Use of zingiberone A in the preparation of a drug for treating acute kidney injury

Abstract

This invention belongs to the field of biomedical technology, specifically relating to the application of gingerone A in the preparation of drugs for treating acute kidney injury. Gingerone A can actively intervene in the process of renal tubular epithelial cell injury. GA directly binds to VCP, upregulating VCP activity; activated VCP specifically promotes K48 ubiquitination at the ACSL4 K148 site, accelerating ACSL4 degradation via the ubiquitin-proteasome pathway, thereby inhibiting ferroptosis in renal tubular epithelial cells and alleviating acute kidney injury.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of gingerone A in the preparation of drugs for treating acute kidney injury. Background Technology

[0002] Acute kidney injury is a common and critical clinical syndrome caused by various factors, including ischemia-reperfusion, sepsis, nephrotoxic drugs, and major surgery. Its core pathological manifestation is progressive damage and dysfunction of renal tubular epithelial cells, and it is clinically characterized by a rapid decline in renal function, significantly elevated serum creatinine and blood urea nitrogen levels, decreased urine output, or even anuria.

[0003] At present, the clinical treatment methods for acute kidney injury are extremely limited, and the main treatment is still supportive care, which includes: (1) actively treating the primary cause, such as anti-infection, discontinuing nephrotoxic drugs, and correcting hypovolemia; (2) maintaining water, electrolyte and acid-base balance; (3) renal replacement therapy, such as hemodialysis and continuous renal replacement therapy, to remove accumulated metabolic waste and excess water in the body, so as to temporarily replace kidney function and buy time for the kidney to repair itself.

[0004] However, the above treatment strategies are all passive support and symptomatic treatment. In addition, in clinical practice, doctors will give adjuvant drug treatment according to the patient's specific condition, such as antioxidants and energy supplements, such as N-acetylcysteine ​​(NAC), vitamin C, trimetazidine and levocarnitine. However, passive support, symptomatic treatment and adjuvant drugs have limited effect on actively intervening in the process of renal tubular epithelial cell damage. Summary of the Invention

[0005] To address the shortcomings of the existing technology, this invention provides the application of gingerone A in the preparation of drugs for treating acute kidney injury, which can solve the technical problem that the existing technology has limited effect on actively intervening in the process of renal tubular epithelial cell injury.

[0006] This invention is achieved by adopting the following technical solution: Application of gingerone A in the preparation of drugs for treating acute kidney injury.

[0007] Furthermore, the kidney injury includes acute kidney injury.

[0008] Furthermore, the drug for treating kidney injury is prepared from gingerone A and medically acceptable pharmaceutical excipients.

[0009] Furthermore, the pharmaceutical excipient is one or more of the following: diluent, disintegrant, precipitation inhibitor, flow aid, binder, dispersant, suspending agent, isotonic agent, thickener, emulsifier, preservative, and stabilizer.

[0010] Furthermore, the pharmaceutical excipient is polyethylene glycol 200.

[0011] Furthermore, the drug for treating kidney injury was prepared by mixing gingerone A and polyethylene glycol 200 at a mass-volume ratio of (2 mg to 3 mg): 1 mL.

[0012] Furthermore, the drug for treating kidney injury was prepared by mixing gingerone A and polyethylene glycol 200 at a mass-volume ratio of 2 mg: 1 mL.

[0013] Furthermore, the drug for treating kidney injury was prepared by mixing gingerone A and polyethylene glycol 200 at a mass-volume ratio of 3 mg: 1 mL.

[0014] Furthermore, the drug for treating kidney injury was prepared by mixing gingerone A and polyethylene glycol 200 at a mass-volume ratio of 2.5 mg: 1 mL.

[0015] A drug for treating kidney injury, wherein the sole active ingredient of the drug is gingerone A.

[0016] Furthermore, the dosage form of the drug includes injections, tablets, capsules, granules, oral liquids, or powder injections.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention relates to the application of gingerone A in the preparation of drugs for treating acute kidney injury. Gingerone A can actively intervene in the process of renal tubular epithelial cell injury. GA directly binds to the Cys184 site of VCP, upregulating the acetylation level of VCP K8 site; activated VCP specifically promotes K48 ubiquitination at the ACSL4 K148 site, accelerating the degradation of ACSL4 via the ubiquitin-proteasome pathway, thereby inhibiting ferroptosis in renal tubular epithelial cells and alleviating acute kidney injury. Attached Figure Description

[0018] Figure 1The study aimed to alleviate acute kidney injury (AKI) by inhibiting ferroptosis through GA intervention. A represents the change in body weight of mice in each group during the experiment (n=6); B represents the ratio of bilateral kidney weight to body weight at the experimental endpoint (n=6); C represents the gross morphological observation of the kidneys in each group; D represents representative images of HE and PAS staining of kidney tissue, scale bar: 100 μm; E represents the semi-quantitative score of renal tubular injury based on HE staining (n=6); F represents the serum blood urea nitrogen (BUN) level of each group of mice (n=6); G represents the serum creatinine (Scr) level of each group of mice (n=6); H represents representative images of Kim-1 and NGAL immunohistochemical staining in kidney tissue, scale bar: 100 μm; I represents the Western chromatogram of Kim-1 and NGAL protein expression in kidney tissue. Blot analysis; J is a volcano plot of differentially expressed genes (DEGs) in Erastin-induced HK-2 cells after GA intervention (n=3); K is a heatmap analysis of ferroptosis-related DEGs (n=3); L is the KEGG pathway enrichment analysis of DEGs between the Erastin group and the Erastin+GA group; M is the cell viability test results of HK-2 cells after treatment with different concentrations of Erastin, RSL3 alone or in combination with GA (n=6); N is the cell viability test results of NRK-52E cells after treatment with different concentrations of Erastin, RSL3 alone or in combination with GA (n=6); all data are expressed as mean ± standard deviation (mean ± SD); one-way ANOVA combined with Tukey post-hoc test was used for comparisons among multiple groups; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0019] Figure 2GA inhibits ferroptosis in renal tubular epithelial cells by regulating lipid peroxidation, iron metabolism, and mitochondrial function. A shows the lipid peroxidation level in HK-2 cells detected using the BODIPY™ 581 / 591 C11 fluorescent probe (red: reduced state; green: oxidized state), scale bar: 100 μm; B shows the lipid peroxidation level in NRK-52E cells detected using the BODIPY™ 581 / 591 C11 fluorescent probe (red: reduced state; green: oxidized state), scale bar: 100 μm; C shows the free Fe²⁺ concentration in HK-2 and NRK-52E cells detected using the RhoNox-6 probe, scale bar: 100 μm; D shows the reactive oxygen species (ROS) level in HK-2 and NRK-52E cells measured using the DCFH-DA probe, scale bar: 100 μm. μm; E: Quantitative detection of ROS content in HK-2 cells by flow cytometry; F: Quantitative detection of ROS content in NRK-52E cells by flow cytometry; G: Quantitative analysis of malondialdehyde (MDA) level in HK-2 cells; H: Quantitative analysis of superoxide dismutase (SOD) level in HK-2 cells; I: Quantitative analysis of glutathione (GSH) level in HK-2 cells; J: Changes in mitochondrial membrane potential of HK-2 and NRK-52E cells detected by JC-1 flow cytometry; K: Changes in mitochondrial morphology of HK-2 cells observed by transmission electron microscopy, scale bar: 2 μm; L: Expression of ACSL4, a key protein in ferroptosis, in HK-2 cells detected by Western blot; M: Expression of ACSL4, a key protein in ferroptosis, in NRK-52E cells detected by Western blot; All data are expressed as mean ± standard deviation (mean ± SD), n=3; One-way ANOVA was used for comparisons among multiple groups. ANOVA combined with Dunnett's multiple comparison test; ****P<0.0001; NS indicates no significant difference.

