Use of vitamin k2 or a pharmaceutical composition thereof in the preparation of a medicament for treating a disease of ovarian dysfunction caused by insulin resistance

Abstract

The application relates to the field of biological medicine, in particular to application of vitamin K2 or a pharmaceutical composition thereof in preparation of a medicine for treating an ovary dysfunction disease caused by insulin resistance. The application discloses that vitamin K2 can regulate FOXO1 / PDK4 (sugar metabolism), AMPK / PGC-1 alpha / TFAM (mitochondrial generation), Nrf2 (antioxidation) and NF-kappa B (anti-inflammation) signal paths by down-regulating / inhibiting over-activated SIRT1 expression, so as to restore mitochondrial function and cell homeostasis. The correlation between serum VK2 level and reproductive indexes (abortion times, AMH) of IR patients is established for the first time, and a new use of VK2 in treating IR ovary dysfunction is provided.

Description

Technical Field

[0001] This application relates to the field of biomedicine, and in particular to the use of vitamin K2 or pharmaceutical compositions thereof in the preparation of medicaments for treating ovarian dysfunction caused by insulin resistance. Background Technology

[0002] Insulin resistance (IR) is a common metabolic feature in patients with polycystic ovary syndrome (PCOS), obesity, and type 2 diabetes. Clinical studies have shown that IR is closely related to decreased female reproductive function, poor egg quality, and increased miscarriage rate. Current treatments mainly focus on improving systemic insulin sensitivity (such as metformin) or lifestyle interventions.

[0003] Disadvantages / deficiencies of existing technology: 1) Existing insulin sensitizers mainly act on the liver and muscle tissue. Their direct protective mechanism on ovarian granulosa cells (GCs) is still unclear, and they have side effects such as gastrointestinal reactions.

[0004] 2) Currently, there is a lack of targeted therapies specifically for mitochondrial dysfunction in ovarian granulosa cells under IR conditions.

[0005] 3) Regarding vitamin K2 (VK2), current technologies mainly focus on its role in osteoporosis and cardiovascular calcification. Although some studies have mentioned VK2 and diabetes, its specific role and molecular mechanisms (especially involving the regulation of the SIRT1 pathway) in IR-induced ovarian reproductive dysfunction have not yet been reported. Summary of the Invention

[0006] To address the aforementioned technical issues, a novel use and pharmaceutical composition of a drug are provided that can effectively improve mitochondrial function of ovarian granulosa cells under insulin resistance conditions, reduce oxidative stress, and restore reproductive function.

[0007] This application provides the use of vitamin K2 or pharmaceutical compositions thereof in the preparation of a medicament for treating ovarian dysfunction caused by insulin resistance.

[0008] This application also provides a pharmaceutical composition comprising vitamin K2 and a pharmaceutically acceptable carrier or excipient.

[0009] The beneficial effects of this application include, but are not limited to: 1) Significantly improve reproductive outcomes: Clinical data confirm that low VK2 levels are associated with high miscarriage rates and low AMH levels; VK2 supplementation can reverse this pathological condition.

[0010] 2) Multi-target repair: VK2 not only improves insulin sensitivity, but more importantly, it can directly repair the mitochondrial ultrastructure of ovarian granulosa cells (reducing swelling and vacuolation) and restore mitochondrial membrane potential (ΔΨm).

[0011] 3) Dual antioxidant and anti-inflammatory effects: significantly reduces MDA and ROS levels while inhibiting NF-κB-mediated inflammatory responses.

[0012] 4) Unique synergistic mechanism: The combined use of SIRT1 inhibitor (EX-527) can further enhance the antioxidant and mitochondrial protective effects of VK2, demonstrating the central role of SIRT1 overactivation in the pathological process and providing a basis for the development of compound drugs. Attached Figure Description

[0013] This application will be further described by way of exemplary embodiments, which will be described in detail with reference to the accompanying drawings. These embodiments are not limiting, wherein: Figure 1 The mouse's body weight (A) and fasting blood glucose (FBG) are shown (B).

[0014] Figure 2 The mouse OGTT (A) and the area under the OGTT curve (AUC of OGTT) (B) are shown.

[0015] Figure 3 The levels of VK2 (A) and MDA (B) in mouse serum are shown.

[0016] Figure 4 This shows the ROS level in mouse ovarian granulosa cells (GCs).

[0017] Figure 5 The ultrastructure of mitochondria in mouse ovarian granulosa cells is shown.

[0018] Figure 6 This shows the mitochondrial membrane potential (ΔΨm) in mouse GCs.

