Application of formononin in preparation of medicine for regulating and controlling BNIP3-mediated excessive mitochondrial autophagy

Abstract

The invention relates to the technical field of biological medicines, and particularly discloses application of formononin in preparation of a medicine for regulating and controlling BNIP3-mediated excessive mitochondrial autophagy. The invention discloses that ononin can be used as a direct small molecule inhibitor of BNIP3 protein, specific binding of ononin with BNIP3 is verified through molecular docking, surface plasmon resonance and molecular dynamics simulation in multiple dimensions, and in an iron overload induced ovarian granulosa cell injury model, the ononin can be used as an iron overload induced ovarian granulosa cell injury model. The formononin effectively inhibits mitochondrial excessive autophagy mediated by a BNIP3-PINK1 / Parkin axis through targeting BNIP3, remarkably recovers mitochondrial membrane potential, reduces mitochondrial active oxygen level and improves cell oxygen consumption rate (OCR), so that stressful high-flux and low-efficiency breathing states are corrected, cell senescence phenotypes are delayed, and cell apoptosis is inhibited. And a new small molecule drug strategy with a clear structure and a clear action mechanism is provided for iron overload related ovarian function impairment.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically the application of gentianin in the preparation of drugs that regulate BNIP3-mediated excessive mitochondrial autophagy. Background Technology

[0002] Iron is an essential trace element for the synthesis of mitochondrial respiratory chain complexes, heme, and iron-sulfur clusters, playing a crucial role in maintaining cellular energy metabolism and redox balance. However, when iron homeostasis is disrupted, elevated levels of intracellular free iron can promote the generation of reactive oxygen species (ROS) through the Fenton reaction, leading to lipid peroxidation and oxidative damage to mitochondrial membranes, proteins, and mitochondrial DNA. This manifests as limited electron transport chain function, disordered energy metabolism, and increased redox stress, ultimately driving cell fate towards functional decline or death. Iron overload has been proven to be closely associated with various diseases, including neurodegenerative diseases, cardiovascular diseases, metabolic diseases, and reproductive system dysfunction.

[0003] Mitochondrial quality control primarily relies on mitophagy, a process that selectively removes damaged or dysfunctional mitochondria, blocks ROS diffusion, and maintains cellular energy homeostasis. The PINK1-Parkin pathway is the most classic regulatory axis, with receptor-mediated pathways such as BNIP3 / NIX and FUNDC1 also participating in regulation. However, the activation level of mitophagy needs to be maintained at an appropriate level—moderate mitophagy has protective significance, but when mitochondrial damage persists, mitophagy may be pushed into an "overactivated" state, leading to excessive mitochondrial clearance and a decline in cellular energy supply, which is particularly detrimental to cells highly dependent on mitochondrial metabolism (such as oocytes, cardiomyocytes, and neurons).

[0004] BNIP3 (BCL2 / adenovirus E1B 19 kDa protein-interacting protein 3) is a member of the Bcl-2 protein family containing the BH3 domain. Located in the outer mitochondrial membrane, it is a downstream target gene of hypoxia-inducible factor-1α (HIF-1α) and is induced to express under hypoxic stress. BNIP3 mediates selective autophagy in mitochondria by directly binding to LC3 and is also a key node connecting stress signals and mitochondrial quality control. Existing research shows that BNIP3 expression is upregulated under various pathological conditions, including myocardial ischemia-reperfusion injury, neurodegenerative diseases, tumors, and osteoporosis. Its abnormal activation is closely related to mitochondrial dysfunction and cell death.

[0005] Currently, the treatment of iron overload mainly relies on iron chelators, such as deferoxamine (DFO), deferiprone, and deferasirox. These drugs work by reducing iron load in the body, but they have the following limitations: First, reducing iron levels does not equate to correcting the imbalance in mitophagy. The damage caused by iron overload comes not only from iron itself but also from iron-mediated mitochondrial damage and the resulting abnormal enhancement of mitophagy. While iron chelators can reduce free iron, they do not directly target this key pathological link of excessive mitophagy. Second, iron deficiency or chelation itself may remodel mitochondrial stress and autophagy programs under different cellular and stress conditions, making the net effect of mitophagy unpredictable. Third, although exploratory studies targeting the ovary attempt to deliver iron chelators to granulosa cells to simultaneously improve local iron load and autophagy / mitochondrial autophagy-related abnormalities, these strategies often rely on complex delivery systems, making them difficult to replace structurally well-defined and easily formulated small molecule drugs.