[0020] Figure 3This study investigated the inhibitory effect of GA on ferroptosis in a mouse model of ischemia / reperfusion (I / R) induced acute kidney injury (AKI). The results showed that: A) ACSL4, SLC7A11, and GPX4 protein expression levels in kidney tissues of each group were detected by Western blotting; B) quantification of ACSL4 protein expression level; C) quantification of SLC7A11 protein expression level; D) quantification of GPX4 protein expression level; E) ACSL4, SLC7A11, and FTH1 protein expression levels were detected by Western blotting with Fer-1 as a positive control; F) quantification of ACSL4 protein expression level; G) quantification of SLC7A11 protein expression level; H) quantification of FTH1 protein expression level; and I) ultrastructural changes of mitochondria in kidney tissue observed by transmission electron microscopy (scale bar: 2). μm; J represents the immunohistochemical staining result of ACSL4 in kidney tissue; K represents the quantitative analysis of ACSL4 positive staining area; L represents the immunohistochemical staining result of FTH1 in kidney tissue; M represents the quantitative analysis of FTH1 positive staining area; N represents the immunohistochemical staining result of GPX4 in kidney tissue; O represents the quantitative analysis of GPX4 positive staining area; P represents the quantitative determination of glutathione (GSH) content in kidney tissue; Q represents the quantitative determination of superoxide dismutase (SOD) content in kidney tissue. All data are expressed as mean ± standard deviation (mean ± SD), n=3; one-way ANOVA combined with Dunnett's multiple comparison test was used for comparisons among multiple groups; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; NS indicates no significant difference.

[0021] Figure 4GA inhibits ferroptosis in Erastin-induced HK-2 and NRK-52E cells by degrading ACSL4 via the ubiquitin-proteasome-dependent pathway. A shows the effect of GA on ACSL4, p62, and LC3B protein expression in Erastin-induced HK-2 cells under CQ autophagy inhibitor intervention, as detected by Western blot. B shows the gray-scale quantitative analysis of ACSL4 protein expression. C shows the gray-scale quantitative analysis of p62 protein expression. D shows the gray-scale quantitative analysis of LC3B protein expression. E shows the effect of GA on ACSL4, p62, and LC3B protein expression in Erastin-induced HK-2 cells under CQ intervention. The effects of GA on ACSL4, p62, and LC3B protein expression in Erastin-induced NRK-52E cells were detected by blot analysis; F represents the gray-scale quantitative analysis of ACSL4 protein expression; G represents the gray-scale quantitative analysis of p62 protein expression; H represents the gray-scale quantitative analysis of LC3B protein expression; I represents the effect of GA on the degradation rate of ACSL4 protein in Erastin-induced HK-2 cells detected and quantitatively analyzed by CHX tracing assay; J represents the effect of GA on the degradation rate of ACSL4 protein in Erastin-induced NRK-52E cells detected and quantitatively analyzed by CHX tracing assay; K represents the quantitative analysis of ACSL4 protein degradation rate in CHX tracing assay (HK-2 and NRK-52E); L represents the effect of GA on the degradation rate of ACSL4 protein in Erastin-induced HK-2 cells under the intervention of the proteasome inhibitor MG-132; M represents the effect of GA on the degradation rate of ACSL4 protein in Erastin-induced HK-2 cells under the intervention of MG-132. The CHX tracing assay was used to detect the effect of GA on the ACSL4 protein degradation rate induced by Erastin in NRK-52E cells; N represents the quantitative analysis of ACSL4 protein degradation rate in the CHX tracing assay (HK-2 and NRK-52E, MG-132 intervention); O represents the co-immunoprecipitation assay to detect the effect of different concentrations of GA on ACSL4 ubiquitination level in Erastin-induced HK-2 cells in the presence of MG-132; P represents the quantitative analysis of ACSL4 ubiquitination level (HK-2); Q represents the quantitative analysis of ACSL4 protein expression level (HK-2); R represents the co-immunoprecipitation assay to detect the effect of different concentrations of GA on ACSL4 ubiquitination level in Erastin-induced NRK-52E cells in the presence of MG-132; S represents the quantitative analysis of ACSL4 ubiquitination level (NRK-52E); T represents the quantitative analysis of ACSL4 protein expression level (NRK-52E). All data are expressed as mean ± standard deviation (mean ± SD), n=3; one-way ANOVA combined with Dunnett's multiple comparison test was used for multiple group comparisons; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; NS indicates no significant difference.

[0022] Figure 5 This study demonstrates the specific interaction between ACSL4 and the protein quality control factor VCP. A shows a schematic diagram of the immunoprecipitation mass spectrometry (IPMS) experiment for ACSL4-interacting proteins in HK-2 cells; B lists the top 10 potential ACSL4-interacting proteins identified by abundance sorting using IMSMS; C verifies the endogenous interaction between ACSL4 and VCP in HK-2 cells using IMSMS; D verifies the endogenous interaction between ACSL4 and VCP in NRK-52E cells using IMSMS; E verifies the interaction between HA-VCP and Flag-ACSL4 in HEK293FT cells using IMSMS; F verifies the interaction between HA-VCP and Flag-ACSL4 in HEK293FT cells using IMSMS (reverse verification); G shows the co-localization of VCP and ACSL4 in HK-2 cells using immunofluorescence staining (scale bar: 20 μm); H shows the co-localization of VCP and ACSL4 in NRK-52E cells using immunofluorescence staining (scale bar: 20 μm). μm; I is a schematic diagram of the construction of a series of truncated variants of VCP and ACSL4 proteins; J is a co-immunoprecipitation assay to detect the interaction between different truncated forms of VCP and ACSL4 to clarify their binding regions; K is a co-immunoprecipitation assay to detect the interaction between different truncated forms of ACSL4 and VCP to clarify their binding regions; L is a schematic diagram of the three-dimensional structure of the VCP-ACSL4 protein interaction and the key hydrogen bond network simulated by AlphaFold (wild type); M is the key amino acid sites and mutant sequences annotated by sequence alignment; N is a schematic diagram of the three-dimensional structure of the VCP-ACSL4 protein interaction and the key hydrogen bond network simulated by AlphaFold (key site mutant); O is a co-immunoprecipitation assay to verify the effect of key binding site mutations on VCP-ACSL4 interaction.