[0019] Figure 7 The mRNA expression of SIRT1, FOXO1, and PDK4 is shown.

[0020] Figure 8 The protein expression of SIRT1, FOXO1, and PDK4 is shown.

[0021] Figure 9 The mRNA expression of AMPK, PGC-1α, and TFAM is displayed.

[0022] Figure 10 The protein expression of AMPK, PGC-1α, and TFAM is displayed.

[0023] Figure 11 The mRNA expression of Nrf2, ND6, and UCP3 is shown.

[0024] Figure 12 The protein expression of Nrf2, ND6, and UCP3 is shown.

[0025] Figure 13 This shows the mRNA and protein expression of NF-κB.

[0026] Figure 14 The plasma VK2 levels were displayed in the IR group and the control group.

[0027] Figure 15 The study showed a correlation between plasma VK2 levels and the number of previous miscarriages.

[0028] Figure 16 This study demonstrates the correlation between plasma VK2 and OGTT blood glucose levels.

[0029] Figure 17 This demonstrates the correlation between plasma VK2 and insulin levels in insulin release assays.

[0030] Figure 18 The correlation between plasma VK2 and the percentage of peripheral blood NK cells was shown.

[0031] Figure 19 This study demonstrates the correlation between plasma VK2 and serum AMH levels. Detailed Implementation

[0032] To more clearly illustrate the technical solutions of the embodiments in this specification, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are merely some examples or embodiments of this specification. For those skilled in the art, these drawings can be applied to other similar scenarios without creative effort. Unless obvious from the context or otherwise specified, the same reference numerals in the drawings represent the same structures or operations.

[0033] As indicated in this specification and claims, unless the context clearly indicates otherwise, the words "a," "an," "an," and / or "the" do not specifically refer to the singular and may also include the plural. Generally speaking, the terms "comprising" and "including" only indicate the inclusion of expressly identified steps and elements, which do not constitute an exclusive list, and the method or apparatus may also include other steps or elements.

[0034] Flowcharts are used in this specification to illustrate the operations performed by the system according to embodiments of this specification. It should be understood that the preceding or following operations are not necessarily performed in exact order. Instead, the steps can be processed in reverse order or simultaneously. Furthermore, other operations can be added to these processes, or one or more steps can be removed from them.

[0035] This application provides the use of vitamin K2 or pharmaceutical compositions thereof in the preparation of a medicament for treating ovarian dysfunction caused by insulin resistance.

[0036] In some embodiments, the vitamin K2 may be different subtypes such as MK-4 and MK-7. In some embodiments, the vitamin K2 or a pharmaceutical composition thereof can improve the oxidative stress state of ovarian granulosa cells in individuals with insulin resistance; In some embodiments, the vitamin K2 or its pharmaceutical composition can reduce serum malondialdehyde and reactive oxygen species levels in granulocytes, while inhibiting NF-κB-mediated inflammatory responses.

[0037] In some embodiments, the vitamin K2 or its pharmaceutical composition can improve insulin sensitivity, repair mitochondrial ultrastructure and membrane potential, and reduce mitochondrial ultrastructural swelling or vacuolation in ovarian granulosa cells.

[0038] In some embodiments, the vitamin K2 or its pharmaceutical composition can modulate the multidimensional signaling pathway downstream of SIRT1 in ovarian granulosa cells, including glucose metabolism, mitochondrial generation, oxidative stress, and inflammatory pathways.

[0039] In some embodiments, the vitamin K2 or its pharmaceutical composition thereof can inhibit the overexpression of SIRT1 in ovarian granulosa cells and downregulate the expression of FOXO1 and PDK4.

[0040] In some embodiments, the vitamin K2 or its pharmaceutical composition can upregulate AMPK activity in ovarian granulosa cells and restore PGC-1α and TFAM levels.

[0041] In some embodiments, the vitamin K2 or its pharmaceutical composition can enhance the Nrf2 signaling pathway in ovarian granulosa cells and upregulate the expression of ND6 and UCP3 to reduce oxidative damage.

[0042] In some embodiments, the vitamin K2 or its pharmaceutical composition may improve ovarian granulosa cell function by inhibiting excessive activation of SIRT1 under insulin resistance conditions, restoring downstream molecular homeostasis.

[0043] In some embodiments, the pharmaceutical composition may further comprise a SIRT1-specific inhibitor. Preferably, in some embodiments, the SIRT1-specific inhibitor may be EX-527.

[0044] This application also provides a pharmaceutical composition comprising vitamin K2 and a pharmaceutically acceptable carrier or excipient.