[0006] In recent years, some research small molecules targeting mitochondrial fission / fusion or pathways such as PINK1 and BNIP3 have been reported to have the ability to regulate mitophagy. However, these molecules are mostly used in models of neurodegenerative diseases, tumors, or cardiovascular diseases, and there are few studies on the specific pathological background of excessive mitophagy in ovarian granulosa cells caused by iron overload in female reproductive damage. For example, hyperoside has been reported to protect cyclophosphamide-induced ovarian damage by inhibiting HIF-1α / BNIP3-mediated autophagy (as described in the reference Hyperoside protects against cyclophosphamide-induced ovarian damage and reduced fertility by suppressing HIF-1α / BNIP3-mediated autophagy), but its mechanism of action remains at the pathway level. Melatonin has been reported to improve the reduced ovarian reserve caused by excessive autophagy damage (as described in CN113350335A). In addition, some studies have used BNIP3 as an intervention target for the treatment of reperfusion injury (see CN114026114A), but these studies used BNIP3-derived peptides. Small molecules from natural products have potential advantages in antioxidation, anti-inflammation, and cell protection, but there is still a significant technological gap in the field of iron overload-related ovarian dysfunction in women.

[0007] Therefore, there is an urgent need to develop small molecule compounds and their applications that can selectively correct excessive mitochondrial autophagy in the context of iron overload, improve mitochondrial ROS and energy metabolism homeostasis, and retain necessary quality control functions as much as possible. These compounds can serve as a supplement to iron-lowering therapy and provide a more direct and controllable mechanism for intervention in iron toxicity-related ovarian dysfunction. Summary of the Invention

[0008] The purpose of this invention is to provide the application of ononin in the preparation of drugs that regulate BNIP3-mediated mitochondrial autophagy. This technical solution reveals that ononin can be used as a direct small molecule inhibitor of BNIP3 protein. The specific binding of ononin to BNIP3 is verified through molecular docking, surface plasmon resonance, and molecular dynamics simulation. In an iron overload-induced ovarian granulosa cell injury model, ononin effectively inhibits BNIP3-PINK1 / Parkin axis-mediated mitochondrial autophagy by targeting BNIP3, significantly restores mitochondrial membrane potential, reduces mitochondrial reactive oxygen species levels, and increases cellular oxygen consumption rate (OCR), thereby correcting the state of "stress-induced high-flux, low-efficiency respiration" and delaying the cellular senescence phenotype. This provides a new strategy for iron overload-related ovarian dysfunction with a well-defined structure and clear mechanism of action of small molecule drugs.

[0009] To achieve the above-mentioned objectives, the present invention provides the following technical solution: the application of gentianin in the preparation of drugs for preventing and / or treating ovarian dysfunction caused by excessive mitochondrial autophagy.

[0010] The basic principle of this technical solution is based on the pathological basis of iron overload leading to mitochondrial dysfunction and imbalance of mitophagy. Iron is an essential trace element for the synthesis of mitochondrial respiratory chain complexes, heme, and iron-sulfur clusters. However, when iron homeostasis is disrupted, the level of free iron in cells increases, which can catalyze the production of large amounts of reactive oxygen species (ROS) through the Fenton reaction. Excessive ROS can induce stress-related signals and be accompanied by upregulation of BNIP3 expression. BNIP3, as an autophagy receptor protein located on the outer mitochondrial membrane, mediates the selective clearance of mitochondria, i.e., mitophagy, by directly binding to LC3 under stress conditions. Under persistent iron overload, BNIP3 is overactivated, causing mitophagy to shift from a protective response of "moderate clearance of damaged mitochondria" to a pathological state of "excessive clearance of mitochondria," leading to a decline in cellular energy supply, disruption of redox homeostasis, and ultimately inducing cellular functional decline or even death. Ovarian granulosa cells are key cells that provide metabolic support to oocytes during follicle development. They are highly dependent on mitochondrial function. Therefore, iron overload-induced BNIP3-mediated excessive mitochondrial autophagy is an important mechanism leading to ovarian dysfunction.

