Application of SIRT5 inhibitor MC3482 in preparation of medicine for treating premature ovarian failure

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of an SIRT5 inhibitor MC3482 in preparation of a medicine for treating premature ovarian failure. According to the invention, a mouse POI model is constructed through the induction of cyclophosphamide (CTX), and the MC3482 is proved to be capable of inhibiting the activity of SIRT5, reducing excessive macrophage infiltration of ovary caused by CTX, reducing M1 type macrophage activation and proinflammatory factor release, further inhibiting the apoptosis of ovarian granular cells, protecting follicular reserve and recovering ovarian functions. The invention clarifies the action effect and application scheme of MC3482 for preventing and treating POI, provides a new treatment strategy for chemotherapy-induced iatrogenic POI, and has important clinical transformation value.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of SIRT5 inhibitor MC3482 in the preparation of drugs for treating premature ovarian failure. Background Technology

[0002] Premature ovarian failure (POF) Premature ovarian insufficiency Ovarian insufficiency (POI) refers to the decline in ovarian function in women under the age of 40, manifested as infrequent or absent menstruation, serum gonadotropin (FSH) levels >25 IU / L, and decreased estrogen levels, accompanied by loss of fertility and endocrine disorders. The global incidence rate has reached 1%-3% and is showing an increasing trend annually. The causes of POI are complex, including genetic factors such as FMR1 gene mutations, autoimmune diseases such as autoimmune thyroiditis, and iatrogenic injury. Among these, ovarian damage induced by chemotherapy drugs such as cyclophosphamide is a major cause of POI after treatment in young cancer patients, severely impacting their quality of life and fertility needs.

[0003] Existing research indicates that chronic inflammation is one of the core pathological mechanisms of POI (Potential Ovarian Inflammation). As a key organ for immuno-endocrine regulation, the ovary's homeostasis depends on the synergistic effects of oocytes, granulosa cells, and immune cells. Among these, macrophages are the most prevalent immune cell population in the ovary, accounting for 30%-50% of the total number of ovarian immune cells. Under physiological conditions, ovarian macrophages support ovarian function by regulating angiogenesis, steroid hormone synthesis, and tissue remodeling. However, current clinical treatment regimens have significant limitations: First, their therapeutic effects lack specificity, acting indiscriminately on all immune cells or macrophage subtypes. While suppressing pro-inflammatory responses, they inevitably disrupt the physiological functions necessary for follicle development, such as macrophage-mediated tissue repair and hormonal regulation, leading to incomplete recovery of ovarian function after treatment. Second, while cell clearance is achieved by destroying macrophage lysosomes, it indiscriminately clears both M1 (pro-inflammatory) and M2 (repair) macrophages, resulting in the loss of physiological functions required for ovarian follicle development. Furthermore, the regeneration of monocytes after depletion may exacerbate inflammatory rebound. Third, traditional hormone replacement therapy carries the risk of increasing thrombosis, while immunosuppressants are prone to causing infection. Both types of drugs have broad targets and non-specific interference with normal physiological processes, making it difficult to meet the long-term treatment needs of POI patients (especially women of childbearing age).

[0004] Further research has shown that under the stimulation of chemotherapeutic drugs such as cyclophosphamide (CTX), peripheral blood monocytes are recruited in large numbers to the ovary and differentiate into M1 macrophages with a pro-inflammatory phenotype. The excessive secretion of pro-inflammatory factors such as TNF-α and IL-1β by these macrophages can activate the apoptosis signaling pathway in granulosa cells, leading to granulosa cell apoptosis, follicle depletion, and ovarian failure. Therefore, regulating monocyte-macrophage differentiation and polarization has become a key target for the prevention and treatment of POI.

[0005] SIRT5 is a member of the Sirtuin family and is a mitochondrial-localized NAD5. + SIRT5, a type of desuccinylation enzyme, regulates protein function by removing succinyl groups from lysine residues of target proteins. Traditional research suggests that SIRT5 primarily participates in energy metabolism, such as ketone body formation and fatty acid β-oxidation. However, recent studies have revealed its important role in immune inflammatory responses—SIRT5 can regulate immune cell function and influence the inflammatory process through its desuccinylation activity.

[0006] MC3482 is a specific SIRT5 inhibitor that can competitively bind to the catalytic domain of SIRT5 to inhibit its desuccinylation activity, and has good in vivo safety and tissue penetration.

[0007] Currently, the role of MC3482 in metabolic diseases has been reported, but its application and efficacy in the prevention and treatment of premature ovarian failure have not yet been studied. Summary of the Invention

[0008] The purpose of this invention is to provide an application of a SIRT5 inhibitor in the preparation of a drug for the prevention and / or treatment of premature ovarian failure. This invention improves the ovarian inflammatory microenvironment from the root by inhibiting SIRT5 activity, blocking the excessive production of macrophages and the polarization of M1 pro-inflammatory macrophages.

[0009] To achieve the above objectives, the present invention provides the following technical solution: The purpose of this invention is to provide the use of a SIRT5 inhibitor in the preparation of a medicament for the prevention and / or treatment of premature ovarian failure.

[0010] Preferably, the SIRT5 inhibitor is compound MC3482.

[0011] Preferably, the premature ovarian failure is chemotherapy-induced premature ovarian failure.

[0012] Preferably, the chemotherapeutic drug is cyclophosphamide or a derivative thereof.

[0013] Preferably, the drug works by inhibiting macrophage infiltration in the ovary and / or reducing the levels of inflammatory factors TNF-α and IL-1β.

[0014] Preferably, the drug exerts its effect by promoting the proliferation of ovarian granulosa cells and / or inhibiting ovarian granulosa cell apoptosis.

[0015] Preferably, the effect is manifested by increasing ovarian weight, increasing the reserve of primordial and growing follicles, and / or increasing MVH. + Total number of oocytes.

[0016] The present invention also aims to provide a pharmaceutical composition for the prevention and / or treatment of premature ovarian failure, wherein the active ingredient of the composition comprises a SIRT5 inhibitor as described above and a pharmaceutically acceptable carrier or excipient.

[0017] Preferably, the drug is administered at a dose of 5 mg / kg via intraperitoneal injection, and the frequency of administration is three times per week.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention demonstrates through systematic animal experiments that the SIRT5 inhibitor MC3482 can effectively prevent and treat cyclophosphamide (CTX)-induced premature ovarian failure (POI) in mice through a multi-dimensional mechanism of action. The core technical effects are as follows: (1) Regulate the ovarian immune microenvironment and alleviate chronic inflammation: MC3482 reduces the excessive macrophage infiltration caused by CTX in the ovary by inhibiting SIRT5 succinylation activity, significantly reduces the activation level of M1 macrophages in ovarian tissue, and downregulates the secretion of pro-inflammatory factors such as TNF-α and IL-1β, thereby effectively alleviating the chronic inflammatory response of the ovary induced by CTX and improving the homeostasis of the local immune microenvironment of the ovary.