[0045] The term “pharmaceutically acceptable” as used in this article generally means a compound, material, composition, and / or dosage form that is suitable, within reasonable medical judgment, for contact with human and animal tissues, organs, and / or body fluids without excessive toxicity, irritation, allergic response, or other problems or complications, in proportion to a reasonable benefit / risk ratio.

[0046] The excipients include various excipients and diluents, which are not essential active ingredients and do not cause adverse reactions after application. The excipients contain sterile water or physiological saline, stabilizers, excipients, antioxidants (ascorbic acid, etc.), buffers (phosphate, citric acid, other organic acids, etc.), preservatives, surfactants (PEG, Tween, etc.), chelating agents (EDTA, etc.), or binders. The excipients also contain other low molecular weight peptides, serum albumin, glycine, glutamine, asparagine, arginine, polysaccharides, monosaccharides, mannitol, or sorbitol. The aqueous solution of the excipients for injection is selected from physiological saline, glucose isotonic solution, D-sorbitol isotonic solution, D-mannose isotonic solution, D-mannitol or sugar alcohol isotonic solution. The aqueous solution for injection contains a solubilizer. The solubilizer is selected from alcohols (ethanol), polyols (propylene glycol or PEG), and / or nonionic surfactants (Tween 80 or HCO-50).

[0047] In the pharmaceutical composition provided in this application, vitamin K2 can be a single active ingredient, or it can be combined with one or more other active ingredients that are useful for the treatment of diseases to form a combined formulation.

[0048] The content of the active ingredient in the pharmaceutical composition is a safe and effective amount, which is understood by those skilled in the art. In general, it should be adjustable; for example, the dosage of the active ingredient in the pharmaceutical composition depends on the patient's weight, the type of application, the condition and severity of the disease.

[0049] In some embodiments, the pharmaceutical composition may further comprise a SIRT1-specific inhibitor. Preferably, in some embodiments, the SIRT1-specific inhibitor may be EX-527.

[0050] A method for preventing and / or treating ovarian dysfunction caused by insulin resistance, the method comprising administering to an individual with ovarian dysfunction caused by insulin resistance a therapeutically effective amount of vitamin K2 or a pharmaceutical composition thereof, as described above.

[0051] The term "effective amount" refers to the amount or dose of a formulation of vitamin K2 or a pharmaceutical composition thereof in this application that, when administered to a patient in a single or multiple doses, produces the intended effect in the treated patient. An effective amount can be determined by a physician skilled in the art by considering a variety of factors, such as: the species of the mammal; its size, age, and general health; the specific disease involved; the degree or severity of the disease; the individual patient's response; the specific formulation administered; the mode of administration; the bioavailability characteristics of the administered formulation; the chosen dosing regimen; and the use of any concomitant therapies.

[0052] Technical solution (core) 1) Core Application: The use of vitamin K2 in the preparation of drugs for the treatment or prevention of ovarian dysfunction, ovarian granulosa cell damage, or improvement of oocyte quality caused by insulin resistance (IR).

[0053] 2) Mechanism of action: This invention reveals that VK2 restores mitochondrial function and cellular homeostasis by downregulating / inhibiting the expression of overactivated SIRT1, thereby regulating the FOXO1 / PDK4 (glucose metabolism), AMPK / PGC-1α / TFAM (mitochondrial generation), Nrf2 (antioxidant) and NF-κB (anti-inflammatory) signaling pathways.

[0054] 3) Pharmaceutical composition: A pharmaceutical composition having an active ingredient comprising: (a) vitamin K2; and (b) a SIRT1 inhibitor (preferably EX-527).

[0055] Data shows that the combined use of VK2 and EX-527 has a significant synergistic effect in restoring mitochondrial membrane potential and reducing ROS and MDA levels.

[0056] Innovation of this invention 1) New application: The correlation between serum VK2 levels and reproductive indicators (number of miscarriages, AMH) in IR patients was established for the first time, and a new application of VK2 in the treatment of IR-related ovarian dysfunction was proposed.

[0057] 2) Novel Mechanism (Reverse Cognition): Existing technologies often aim to activate SIRT1, but this invention has found that SIRT1 is pathologically overexpressed in IR ovarian granulosa cells. The mechanism of action of VK2 is to inhibit the overactivation of SIRT1, which has unexpected technical effects.

[0058] 3) Synergistic effect: The superiority of combining VK2 with SIRT1 inhibitors was confirmed.

[0059] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were all purchased from conventional biochemical reagent companies. All quantitative experiments in the following examples were performed in triplicate, and the results were averaged.