[0023] Figure 6VCP mediates K48-linked polyubiquitination of ACSL4 at the K148 site, while GA promotes this modification in a VCP-dependent manner. A shows the effect of VCP knockdown on the degradation rate of endogenous ACSL4 in HK-2 cells as detected by the CHX tracking assay; B shows the effect of VCP knockdown on the degradation rate of endogenous ACSL4 in HEK293FT cells as detected by the CHX tracking assay; C shows the quantitative analysis of ACSL4 degradation rate after VCP knockdown; D shows the effect of VCP overexpression on the degradation rate of endogenous ACSL4 in HK-2 cells as detected by the CHX tracking assay; E shows the effect of VCP overexpression on the degradation rate of endogenous ACSL4 in HEK293FT cells as detected by the CHX tracking assay; F shows the quantitative analysis of ACSL4 degradation rate after VCP overexpression; G shows the identification of ubiquitin mutants through co-immunoprecipitation screening. The study identified VCP-regulated ubiquitin chain types in HK-2 cells and tested various single-lysine ubiquitin mutants (K6O, K11O, K27O, K29O, K33O, K63O). H shows the identification of VCP-regulated ubiquitin chain types through co-immunoprecipitation screening of ubiquitin mutants (HEK293FT cells), with testing of various single-lysine ubiquitin mutants (K6O, K11O, K27O, K29O, K33O, K63O). I demonstrates that VCP specifically induces K48-linked polyubiquitination in ACSL4 using a combination of K48R and K48O ubiquitin mutants. J shows that co-immunoprecipitation experiments indicate that GA selectively enhances K48-linked ubiquitination in ACSL4. K confirms that in the presence of wild-type or K48-only ubiquitin, VCP knockdown completely eliminates GA-induced ACSL4. K48 polyubiquitination (HK-2 cells); L: co-immunoprecipitation assay confirms that knockdown of VCP completely eliminates GA-induced ACSL4 K48 polyubiquitination in the presence of wild-type or K48-only ubiquitin (HEK293FT cells); M: co-immunoprecipitation assay shows that inhibition of VCP by CB-5339 completely eliminates GA-stimulated ACSL4 K48 ubiquitination; N: co-immunoprecipitation results confirm that overexpression of full-length VCP or its A1 truncated mutant significantly enhances ACSL4 K48-linked ubiquitination; O: co-immunoprecipitation assay shows that the VCP-binding motif (BS-Mut) on the mutant VCP completely eliminates VCP-mediated ACSL4 K48 ubiquitination (HK-2 cells); P: co-immunoprecipitation assay shows that the VCP-binding motif (BS-Mut) on the mutant VCP completely eliminates VCP-mediated ACSL4 K48 ubiquitination (HEK293FT cells).Q represents the screening of ACSL4 lysine residues responsible for VCP-mediated ubiquitination using a series of ACSL4 mutants containing only a single lysine (K124O, K148O, K512O, K536O, K596O). Only the K148O mutant retained the VCP-induced ubiquitination signal. R represents the confirmation by co-immunoprecipitation analysis in HK-2 cells that the ACSL4 K148R mutant completely blocks VCP-dependent ACSL4 K48-linked ubiquitination. S represents the confirmation by co-immunoprecipitation analysis in HEK293FT cells that the ACSL4 K148R mutant completely blocks VCP-dependent ACSL4 K48-linked ubiquitination. All data are expressed as mean ± standard deviation (mean ± SD), n=3. Multiple comparisons were performed using one-way ANOVA combined with Dunnett's multiple comparison test.

[0024] Figure 7Multiple experiments have confirmed the direct binding of GA and VCP. A shows a schematic diagram of the experimental procedure for screening direct targets of GA using a combination of DARTS-MS and LiP-MS techniques; B shows the Venn diagram and list of overlapping candidate targets from the DARTS-MS and LiP-MS screening results, with VCP marked as a core potential target; C verifies the direct binding of GA and VCP using DARTS experiments, showing that after GA intervention under gradient Pronase E digestion conditions, SDS-PAGE detected enhanced resistance of VCP to Pronase E hydrolysis; D verifies the direct binding of GA and VCP using DARTS experiments, showing that after GA intervention under gradient Pronase E digestion conditions, Western blot detected enhanced resistance of VCP to Pronase E hydrolysis. E shows enhanced resistance to enzyme hydrolysis; E is a small molecule pull-down experiment verifying the direct interaction between GA and VCP, where excess unlabeled GA competitively inhibits the binding of GA-biotin to VCP in HK-2 cells; F is a small molecule pull-down experiment verifying the direct interaction between GA and VCP, where excess unlabeled GA competitively inhibits the binding of GA-biotin to VCP in NRK-52E cells; G is the SPR analysis of the binding response curves of different concentrations of GA to immobilized VCP and the calculation of the dissociation constant KD value; H is the effect of CETSA on the thermal stability of VCP in HK-2 cells; I is the effect of CETSA on the thermal stability of VCP in HK-2 cells (thermal denaturation curve); J is the effect of CETSA on the thermal stability of VCP in NRK-52E cells; K is the effect of CETSA on the thermal stability of VCP in NRK-52E cells (thermal denaturation curve), where the thermal denaturation curve of VCP protein shifted significantly to the right after GA intervention. All data are expressed as mean ± standard deviation (mean ± SD), n=3; multiple comparisons were performed using one-way ANOVA combined with Dunnett's multiple comparison test.