[0019] (2) Protecting the function of ovarian granulosa cells and maintaining cell homeostasis: MC3482 can target and inhibit the caspase-dependent apoptosis pathway of ovarian granulosa cells, while promoting the proliferation activity of granulosa cells and reducing the cellular senescence process; by maintaining the normal physiological function of granulosa cells, it ensures their nutritional support and development regulation of oocytes, and provides the necessary conditions for normal follicle development.

[0020] (3) Maintaining follicular reserve and function: MC3482 can reduce follicular atresia and restore genes related to follicular development ( Amh、 Foxl2 etc.) and estrogen synthesis genes ( Cyp19a1 The expression of ) promotes the secretion of ovarian estrogen, maintains the homeostasis of ovarian endocrine function, and thus protects the overall reproductive function reserve of the ovary.

[0021] This invention also improves the ovarian inflammatory microenvironment from the root by inhibiting SIRT5 to block the initiation of macrophage pro-inflammatory differentiation. After drug withdrawal, ovarian immune homeostasis can be maintained for a long time, solving the problems of short-term efficacy and easy relapse of existing technologies. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 A is a schematic diagram of POI model construction, in which CTX (50 mg / kg / day) was injected intraperitoneally for 15 consecutive days to induce modeling: WT and CTX-POI groups; B shows the H&E staining results of ovarian tissue from mice in the WT and CTX-POI groups; scale bar: 200 μm; CF shows the detection of follicle development-related genes in the ovaries of WT and CTX-POI mice by quantitative PCR (qRT-PCR). Amh, Foxl2, Gdf9 Fshr C) Hormone synthase gene ( Cyp19a1 D) Antioxidant-related genes ( Gpx1, Sod1 ;E) and apoptosis regulators ( Bax, Bcl2 The expression levels of F4 / 80 in macrophages in the ovaries of WT and CTX-POI mice were measured by immunofluorescence (n=3 per group). G is a schematic diagram showing the expression levels of F4 / 80 in the ovaries of WT and CTX-POI mice, with the right side representing the F4 / 80 levels in the ovaries. + The proportion of macrophages, scale bar: 50 μm; H is the flow cytometry plot, top: immune cell subsets (CD45) in ovarian single-cell suspension. + A schematic diagram of the flow cytometry sorting strategy; the figure below shows the quantitative analysis of immune cell populations in the ovaries of the WT group and the CTX-POI treatment group, including plasma cells (B220). - CD138 + ), dendritic cells (CD11c + MHC-II + ), macrophages (CD11b + F4 / 80 + ), B cells (B220) + ) and T cells (CD3) + The statistical graph shows the proportion of immune cell subsets in ovarian single-cell suspension; data are expressed as mean ± standard deviation (Mean ± SD), and statistical analysis was performed using unpaired t-tests. p < 0.05; p < 0.01; p < 0.001.

[0024] Figure 2 A dataset of human ovarian single-cell RNA sequencing (scRNA-seq) from the Human Cell Landscape was obtained using the CELLxGENE Discover platform; gene expression levels were expressed in ln (CPTT + 1) Standardization (left); Single-cell RNA sequencing analysis of adult mouse ovarian tissue shows... Sirt5 mRNA expression levels in different ovarian cell populations (log2) [TPM+1] (Right); B is a schematic diagram of the immunofluorescence co-staining images of SIRT5 (red), F4 / 80 (green), and AMH (white) in WT mouse ovarian sections, scale bar: 10μm; CD shows qRT-PCR (C) and Western blot (WB; D) detection. Sirt5 + / + and Sirt5 - / - mouse ovaries Sirt5 Schematic diagram of mRNA and protein expression levels, with β-actin as an internal control (n=3 per group); EF represents 8 weeks of age. Sirt5 + / + and Sirt5 - / - Gross morphology (E) and H&E staining results of mouse ovaries (scale bar: 200 μm; F); G represents qRT-PCR detection. Sirt5 + / + and Sirt5 - / - Key luteinizing steroid genes in mouse ovaries ( Star, Cyp11a1, Cyp17a1, Hsd3b1 / 2 The expression levels of ) (n=4 per group); H represents adults Sirt5 + / + and Sirt5 - / - Statistical analysis of the number of offspring in the first litter of female mice (n=3 per group). Statistical significance was analyzed using unpaired t-tests (C, G, H). Data are expressed as Mean ± SD. p <0.0001; ns, no statistical significance.

[0025] Figure 3 A represents flow cytometry analysis. Sirt5 + / + and Sirt5 - / - Total macrophages (CD11b) in mouse ovaries + F4 / 80 + M1 subtype (CD86) + CD11b + F4 / 80 + ) and M2-like subtype (CD206)+ CD11b + F4 / 80 + The proportion of ); the left figure of BD is Sirt5 + / + and Sirt5 - / - Macrophages (B, F4 / 80) in mouse ovarian sections + Cells, green, n=5 per group), M1 macrophages (C, CD86) + Red; F4 / 80 + (green, n=3 per group) or M2 macrophages (D, CD206) + Red; F4 / 80 + Immunofluorescence staining images (green, n=4 per group); the right side shows F4 / 80. + CD86 + F4 / 80 + Or CD206 + F4 / 80 + Quantitative results of cell proportions, scale bar: 50 μm; E represents qRT-PCR analysis. Sirt5 + / + and Sirt5 - / - Pan-macrophage markers in mouse ovarian tissue ( Adgre1, Cd68, Fcgr1, Cx3cr1 ), M1 or M2 polarization markers (M1 type: Nos2, Cd80, Cd86; M2 type: Arg1、 Mrc1 mRNA expression levels of ) (n=6 per group); F represents qRT-PCR detection. Sirt5 - / - mouse ovaries and Sirt5 + / + Compared with mice, cytokine gene expression was reduced (n=3 per group); statistical significance was analyzed by unpaired t-test (BF), and data are expressed as Mean ± SD; p <0.05; p <0.01; p <0.001; p <0.0001; ns, no statistical significance.

[0026] Figure 4 The image above is... Sirt5 + / + , Sirt5 - / - CTX processing Sirt5 + / + and CTX processing Sirt5 - / -Immunofluorescence staining images of CD86 (red) and F4 / 80 (green) in mouse ovaries (used to identify M1 macrophages); the following figure shows the quantitative analysis results (n=3 per group); B shows the immunofluorescence staining images of CD206 (red) and F4 / 80 (green) in the ovaries of four groups of mice (used to identify M2 macrophages); the following figure shows the quantitative analysis results (n=4 per group); C shows the qRT-PCR analysis of the four groups of mouse ovaries. Tnf and Il1b D represents the expression levels of TNF-α and IL-1β in the ovaries of four mice analyzed by immunofluorescence staining (n=3 per group), scale bar: 50μm; E represents the protein levels of TNF-α and IL-1β in the ovarian homogenates and serum of four groups of mice as determined by enzyme-linked immunosorbent assay (ELISA) (n=5 per group); all quantitative data are expressed as Mean±SD, and statistical significance was analyzed by one-way ANOVA combined with Tukey's multiple comparison test (A, B, C, E); p <0.05; p <0.01; p <0.001; p <0.0001; ns, no statistical significance.

