Application of hydrogen sulfide donor SPRC in improving early ovarian dysfunction caused by chemotherapy drugs

Abstract

The application provides application of a hydrogen sulfide donor SPRC in improving early-onset ovarian dysfunction caused by a chemotherapy drug, and relates to the technical field of biomedicine, and the technical key points are as follows: the application provides application of the hydrogen sulfide donor SPRC in preparing a drug for improving early-onset ovarian dysfunction. The present application aims at the technical problems of early-onset ovarian dysfunction causing the decline of female ovarian function and reproductive capacity, including the decline of ovarian index, the reduction of the number of follicles at all levels, and the significant increase of the number of apoptotic cells in ovarian tissue. The specific verification experiments prove that supplementing the hydrogen sulfide donor SPRC in a mouse model of early-onset ovarian dysfunction can improve the body weight and ovarian index of the mouse, and maintain the normal morphology of the ovary. Specifically, the SPRC can improve the number and quality of follicles in the ovary by reducing cell apoptosis, and ultimately rescue the ovarian damage caused by POI.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more particularly to the application of hydrogen sulfide donor SPRC in improving early-onset ovarian insufficiency caused by chemotherapy drugs. Background Technology

[0002] Premature ovarian insufficiency (POI) refers to a condition in which women experience a decline in ovarian function before the age of 40. The main symptoms include amenorrhea, elevated gonadotropin levels, and decreased estrogen levels. Potential causes of POI include iatrogenic factors such as ovarian surgery, radiation therapy, and chemotherapy, as well as environmental factors and viral infections. Hormone replacement therapy (HRT) is currently one of the main treatment methods, considered a physiological replacement of estrogen, but it cannot restore ovarian function, and long-term use of hormones may increase the risk of diseases such as endometrial cancer and breast cancer.

[0003] Currently, there is a lack of relevant research on early-onset ovarian insufficiency in women, especially early-onset ovarian insufficiency induced by chemotherapy drugs such as cyclophosphamide and busulfan. Discovering a method to salvage early-onset ovarian insufficiency and thus its impact on female fertility would provide valuable insights for women with this condition in clinical practice. Summary of the Invention

[0004] The purpose of this application is to address the technical limitations of current treatments for early-onset ovarian insufficiency.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] Application of hydrogen sulfide donor SPRC in the preparation of drugs to improve early-onset ovarian insufficiency.

[0007] Preferably, the premature ovarian insufficiency is premature ovarian insufficiency caused by chemotherapy drugs.

[0008] Preferably, the chemotherapy drugs are cyclophosphamide and busulfan.

[0009] Preferably, the hydrogen sulfide donor SPRC improves ovarian damage caused by early-onset ovarian insufficiency by providing a high ovarian index, improving ovarian morphology, and inhibiting cell apoptosis.

[0010] This application also provides a drug for improving early-onset ovarian insufficiency, wherein the active ingredient of the drug is a hydrogen sulfide donor SPRC.

[0011] Preferably, the drug also includes other pharmaceutically acceptable excipients.

[0012] Preferably, the drug is an oral dosage form or a non-oral dosage form.

[0013] Preferably, the drug is one of the following: tablets, capsules, powders, pills, granules, solutions, suspensions, syrups, injections, suppositories, inhalers, or sprays.

[0014] Compared with the prior art, this application has the following beneficial effects:

[0015] 1. This application addresses the technical problem of decreased ovarian function and reproductive capacity in women caused by premature ovarian insufficiency, including decreased ovarian index, reduced number of follicles at all stages, and significantly increased number of apoptotic cells in ovarian tissue. It provides a new technical solution that can improve the effect of POI on ovarian reproductive capacity by supplementing SPRC in vivo, including reversing the significant increase in apoptotic cells and the decrease in the number of follicles in the POI group, protecting the ovary from inflammatory damage and improving follicle quality.

[0016] 2. The hydrogen sulfide donor SPRC in this application can be used to provide a new approach or reference for the protection of ovaries and fertility in patients with early-onset ovarian insufficiency. Attached Figure Description

[0017] Figure 1 The effects of the control group, the cyclophosphamide combined with busulfan group, and the rescue group on the body weight and ovarian weight of mice in Example 1 of this invention are shown in the following figures: a) is a bar chart of body weight statistics for each group; b) is a bar chart of ovarian weight statistics for each group; c) is a statistical chart of ovarian index for each group; where *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

[0018] Figure 2 The effects of POI and SPRC on the number of follicles at each stage of female ovaries in Example 2 of this invention are shown in Figure a: a) representative ovarian slices of each group; b) representative images of primordial follicles, primary follicles, secondary follicles, and antral follicles; c) statistical chart of the number of follicles at each stage of each group; where *P<0.05, **P<0.01, ***P<0.005, and ns represent no significant difference.