[0011] This technology, through molecular docking, molecular dynamics simulation, and surface plasmon resonance, has for the first time discovered and verified that the natural isoflavone glycoside, gentianin, can directly bind to the BNIP3 protein, with a dissociation constant KD of 10. -5 ~10 -7 M. This direct binding allows mangosteen to directly bind to BNIP3 and inhibit BNIP3-mediated excessive mitochondrial autophagy signaling. In an iron overload-induced ovarian granulosa cell injury model, mangosteen treatment significantly downregulated BNIP3 protein expression and the activation level of its downstream PINK1-Parkin pathway, effectively curbing excessive mitochondrial clearance. Further functional assays showed that after mangosteen intervention, mitochondrial membrane potential was restored, mitochondrial reactive oxygen species levels decreased, and cellular oxygen consumption rate (OCR) increased, indicating improved mitochondrial oxidative phosphorylation function and energy metabolism efficiency. Simultaneously, the positivity rate of the cellular senescence marker SA-β-gal was significantly reduced, suggesting that mangosteen delays the iron overload-induced cellular senescence process by restoring mitochondrial quality control balance.

[0012] Mangosteen glycosides specifically target and bind to the BNIP3 protein, inhibiting excessive mitochondrial autophagy mediated by the BNIP3-PINK1-Parkin axis induced by iron overload, thereby restoring the homeostasis of mitochondrial quantity and function, reducing oxidative stress damage, improving cellular energy metabolism efficiency, and ultimately achieving a protective effect on ovarian granulosa cells. This provides a well-defined intervention strategy with a clear mechanism and target for iron overload-related ovarian dysfunction.

[0013] The specific research and development process is as follows: The research and development process of this technical solution is to systematically verify the role of mangiferin in improving excessive mitochondrial autophagy and ovarian granulosa cell damage by targeting BNIP3 under the background of iron overload through multi-dimensional experiments such as computational biology, biophysics and cell biology.

[0014] 1. Molecular docking selection

[0015] (1) Docking method: The three-dimensional structures of candidate small molecules were obtained (batch acquisition using PubChemPy) and preprocessed using PyMOL (conformation checking, hydrogenation, and structure cleaning).

[0016] Molecular docking was performed using AutoDock Vina, and binding sites and conformational scoring were performed on each ligand with the BNIP3 protein to record the optimal binding energy.

[0017] Visualize and analyze the interaction characteristics (hydrogen bonds, hydrophobic interactions, etc.) of docking conformations.

[0018] (2) Docking results: The predicted binding energy of Ononin and BNIP3 is 7.4 kcal / mol is the best among the candidate molecules.

[0019] Docking conformations show that Ononin can form stable conformations in potential binding regions of BNIP3 (such as...). Figure 1 A, 1B and Figure 2 (As shown).

[0020] 2. Molecular dynamics simulation (1) Simulation method: Molecular dynamics simulations for 100 ns were performed using GROMACS (v2025.4).

[0021] Force field: AMBER14SB; Solvent: TIP3P water model; Added ion neutralization system.

[0022] First, energy minimization is performed to eliminate unreasonable contacts and high-energy conformations. Then, 100 ps NPT equilibrium is performed at 298.15 K, and finally, a 100 ns production period simulation is performed.

[0023] Analyze RMSD, RMSF, Rg, SASA, hydrogen bond quantity, and Gibbs free energy landscape.

[0024] (2) Simulation results: After approximately 35 ns, the RMSD enters a plateau phase, and the system reaches a kinetic steady state (e.g., Figure 3 (As shown in A).

[0025] RMSF showed high flexibility near site 153 of the protein, which matched the binding region (e.g. Figure 3 As shown in B).