[0027] Figure 5 A is Sirt5 + / + , Sirt5 - / - CTX processing Sirt5 + / + and CTX processing Sirt5 - / - A) Immunohistochemical (IHC) staining image of Ki67 in mouse ovarian sections, scale bar: 100 μm; B) Immunofluorescence staining image of Cleaved-Caspase-3 in ovarian sections of four groups of mice, scale bar: 50 μm; C) TUNEL detection image of apoptotic cells (green) in ovarian sections of four groups of mice, scale bar: 50 μm; D) Staining image of aging-associated β-galactosidase (SA-β-Gal) in ovarian tissue of four groups of mice, scale bar: 20 μm.

[0028] Figure 6 AB is Sirt5 + / + , Sirt5 - / - CTX processing Sirt5 + / + and CTX processing Sirt5 - / -Morphology of mouse ovaries (A) and hematoxylin-eosin (H&E) staining images (B), scale bar: 100 μm; C: Immunofluorescence staining images of MVH (green) in the ovaries of four groups of mice, scale bar: 100 μm; D: Quantitative analysis of the number of follicles in the ovaries of four groups of mice, including the number of primordial follicles, the number of growing follicles, and the total number of follicles (n=3 per group); EH: qRT-PCR analysis: E, genes related to follicle development (… Amh, Foxl2, Gdf9, Fshr F, hormone synthase gene ( Cyp19a1 G, antioxidant-related genes ( Gpx1、 Sod1 H, apoptosis regulator ( Bax, Bcl2 The expression levels of β-actin in the ovaries of four groups of mice (n=3 per group) were determined. Data were normalized with β-actin as an internal reference and expressed as Mean±SD. Statistical significance was determined by one-way ANOVA combined with Tukey's multiple comparison test (DH). p <0.05; p <0.01; p <0.001; p < 0.0001; ns, no statistical significance.

[0029] Figure 7 A is a schematic diagram of the drug administration timeline, showing the administration regimens of MC3482 (5 mg / kg) or the vehicle in a CTX (50 mg / kg)-induced POI model; B shows the succinylation level in mouse ovaries as detected by Western blotting after intraperitoneal injection of MC3482 for one week (5 mg / kg, 3 times / week); C shows the macrophage (F4 / 80) levels in the ovarian tissue of mice in the control group, POI+Vehicle group, and POI+MC3482 group. + Immunofluorescence staining images of cells, scale bar: 50 μm; DE represents immunofluorescence staining detection of ovarian tissue in three groups of mice (D) and ELISA (E) quantitative detection of TNF-α and IL-1β levels in ovarian tissue homogenate and serum (n=5 per group); F represents qRT-PCR (n=3 per group) showing the levels of inflammatory cytokines TnF and IL-1β in ovarian tissue of designated groups of mice. Il1b Transcriptional levels; data are expressed as mean ± standard deviation (Mean ± SD), and statistical significance was determined using one-way ANOVA combined with Tukey's multiple comparison test (D, E). p <0.01; p <0.001; p <0.0001.

[0030] Figure 8 A shows representative immunohistochemical staining of Ki67 in ovarian sections of mice in the control group, POI+Vehicle group, and POI+MC3482 group. Scale bar: 100 μm; B shows representative immunohistochemical staining of Cleaved-Caspase-3 in ovarian sections of mice in the three groups. Scale bar: 50 μm; C shows TUNEL assay to assess granulosa cell apoptosis in ovarian tissues of mice in the three groups. Scale bar: 50 μm.

[0031] Figure 9 AB shows representative morphological (A) and H&E staining images (B) of the ovaries of mice in the control group, POI+Vehicle group, and POI+MC3482 group; C shows immunofluorescence staining images of MVH (green) in the ovaries of the three groups of mice, scale bar: 100μm; D shows quantitative analysis of the number of primordial follicles, the number of growing follicles, and the total number of follicles in the ovaries of the three groups of mice (n=4 per group), scale bar: 100μm; EH shows qRT-PCR detection of follicle development-related genes in the ovarian tissue of each group of mice. Amh, Foxl2, Gdf9 and Fshr E) Hormone biosynthesis-related genes ( Cyp19a1 F), apoptosis-related genes ( Bax, Bcl2 ;G) and genes related to antioxidant responses ( Gpx1, Sod1 ; H) expression level; data in β-actin The values ​​are internal references, expressed as Mean ± SD. Statistical analysis was performed using one-way ANOVA combined with Tukey's multiple comparison test, with n=3 for each group. p <0.01; p <0.001. Detailed Implementation

[0032] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0033] Unless otherwise specified, the production processes, experimental methods, or testing methods involved in the embodiments of this invention are all conventional methods in the prior art, and their names and / or abbreviations are all conventional names in the field, which are very clear and distinct in the relevant application areas. Those skilled in the art can understand the conventional process steps based on the names and apply the corresponding equipment, and implement them according to conventional conditions or the conditions recommended by the manufacturer.

[0034] There are no special restrictions on the sources of various instruments, equipment, raw materials or reagents used in the embodiments of the present invention. They are all conventional products that can be obtained through regular commercial channels, or can be prepared according to the conventional methods well-known to those skilled in the art.

[0035] Example 1 Construction and verification of a mouse model of premature ovarian failure 1.1 Experimental animals 8-week-old SPF-grade female C57BL / 6J mice, weighing 20-22 g, were purchased from Shanghai Jiesijie Experimental Animal Co., Ltd., and the animal license number is SCXK (Shanghai) 2022-0006.

[0036] 1.2 Model construction The schematic diagram of model construction is as shown in Figure 1 Figure A. After acclimatizing the mice for one week, the experiment was started, and the mice were randomly divided into a control group (WT, n = 6) and a model group (CTX-POI, n = 6). The CTX-POI group was intraperitoneally injected with 50 mg / kg cyclophosphamide daily, and the WT group was injected with an equal volume of 200 μL of sterile saline. This was done for 15 consecutive days, with the injection frequency and cycle the same as those of the CTX-POI group. At the end of the experiment, the mice were sacrificed by carbon dioxide asphyxiation, and the bilateral ovaries were quickly dissected. One side of the ovary was fixed in 4% paraformaldehyde (PFA) for histological detection, and the other side was frozen in liquid nitrogen and then transferred to an -80°C refrigerator for storage for molecular biology analysis.