[0019] Figure 3 The effects of POI and SPRC on female ovarian cell apoptosis in Example 3 of this invention are shown in the following figures: a) Representative TUNEL distribution of ovarian sections in each group; b) Statistical chart of TUNEL content in ovarian sections in each group; c) Representative immunoblot distribution of Bcl-2, BAX, and GAPDH in each group; d) Bar chart of relative expression levels of Bcl-2, BAX, and GAPDH in each group; where *P < 0.05. Detailed Implementation

[0020] The present invention will be further described in detail below with reference to specific embodiments.

[0021] The application of hydrogen sulfide donor SPRC in the preparation of drugs to improve early-onset ovarian insufficiency, wherein the early-onset ovarian insufficiency is caused by chemotherapy drugs, specifically, the chemotherapy drugs are cyclophosphamide and busulfan.

[0022] In one embodiment, the hydrogen sulfide donor SPRC improves ovarian damage caused by early-onset ovarian insufficiency by providing a high ovarian index, improving ovarian morphology, and inhibiting apoptosis.

[0023] The hydrogen sulfide donor SPRC is S-propynylcysteine, an organic compound extracted from garlic. Its chemical formula is shown below:

[0024]

[0025] In addition, this application also provides a drug for improving early-onset ovarian insufficiency, wherein the active ingredient of the drug is a hydrogen sulfide donor SPRC. In one embodiment, the drug further includes other pharmaceutically acceptable excipients. Furthermore, the drug is an oral or non-oral dosage form, specifically, the drug is one of the following: tablets, capsules, powders, pills, granules, solutions, suspensions, syrups, injections, suppositories, inhalers, or sprays.

[0026] The above content will be explained in conjunction with specific verification experiments:

[0027] Experimental materials and their sources:

[0028] Unless otherwise specified, all materials and reagents used in this application are commercially available.

[0029]

[0030]

[0031] The animals used in this application were 8-week-old female ICR mice from the Experimental Animal Center of Nantong University. The animals were housed in a constant temperature environment of 23±2℃ and humidity of 40±10%, with a 12-hour day-night cycle. The feed used was ordinary irradiated feed (AIN-93).

[0032] Example 1: Effects of hydrogen sulfide donor SPRC on body weight and ovarian index in mice with early-onset ovarian insufficiency

[0033] 1. Model Establishment

[0034] Eight-week-old female ICR mice were randomly divided into three groups.

[0035] Control group (CON group): Mice were injected intraperitoneally with physiological saline.

[0036] Model group (POI group) and rescue group (SPRC group): Mice were injected intraperitoneally with cyclophosphamide 120mg / kg and busulfan 30mg / kg 0.1mL each.

[0037] In this application, two drugs were used in combination to create a POI model in ICR mice.

[0038] On the second day after injection, mice in the rescue group were intraperitoneally administered SPRC solutions at three different concentration gradients (SPRC-Low 25 mg / kg, SPRC-Middle 50 mg / kg, SPRC-High 100 mg / kg) daily for two weeks. Mice in the other two groups were intraperitoneally injected with 0.1 mL of physiological saline daily. Ovarian tissue was collected from the mice after euthanasia.

[0039] 2. Weight and ovarian index

[0040] Two weeks after SPRC injection, mice were sacrificed, and their weight was recorded. The mice's abdomens were then disinfected with 75% alcohol, and the ovaries were removed. Surrounding adipose tissue was removed to obtain purified bilateral ovaries, which were then weighed and recorded. The ovarian organ index was calculated using the following formula: Ovarian organ index = Ovarian weight / Body weight × 1000%.

[0041] The results after comparing the weight and ovarian index of each group are as follows: Figure 1 As shown in the figure, compared with the CON group, the body weight of mice in the POI group was ( Figure 1 a) Ovarian index ( Figure 1 b) and c) were significantly smaller. However, SPRC injection significantly improved the effects of POI on mouse body weight and ovarian index. Compared with the POI group, the SPRC group had significantly larger body weight and ovarian index.