[0025] Figure 8GA enhances VCP-mediated ACSL4 ubiquitination and degradation, alleviating renal tubular ferroptosis and ischemia / reperfusion-induced AKI both in vitro and in vivo. Specifically: A) HK-2 cells transfected with Si-NC or Si-VCP, followed by Erastin and GA treatment, with MG-132 cells pre-incubated to block proteasome degradation; total ACSL4 ubiquitination levels were detected using co-immunoprecipitation. B) NRK-52E cells transfected with Si-NC or Si-VCP, followed by Erastin and GA treatment, with MG-132 cells pre-incubated to block proteasome degradation; total ACSL4 ubiquitination levels were detected using co-immunoprecipitation. C) Immunofluorescence staining was used to detect the presence of lipid reactive oxygen species (ROS) in HK-2 and NRK-52E cells (scale bar: 100 μm). D) Immunofluorescence staining was used to detect the presence of ferrous ions (Fe²⁺) in HK-2 and NRK-52E cells (scale bar: 100 μm). μm; E: Endogenous reverse co-immunoprecipitation assay in HK-2 cells confirms that GA enhances the endogenous interaction between VCP and ACSL4, with normal IgG as a negative control; F: Endogenous reverse co-immunoprecipitation assay in NRK-52E cells confirms that GA enhances the endogenous interaction between VCP and ACSL4, with normal IgG as a negative control; G: Exogenous co-immunoprecipitation assay in HEK293FT cells transfected with HA-VCP and Flag-ACSL4 plasmids further verifies that GA promotes the physical binding of VCP and ACSL4; H: Exogenous co-immunoprecipitation assay in HEK293FT cells transfected with HA-VCP and Flag-ACSL4 plasmids further verifies that GA promotes the physical binding of VCP and ACSL4 (reverse verification); I: Effects of Erastin stimulation, GA alone or in combination with... Western blot analysis of ferroptosis-related proteins (ACSL4, HO-1, GPX4, FSP1, FTH1) and kidney injury markers (KIM-1, NGAL) in HK-2 cells treated with the VCP inhibitor CB-5339, with β-actin as a loading control; J shows the in vivo co-immunoprecipitation ubiquitination assay of ACSL4 in kidney tissue of ischemia-reperfusion (I / R) mice treated with GA and / or CB-5339; K shows representative HE-stained images in kidney sections of I / R-AKI model mice; L shows representative PAS-stained images in kidney sections of I / R-AKI model mice; M shows representative Kim-1 / NGAL immunohistochemical staining images in kidney sections of I / R-AKI model mice; N shows the quantitative measurement of serum creatinine (Scr) in each group of I / R mice to assess kidney function; O shows the quantitative measurement of serum blood urea nitrogen (BUN) in each group of I / R mice to assess kidney function.P shows the immunofluorescence co-localization of VCP (red) and ACSL4 (green) in normal human kidney tissue and kidney specimens from AKI patients. DAPI was used to stain cell nuclei (blue). Scale bar: 100 μm. Q is a schematic diagram of the molecular mechanism of action: GA enhances the binding between VCP and ACSL4, promotes K48-linked ubiquitination modification and proteasomal degradation of ACSL4, thereby inhibiting ferroptosis in renal tubular epithelial cells and ultimately improving ischemia / reperfusion-induced AKI. This schematic diagram was created by BioRender.com. All data are expressed as mean ± standard deviation (mean ± SD), n=3. Multiple comparisons were performed using one-way ANOVA combined with Dunnett's multiple comparison test. Detailed Implementation

[0026] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods. Specific explanations are needed as follows:

[0027] Acute kidney injury (AKI); valine peptide protein (VCP); gingerenone A (GA), purchased from Santa Cruz Biotechnology, Cat# sc-490979, is an oily liquid, prepared into a 2.5 mg / mL solution using medically acceptable excipient polyethylene glycol 200 as a solvent, and administered to mice by gavage. The GA to polyethylene glycol 200 concentration ratio was 2.5 mg / mL; long-chain acyl-CoA synthetase 4 (ACSL4); I / R represents renal ischemia-reperfusion, used to construct an acute kidney injury mouse model.

[0028] Example 1: Application of gingerone A in the preparation of drugs for treating acute kidney injury. The specific steps are as follows: S1. Mice were administered gingerol A via gavage for 7 consecutive days, with each gavage dose being 5 mg / kg, to obtain pretreated mice.

[0029] S2. Bilateral renal pedicles of pretreated mice were clamped with non-invasive arterial clamps. After 45 minutes of ischemia, the arterial clamps were released for reperfusion to obtain pretreated mice with acute kidney injury. These mice were the normal drug administration group and were labeled as I / R+GA (5 mg / kg).

[0030] Shogaol A can significantly improve renal function abnormalities and reduce renal tubular pathological damage in pretreated mice with acute kidney injury, while inhibiting lipid peroxidation, ferrous ion accumulation, and mitochondrial structural and functional disorders.

[0031] Example 2: Application of gingerone A in the preparation of drugs for treating acute kidney injury, the specific steps are as follows: S1. Mice were administered gingerol A via gavage for 7 consecutive days, with each gavage administered at a dose of 20 mg / kg, to obtain pretreated mice.

[0032] S2. Bilateral renal pedicles of pretreated mice were clamped with non-invasive arterial clamps. After 45 minutes of ischemia, the arterial clamps were released for reperfusion to obtain pretreated mice with acute kidney injury. These mice were the normal drug administration group and were labeled as I / R+GA (20 mg / kg).

[0033] After anesthetizing the mice, the sham-operated group was obtained by simply freeing the bilateral renal pedicles without clamping them, and the mice were denoted as Sham.

[0034] Mice were administered gingerone A via gavage for 7 consecutive days at a dose of 20 mg / kg each time to obtain pretreated mice. The pretreated mice were then subjected to sham surgery, denoted as Sham++GA (20 mg / kg).

[0035] The mice treated with I / R are the model group mice, referred to as I / R in the figure.

[0036] Evidence 1: The intervention effect of GA on AKI was investigated by establishing an AKI model through the construction of a mouse model of renal ischemia-reperfusion injury. Figure 1 As shown in Figure A, the body weight of mice in the sham-operated group continued to increase; after continuous intragastric administration of GA (5 mg / kg or 20 mg / kg), the body weight change was not significantly different from that in the sham-operated group for the first 4 days, but gradually decreased from the 5th day. The kidney weight-to-body weight ratio is a direct indicator reflecting the degree of renal edema and proliferation in AKI; the results showed no significant difference in this indicator among the drug administration groups. After establishing the AKI model, as... Figure 1 As shown in B and C, the kidney weight-to-body weight ratio in mice was significantly increased, and GA intervention significantly reduced the pathological damage of AKI in a dose-dependent manner. Furthermore, the experimental mice with AKI exhibited severe kidney damage, such as... Figure 1 As shown in Figure D, HE and PAS staining revealed the absence of the brush border in the renal tubules, luminal dilation, protein casts, and epithelial cell necrosis. Simultaneously, the renal injury score increased, and serum urea nitrogen (BUN) and creatinine (Scr) levels rose synchronously. These injuries were alleviated in the GA (5 mg / kg) intervention group; as shown in Figure D. Figure 1 As shown in E, F, and G, the GA (20 mg / kg) intervention group further improved renal tubular pathological changes and significantly reduced renal injury scores, BUN, and Scr levels. Figure 1As shown in H and I, kidney injury molecule-1 (Kim-1) and neutrophil gelatinase-associated lipocalin (NGAL) are specific markers of renal tubular injury, sensitively reflecting the degree of early renal parenchymal injury in the AKI model. To verify the nephroprotective effect of GA, the inventors further examined the protein expression levels of Kim-1 and NGAL in tissues and the areas of immunohistochemical staining. The results showed that the expression of both markers was significantly upregulated in the renal ischemia / reperfusion model group; while in the ischemia / reperfusion combined with GA intervention group, their expression levels and the areas of positive staining were reduced. In addition, GA effectively inhibited the mRNA expression levels of IL-1β, IL-6, and TNF-α in the renal tissue of AKI mice. In summary, this invention confirms that GA intervention can effectively improve renal dysfunction and pathological damage in the AKI model.