[0037] 1.3 Model verification 1.3.1 Ovarian morphological detection After the fixed ovarian tissues were dehydrated with gradient ethanol (30%, 50%, 75%, 95%, 100%, and the incubation time for each gradient was 1 hour) in sequence, they were treated with xylene for transparency twice, with each treatment for 8 minutes. Subsequently, the tissues were embedded in paraffin to obtain paraffin blocks. The above paraffin blocks were cut into 5-μm-thick continuous sections using a Leica RM2126 microtome, and stained using a hematoxylin-eosin (H&E) staining kit (Solarbio, product number G1120): After the sections were dewaxed to water, they were incubated with hematoxylin staining solution for 5 minutes, differentiated with 0.1% hydrochloric acid alcohol for 30 s, incubated with eosin staining solution for 10 s, dehydrated and made transparent again with gradient, and then sealed with neutral resin; images were obtained using a Zeiss Axio Scan 7 slide scanner.

[0038] The results of H&E staining are as shown in Figure 1 Figure B. The ovarian volume of the mice in the model group was significantly reduced, and the ovarian weight was significantly lower than that of the control group; the numbers of primordial follicles and growing follicles in the ovaries of the model group were significantly reduced compared with the control group, and the follicular atresia rate was significantly increased.

[0039] 1.3.2 Molecular biology detection Total RNA was extracted from ovarian tissue using the FastPure® Complex Tissue Total RNA Extraction Kit (Vazyme, catalog number RC113): Ovarian tissue was collected, 600 μL of SRL lysis buffer was added, and homogenization was performed using a Servicebio mechanical homogenizer until no obvious tissue blocks were observed. After centrifugation at 12000×g for 5 minutes, 500 μL of supernatant was transferred to a new centrifuge tube, 0.5 times the volume of anhydrous ethanol was added and mixed thoroughly. The mixture was then transferred to FastPure RNA Columns VI in the kit, centrifuged, and the liquid was discarded. The tissue was washed once with 700 μL Buffer RWA and once with 500 μL Buffer RWA, and finally dissolved in RNase-free water. RNA purity (A260 / A280 ratio of 1.8-2.0 is acceptable) and concentration were determined using a NanoDrop 2000 spectrophotometer; 1 μg of total RNA was used to synthesize cDNA using a PrimeScript™ RT kit (Takara, catalog number RR047A) (reaction conditions: 37℃ for 15 minutes, 85℃ for 5 seconds); qRT-PCR was performed using TB Green® Premix Ex Taq™ II (Takara, catalog number RR820A) to detect genes related to follicle development (…). Amh, Foxl2, Gdf9, Fshr ), estrogen synthase gene ( Cyp19a1 Apoptosis-related genes ( Bax, Bcl2 ) and oxidative stress markers ( Gpx1, Sod1 mRNA expression of ) to β- actin For internal reference, use 2 -ΔΔCt The relative expression level is calculated using this method.

[0040] The results are as follows Figure 1 As shown in CF, genes related to follicle development in the CTX-POI group Amh, Foxl2, Gdf9, Fshr The mRNA expression levels of the estrogen synthase gene were significantly downregulated compared to the WT group; Cyp19a1 Significantly downregulated expression; upregulated expression of the pro-apoptotic gene Bax, downregulated expression of the anti-apoptotic gene Bcl2, and a significantly increased Bax / Bcl2 ratio; oxidative stress markers Gpx1, Sod1 The expression was significantly upregulated.

[0041] 1.3.3 Ovarian Immune Microenvironment Detection Immunofluorescence staining: Ovarian tissue was fixed with 4% PFA for 1 hour, then immersed in 30% sucrose solution overnight at 4°C, and embedded in OCT (Sakura Finetek, catalog number 4583). Frozen sections were then prepared as 5μm thick sections using a cryosectioning mechanism. Sections were permeabilized with 0.3% Triton X-100 for 20 minutes, blocked with 5% goat serum for 1 hour, and then incubated overnight at 4°C with F4 / 80 primary antibody (Thermo, catalog number 14-4801-85, 1:600). After washing 5 times with PBS, sections were incubated with Alexa Fluor 488 conjugated secondary antibody (CST, catalog number 4416, 1:1200) at 4°C for 2 hours, followed by DAPI staining of the nuclei for 5 minutes. After mounting with antifluorescence quenching mounting solution, the sections were observed using a Zeiss LSM 880 confocal microscope, and the F4 / 80 concentration was quantified using ImageJ software. + Number of positive cells.

[0042] Flow cytometry: Fresh ovarian tissue was obtained and minced into 1mm pieces using a sterile scalpel. 3 Digest the cells into small pieces, add HBSS buffer containing 1 mg / mL type IV collagenase (Sigma, catalog number C5138) and 1 U / mL DNase I (Thermo, catalog number EN0521), incubate at 37°C with shaking for 30 minutes, add 5 mM EDTA to stop digestion, filter through a 70 μm cell sieve to remove residue; lyse red blood cells with red blood cell lysis buffer (BioLegend, catalog number 420301) for 5 minutes, centrifuge at 300×g for 5 minutes to collect cells, resuspend in HBSS containing 2% fetal bovine serum (FBS) and 2 mM EDTA; take 1×10 6 Cells were blocked at 4°C for 10 minutes with TruStain FcX™ (BioLegend, catalog number 101320), followed by incubation at 4°C for 30 minutes with fluorescein-conjugated antibodies (CD45.2-Alexa Fluor 700, CD11b-PerCP / Cyanine 5.5, F4 / 80-FITC, CD11c-PECy7, CD138-PE, B220-Super Bright 436, CD3-Super Bright 600, and MHC-II-APC, all derived from BioLegend and diluted 1:200). Dead cells were then labeled with eFluor™ 780 dye. CytoFLEX flow cytometry was used to analyze ovarian immune cell infiltration, such as plasma cells (B220...). - CD138 + ), dendritic cells (CD11c + MHC-II + ), macrophages (CD11b + F4 / 80+ ), B cells (B220) + ), T cells (CD3) + )Proportion.

[0043] Depend on Figure 1 The experimental data shown in G and 1H indicate that: Immunofluorescence staining results showed that the F4 / 80 ratio of ovarian stroma and perifollicular tissue in the CTX-POI group was [not specified]. + Macrophages aggregated in large numbers, and the number of positive cells was significantly higher than that of the control group.

[0044] Flow cytometry results showed that macrophages (CD11b) were present in the ovarian tissue of the CTX-POI group. + F4 / 80 + The proportion of () showed a significant upward trend compared to the WT group; The above results indicate that CTX successfully induced a mouse model with typical POI pathological features, which can be used to evaluate the subsequent POI prevention and treatment effects.

[0045] Example 2: Effects of SIRT5 deficiency on macrophage infiltration and fertility in mouse ovaries 2.1 Laboratory Animals Choose 8 weeks old Sirt5 + / + and Sirt5 - / - Six mice from the same littermate were used in each group.