[0026] A transient conformational rearrangement occurs at approximately 70 ns (a brief increase in Rg and SASA), indicating a temporary opening of the binding pocket (e.g., Figure 3 (As shown in C, 3D).

[0027] The system can form 1 to 2 relatively stable hydrogen bond networks (such as...) Figure 3 E is shown.

[0028] The Gibbs free energy landscape shows significant low-energy valleys in the Rg 1.5–1.6 nm and RMSD 0.7–0.9 nm ranges, indicating that the complex possesses a preferred set of stable binding conformations (e.g., ...). Figure 4 (As shown in A and 4B).

[0029] The above results demonstrate that Ononin and BNIP3 can maintain stable binding in dynamic environments.

[0030] 3. Surface plasmon resonance (SPR) combined with kinetic detection (1) Detection method: Instrument: Biacore 1K; Chip: CM5 sensor chip.

[0031] BNIP3 protein was immobilized using amine coupling (diluted to 50 μg / mL with acetate buffer, pH 4.5), while the reference channel was activated but the protein was not immobilized.

[0032] Run buffer: 1× PBS + 5% DMSO; temperature: 25°C.

[0033] Ononin was serially diluted and injected at a flow rate of 30 μL / min for 60 s; then regenerated with 10 mM glycine-HCl (pH 2.0).

[0034] A 1:1 Langmuir model was used for global fitting to calculate the dissociation constant KD.

[0035] (2) Test results Ononin exhibits a specific, concentration-dependent binding response to BNIP3 (e.g., Figure 5 (As shown).

[0036] Global fitting yielded the dissociation constant KD = 7.85 × 10⁻⁶. -6 M indicates detectable binding affinity.

[0037] The above results indicate that Ononin binds directly to the BNIP3 protein, and the binding force is at the micromolar level.

[0038] Furthermore, the iron overload-induced ovarian dysfunction manifests as excessive mitochondrial autophagy.

[0039] Furthermore, the gentianin inhibits excessive mitochondrial autophagy by suppressing the activity of the BNIP3 protein.

[0040] Furthermore, the gentianin directly binds to the BNIP3 protein, with a binding dissociation constant KD of 7.85 × 10⁻⁶. -6 M.

[0041] Furthermore, the gentianin is used to improve at least one of the following indicators: mitochondrial membrane potential, mitochondrial reactive oxygen species level, cellular oxygen consumption rate, and cellular senescence markers.

[0042] Furthermore, the gentianin is used to restore mitochondrial membrane potential, reduce mitochondrial ROS levels, increase cellular oxygen consumption, or reduce the rate of SA-β-gal positive cells.

[0043] Application of gentianin as a BNIP3 inhibitor in the preparation of drugs for the prevention and / or treatment of BNIP3-mediated mitochondrial autophagy-related diseases.

[0044] Furthermore, the BNIP3-mediated excessive mitophagy-related diseases include ovarian dysfunction caused by iron overload.

[0045] A pharmaceutical composition for preventing and / or treating ovarian dysfunction caused by excessive mitochondrial autophagy, comprising an effective amount of mangiferin and a pharmaceutically acceptable carrier.

[0046] Furthermore, the dosage form of the pharmaceutical composition is an oral formulation or an injectable formulation.

[0047] The beneficial effects of this invention are: (1) By employing molecular docking, molecular dynamics simulation, and surface plasmon resonance technology, it is demonstrated for the first time that gentianin directly binds to the BNIP3 protein, providing a small molecule inhibitor with a well-defined structure and mechanism for BNIP3. Compared with existing reports, this technical solution has a significant advantage in the depth of target validation, providing a new chemical entity for the development of drugs targeting BNIP3.

[0048] (2) This technical solution found that iron overload-induced excessive mitochondrial autophagy not only involves the upregulation of BNIP3, but also the activation of PINK1 and Parkin. Mangosteen glycoside can simultaneously inhibit this pathway, revealing the synergistic regulatory mechanism between BNIP3 and the classical mitochondrial autophagy pathway. After intervention with mangosteen glycoside, the cellular oxygen consumption rate further increased, indicating that its effect is not simply to inhibit autophagy, but to restore mitochondrial respiratory efficiency, achieving a mechanistic transformation.