[0037] In the development of AKI, to elucidate the potential mechanism of action of GA in treating AKI, network pharmacology analysis was performed. The results showed that GA can delay AKI progression by inhibiting ferroptosis. Further RNA sequencing analysis of HK-2 cells was conducted. The results showed that in an Erastin-induced HK-2 cell ferroptosis model, as... Figure 1 As shown in j, a total of 476 differentially expressed genes (DEGs) were screened after GA intervention, of which 295 were upregulated genes and 181 were downregulated genes. Figure 1 As shown in K, the data indicate that GA upregulates five ferroptosis-suppressing genes (FTH1, SQSTM1, GSS, GPX4, and SLC7A11) and downregulates seven ferroptosis-promoting genes (HMOX1, NCOA4, LIPG, TFRC, PTGES, LPCAT3, and ALOX5). KEGG enrichment analysis further confirms that the ferroptosis pathway is a key regulatory node, consistent with previous network pharmacology predictions. Furthermore, transcriptome sequencing revealed that arachidonic acid metabolism, cholesterol metabolism, and steroid hormone biosynthesis are all directly related to lipid metabolism pathways. Figure 1 As shown in Figure L, given that the ferroptosis pathway is centered on lipid peroxidation and is closely related to lipid metabolism regulation, it can be inferred that lipid metabolism disorders promote ferroptosis. To further verify that ferroptosis is a key pathway for GA to alleviate AKI, ferroptosis models were constructed in HK-2 and NRK-52E cells using two classic ferroptosis agonists, Erastin and RSL3. Erastin and RSL3 interventions significantly reduced cell viability, confirming the successful establishment of the models; while GA intervention increased cell viability in both ferroptosis models. Figure 1As shown by M and N in the figure, in the Erastin and RSL3-induced experimental groups, both 8 μM and 16 μM GA significantly improved cell survival (with better effects at higher concentrations), and the differences between groups were statistically significant. Therefore, GA can inhibit the ferroptosis signaling pathway, improve renal function, and alleviate AKI pathological damage.

[0038] Evidence 2: Based on the significant enrichment of ferroptosis and lipid metabolism pathways, the inhibitory effect of GA on ferroptosis was systematically verified at the morphological, biochemical, and key protein levels. Lipid peroxidation is a key biomarker distinguishing ferroptosis from other forms of cell death. The level of lipid peroxidation was detected using the BODIPY-C11 fluorescent probe, such as... Figure 2 As shown in A and B, Erastin and RSL3 treatments significantly increased cellular lipid peroxidation levels; while GA intervention significantly reduced the redox fluorescence ratio, effectively reversing the lipid peroxidation induced by the two inducers, with statistically significant differences between groups. Iron metabolism imbalance is a core driver of ferroptosis. Figure 2 As shown in S and D, after Erastin and RSL3 intervention, intracellular free Fe in HK-2 and NRK-52E cells was reduced. 2+ Significant accumulation of these ions can catalyze the production of large amounts of reactive oxygen species (ROS) through the Fenton reaction, thereby exacerbating oxidative stress imbalance and lipid peroxidation chain reactions, and promoting ferroptosis. For example... Figure 2 As shown in E and F, GA intervention can simultaneously reduce Fe 2+ ROS and lipid peroxidation levels. Further detection of malondialdehyde (MDA), a lipid peroxidation product, revealed that... Figure 2 As shown in G in the figure. Erastin treatment significantly increased MDA levels in HK-2 cells, while GA intervention effectively inhibited intracellular MDA levels. Furthermore, as shown in the figure... Figure 2 As shown in H and i in the figure. GA intervention can restore the levels of antioxidants superoxide dismutase (SOD) and glutathione (GSH) and reduce MDA levels, thereby improving Erastin-induced lipid peroxidation and oxidative stress.

[0039] Mitochondrial dysfunction and morphological changes are important downstream events of ferroptosis. For example... Figure 2 As shown in J in the figure. JC-1 assay showed that treatment with Erastin and RSL3 significantly reduced mitochondrial membrane potential in cells, and GA reversed this phenomenon. Furthermore, as shown in the figure... Figure 2As shown in K in the figure. Transmission electron microscopy (TEM) observation of the model group showed mitochondrial shrinkage, increased membrane density, and reduced or absent cristae, all consistent with typical mitochondrial morphological characteristics of ferroptosis. GA intervention significantly alleviated the aforementioned mitochondrial structural damage. Finally, the expression of the key ferroptosis regulatory protein ACSL4 was detected: as shown in the figure. Figure 2 As shown in L and M, Erastin treatment significantly upregulated ACSL4 expression, while GA intervention inhibited its overexpression in a dose-dependent manner. In summary, GA effectively inhibits ferroptosis in renal tubular epithelial cells by interfering with three key processes: lipid peroxidation, iron metabolism disorder, and mitochondrial dysfunction.

[0040] Evidence 3: Although GA can inhibit lipid peroxidation in renal tubular epithelial cells, downregulate ACSL4 expression, and block ferroptosis in vitro, its in vivo effects in an AKI model remain to be verified. Therefore, a mouse AKI model was established, such as... Figure 3 As shown in Figure A, patients were pretreated for one week with gradient doses of GA (5, 20 mg / kg) or a solvent control, and the expression level of key ferroptosis protein was detected. Figure 3 As shown in B, C, and D, Western blot results revealed that, compared to the sham-operated group, ACSL4 expression was significantly upregulated in the renal tissue of the ischemia / reperfusion group, while SLC7A11 and GPX4 expression was significantly decreased. GA intervention inhibited ACSL4 overexpression and increased SLC7A11 and GPX4 levels, indicating that GA exerts its anti-AKI effect in vivo by regulating key ferroptosis proteins. Figure 3 As shown in E, Fer-1 was used as a positive control to verify the mechanism of action of GA: Figure 3 As shown in F, G, and H, Western blot analysis revealed significantly increased ACSL4 expression and significantly decreased SLC7A11 and FTH1 expression in the renal tissue of the ischemia / reperfusion group. Fer-1 (2 mg / kg) treatment effectively reversed these changes, and the intervention effect of GA (20 mg / kg) was consistent with that of Fer-1, indicating that GA exerts a renal protective effect similar to ferroptosis inhibitors by regulating key ferroptosis proteins. Transmission electron microscopy (TEM) observation of the renal tissue ultrastructure showed that the renal tissue of the ischemia / reperfusion (I / R) group exhibited mitochondrial shrinkage, increased membrane density, and reduced or absent cristae structures, presenting typical ferroptosis mitochondrial morphological characteristics. Figure 3 As shown in Figure I, Fer-1 treatment significantly improved the aforementioned pathological changes; while GA intervention alleviated mitochondrial damage, reduced membrane density, and maintained structural integrity, further confirming its role in inhibiting ferroptosis. Figure 3 As shown in J, the immunohistochemical results are consistent with the Western blot analysis results. Figure 3As shown in K, L, and M, in the I / R group, both Fer-1 and GA intervention downregulated ACSL4 expression while upregulating FTH1 and GPX4 levels with similar effects, which histologically validates the regulatory role of GA on key ferroptosis proteins. Furthermore, as... Figure 3 As shown in N and O, the levels of key intracellular antioxidant molecules GSH and SOD were detected: GSH content and SOD activity in the kidney tissue of the I / R group were significantly reduced, while GA intervention could effectively reverse these changes, with an effect comparable to Fer-1. This indicates that GA can restore the antioxidant capacity of kidney tissue and alleviate I / R-induced oxidative stress damage, thereby supporting its role in inhibiting ferroptosis at the functional level.