[0060] Example 1: The effect of vitamin K2 (VK2) on reversing the metabolic phenotype of insulin resistance (IR) mice induced by a high-fat diet. This embodiment verifies the efficacy of VK2 by constructing a C57BL / 6J mouse IR model.

[0061] Experimental Materials and Grouping Animals: C57BL / 6J female mice, 8-12 weeks old, weighing 20-25g, purchased from Shanghai JessJ Experimental Animal Co., Ltd.

[0062] Reagents: VK2 (SM9450, Beijing Solarbio Science & Technology Co., Ltd.); SIRT1 specific inhibitor EX-527 (MB3761-2, Dalian Meilun Biotechnology Co., Ltd.); high-fat diet (60% fat for energy, 0296044901-i, MP Biomedicals).

[0063] Grouping and Modeling: After 1 week of adaptive feeding, the participants were randomly divided into 4 groups (n=5 / group): (1) Control group: fed with normal maintenance diet for 16 weeks; (2) Model group (HFD / IR): fed with high-fat diet for 16 weeks to induce IR model; (3) Vitamin K2 group (VK2): fed with high-fat diet for 16 weeks, and VK2 (60 mg / kg / day) was administered by gavage from week 9 (i.e. the last 8 weeks); (4) Combined intervention group (VK2+EX-527): in addition to the VK2 group, EX-527 (5 mg / kg / day) was injected intraperitoneally at the same time.

[0064] Drug preparation and administration: Since VK2 is lipid-soluble and poorly soluble in water, we use a "DMSO-Tween 80-physiological saline" co-solvent system for preparation to ensure safety and absorption efficiency.

[0065] VK2 Preparation and Administration: Due to the lipid solubility of VK2, a DMSO-Tween 80-physiological saline co-solvent system was used for preparation. First, VK2 powder was dissolved in dimethyl sulfoxide (DMSO) to prepare a high-concentration stock solution. Just before use, an appropriate amount of the stock solution was added to Tween 80 to aid dissolution, followed by slow addition of warm sterile physiological saline and vigorous vortexing to prepare a homogeneous emulsion. The final gavage working solution had a volume ratio of DMSO : Tween 80 : physiological saline = 5 : 5 : 90. During the last 8 weeks of the experiment, mice in the IR + VK2 group and the IR + VK2 + EX-527 group were administered the drug via gavage (ig) daily at a dose of 60 mg / kg body weight.

[0066] EX-527 Preparation and Administration: The preparation method is the same as above. EX-527 is dissolved in DMSO, and then diluted with sterile physiological saline (or physiological saline containing a small amount of Tween 80) to control the final concentration of DMSO in the injection solution to < 10% and without any visible precipitation. Mice in the IR + VK2 + EX-527 group were administered intraperitoneal injection (ip) daily for the last 8 weeks of the experiment at a dose of 5 mg / kg body weight.

[0067] Solvent control treatment: To eliminate the interference of the solvent system on the experimental results, the control group was given an equal volume of solvent control solution (physiological saline containing 5% DMSO and 5% Tween 80) via the corresponding administration route (gavage or intraperitoneal injection).

[0068] Metabolic index testing Blood glucose and insulin monitoring: Weight and fasting blood glucose (FBG, Roche blood glucose meter) were monitored weekly during the experiment. At the end of the experiment, serum was collected, and insulin levels were measured using an ELISA kit (Sangon Biotech (Shanghai) Co., Ltd.).

[0069] Oral glucose tolerance test (OGTT): In week 16 of the experiment, mice were fasted for 12 hours and then injected intraperitoneally with glucose (2 g / kg). Blood was collected from the tail vein at 0, 30, 60, 90 and 120 min and blood glucose levels were measured using a glucometer.

[0070] Insulin tolerance test (ITT): After fasting for 4 hours, insulin (0.75 U / kg) is injected intraperitoneally, and blood glucose is measured at the above time points.

[0071] Experimental results Weight and fasting blood glucose: such as Figure 1As shown, the body weight of mice in the model group reached 43.74 ± 1.54 g at week 16, which was significantly higher than that of the control group (30.96 ± 0.68 g). p <0.001). After VK2 intervention, weight gain was suppressed, and the final weight decreased to 41.46 ± 1.71g. The fasting blood glucose (FBG) in the model group was 10.80 ± 0.84 mmol / L, while that in the VK2 group it significantly decreased to 8.80 ± 0.84 mmol / L.