[0049] (3) Based on its mechanism against iron overload-induced excessive autophagy in mitochondria, cytosine can simultaneously restore mitochondrial membrane potential, reduce mitochondrial reactive oxygen species levels, increase cellular oxygen consumption, and inhibit cellular senescence phenotypes, thus significantly improving symptoms.

[0050] (4) Existing iron chelators (such as deferoxamine) only reduce free iron levels and do not directly target the key pathological link of excessive mitochondrial autophagy. Mangosteen glycoside directly intervenes in the imbalance of mitochondrial autophagy by targeting BNIP3, blocking the vicious cycle at the mechanistic level. Currently, there are no small molecule drugs targeting excessive mitochondrial autophagy in ovarian granulosa cells under the background of iron overload, and this technical solution fills this gap.

[0051] (5) Mangguisin is a natural isoflavone glycoside with a well-defined structure and stable properties, making it easy to develop into oral or injectable formulations and showing good prospects for drug development. This technical solution demonstrates the inhibitory effect of mangguisin on BNIP3-mediated excessive mitochondrial autophagy, providing a theoretical basis for its application in BNIP3-related diseases such as chemotherapy-induced ovarian injury, ischemia-reperfusion injury, and osteoporosis. Attached Figure Description

[0052] Figure 1 A. Chemical structural formula of Ononin; B. Schematic diagram of the conformation of the BNIP3–Ononin complex in molecular docking.

[0053] Figure 2 The binding states of the BNIP3–Ononin complex were selected at representative time points in molecular dynamics simulations from 0 to 100 ns.

[0054] Figure 3 Time series analysis of key molecular dynamics parameters, including root mean square shift (RMSD), root mean square fluctuation (RMSF), radius of gyration (Rg), solvent accessible surface area (SASA), and changes in the number of hydrogen bonds.

[0055] Figure 4 A. Three-dimensional and B. Two-dimensional Gibbs free energy landscape of the BNIP3–Ononin complex.

[0056] Figure 5 Sensing curves obtained by injecting different concentrations of Ononin onto a BNIP3 immobilized chip were used to evaluate the binding specificity and concentration dependence of Ononin to BNIP3.

[0057] Figure 6 To compare the activity of granulocytes under different doses of Ononin intervention under iron overload conditions.

[0058] Figure 7 A. Representative immunofluorescence images of lysosomes (green), mitophagy signals (red), and nuclei (blue) in granule cells of each group; B. Quantitative analysis of the positive area of ​​mitophagy in each group.

[0059] Figure 8 A. Representative Western blot bands of mitophagy-related proteins in each group; B. Quantitative comparison of the expression levels of BNIP3, PINK1, and Parkin1 proteins.

[0060] Figure 9 A. Representative fluorescence images for mitochondrial membrane potential detection; B. Quantitative analysis of the fluorescence intensity ratio of JC-1 aggregates / JC-1 monomers.

[0061] Figure 10 A. Representative images of ROS fluorescent staining; B. Quantitative comparison of relative fluorescence intensity.

[0062] Figure 11 A. Measure the mitochondrial oxygen consumption rate (OCR) of granulocytes in different groups; B. Quantitatively analyze the respiration rate of granulocytes in each group.

[0063] Figure 12 A. Representative images of SA-β-gal staining in granulocytes of each group; B. Quantitative analysis of the relative activity level of SA-β-gal. Detailed Implementation

[0064] The specific implementation method is described below with reference to the accompanying drawings.

[0065] Example 1 This embodiment provides the establishment and grouping of an iron overload model. (1) The human granulocyte cell line KGN was cultured in DMEM / F12 medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin antibiotics. The cells were cultured routinely in a 37°C, 5% CO2, saturated humidity incubator and passaged periodically according to the cell growth status. Cells in the logarithmic growth phase were used in the experiment.

[0066] (2) Model building method: Iron overload was induced by treating KGN cells with ferric ammonium citrate (FAC, 2.5 mM) for 48 h.