[0041] Evidence 4: To elucidate the molecular mechanism by which GA regulates ACSL4 expression, this study further investigated whether GA directly promotes ACSL4 protein degradation. The ubiquitin-proteasome system (UPS) and the autophagy-lysosome pathway play crucial roles in the selective and specific degradation of most cellular proteins. First, quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the effect of Erastin-induced GA on ACSL4 transcription. The results showed that in HK-2 and NRK-52E cells, Erastin treatment significantly upregulated ACSL4 mRNA levels; however, combined use of GA (4, 8, and 16 μM) did not significantly alter ACSL4 mRNA levels, indicating that GA does not regulate ACSL4 at the transcriptional level, but may exert its effect through post-translational modification or degradation pathways. Further Western blot analysis showed that Erastin treatment significantly increased ACSL4 protein levels, while GA (8 and 16 μM) reversed this effect in a concentration-dependent manner, accompanied by a decrease in LC3B levels and accumulation of P62 protein, suggesting that GA may inhibit autophagic flux. To exclude the involvement of the autophagy-lysosomal pathway, cells were treated with the autophagy inhibitor chloroquine (CQ). Figure 4 As shown in A, B, C, D, E, F, G, and H, CQ treatment failed to reverse the downregulation of ACSL4 protein by GA; furthermore, after the autophagic flux was blocked, the regulatory trends of GA on p62 and LC3B did not change significantly, thus ruling out the role of the autophagy-lysosomal pathway in GA-mediated ACSL4 degradation. To directly verify the effect of GA on the rate of ACSL4 protein degradation, as shown in Figure 1, Figure 2 shows that CQ treatment failed to reverse the downregulation of ACSL4 protein by GA; furthermore, after the autophagic flux was blocked, the regulatory trends of GA on p62 and LC3B did not change significantly, thus ruling out the role of the autophagy-lysosomal pathway in GA-mediated ACSL4 degradation. Figure 4As shown by I, J, K, L, M, and N in the figure, a cyclohexane (CHX) tracking experiment was performed. The results showed that in Erastin-induced HK-2 and NRK-52E cells, GA intervention significantly accelerated the degradation rate of ACSL4 protein, with its protein level decreasing more rapidly over time. However, the downregulation effect of GA on ACSL4 protein was significantly blocked upon the addition of the proteasome inhibitor MG-132, and the degradation rate of ACSL4 protein was significantly slowed, indicating that the effect of GA depends on the ubiquitin-proteasome system. To further verify this conclusion, an ACSL4 ubiquitination experiment was conducted: under the condition of MG-132, as shown... Figure 4 As shown in the O, P, Q, R, S, and T values, GA treatment significantly increased ACSL4 ubiquitination levels in Erastin-induced HK-2 and NRK-52E cells in a concentration-dependent manner. These results collectively indicate that GA reduces ACSL4 protein levels not through transcriptional regulation or the autophagy-lysosomal pathway, but rather by enhancing ACSL4 ubiquitination and promoting its degradation via the ubiquitin-proteasome pathway.

[0042] Evidence 5: Given that GA significantly regulates lipid peroxidation during ferroptosis, and that ACSL4 is a core protein regulating lipid metabolism in ferroptosis, immunoprecipitation-mass spectrometry (IP-MS) was used to identify potential ACSL4-interacting proteins in HK-2 cells. A total of 620 proteins were identified in the IP-ACSL4 and IP-IgG groups, of which 524 were ACSL4-specific enriched proteins. Figure 5 As shown in Figure A, the most abundant protein is VCP, indicating that it may participate in the regulation of ferroptosis as a key interacting molecule with ACSL4. Subsequently, the interaction between VCP and ACSL4 was systematically verified: as follows... Figure 5 As shown in B and C, endogenous VCP was detected in ACSL4 immunoprecipitates from both HK-2 cells and NRK-52E cells, and vice versa; Figure 5 As shown in D and E, after overexpressing HA-tagged VCP and Flag-tagged ACSL4 in HEK293FT cells, as Figure 5 As shown in F and G, the specific interaction between the two proteins was further confirmed by exogenous co-immunoprecipitation assay; immunofluorescence co-localization assay also showed that the two proteins had obvious spatial co-localization. Based on the fact that VCP belongs to the AAA+ ATPase family and its C-terminal region (767–806 amino acids) is a key conserved fragment regulating ubiquitination, as... Figure 5 As shown in H, a series of truncated variants of VCP and ACSL4 were constructed to clarify their binding sites. Figure 5As shown in Figures I and J, co-immunoprecipitation experiments revealed that the C-terminal fragment of the VCP protein (amino acids 767–806) is the core region interacting with ACSL4; while the region responsible for binding VCP in ACSL4 is the M1 region (amino acids 1–474), and deletion of the C-terminal 475–711 amino acid sequence does not affect the interaction between the two proteins. Sequence information of VCP and ACSL4 was extracted from the UniProt database, and protein docking simulations were performed using the Alphafold tool, as shown in Figures I and J. Figure 5 As shown in K, the results indicate that residues S794, Y796, T797, E798, N800, D801, D802, L804, and Y805 in the C-terminal 767–806 amino acid region of the VCP protein can form multiple hydrogen bonds with residues E187, T278, S281, T282, R284, K286, and G467 in the 1–474 amino acid region of the ACSL4 protein. This hydrogen bond network is a major force stabilizing protein-protein interactions, and these sites are potentially key binding regions for both proteins. Subsequently, as... Figure 5 As shown by L in the diagram, the predicted key site in the VCP and ACSL4 binding region was mutated to alanine, and protein docking analysis was performed again. Figure 5 As shown in M, the results indicate that the number of hydrogen bond interactions between VCP and ACSL4 is significantly reduced after mutation, suggesting that the aforementioned residues are key sites for maintaining the stability of the interaction interface between the two proteins. Figure 5 As shown in N, Western blot experiments further confirmed that mutations in either the VCP binding site (HA-VCP-BS-Mut) or the ACSL4 binding site (Flag-ACSL4-BS-Mut), whether occurring alone or in combination, significantly weakened the interaction between these two proteins. In summary, ACSL4 can specifically bind to VCP.