[0072] Glucose tolerance curve (AUC): as follows Figure 2 As shown, the area under the OGTT curve (AUC) of the model group increased significantly ( p <0.001. The AUC of the VK2 group was approximately 10% lower than that of the model group ( p <0.001 indicates that glucose clearance capacity has partially recovered.

[0073] Synergistic effect: such as Figure 1 Figure 2 As shown, the levels of FBG and AUC in the combined intervention group (VK2+EX-527) were further reduced, suggesting that inhibiting SIRT1 did not counteract the effect of VK2, but instead showed a synergistic effect.

[0074] Example 2: VK2 improves oxidative stress in ovarian granulosa cells (GCs) of IR mice This embodiment analyzes the microenvironment of the target tissue (ovary) and target cells (granulosa cells).

[0075] Sample Collection and Processing: Bilateral ovaries were rapidly removed from mice after anesthesia and euthanasia. Five units of pregnant mare serum gonadotropin (PMSG; M2620, Shanghai Aibei Biotechnology Co., Ltd.) were injected intraperitoneally to stimulate follicular development. Forty-eight hours later, the ovaries were removed and placed in pre-warmed follicle collection medium (MEM / PVP + M) containing 2.5 μM milrinone (SM9050, Beijing Solarbio Science & Technology Co., Ltd.) to prevent meiotic recovery. Large antral follicles were then punctured under a microscope to release cumulus-oocyte complexes (COCs). Cumulus cells were gently removed with a pipette. After cumulus cell removal, the naked oocytes were cultured at 37°C in a 5% CO2 environment for at least 1 hour before use. GCs were isolated by mechanically dispersing the ovaries in DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. Follicles were punctured using a 26G needle, and the released cells were collected over 5 minutes by centrifugation at 1000×g. The purity of the granulosa cells was assessed under an optical microscope.

[0076] Biochemical indicator testing Serum VK2 detection: Serum VK2 levels in mice were quantitatively measured using an enzyme-linked immunosorbent assay (ELISA) kit (JLC3850, Shanghai Jingkang Biotechnology Co., Ltd.).

[0077] Reactive oxygen species (ROS) detection: ROS levels in GCs were detected using the fluorescent probe 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA, S0033M, Beyotime Biotechnology Co., Ltd., Shanghai, China). Cell suspension and probe (10 μM) were incubated at 37°C in the dark for 30 min. After washing three times with PBS, fluorescence intensity was detected using a fluorescence microplate reader (Ex = 488nm, Em = 525nm).

[0078] Malondialdehyde (MDA) detection: MDA in mouse serum was measured using the Thiobarbituric Acid Reactive Substance (TBARS) assay kit (G4300, Cyagen (Guangzhou) Biotechnology Co., Ltd.) as a marker of lipid peroxidation.

[0079] Experimental results Serum VK2 levels: such as Figure 3 As shown in Figure A, compared with the control group, the serum VK2 level in the model group was significantly lower ( p <0.001), serum VK2 levels in mice were significantly increased after VK2 supplementation in the VK2 group ( p <0.001).

[0080] MDA level: such as Figure 3 As shown in B, the MDA level in granulocytes of the model group was significantly increased ( p <0.001. MDA levels were significantly downregulated in the VK2 group ( p <0.001).

[0081] ROS level: such as Figure 4 As shown, the ROS fluorescence intensity of granulosa cells in the model group was significantly increased. The ROS level in the VK2 group was significantly downregulated.

[0082] Effects of SIRT1 inhibitors: such as Figure 3 B and Figure 4 As shown, in the combined group (VK2+EX-527), the serum MDA level in mice was further reduced ( Figure 3 B, p <0.01), the ROS removal rate in GCs was further improved compared to the VK2 group alone. Figure 4 This confirms that inhibition of the SIRT1 pathway is beneficial for the reconstruction of the antioxidant environment.

[0083] Example 3: VK2 repairs mitochondrial ultrastructure and membrane potential Transmission electron microscopy (TEM) was used to observe the ultrastructure of granulosa cells in fresh ovarian tissue, which was cut into 1 mm³ pieces and fixed overnight in 2.5% glutaraldehyde fixative (4°C). After washing with PBS, the tissue was fixed with 1% osmium tetroxide at room temperature for 2 h, followed by dehydration with a gradient of ethanol and acetone, and then embedded in epoxy resin. After polymerization at 60°C for 48 h, 60-80 nm sections were cut using an ultramicrotome. The sections were double-stained with uranium acetate and lead citrate, and then the ultrastructure of granulosa cells was observed and photographed under a transmission electron microscope (Hitachi H-7650, Japan).