[0067] The study set up a negative control group (NC), a positive control group (FAC+MDIVI-1), an FAC model group, and a FAC+ononin group with different concentrations (dose exploration) to evaluate the effect of ononin on cell viability under iron overload.

[0068] Experimental Example 1 This experimental example demonstrates the effect of Ononin on cell viability under iron overload conditions.

[0069] (1) Detection method: The cell viability of each group of cells treated in Example 1 was detected by CCK-8 method, and the absorbance was read by microplate reader and normalized to the control group.

[0070] (2) Detection results: Under the background of FAC-induced iron overload, treatment with 20 μM Ononin significantly improved the survival level of granulosa cells, and the cell status was improved compared with the model group (e.g., Figure 6 (As shown). 20 μM was determined to be the preferred working concentration for subsequent mechanism verification.

[0071] Experimental Example 2 This experimental example demonstrates the effects of iron overload and Ononin intervention on mitophagy.

[0072] (1) Experimental method: Immunofluorescence staining: Cells were seeded in confocal culture dishes and divided into four groups: control group, FAC group, FAC+Ononin group, and FAC+Mdivi-1 group (positive control). Mitophagy Detection Kit was used for staining, Hoechst 33342 was used for nucleus staining, and images were acquired using a laser confocal microscope to quantify the area of ​​positive mitochondrial autophagy.

[0073] Western Blot: Total cellular protein was extracted, quantified, and then subjected to SDS-PAGE electrophoresis. The protein was transferred to a PVDF membrane, blocked, and incubated with primary antibodies (BNIP3, PINK1, Parkin, β-actin). After incubation with secondary antibodies, ECL was developed, and grayscale analysis was performed.

[0074] (2) Experimental results: Immunofluorescence: Mitochondrial autophagy signaling was significantly enhanced in the FAC group; ononin treatment significantly alleviated the signaling, with the inhibition level being similar to that of the positive control group using the mitochondrial division inhibitor Mdivi-1 (e.g., Figure 7 (As shown in A and 7B).

[0075] Western Blot: In the FAC group, the levels of BNIP3, PINK1, and Parkin proteins were all upregulated; Ononin effectively inhibited the upregulation of BNIP3 and simultaneously reduced the levels of PINK1 and Parkin (e.g., Figure 8 (As shown in A and 8B).

[0076] The above results demonstrate that Ononin can inhibit iron overload-induced BNIP3-PINK1 / Parkin-mediated excessive mitochondrial autophagy.

[0077] Experimental Example 3 This experimental example demonstrates the effects of iron overload and ononin intervention on mitochondrial function. (1) Experimental method: JC-1 staining to detect membrane potential: Cells were seeded in confocal dishes, grouped and treated, and then incubated with JC-1 working solution. Red / green fluorescence was collected by laser confocal microscope, and the ratio was calculated to reflect the change in membrane potential.

[0078] Reactive oxygen species (ROS) detection: CellROX™ Deep Red reagent was used for staining, Hoechst 33342 was used for nuclei staining, and fluorescence intensity was acquired by confocal microscopy.

[0079] Cellular oxygen consumption rate (OCR) assay: An Agilent Seahorse XFe96 analysis platform was used. Cells were treated with FAC and then re-coated into XF96 microplates, allowing them to recover overnight. After basal OCR was measured, oligomycin (1.5 μM), the uncoupling agent trifluoromethoxyphenylhydrazone (FCCP) (0.5 μM), rotenone, and antimycin A (1 μM) were injected sequentially. Basal respiration, ATP production, and maximal respiration were calculated and normalized to the cell count.

[0080] (2) Experimental results: JC-1: The red / green fluorescence ratio decreased in the FAC group (membrane potential decreased); the ratio partially recovered in the Ononin group (e.g., ...). Figure 9 (As shown in A and 9B).

[0081] Reactive oxygen species (ROS): Fluorescence intensity increased in the FAC group; ROS levels decreased in the Ononin group (e.g., Figure 10 (As shown in A and 10B).

[0082] Cellular oxygen consumption rate (OCR): The OCR in the FAC group showed an increasing trend; after Ononin intervention, the OCR further increased relative to the FAC group, suggesting adjustment of respiratory metabolic status (e.g., Figure 11 (As shown in A and 11B).