[0043] Evidence 6: Given that VCP regulates ACSL4 ubiquitination, an actinomycete ketone tracking assay was used to assess ACSL4 protein turnover. To investigate whether VCP mediates ACSL4 ubiquitination, Flag-ACSL4, His-Ub, and wild-type or binding-deficient HA-VCP mutants were co-transfected into HK-2 and HEK293FT cells. After treating with MG132 to block proteasome degradation, anti-Flag immunoprecipitation was performed on cell lysates to enrich ACSL4 protein, and significant polyubiquitination of ACSL4 was clearly detected in the precipitate. Figure 6As shown in A, B, and C, treatment with the protein synthesis inhibitor actinomycete significantly delayed the degradation of ACSL4 protein and prolonged its half-life after silencing endogenous VCP. Conversely, as... Figure 6 As shown in D, E, and F, ectopic overexpression of the HA tag VCP accelerates the clearance of ACSL4 protein and shortens its half-life, confirming that VCP mainly regulates ACSL4 levels through a proteasome-dependent degradation pathway.

[0044] To elucidate the chain-type specificity of VCP-mediated ACSL4 ubiquitination, the variations in different polyubiquitin linkages were systematically analyzed. Polyubiquitin chains are assembled via isopeptide bonds formed between different lysine residues on the ubiquitin, and each linkage imparts unique structural and functional properties. K48-linked and K63-linked polyubiquitin chains are the two most thoroughly investigated subtypes: K48-linked chains act as typical targeting signals, driving proteasome-dependent substrate degradation; while K63-linked chains primarily regulate non-proteolytic cellular processes, including DNA damage repair and signal transduction cascades. The physiological roles of other ubiquitin linkages (K6, K11, K27, K29, K33) remain unclear and require further mechanistic investigation. Subsequently, the mechanism by which VCP regulates different polyubiquitin chain subtypes binding to ACSL4 was explored: such as... Figure 6 As shown in G and H, immunoprecipitation assay revealed that VCP overexpression significantly enhanced K48-linked ubiquitin signaling on ACSL4, but had little effect on ubiquitin modifications linked to K6, K11, K27, K29, K33, and K63. Furthermore, combined experiments using K48O and K48R ubiquitin mutants further confirmed that GA exerts its biological function by enhancing ACSL4 ubiquitination and accelerating its degradation via the ubiquitin-proteasome pathway. Figure 6 As shown in I and J, the ubiquitin mutant immunoprecipitation assay confirmed that GA specifically triggers K48-linked ubiquitination of ACSL4. Figure 6 As shown by K, L, and M, knockdown of endogenous VCP significantly reversed GA-induced K48 ubiquitination and accumulation of ACSL4. Blocking with the selective VCP inhibitor CB-5339 completely eliminated the promoting effect of GA on ACSL4 K48 ubiquitination. Figure 6 As shown in N, overexpression of full-length VCP or its A2 truncated mutant interacting with ACSL4 significantly enhanced the K48-linked ubiquitination level of ACSL4. This result further confirms that the A2 domain mediating the VCP-ACSL4 interaction is the core functional region responsible for this ubiquitination modification. In summary, these data indicate that GA promotes ACSL4 K48-linked ubiquitination and subsequent degradation in a VCP-dependent manner.

[0045] To verify whether the physical interaction between VCP and ACSL4 is indispensable for the regulation of this ubiquitination process, such as... Figure 6 As shown in O and P, a VCP binding-deficient mutant (HA-VCP-BS-Mut) was constructed and co-transfected for ubiquitination. Disruption of the VCP-ACSL4 binding interface significantly reduced VCP's ability to enhance K48-linked polyubiquitination of ACSL4, indicating that direct binding of VCP to ACSL4 is a necessary condition for this degradative ubiquitination modification.

[0046] Previous structural and biochemical studies have identified five evolutionarily conserved lysine residues (K124, K148, K512, K536, and K596) in the ACSL4 peptide as potential ubiquitination receptor sites. Therefore, as... Figure 6 As shown in Q, a series of single-lysine-to-glutamine mutants of ACSL4 (K124O, K148O, K512O, K536O, K596O) were constructed to identify the main ubiquitination target of VCP. Ubiquitination profiling analysis after co-expression with HA-VCP showed that only the K148O mutant could completely inhibit VCP-mediated polyubiquitination linked to the ACSL4 K48 site; substitutions of the other four lysine sites failed to block VCP-mediated ubiquitin chain deposition. After screening ACSL4 lysine-only mutants to confirm that K148 is the only key residue mediating VCP-promoted polyubiquitination linked to the ACSL4 K48 site, as shown in Q, ... Figure 6 As shown by R and S in the figure, parallel immunoprecipitation ubiquitination experiments in HK-2 cells and HEK293FT cells further confirmed that the K148R point mutation completely eliminates VCP-stimulated ACSL4 K48-linked polyubiquitination. In both cell models, VCP failed to enhance ACSL4 ubiquitination levels in the K148R mutant. In summary, these results indicate that VCP is a key mediator of GA regulation of ACSL4 homeostasis. GA enhances the binding interaction between VCP and ACSL4, thereby specifically promoting K48-linked ubiquitination modification of the ACSL4 protein at the K148 site, promoting proteasome-dependent degradation, and ultimately precisely regulating intracellular ACSL4 protein expression levels. (ACSL4 K48-linked ubiquitination modification.)

[0047] Evidence 7: To screen for direct targets of GA, two classic drug target screening techniques were combined: such as Figure 7As shown in Figure A, Drug Affinity Responsive Target Stability Mass Spectrometry (DARTS-MS) identified 31 candidate targets; Limiting Proteolytic Enzyme Digestion Mass Spectrometry (LiP-MS) identified 190 candidate targets, including 10 overlapping targets such as Hspa8, Psdm2, Vcl, Hspa5, Vcp, Gdi2, Rpsa, Uba1, Cltc, and Actn4. VCP specifically interacts with ACSL4, thus VCP was identified as a core potential target. The status of VCP as a key target was further validated by DARTS-WB experiments, small molecule pull-down experiments, surface plasmon resonance (SPR) analysis, and cellular thermal shift assay (CETSA). Firstly, as... Figure 7 As shown in B and C, the DARTS-WB experiment demonstrated that GA significantly enhanced the resistance of VCP to Pronase E enzyme hydrolysis, such as... Figure 7 As shown in D and E, this suggests a direct binding between the two; both small molecule pull-down assays and Western blotting results show that GA-biotin can directly bind to VCP protein in HK-2 and NRK-52E cells, and that excess unlabeled GA can competitively inhibit this binding in a concentration-dependent manner; Figure 7 As shown in F, SPR analysis confirmed the direct interaction between GA and VCP—the response value of GA binding to immobilized VCP at different concentrations increased with increasing GA concentration, and its dissociation constant (KD) was 5.02 × 10⁻⁶. -6 .like Figure 7 As shown in G, H, I, and J, CETSA analysis revealed that, compared to the control group, the GA-treated group exhibited significantly higher relative band intensities of VCP protein at all temperature points, with the curves shifting to the right, indicating that GA binding enhances the thermal stability of VCP. These results collectively demonstrate that GA specifically targets VCP.