[0084] Result: As Figure 5 As shown, the mitochondria in the control group had normal morphology, mostly appearing as typical elliptical or elongated shapes. The double membrane structure was intact, the internal mitochondrial cristae were tightly arranged, the matrix electron density was normal (darker in color), and no obvious swelling or vacuolation was observed.

[0085] The model group showed significant ultrastructural damage to mitochondria, manifested as obvious swelling and rounding, increased volume, decreased matrix electron density, cristae breakage, sparseness, or vacuolation.

[0086] The VK2 group significantly improved IR-induced damage, with mitochondria regaining their elongated or oval shape, intact mitochondrial membranes, reappearing internal cristae structures, and some recovery of matrix electron density.

[0087] The ultrastructure of mitochondria in the combined group was further optimized, with restored morphology, showing healthy elongated shapes, high matrix electron density, and dense and regular arrangement of internal mitochondrial cristae, with almost no vacuolation. This indicates that EX-527 and VK2 exhibit a synergistic protective effect.

[0088] Mitochondrial membrane potential (ΔΨm) was assessed using the JC-1 probe kit (C2006, Beyotime Biotechnology Co., Ltd., Shanghai, China). Cells from each experimental group were incubated with JC-1 working solution at 37°C in the dark for 20 minutes. After incubation, cells were gently washed twice with pre-chilled JC-1 staining buffer to remove excess dye. Subsequently, 2 mL of fresh culture medium was added to each well, and fluorescence signals were observed and recorded using an inverted fluorescence microscope.

[0089] Result: As Figure 5 As shown in Figures A and 5B, under a fluorescence microscope, compared to the control group, the red / green fluorescence ratio in the model group significantly decreased from 1.72±0.08 to 0.20±0.01. p <0.001 indicates membrane potential collapse. The VK2 group ratio recovered to 0.47±0.02 ( p <0.001). The combined group ratio further improved to 0.71±0.04 ( pThe differences were all statistically significant (<0.01).

[0090] Example 4: Mechanism Verification – VK2 Modulates the Multidimensional Signaling Pathway Downstream of SIRT1 This embodiment uses molecular biology techniques (qPCR and Western Blot) to confirm the core mechanism of the invention: VK2 exerts its effects by "inhibiting" rather than "activating" SIRT1, and by regulating downstream pathways of SIRT1 such as glucose metabolism, mitochondrial generation, oxidative stress, and inflammation.

[0091] 1. Western Blotting: GCs were lysed in RIPA lysis buffer (Cyagen (Guangzhou) Biotechnology Co., Ltd.) containing protease and phosphatase inhibitors. Protein concentration was determined using a BCA protein assay kit (Beyotime Biotechnology Co., Ltd., Shanghai). 30 μg of protein was loaded onto a PVDF membrane, separated by SDS-PAGE, and transferred to the membrane. The blot was blocked with 5% skim milk at room temperature for 1 hour, then incubated overnight at 4°C with primary antibodies against SIRT1, NF-κB, FOXO1, PDK4, AMPK, Nrf2, PGC-1α, TFAM, UCP3, GAPDH (1:1000 dilution, Proteintech), and ND6 (1:1000 dilution, Abcam). The following day, the blot membrane was incubated with a horseradish peroxidase (HRP) conjugated secondary antibody (dilution ratio 1:8000, Shanghai Beyotime Biotechnology Co., Ltd.) at room temperature for 1 hour. Finally, the protein bands were developed using Super ECL chemiluminescence detection reagent (Shanghai Yisheng Biotechnology Co., Ltd.).

[0092] 2. RNA extraction and qPCR detection Total RNA was extracted from GCs and reverse transcribed into cDNA. qPCR analysis was performed using a SYBR Green PCR mix on a real-time PCR instrument. Target gene ( Sirt1, NF-κB, FOXO1, PDK4, AMPK, Nrf2, PGC-1α, TFAM, UCP3, ND6 The primer sequences are listed in Table 1.

[0093] Table 1 Experimental results SIRT1 and glucose metabolism pathway (FOXO1 / PDK4): such as Figure 7 and Figure 8 As shown, SIRT1 expression (mRNA and protein) was significantly upregulated in the IR group (overactivation under IR pathology). Figure 7 A, 8A, p<0.001), accompanied by FOXO1 ( Figure 7 B, 8B, p <0.001) and PDK4 ( Figure 7 C, 8C, p Increased expression of <0.001 indicates impaired glucose oxidation. VK2 treatment significantly inhibited SIRT1 overexpression and downregulated FOXO1 and PDK4 ( p <0.001). Combined with EX-527, this inhibitory effect was further enhanced ( p <0.001).