[0083] The above results indicate that Ononin can partially restore mitochondrial membrane potential, reduce oxidative stress, and improve mitochondrial respiratory efficiency.

[0084] Experiment Example 4 This experimental example provides the detection of the effects of iron overload and Ononin intervention on cellular senescence. (1) Experimental method: SA-β-gal staining: Cells were seeded in 6-well plates, grouped and fixed, and then incubated overnight at 37°C with staining working solution. The proportion of positive cells was observed under a microscope and counted.

[0085] (2) Experimental results: The proportion of SA-β-gal positive cells was significantly increased in the FAC group; the proportion of positive cells was significantly reduced after Ononin treatment, with an improvement similar to that in the positive control group (e.g., Figure 12 (As shown in A and 12B).

[0086] The above results indicate that Ononin can inhibit iron overload-induced cell senescence.

[0087] Based on the above systematic validation experiments at the molecular and cellular levels, the following conclusions can be drawn from this validation process: Ononin can bind directly to the BNIP3 protein, with a binding affinity of KD ≈ 7.85 × 10⁻⁶. -6 In an iron-overloaded KGN cell model, Ononin was able to: inhibit BNIP3 expression and PINK1 / Parkin pathway activation; reduce mitophagy levels; improve mitochondrial membrane potential, reduce ROS, and increase OCR; and inhibit cellular senescence phenotypes. These effects indicate that Ononin exerts a protective effect on ovarian granulosa cells by targeting BNIP3 to correct iron overload-induced excessive mitophagy and restore mitochondrial functional homeostasis.

Claims

1. Application of gentianin in the preparation of drugs for the prevention and / or treatment of ovarian dysfunction caused by excessive mitochondrial autophagy.

2. The use of the gentianin according to claim 1 in the preparation of a medicament for preventing and / or treating ovarian dysfunction caused by excessive mitophagy, characterized in that, The iron overload-induced ovarian dysfunction manifests as excessive mitochondrial autophagy.

3. The use of the gentianin according to claim 2 in the preparation of a medicament for preventing and / or treating ovarian dysfunction caused by excessive mitochondrial autophagy, characterized in that, The gentianin inhibits excessive mitochondrial autophagy by suppressing the activity of the BNIP3 protein.

4. The use of the gentianin according to claim 3 in the preparation of a medicament for preventing and / or treating ovarian dysfunction caused by excessive mitophagy, characterized in that, The gentianin directly binds to the BNIP3 protein, with a binding dissociation constant KD of 7.85 × 10⁻⁶. -6 M.

5. The use of the gentianin according to claim 1 in the preparation of a medicament for preventing and / or treating ovarian dysfunction caused by excessive mitophagy, characterized in that, The gentian glycoside is used to improve at least one of the following indicators: mitochondrial membrane potential, mitochondrial reactive oxygen species level, cellular oxygen consumption rate, and cellular senescence markers.

6. The use of the gentianin according to claim 5 in the preparation of a medicament for preventing and / or treating ovarian dysfunction caused by excessive mitophagy, characterized in that, The gentian glycosides are used to restore mitochondrial membrane potential, reduce mitochondrial ROS levels, increase cellular oxygen consumption, or reduce the rate of SA-β-gal positive cells.

7. Application of gentianin as a BNIP3 inhibitor in the preparation of drugs for the prevention and / or treatment of BNIP3-mediated mitochondrial autophagy-related diseases.

8. The application of the gentianin according to claim 7 as a BNIP3 inhibitor, characterized in that, The BNIP3-mediated excessive mitophagy-related diseases include ovarian dysfunction caused by iron overload.

9. A pharmaceutical composition for preventing and / or treating ovarian dysfunction caused by excessive mitophagy, characterized in that, It contains an effective amount of gentianin and a pharmaceutically acceptable carrier.

10. The pharmaceutical composition for preventing and / or treating ovarian dysfunction caused by excessive mitophagy according to claim 9, characterized in that, The dosage form of the pharmaceutical composition is an oral formulation or an injectable formulation.

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