[0046] 2.2 Localization detection of SIRT5 in the ovary 2.2.1 Analysis of SIRT5 expression in the ovary using single-cell RNA sequencing data Human cell atlas was analyzed using the CELLxGENE Discover platform (https: / / cellxgene.cziscience.com); adult mouse ovarian single-cell transcriptome datasets were downloaded from Mouse Cell Atlas 2.0 (MCA 2.0, https: / / bis.zju.edu.cn / MCA / ), and analyzed in human and mouse ovaries respectively. Sirt5 The transcriptional expression levels were measured. Both datasets contain gene expression information from various cell types, including oocytes, granulosa cells, theca cells, macrophages, and luteal cells. For human ovarian single-cell sequencing analysis, gene expression was pre-normalized to ln(CPTT+1), where CPTT represents the number of counts per 10,000. The average ln(CPTT+1) value of SIRT5 in each cell cluster was calculated to assess its expression distribution in the human ovary. For mouse ovarian single-cell sequencing data, the transcriptional expression levels of SIRT5 in each cell cluster were extracted. Sirt5 Transcription per million (TPM) values ​​were used to calculate the average expression level and generate a heatmap analysis. Sirt5 Cell-specific distribution.

[0047] like Figure 2 The experimental results shown in Figure A indicate that: Single-cell RNA sequencing results from mouse ovarian tissue showed that Sirt5 The gene is highly expressed in ovarian macrophages (ranking among the top three in expression levels), but almost not expressed in oocytes, granulosa cells, and theca cells. Analysis of single-cell RNA sequencing (scRNA-seq) datasets from human ovarian tissue showed that the expression distribution pattern of the SIRT5 gene was highly consistent with that in mice, with high expression in macrophages.

[0048] 2.2.2 Immunofluorescence co-staining analysis of SIRT5 localization in the ovary The frozen section preparation method is the same as in Example 1.3.3. The fluorescence co-staining protocol is as follows: the sections were permeated with 0.3% Triton X-100 at room temperature for 20 minutes, washed 3 times, and then blocked with H2O2 and goat serum from the kit for 1 hour at room temperature, according to the Thermo Fisher SuperBoost TSA kit (catalog number B40955) instructions. After that, they were incubated with SIRT5 primary antibody (NOVUS, catalog number NBP1-89497, 1:500) at 4°C overnight. The next day, they were washed 5 times with 1×PBS (5 minutes each time), and then incubated with the kit's HRP anti-rabbit secondary antibody at room temperature for 1 hour, followed by 5 washes. The Alexa Fluor 594 fluorescent tyrosine working solution was prepared according to the kit instructions (the stock solution was diluted 1:100 with the kit's diluent and 0.003% fresh H2O2 was added), and 200 μL was used to cover the sections and incubated at room temperature in the dark for 4 minutes. The staining was terminated with the stop solution from the kit. After antigen retrieval with sodium citrate, F4 / 80 and CST Alexa Fluor 488 anti-rat secondary antibody were incubated with immunofluorescence as described in Example 1.3.3. Then, AMH primary antibody (Abcam, catalog number ab27969, 1:500) was incubated overnight at 4°C. After 5 washes, Thermo Fisher Alexa Fluor 647 anti-goat secondary antibody (1:1000) was added and incubated at room temperature in the dark for 1 hour, followed by 5 washes. DAPI (1:1000) was added and stained the nuclei at room temperature for 5 minutes. After 3 washes, the slides were mounted with anti-fluorescence quenching mounting medium. Images were acquired using a Zeiss LSM 880 confocal microscope (excitation wavelengths of 405nm, 488nm, 561nm, and 640nm) to analyze the localization relationship between SIRT5 and F4 / 80 and AMH.

[0049] like Figure 2The experimental results shown in B indicate that the immunofluorescence co-staining experiment further confirmed that SIRT5 protein (red fluorescent label) and F4 / 80 macrophages (green label) have a clear co-localization relationship, and both are mainly distributed in the ovarian stromal region surrounding AMH-positive granulosa cells (white label). This result verifies the specific enrichment of SIRT5 in ovarian macrophages.

[0050] 2.2.3 Analysis of Follicular Development and Fertility To assess ovarian macrophage infiltration, this invention employs existing systemic methods. Sirt5 Knockout mice ( Sirt5 - / - ) and its nestmate wild-type control ( Sirt5 + / + The study investigated follicular development and fertility-related indicators in two groups of mice under physiological conditions. The specific experimental steps are as follows: Ovarian tissues from two groups of mice were collected, and the relative expression level of SIRT5 mRNA was detected by qRT-PCR. Simultaneously, the expression level of SIRT5 protein was detected by Western blotting for double verification. Sirt5 In vivo gene knockout effect. The expression levels of hormone-related genes in the ovaries of two groups of mice were detected using qRT-PCR, such as... Star, Cyp11a1, Cyp17a1, Hsd3b1 and Hsd3b2 After both groups of mice reached 8 weeks of age, they were naturally mated with wild-type (WT) male mice. The number of offspring in the first litter of each group was counted to quantify the impact. Sirt5 The effect of gene knockout on fertility in mice.

[0051] The results are as follows Figure 2 As shown in CD, Sirt5 - / - mouse ovaries Sirt5 Both mRNA and protein levels were significantly reduced; and such Figure 2 E and Figure 2 F shows that histological results indicate Sirt5 - / - The mouse ovaries were morphologically normal, containing follicles at all stages from primordial to antral follicles, and the theca cell layer structure was intact. Although SIRT5 was also expressed in luteal cells, Sirt5 - / - Key steroid-producing enzymes in the ovaries of mice and control groups ( Star, Cyp11a1, Cyp17a1, Hsd3b1 and Hsd3b2 The similar mRNA levels of SIRT5 indicate that SIRT5 deficiency has little effect on progesterone synthesis. Figure 2 G). Consistent with this, fertility tests show Sirt5 - / - There was no significant difference in litter size between female and wild-type female mice. Figure 2 H).

[0052] These results indicate that, under basal physiological conditions, the absence of SIRT5 does not affect the normal development of mouse ovaries or fertility.

[0053] 2.3 Analysis of ovarian macrophage infiltration Total macrophages (CD45-PE / Cy7, CD11b-FITC, F4 / 80-APC, CD86-PE, CD206-PerCP / Cy5.5) were quantified using flow cytometry. + CD11b + F4 / 80 + M1 macrophages (CD45) + CD11b + F4 / 80 + CD86 + M2 macrophages (CD45) + CD11b + F4 / 80 + CD206 + The proportion of ); Immunofluorescence detection Sirt5 + / + and Sirt5 - / - Total macrophages in the ovaries of littermates (F4 / 80) + M1 macrophages (F4 / 80) + CD86 + M2 macrophages (F4 / 80) + CD206 + The expression of ) was analyzed and quantitative statistics were performed.