[0094] Mitochondrial biosynthetic pathway (AMPK / PGC-1α / TFAM): IR leads to inhibition of AMPK phosphorylation ( Figure 9 A, 10A, p <0.001), PGC-1α ( Figure 9 B, 10B, p <0.001) and TFAM ( Figure 9 C, 10C, p <0.001) expression decreased. VK2 supplementation upregulated AMPK activity and restored PGC-1α and TFAM levels, and the combined effect with EX-527 was more significant. p <0.05).

[0095] Antioxidant pathways (Nrf2 / ND6 / UCP3): IR group Nrf2 ( Figure 11 A, 12A, p <0.001) and its downstream target ND6 ( Figure 11 B, 12B, p <0.001), UCP3 ( Figure 11 C, 12C, p <0.001) expression was suppressed. VK2 enhanced the Nrf2 signaling pathway, upregulating ND6 and UCP3 expression to alleviate oxidative damage. EX-527 further enhanced this protective effect. p <0.001).

[0096] Inflammatory pathways (NF-κB): such as Figure 13 IR significantly increased NF-κB. VK2 treatment significantly inhibited NF-κB expression, and this inhibitory effect was more pronounced when combined with EX-527.

[0097] Conclusion: VK2 restores downstream molecular homeostasis and improves ovarian granulosa cell function by inhibiting excessive activation of SIRT1 under IR conditions.

[0098] Example 5: Clinical Correlation Analysis – Correlation between Serum VK2 Levels and Metabolic Indicators in IR Patients 1. Study participants were women of childbearing age who visited the Department of Reproductive Immunology at Shanghai First Maternity and Infant Hospital between May and October 2024. Participants meeting the following two criteria were defined as IR patients: ① Homeostasis Model of Insulin (HOMA-IR) > 2.5; ② Clinically diagnosed IR based on oral glucose tolerance test (OGTT) and insulin release test (IRT) (fasting insulin level exceeding 10 mmol / L, postprandial insulin level reaching 5 to 10 times the fasting level at 0.5 or 1 hour, or delayed insulin peak at 2 to 3 hours without returning to fasting levels). Exclusion criteria for IR patients included: use of hormonal drugs and drugs affecting insulin within the past three months, and individuals with incomplete information. A total of 42 IR patients were enrolled, and 40 healthy women who underwent physical examinations during the same period served as controls.

[0099] 2. Detection Methods Plasma samples were collected from 82 enrolled participants to assess various biomarkers. 5 mL of fasting venous blood was collected and centrifuged to separate the plasma. Levels of VK2, cholesterol (CHO), triglycerides (TG), anti-Müllerian hormone (AMH), blood glucose, and insulin were measured at multiple time points (fasting 0 h, 0.5 h, 1 h, 2 h, 3 h) during the oral glucose tolerance test (OGTT) and insulin release test (IRT), following the manufacturer's instructions. The relative counts of T, B, NK, and regulatory T lymphocytes (Treg), as well as the cytokine levels of interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-10 (IL-10), interleukin-17A (IL-17A), tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ) were determined by flow cytometry (BD Biosciences). The IR index (HOMA-IR) was calculated for each participant (HOMA-IR: fasting serum insulin (Ins 0 h) mU / ml × fasting blood glucose (Glu 0 h) mmol / L / 22.5).

[0100] 3. Statistical analysis data were analyzed using SPSS 26.0 software. Independent samples t-tests were used for comparisons between groups, and Spearman correlation analysis was used for correlations.

[0101] 4. Results Differences in serum VK2 levels: such as Figure 14 The serum VK2 concentration in the IR group was 3.93±2.30 nmol / L, significantly lower than that in the healthy control group (6.28±2.96 nmol / L). p<0.001).

[0102] Correlation: such as Figure 15 Correlation analysis showed that plasma VK2 levels were negatively correlated with the number of previous miscarriages (r=-0.262, p =0.017); like Figure 16 Plasma VK2 and OGTT at 0.5 h (r=-0.420, p =0.002), 1 h (r=-0.345, p =0.011) and 2 h (r=-0.427, p The blood glucose level was negatively correlated with the value of 0.001. like Figure 17 Plasma VK2 was negatively correlated with insulin levels 2 hours after the insulin release test (r=-0.268). p =0.040); like Figure 18 Plasma VK2 was negatively correlated with the percentage of NK cells in peripheral blood (r=-0.240, p =0.030).

[0103] On the contrary, such as Figure 19 Plasma VK2 levels were positively correlated with serum AMH levels (r=0.361, p =0.026).

