit inflammatory response in the kidneys of mice with cisplatin-induced acute kidney injury, and its effect is comparable to that of the ferroptosis inhibitor Fer-1.

[0052] Example 4: DHE inhibits ferroptosis in cisplatin-induced acute kidney injury. To determine whether DHE alleviates cisplatin-induced AKI by inhibiting ferroptosis, this invention first evaluated redox homeostasis and ferroptosis-related markers in renal tissue. Compared with the sham group, the cisplatin model group showed significantly decreased GSH and SOD levels, while MDA levels were significantly increased, suggesting exacerbated oxidative stress and lipid peroxidation. Figure 5 (AC). DHE treatment dose-dependently restored GSH and SOD levels and reduced MDA, with high doses producing effects comparable to Fer-1. Correspondingly, cisplatin significantly increased the proportion of TUNEL-positive renal tubular cells, while both DHE and Fer-1 significantly reduced renal tubular cell death (AC). Figure 5 (D and E). Western blot and qPCR results further showed that cisplatin significantly downregulated the expression of ferroptosis-related antioxidant systems, manifested as decreased levels of GPX4 and SLC7A11 protein and mRNA, while DHE reversed these changes, with effects similar to Fer-1 ( Figure 5 (FJ). Furthermore, the ferrous iron content and the lipid peroxidation marker 4-HNE in the renal tissue of the cisplatin model group were significantly elevated, while DHE and Fer-1 could effectively alleviate these elevations (FJ). Figure 5 (Middle KM). This indicates that DHE treatment can inhibit cisplatin-induced renal ferroptosis caused by AKI.

[0053] Example 5: DHE inhibits ferroptosis in renal tubular cells This invention measured changes in lipid peroxidation in an in vitro model of ferroptosis in renal tubular epithelial cells HK-2 and NRK-52E. Compared with the control group, the erastin-induced group showed significantly decreased GSH and SOD levels, while significantly increased MDA levels, suggesting exacerbated oxidative stress and lipid peroxidation. DHE treatment dose-dependently restored GSH and SOD levels and reduced MDA, with effects comparable to Fer-1. Figure 6(AF). Simultaneously, both Fer-1 and DHE reduced erastin-induced ferrous overexpression (AF). Figure 6 G, H). BODIPY-C11 staining combined with flow cytometry analysis of lipid peroxidation results also showed that DHE significantly reduced erastin-induced lipid peroxidation accumulation (G, H). Figure 6 (Middle IL). Western blot results showed that DHE reversed the Erastin-induced decrease in GPX4 and SLC7A11 protein levels ( Figure 6 (M, N). Similarly, qPCR results showed that DHE had the same effect at the GPX4 and SLC7A11 mRNA levels (M, N). Figure 7 (Chinese AD).

[0054] Example 6: ABPP proteomics analysis revealed that ACSL4 is a target protein of DHE anti-ferroptosis. ABPP proteomics analysis confirmed that ACSL4 is a target protein of DHE. Given the significant anti-ferroptosis effect of DHE, this invention further explored the target proteins and molecular mechanisms of DHE based on ABPP analysis technology. Figure 8 (A). In short, a photoactive DHE Probe ( Figure 8 (Middle B). After incubation with HK-2 cell lysates, the probe binds to proteins upon UV irradiation, and subsequently, the fluorescent dye TAMRA or biotin can be linked to the probe via a click chemistry reaction to label the direct-bound protein target of DHE. The target protein can then be identified by mass spectrometry or visualized by fluorescence imaging. Figure 8 (A) There was no significant difference in the antiferroptosis effect between DHE Probe and DHE. Figure 8 Next, different concentrations of DHE Probe were incubated with activated HK-2 cells for 4 hours. After binding to proteins under UV irradiation, the proteins were linked to the fluorescent dye TAMRA via click chemistry, separated by SDS-PAGE electrophoresis, and visualized by fluorescence imaging. The results showed that a concentration of 40 μM DHE Probe could label most cellular proteins (…). Figure 8 (C). Then, a competition experiment was performed between DHE Probe and DHE. HK-2 cells were incubated with DHE Probe for 4 h, with or without DHE (80 μM) pretreatment for 3 h, using DMSO as a control. Fluorescence imaging was then performed for visualization. The results showed that the DHE Probe-labeled protein competed with pre-incubated 80 μM DHE in HK-2 cells, indicating that the DHE Probe-labeled protein has specificity (C). Figure 8(D). The labeled protein was then coupled with biotin-azide to form a DHE probe-biotin complex, which was purified by streptavidin magnetic beads and identified by LC-MS / MS analysis (n=3). Among the identified DHE candidate target proteins, ACSL4 was found to be a key protein in the ferroptosis pathway, showing great DHE binding potential (Probe / control logFC=7.39, P <0.01)( Figure 8 (E). Immunoblot assays using the DHE active probe successfully confirmed that ACSL4 is a potential direct target of DHE. Significant competitive inhibition was detected upon pretreatment with DHE (80 μM), indicating a specific interaction between DHE and ACSL4. In conclusion, ACSL4 is a potential direct target of DHE.