[0054] Total macrophage marker genes in ovarian tissue were detected by qRT-PCR. Adgre1, Cd68, Fcgr1 Cx3cr1 M1 Nos2, Cd80, Cd86 ) or M2 polarization ( Arg1, Mrc1 The expression status of marker genes; and the detection of inflammatory factors in the ovary, such as... Tnf, Il1b, Il1a, Il10, Il34, Cxcl9, Cxcl10, Wnt4, Inhbb mRNA expression levels.

[0055] like Figure 3 The experimental results shown in AE indicate that: Flow cytometry showed that Sirt5 - / - Total macrophages (CD45) in mouse ovaries + CD11b + F4 / 80 + The proportion is relatively high Sirt5 + / +In mice, the number of M1 macrophages (CD86) decreased. + CD11b + F4 / 80 + ) and M2 macrophages (CD206) + CD11b + F4 / 80 + The proportions decreased respectively; immunofluorescence staining showed that... Sirt5 - / - F4 / 80 in mouse ovaries + Positive cell count, CD86 + F4 / 80 + Positive cell count, CD206 + F4 / 80 + The number of positive cells was higher than Sirt5 + / + The number of mice was significantly reduced; at the same time, such as Figure 3 E shows, Sirt5 - / - Total macrophage marker genes in mouse ovaries ( Adgre1, Cd68, Fcgr1, Cx3cr1 M1 Nos2, Cd80 Cd86 ) or M2 ( Arg1, Mrc1 The expression level of marker genes is relatively high. Sirt5 + / + The effect was significantly reduced in mice.

[0056] like Figure 3 F qRT-PCR showed that Sirt5 - / - mouse ovaries Tnf, Il1b, Il1a, Il10, Cxcl9 and Cxcl10 The expression levels of cytokine mRNAs of [various species] were respectively compared to Sirt5 + / + The effect was significantly reduced in mice.

[0057] This indicates that under physiological conditions, SIRT5 deficiency can significantly reduce ovarian macrophage infiltration and the production of inflammatory factors, but does not affect follicle development and fertility.

[0058] Example 3: Three protective effects of SIRT5 deficiency in the CTX-POI model 3.1 Grouping of experimental animals and construction of experimental models Choose 8 weeks old Sirt5 + / + and Sirt5 - / - The littermates were divided into 4 groups: Sirt5 + / + ; Sirt5 - / - CTX processing Sirt5 + / + Mice; CTX-treated Sirt5- / - Mice; 6 mice per group. The mouse model was established as described in Example 1.2, and ovarian tissue was collected at the end of the experiment.

[0059] 3.2 Assessment of changes in ovarian macrophages and inflammatory factors after CTX treatment Detection of macrophage count in the ovary: Collected ovarian tissue was divided into two groups for macrophage immunofluorescence staining to detect different combinations of markers: Group 1: F4 / 80 and CD86 co-stained; Group 2: F4 / 80 and CD206 co-stained. Results are as follows. Figure 4 As shown in AB.

[0060] Detection of inflammatory factor mRNA expression (qRT-PCR): Ovarian tissue was collected, and total RNA was extracted and cDNA was synthesized using the method described in Example 1 above; inflammatory factors were detected using TB Green® Premix Ex Taq™ II (Takara, catalog number RR820A). Tnf, Il1b mRNA expression levels; β-actin For internal reference, 2 -ΔΔCt Relative expression levels were calculated using a method with three technical replicates for each sample. Results are as follows: Figure 4 As shown in C.

[0061] Detection of inflammatory cytokine protein concentrations (immunofluorescence staining and ELISA): Mouse ovarian tissue and blood were collected separately. Each mouse ovary was treated with 300 μL of pre-chilled PBS (containing 1× protease inhibitor mixture (Roche, catalog number 04693116001)). After sonication, the tissue was centrifuged at 5000×g for 10 minutes at 4°C, and the supernatant was collected. Blood was allowed to stand at room temperature for 2 hours, then centrifuged at 1500×g for 10 minutes, and the supernatant serum was collected for testing. The Elabscience® high-sensitivity ELISA kit (catalog number E-EL-M0049c for TNF-α detection, E-EL-M0037c for IL-1β detection) was used according to the manufacturer's instructions. 100 μL of tissue supernatant and 100 μL of mouse serum were used for detection, respectively. The samples were incubated at 37°C for 90 minutes. After TMB color development, the values ​​were read at 450 nm using a microplate reader. Protein concentrations were calculated based on the standard curve (tissue inflammatory factor protein results are expressed as "pg / mg protein", and protein concentration was determined by the BCA method; serum inflammatory factor protein results are expressed as "pg / ml"). For immunofluorescence staining: after fixation, permeabilization, and blocking, ovarian frozen sections were incubated overnight at 4°C with either TNF-α primary antibody (Abcam, catalog number ab6671, 1:500) or IL-1β primary antibody (Abcam, catalog number ab9722, 1:500). After washing five times with 1×PBS, Alexa Fluor 488 conjugated with anti-rabbit secondary antibody (1:1000) was added and incubated at room temperature in the dark for 1 hour. After nuclear staining with DAPI, the sections were mounted, and the expression levels of TNF-α and IL-1β in ovarian tissue were observed and quantified using a confocal microscope. The results are as follows: Figure 4 As shown in D and E.

[0062] Based on the above results of ovarian macrophages and inflammatory factors after CTX treatment Figure 4 As can be seen in AE, CTX- Sirt5 - / - Macrophage infiltration was not excessively increased in mouse ovaries, and immunofluorescence confirmed that the total number of macrophages and the number of M1 and M2 subtypes were significantly lower than those in CTX- Sirt5 + / + Mice; ELISA, immunofluorescence and qRT-PCR showed that CTX- Sirt5 - / - The levels of TNF-α and IL-1β in mouse ovaries were significantly reduced, indicating that SIRT5 deficiency can effectively block CTX-induced excessive ovarian inflammation.

[0063] 3.3 Assessment of Granulosa Cell Proliferation and Apoptosis 3.3.1 Proliferation detection (Ki67 immunohistochemistry) After dewaxing ovarian paraffin sections to water, antigen retrieval was performed by microwave heating in citrate buffer (pH 6.0) for 10 minutes, followed by blocking with 5% goat serum for 1 hour. Ki67 primary antibody (Abcam, catalog number ab16667, 1:800) was added and incubated overnight at 4°C. HRP-conjugated secondary antibody was then added. Jackson ImmunoResearch Catalog number 111-035-003, 1:1000) Incubate at room temperature for 1 hour; DAB chromogenic reagent kit ( Maixin Biotech The follicles were developed, counterstained with hematoxylin, dehydrated, and mounted. Images were acquired using a Zeiss Axio Scan.Z1 scanner to detect the number of Ki67-positive cells in the follicles.