[0104] Conclusion: Human clinical data show that VK2 deficiency is positively correlated with the degree of IR and decreased ovarian reserve function, supporting the clinical applicability of the animal experimental conclusions in Examples 1-4.

[0105] The basic concepts have been described above. Obviously, for those skilled in the art, the detailed disclosure above is merely illustrative and does not constitute a limitation of this specification. Although not explicitly stated herein, those skilled in the art may make various modifications, improvements, and corrections to this specification. Such modifications, improvements, and corrections are suggested in this specification and therefore remain within the spirit and scope of the exemplary embodiments described herein.

[0106] Furthermore, this specification uses specific terms to describe embodiments thereof. For example, "an embodiment," "one embodiment," and / or "some embodiments" refer to a particular feature, structure, or characteristic associated with at least one embodiment of this specification. Therefore, it should be emphasized and noted that references to "an embodiment," "one embodiment," or "an alternative embodiment" in different locations throughout this specification do not necessarily refer to the same embodiment. Moreover, certain features, structures, or characteristics in one or more embodiments of this specification can be appropriately combined.

[0107] In some embodiments, numbers describing the quantity of components and attributes are used. It should be understood that such numbers used in the description of embodiments are modified in some examples with the terms "approximately," "approximately," or "generally." Unless otherwise stated, "approximately," "approximately," or "generally" indicates that the numbers are allowed to vary by ±20%. Accordingly, in some embodiments, the numerical parameters used in the specification and claims are approximate values, which may be changed depending on the characteristics required by individual embodiments. In some embodiments, numerical parameters should take into account specified significant digits and employ a general method of digit reservation. Although the numerical ranges and parameters used to confirm their breadth of range in some embodiments of this specification are approximate values, in specific embodiments, such values ​​are set as precisely as feasible.

[0108] Finally, it should be understood that the embodiments described in this specification are merely illustrative of the principles of the embodiments described herein. Other variations may also fall within the scope of this specification. Therefore, alternative configurations of the embodiments described herein are intended to be illustrative rather than limiting, and should be considered consistent with the teachings of this specification. Accordingly, the embodiments described herein are not limited to those explicitly introduced and described herein.

Claims

1. The use of vitamin K2 or pharmaceutical compositions thereof in the preparation of drugs for treating ovarian dysfunction caused by insulin resistance.

2. The application as described in claim 1, characterized in that, The vitamin K2 or its pharmaceutical composition improves the oxidative stress state of ovarian granulosa cells in individuals with insulin resistance; And / or, the vitamin K2 or its pharmaceutical composition thereof reduces serum malondialdehyde and reactive oxygen species levels in granulocytes, while inhibiting NF-κB-mediated inflammatory responses.

3. The application as described in claim 1, characterized in that, The vitamin K2 or its pharmaceutical composition improves insulin sensitivity, repairs mitochondrial ultrastructure and membrane potential, and reduces mitochondrial ultrastructural swelling or vacuolation in ovarian granulosa cells.

4. The application as described in claim 1, characterized in that, The vitamin K2 or its pharmaceutical composition regulates the multidimensional signaling pathway downstream of SIRT1 in ovarian granulosa cells, including glucose metabolism, mitochondrial generation, oxidative stress, and inflammation pathways.

5. The application as described in claim 1, characterized in that, The vitamin K2 or its pharmaceutical composition thereof inhibits the overexpression of SIRT1 in ovarian granulosa cells and downregulates the expression of FOXO1 and PDK4; And / or, the vitamin K2 or its pharmaceutical composition upregulates AMPK activity in ovarian granulosa cells and restores PGC-1α and TFAM levels.

6. The application as described in claim 1, characterized in that, The vitamin K2 or its pharmaceutical composition enhances the Nrf2 signaling pathway in ovarian granulosa cells and upregulates the expression of ND6 and UCP3 to reduce oxidative damage.

7. The application as described in claim 1, characterized in that, The vitamin K2 or its pharmaceutical composition improves ovarian granulosa cell function by inhibiting excessive activation of SIRT1 under insulin resistance conditions, restoring downstream molecular homeostasis.

8. The application as described in claim 1, characterized in that, The pharmaceutical composition further includes a SIRT1-specific inhibitor, preferably EX-527.

9. A pharmaceutical composition comprising vitamin K2 and a pharmaceutically acceptable carrier or excipient.

10. The pharmaceutical composition according to claim 9, characterized in that, The pharmaceutical composition further includes a SIRT1-specific inhibitor, preferably EX-527.

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