[0048] Evidence 8: To further verify that GA exerts a nephroprotective effect through VCP, such as... Figure 8 As shown in A and B, VCP expression was knocked down in HK-2 and NRK-52E cells. The results showed that in the Si-NC control group, GA significantly upregulated Erastin-induced ACSL4 ubiquitination; however, after VCP knockdown, the regulatory effect of GA on ACSL4 ubiquitination was completely eliminated. Figure 8 As shown in C and D, functional experiments indicate that under Si-NC conditions, GA significantly reduces Erastin-induced lipid reactive oxygen species and Fe. 2+Accumulation and effective reversal of ferroptosis phenotype; to further investigate whether GA promotes the interaction between VCP and ACSL4, co-immunoprecipitation experiments were performed in HK-2 and HEK293FT cells using antibodies targeting VCP and ACSL4. Figure 8 As shown in Figures E, F, G, and H, GA significantly enhances the binding ability of VCP to ACSL4; this result was further validated by immunofluorescence co-localization assays. Knockdown of VCP weakens the interaction between VCP and ACSL4, while GA treatment restores and enhances their binding. In summary, these results indicate that GA can promote the intracellular physical interaction between VCP and ACSL4. To promote the clinical application of VCP-targeted intervention strategies, the second-generation VCP inhibitor CB-5339, which has entered clinical trials and shown therapeutic potential in various disease models, was introduced, and rescue experiments were conducted using this inhibitor. Figure 8 As shown in Figure I, Erastin treatment induced ferroptosis in HK-2 and NRK-52E cells, upregulating the expression of ferroptosis-promoting proteins ACSL4 and HO-1, as well as damage markers KIM-1 and NGAL, while downregulating the expression of antiferroptosis proteins GPX4, FSP1, and FTH1; GA reversed this phenotype and inhibited ferroptosis. Combined use of CB-5339 completely eliminated the protective effect of GA, indicating that GA inhibits ferroptosis in renal tubular epithelial cells by downregulating ACSL4 in a VCP-dependent manner. Figure 8 As shown in J, CB-5339 can block GA-induced increases in ACSL4 ubiquitination levels and reverse its protective effect against AKI. (As indicated by...) Figure 8 As shown in K, L, and M, HE and PAS staining revealed that after CB-5339 treatment, the ameliorative effect of GA on renal tubular structural disorder disappeared; the positive expression of Kim-1 and NGAL was significantly increased; as shown in K, L, and M, HE staining and PAS staining revealed that after CB-5339 treatment, the ameliorative effect of GA on renal tubular structural disorder disappeared; the positive expression of Kim-1 and NGAL was significantly increased; Figure 8 As shown by N and O in the figure, serum Scr and BUN levels also increased synchronously, suggesting exacerbated renal impairment. However, no improvement in impairment was observed in the CB-5339 combined treatment group with GA. These results indicate that GA exerts a renal protective effect through the VCP pathway. Figure 8 As shown in Figure P, clinical sample analysis revealed that compared to adjacent normal kidney tissue, VCP expression was significantly downregulated and ACSL4 expression was significantly upregulated in the damaged kidney tissue of AKI patients. This suggests that decreased VCP levels and increased ACSL4 levels jointly contribute to the pathological progression of AKI, providing clinical evidence for targeting the VCP-ACSL4 pathway to intervene in renal ferroptosis. In summary, this demonstrates that GA achieves renal protection by targeting and activating the VCP-ACSL4 pathway, providing solid experimental support for targeted therapy of AKI.

[0049] All paraffin-embedded tissue specimens were obtained from patients at the First Hospital of Lanzhou University. Specimen collection did not affect the patients' clinical diagnosis and treatment plans, and did not cause any psychological or physical burden on the subjects. A total of 12 specimens from patients with acute kidney injury and 15 specimens from normal kidney tissue adjacent to renal cell carcinoma were included. All experimental procedures involving human specimens followed the Declaration of Helsinki, and all patients signed informed consent forms. The experiments were approved by the Ethics Committee of the First Hospital of Lanzhou University (ethics approval number: LDYYLL2026-668).

[0050] The protective effect and molecular mechanism of GA against acute kidney injury are as follows: This invention, through the construction of in vitro ferroptosis and in vivo acute kidney injury models, demonstrates that GA can improve renal dysfunction and pathological damage induced by acute kidney injury. Mechanistically, GA directly binds to the Cys184 site of venous cytoplasm (VCP), upregulating the acetylation level of VCP at the K8 site; activated VCP specifically promotes K48 ubiquitination at the ACSL4 K148 site, accelerating ACSL4 degradation via the ubiquitin-proteasome pathway, thereby inhibiting ferroptosis in renal tubular epithelial cells and alleviating acute kidney injury. This invention comprehensively elucidates the therapeutic value of GA for acute kidney injury, providing a potential new strategy for the clinical treatment of acute kidney injury.

[0051] The above descriptions are merely embodiments of the present invention, and common knowledge such as specific technical solutions and / or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. Application of gingerone A in the preparation of drugs for treating acute kidney injury.

2. The application according to claim 1, characterized in that, The kidney injury mentioned includes acute kidney injury.

3. The application according to claim 1, characterized in that, The sole active ingredient of the drug is gingerone A.

4. The application according to claim 3, characterized in that, The dosage forms of the drug include injections, tablets, capsules, granules, oral liquids, or powder injections.

5. The application according to claim 3, characterized in that, The drug for treating kidney injury is prepared from gingerone A and medically acceptable pharmaceutical excipients.

6. The application according to claim 5, characterized in that, The pharmaceutical excipients are one or more of the following: diluents, disintegrants, precipitation inhibitors, flow aids, binders, dispersants, suspending agents, isotonic agents, thickeners, emulsifiers, preservatives, and stabilizers.

7. The application according to claim 5, characterized in that, The pharmaceutical excipient is polyethylene glycol 200.

8. The application according to claim 7, characterized in that, The drug for treating kidney injury was prepared by mixing gingerone A and polyethylene glycol 200 at a mass-volume ratio of (2 mg to 3 mg): 1 mL.

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