[0064] 3.3.2 Apoptosis and Senescence Detection Apoptosis detection: Cleaved-Caspase-3 immunofluorescence: Ovarian frozen sections were permeabilized with 0.3% Triton X-100 for 20 minutes, blocked with 5% goat serum for 1 hour, and then incubated overnight at 4°C with Cleaved Caspase-3 primary antibody (CST, catalog number 9664, 1:500). After incubation with Alexa Fluor 488 conjugated with rabbit secondary antibody for 2 hours, nuclei were stained with DAPI. The number of positive granulosa cells was observed and quantified under a Zeiss LSM 880 confocal microscope. TUNEL detection: The Promega TUNEL apoptosis detection kit (Promega, catalog number G3250) was used. Frozen sections were fixed with 4% PFA at 4°C for 10 minutes, permeabilized with 0.2% Triton X-100 for 20 minutes, and incubated at 37°C in the dark for 60 minutes with TdT reaction mixture. The reaction was terminated by washing with 2×SSC. Nuclei were stained with DAPI, and TUNEL was observed under a Zeiss LSM880 confocal microscope. + Positive granular cells.

[0065] Senescence detection: The senescence of ovarian granulosa cells was detected using the SA-β-galactosidase staining kit (Beyotime, catalog number C0602). Ovarian frozen sections were fixed at room temperature for 10 minutes, washed three times with PBS, incubated with SA-β-galactosidase working solution at 37°C for 4 hours, washed with PBS, mounted, and images were acquired using a Zeiss Axio Scan.Z1 scanner to observe SA-β-Gal positive cells in the ovaries of four groups of mice.

[0066] The above results are as follows Figure 5 As shown in AD, in CTX-POI model mice, Ki67 immunohistochemical detection results showed that CTX-POI... Sirt5 - / - Ki67 positive in mouse growing follicles (Ki67 + The proportion of granulocytes, compared to CTX- Sirt5+ / + The mice showed a significant increase, which suggests that... Sirt5 Gene deletion can enhance the proliferative activity of granulocytes; further results from Cleaved-Caspase-3 immunofluorescence and TUNEL assays showed that CTX- Sirt5 - / - The number of apoptotic positive granulosa cells in mouse ovarian tissue was higher than that of CTX- Sirt5 + / + The number of mice was significantly reduced; meanwhile, SA-β-galactosidase staining results showed that CTX- Sirt5 - / - The proportion of senescent granulosa cells in mouse ovaries was significantly reduced. In summary, the above results collectively confirm that the loss of SIRT5 protein can effectively inhibit apoptosis and senescence in granulosa cells, thereby maintaining their functional homeostasis.

[0067] 3.4 Assess follicular reserve and development 3.4.1 Follicle count After H&E staining, the number of primordial follicles (containing only a single layer of flattened granulosa cells), growing follicles (including primary follicles containing a single layer of cuboidal granulosa cells, secondary follicles containing two or more layers of granulosa cells, and antral follicles containing follicular cavities) was counted. Simultaneously, MVH immunofluorescence staining (an oocyte-specific marker) was used for verification: frozen ovarian sections were incubated overnight at 4°C with MVH primary antibody (Abcam, catalog number ab13840, 1:500), followed by incubation at 4°C for 2 hours with Alexa Fluor 488 conjugated with secondary antibody. Nuclei were then stained with DAPI, and MVH was observed using a Zeiss LSM 880 confocal microscope. + The number of follicles corresponding to an oocyte.

[0068] 3.4.2 Follicle function gene testing qRT-PCR was used to detect genes related to follicle development ( Amh, Foxl2, Gdf9, Fshr ) and genes related to estrogen synthesis ( Cyp19a1 The mRNA expression level of ) was measured; RNA extraction, cDNA synthesis and qRT-PCR statistics were performed according to Example 1.3.2.

[0069] 3.4.3 Assessment of Oxidative Stress and Apoptosis Pathways qRT-PCR was used to detect biomarkers of oxidative stress ( Gpx1, Sod1 ) and apoptosis-related genes ( Bax, Bcl2 The mRNA expression level of ) was measured, and the Bax / Bcl2 ratio was calculated.

[0070] like Figure 6 AD, H&E staining and MVH immunofluorescence detection results showed: CTX- Sirt5 - / -In mouse ovarian tissue, the number of primordial follicles, growing follicles, and total oocytes were all significantly higher than those obtained with CTX- Sirt5 + / + Mice; Figure 6 qRT-PCR results from EH showed changes in the mRNA expression levels of key functional genes in ovarian tissue: compared with CTX- Sirt5 + / + Compared to mice, CTX- Sirt5 - / - Genes related to the regulation of follicle development in mouse ovaries ( Amh, Foxl2, Gdf9, Fshr ) and hormone synthesis-related genes ( Cyp19a1 The mRNA expression levels of CTX- were significantly upregulated; meanwhile, CTX- Sirt5 - / - The apoptosis-regulating Bax / Bcl2 ratio in mouse ovarian tissue was significantly decreased, and markers of oxidative stress defense ( Gpx1, Sod1 The mRNA expression level of ) was significantly upregulated.

[0071] In summary, the above series of test results collectively confirm that in the CTX-POI mouse model, SIRT5 protein deficiency can upregulate the expression of key genes involved in follicle development and hormone synthesis, optimize apoptosis regulation pathways (reducing the Bax / Bcl2 ratio), and enhance oxidative stress defense capabilities (upregulating...). Gpx1, Sod1 This method effectively protects the ovarian follicle reserve, restores the normal development process of follicles and the ovarian hormone synthesis function, and significantly reduces the damage to ovarian tissue caused by oxidative stress.

[0072] Example 4: Validation of the preventive and therapeutic effects of MC3482 on POI mice 4.1 Experimental Grouping Eight-week-old female C57BL / 6J mice were randomly divided into three groups: sham-operated group (Control group), solvent + CTX group (POI + Vehicle group), and MC3482 + CTX group (POI + MC3482 group), with six mice in each group. The administration times were as follows: Figure 7 As shown in A: Experimental group (POI+MC3482 group): In week 1, MC3482 (5 mg / kg, dissolved in a mixed solvent containing 10% DMSO, 40% PEG300, 5% Tween-80, and 45% normal saline) was administered intraperitoneally three times a week, 200 μL each time; in weeks 2–3, MC3482 was continued, and CTX (50 mg / kg) was administered intraperitoneally daily for 15 days. Control group (POI+Vehicle group): Administered only an equal volume of solvent + CTX; Sham surgery group (Control group): only solvent + normal saline was administered.

[0073] 4.2 Detection of the inhibitory effect of MC3482 on SIRT5 activity To evaluate the inhibitory effect of MC3482 on SIRT5 desuccinylation activity, mice were intraperitoneally injected with MC3482 (5 mg / kg, 3 times / week), and bilateral ovarian tissues were collected for immunoblotting. The method was the same as in Example 2.2.3. The primary antibodies used included anti-succinyl lysine antibody (SuccK, Abclonal, catalog number: A18106, 1:1000), anti-SIRT5 antibody (Proteintech Group, catalog number: 17124-1-AP, 1:1000), and anti-β-actin antibody (Proteintech Group, catalog number: 66009-1-Ig, 1:5000) as an internal control.