[0055] Example 7: DHE directly targets ACSL4 protein and binds to its lysine 690. Western blot analysis showed that ACSL4 protein levels were significantly upregulated in the kidney tissue of CDDP model mice, and these changes were reversed after DHE treatment. Figure 9 (A, B). ACSL4-mediated PUFA metabolism promotes the synthesis of arachidonic acid (AA)-containing phospholipids and lipid peroxidation, ultimately leading to ferroptosis. Therefore, AA levels in cells with and without DHE treatment were measured in the presence of erastin. ELISA assays confirmed that DHE reversed erastin-mediated AA production in cell supernatants, indicating that the binding of DHE to ACSL4 inhibits its mediated PUFA metabolism (…). Figure 9 (C)

[0056] To further confirm the binding of DHE to ACSL4, this invention verified the in situ binding of DHE to ACSL4 using a Drug Affinity Responsive Target Stability (DARTS) assay. This assay is a method for recognizing label-free small molecule targets based on the decreased sensitivity of target protein proteases upon drug binding. The results showed that DHE protects ACSL4 from degradation by prostate enzymes in HK-2 cells. Figure 9 In addition, cellular thermal displacement assays (CETSA) showed that DHE significantly improved the thermal stability of ACSL4 compared to the control group. Figure 9 (E, F). Surface plasmon resonance imaging (SPRi) results also showed that DHE has a good binding ability with ACSL4, with an equilibrium dissociation constant (KD) of 8.6 μM (E, F). Figure 9(G, H). Similarly, molecular docking results showed that DHE and ACSL4 can spontaneously bind, interacting through hydrogen bonds and π forces, with a binding energy of -8.4 kcal / mol (G, H). Figure 9 To further deduce the dynamic behavior of ACSL4 active site residues, this invention also used Gromacs 2024.4 software to perform a 100 ns molecular dynamics simulation under real solution conditions and analyzed their molecular dynamic trajectories. Finally, the root mean square deviation (RMSD), root mean square fluctuation (RMSF), radius of gyration (Rg), solvent accessible surface area (SASA), number of hydrogen bonds between the protein and ligand in the complex, relative free energy distribution, and structural comparison of the complex at five time points (0, 25, 50, 75, and 100 ns) were analyzed in the molecular dynamics simulation trajectory of the ACSL4 protein-DHE complex. Furthermore, the average binding free energy between the protein and ligand was calculated using the MM / GBSA method. The results show that the ACSL4 protein-DHE complex has good stability and strong binding (…). Figure 9 Zhong K, Figure 10 AB in the middle Figure 11 (AD). Furthermore, in kinetic simulations, the key amino acid residues for DHE binding to ACSL4 were LYS-690, LEU-691, and ARG-693, with binding energies of -3.4, -2.5, and -1.7, respectively. Figure 9 To determine which residues in ACSL4 bind to DHE, DARTS was performed in HK-2 cells overexpressing WT or ACSL4 mutant plasmids. The results showed that only the LYS690A mutation eliminated its binding to DHE (…). Figure 9 This is consistent with the results of kinetic simulations, where LYS690 exhibits the highest binding energy. To further clarify this result, CETSA analysis was performed, revealing that, compared to the control, DHE had no effect on the thermal stability of ACSL4 upon the LYS690A mutation. Figure 9 (N). This indicates that DHE directly binds to LYS690 of ACSL4 to inhibit ferroptosis.

[0057] Example 8: DHE inhibits ferroptosis by suppressing ACSL4 dimerization and activation. ACSL4 is a key protein in ferroptosis. To confirm whether DHE inhibits ferroptosis by suppressing ACSL4 activation, this invention first overexpressed ACSL4 in HK-2 cells. Figure 12(A, B). Then, BODIPY-C11 staining combined with flow cytometry was used to detect the lipid peroxidation levels of cells under different treatments. It was found that ACSL4 increased cellular lipid peroxidation, and ACSL4 overexpression impaired the protective effect of DHE against Erastin-induced ferroptosis in HK-2 cells. Figure 12 (C, E). Furthermore, CCK8 assay results also showed that, under ACSL4 saturation conditions, DHE could not rescue Erastin-induced ferroptosis in HK-2 cells (C, E). Figure 12 Similarly, Western blotting showed that DHE could not reverse the expression of ferroptosis-associated proteins GPX4 and SLC7A11 in HK-2 cells overexpressing ACSL4. Ferric ion assays showed that ACSL4 overexpression alleviated the effect of DHE on erastin-induced reduction of ferrous ions in HK-2 cells. DHE treatment reduced erastin-induced ACSL4 dimerization in HK-2 cells. Figure 12 Similarly, this invention also found that in mouse models, DHE treatment reduced CDDP-induced ACSL4 dimerization in mouse kidney tissue (H). Figure 12 (H). In summary, DHE inhibits ferroptosis by suppressing ACSL4 dimerization.

[0058] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Use of dehydrodiisoeugenol in the preparation of an inhibitor of cellular ferroptosis.

2. Use of dehydrodiisoeugenol in the preparation of a medicament for treating acute kidney injury.

3. Use according to claim 2, characterized in that, The acute kidney injury includes acute kidney injury induced after cisplatin.

4. Use according to claim 3, characterized in that, The therapeutic effect of the medicament includes at least one of the following: targeting inhibition of ACSL4, inhibition of renal tubular cell ferroptosis, recovery of renal tubular antioxidant defense function, reduction of lipid peroxidation, reduction of iron deposition, improvement of renal function, improvement of histological injury, reduction of renal tubular injury and reduction of renal tubular inflammatory response.

5. Use of an inhibitor of cellular ferroptosis in the preparation of a medicament for treating acute kidney injury.

6. Use according to claim 5, characterized in that, The inhibitor of cellular ferroptosis includes dehydrodiisoeugenol.

7. A medicament for treating acute kidney injury, characterized by, The active ingredient includes dehydrodiisoeugenol, and also includes a pharmaceutically acceptable excipient.

8. The medicament according to claim 7, characterized in that, The acute kidney injury includes acute kidney injury induced after cisplatin.

9. The medicament according to claim 7, characterized in that, The dosage form of the medicament includes a solid preparation or a liquid preparation.

10. The medicament according to claim 7, characterized in that, The mass percentage of dehydrodiisoeugenol in the medicament is 0.01-99.9%.

Images