[0074] The results are as follows Figure 7 B showed that the overall protein succinylation level in the ovaries of mice treated with MC3482 was significantly increased, while the expression of SIRT5 protein did not change significantly, indicating that MC3482 effectively inhibited the desuccinylation activity of SIRT5, rather than by regulating its expression level.

[0075] 4.3 Detection of ovarian inflammation and oxidative stress 4.3.1 Detection of ovarian macrophage count and inflammation Macrophage infiltration detection: F4 / 80 immunofluorescence staining was used to observe the distribution of ovarian macrophages and compare the F4 / 80 ratio. + Number of positive cells. The expression levels of inflammatory factors in ovarian tissue were detected in multiple dimensions using ELISA, immunofluorescence staining, and qRT-PCR.

[0076] Results of macrophage count detection, such as Figure 7 As shown in C, the F4 / 80 ratio in the ovaries of the POI+MC3482 group was... + The number of macrophage-positive cells was significantly lower than that in the POI+Vehicle group, indicating that MC3482 can effectively inhibit macrophage infiltration in ovarian tissue induced by CTX in the POI model.

[0077] Results of inflammatory factor detection in the ovaries, such as Figure 7 DF showed that ELISA and immunofluorescence assays revealed significantly lower concentrations of TNF-α and IL-1β in the ovarian tissue of the POI+MC3482 group compared to the solvent+CTX group; qRT-PCR showed that... Tnf, Il1b The mRNA expression level was also significantly downregulated.

[0078] In summary, MC3482 can effectively inhibit CTX-induced ovarian inflammatory response by suppressing macrophage infiltration and reducing the transcription and translation levels of inflammatory factors (TNF-α, IL-1β).

[0079] 4.3.2 Detection of Ovarian Granulosa Cell Proliferation and Apoptosis Proliferation detection: Ki67 immunohistochemistry was used to detect the proliferation of granulosa cells in follicles and to quantify Ki67 levels. + The proportion of granulocytes; Apoptosis detection: Granulocyte apoptosis was assessed by Cleaved-Caspase-3 immunofluorescence and TUNEL assay; according to Figure 8 Results A showed that, compared with the POI+Vehicle group, the POI+MC3482 group had higher levels of Ki67 in growing follicles. + The proportion of granulocytes increased significantly.

[0080] Cleaved-Caspase-3 immunofluorescence ( Figure 8 B) shows that, compared with the POI+Vehicle group, the number of positive granulocytes in the POI+MC3482 group was significantly reduced. TUNEL test results ( Figure 8 C) showed that the proportion of apoptotic positive granulocytes was significantly lower in the POI+MC3482 group compared with the POI+Vehicle group.

[0081] The above results indicate that MC3482 can significantly restore the proliferation capacity of ovarian granulosa cells in the CTX-induced POI model and can also effectively block CTX-induced apoptosis of ovarian granulosa cells.

[0082] 4.3.3 Ovarian morphology and follicular function detection Ovarian weight and morphology: At the experimental endpoint, bilateral ovaries of mice were isolated and their size was observed under a stereomicroscope. Ovarian tissue was fixed with 4% PFA, embedded in paraffin, and H&E staining was used to observe the structural integrity of the ovary. The number of follicles at each stage was counted. Quantitative MVH was verified by MVH immunofluorescence staining. + The number of oocytes is determined by referring to Example 3.4.1.

[0083] Follicle function gene detection: qRT-PCR was used to detect genes related to follicle development ( Amh, Foxl2, Gdf9, Fshr ) and genes related to estrogen synthesis ( Cyp19a1 The mRNA expression level of ).

[0084] Detection of oxidative stress and apoptosis pathways: qRT-PCR was used to detect oxidative stress markers ( Gpx1, Sod1 ) and apoptosis-related genes ( Bax, Bcl2 The mRNA expression level of ) was measured, and the Bax / Bcl2 ratio was calculated.

[0085] Ovarian volume measurement results ( Figure 9 A) shows that the ovarian volume of mice in the POI+MC3482 group was significantly larger than that in the POI+Vehicle group; H&E staining and MVH ( Figure 9 BD immunofluorescence showed that the number of primordial follicles and growing ovaries in the POI+MC3482 group was significantly increased compared to the POI+Vehicle group, and the total MVH was also significantly higher. + The number of oocytes increased significantly.

[0086] Results of follicular function and oxidative stress level tests are as follows: Figure 9 EH showed that in the POI+MC3482 group, ovaries Amh、 Foxl2, Gdf9, Fshr and Cyp19a1 The mRNA expression levels of these cells were significantly upregulated compared to the POI+Vehicle group; simultaneously, the Bax / Bcl2 ratio decreased, and oxidative stress markers were also observed. Gpx1, Sod1 The downregulation indicates that MC3482 can effectively restore follicle development and hormone synthesis functions and reduce oxidative stress damage.

[0087] In summary, MC3482 can increase ovarian weight, the reserve of primordial and growing follicles, and MVH. + The total number of oocytes effectively improves the reproductive reserve of the ovary in the CTX-induced POI model.

[0088] In summary, MC3482 effectively mimics the activity of SIRT5 desuccinylation by inhibiting its desuccinylation. Sirt5 The protective effect of gene knockout significantly reduces ovarian damage caused by CTX, providing an effective prevention and treatment strategy for cyclophosphamide-induced premature ovarian failure.

[0089] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Use of a SIRT5 inhibitor in the preparation of a medicament for preventing and / or treating premature ovarian failure.

2. Use according to claim 1, characterized in that, The SIRT5 inhibitor is compound MC3482.

3. Use according to claim 1, characterized in that, The premature ovarian failure is chemotherapy drug-induced premature ovarian failure.

4. Use according to claim 3, characterized in that, The chemotherapy drug is cyclophosphamide or a derivative thereof.

5. Use according to any one of claims 1 to 4, characterized in that, The medicament exerts its effect by inhibiting macrophage infiltration in the ovary and / or reducing the levels of inflammatory factors TNF-a and IL-1β.

6. Use according to claim 5, characterized in that, The medicament exerts its effect by promoting proliferation of ovarian granulosa cells and / or inhibiting apoptosis of ovarian granulosa cells.

7. Use according to claim 6, characterized in that, The effect is manifested by increasing the ovarian weight, increasing the reserve of primordial and growing follicles and / or increasing the MVH + Total number of oocytes.

8. A pharmaceutical composition for preventing and / or treating premature ovarian failure, characterized by, The effective component of the composition comprises the SIRT5 inhibitor in the use of claim 1 and a pharmaceutically acceptable carrier or excipient.

9. The pharmaceutical composition according to claim 8, wherein The dosage of the medicament is 5 mg / kg, the administration mode is intraperitoneal injection, and the administration frequency is three times a week.

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