[0042] Example 2: Effects of hydrogen sulfide donor SPRC on follicular development in mice with early-onset ovarian insufficiency

[0043] 1. Ovarian histological analysis

[0044] To investigate the reasons for the above results, in this embodiment, mouse ovaries in each group were fixed overnight in 4% paraformaldehyde, graded, dehydrated, and embedded in paraffin. Sections were cut to 5 μm thickness, numbered sequentially, dewaxed, and then graded with alcohol for hydration before hematoxylin-eosin (HE) staining. Tissue images were taken under a 5X microscope, and follicles at different developmental stages were counted according to their classification as primordial follicles, primary follicles, secondary follicles, and antral follicles.

[0045] The results are as follows Figure 2Figure a shows HE staining of ovarian sections from mice in each group. It is clear from the figure that the SPRC group had larger ovaries and a greater number of follicles compared to the POI group. Figure 2 b presents typical diagrams of primordial follicles, primary follicles, secondary follicles, antral follicles, and atretic follicles in sequence to facilitate the statistical analysis of different types of follicles in each group.

[0046] 2. Follicle count

[0047] To avoid double counting, follicles are counted every two tissue samples, which effectively shows the number of follicles at each stage in the ovarian tissue.

[0048] The results are as follows Figure 2 As shown in Figure c, compared with the CON group, the number of follicles at each stage was significantly reduced and the number of atretic follicles increased in the POI group. However, SPRC injection significantly alleviated the reduction in the number of follicles at each stage and the increase in the number of atretic follicles caused by POI, and the rescue effect was more significant with increasing SPRC concentration.

[0049] Example 3: Effect of hydrogen sulfide donor SPRC on ovarian cell apoptosis in mice with early-onset ovarian insufficiency

[0050] 1. Immunofluorescence of paraffin sections of ovaries

[0051] To further investigate the POI-induced apoptosis mechanism, in this embodiment, ovarian tissue from each of the above groups was collected, and mouse ovarian paraffin sections were stained with TUNEL. Paraffin sections were prepared according to the above method. After dewaxing and hydration with gradient alcohols, 20 μg / ml proteinase K was added to each section and incubated at room temperature for 20 minutes. The sections were washed three times with PBS to thoroughly remove the proteinase K. 50 μL of TUNEL detection solution was added to each sample, and a sealing film of appropriate size was cut to cover the sample to prevent the TUNEL detection solution from evaporating. The sections were incubated at 37°C in the dark for 60 minutes. Finally, the sections were mounted with an anti-fluorescence quencher containing DAPI, observed under an immunofluorescence microscope, and scanned. The apoptosis was then counted using ImageJ software.

[0052] The results are as follows Figure 3 As shown in a and b, the number of apoptotic cells in the POI group was significantly higher than that in the CON group after TUNEL staining, but the number of apoptotic cells in the SPRC group was significantly lower.

[0053] 2. Ovarian immune protein imprinting

[0054] Mouse ovaries were collected and homogenized in 200 μL of RIPA lysis buffer and 2 μL of protease inhibitor. The supernatant was collected after centrifugation. Protein concentration and required lysis buffer volume were determined using a BCA protein kit, and protein and lysis buffer were added according to the measured data. 7 μL of LDS loading buffer and 0.5 μL of β-mercaptoethanol were added to each tube, and the proteins were incubated at 100°C for 5 minutes and stored at -20°C. Electrophoresis buffer and transfer buffer were prepared. The electrophoresis tank was placed in a pre-prepared ice bath. A constant current of 120V was used for 1 hour and 10 minutes. Appropriately sized PVDF membranes were used for transfer at 0.2A for 1.5 hours. After transfer, the membranes were blocked with blocking buffer. BAX, Bcl-2, and GAPDH antibodies were diluted 1:1000 with Western blotting buffer, and the membranes were incubated overnight (4°C). The next day, the membranes were incubated with secondary antibody for 1 hour in the dark. Finally, the membrane was placed on an immunoprotein imaging system for scanning, development, and exposure to detect the protein expression levels of apoptosis-related factors BAX and Bcl-2.

[0055] The results are as follows Figure 3 As shown in c and d, compared with the CON group, the Bcl-2 / Bax protein expression ratio in the POI group was significantly decreased. However, compared with the POI group, the ovarian Bcl-2 / Bax protein expression ratio was significantly increased in the medium- and high-dose SPRC groups.

[0056] Based on the above verification experiment analysis, it can be concluded that the hydrogen sulfide donor SPRC can increase the number and quality of follicles in the ovary by reducing cell apoptosis, and ultimately salvage ovarian damage caused by POI.

[0057] In summary, this application provides the application of hydrogen sulfide donor SPRC in improving premature ovarian insufficiency induced by the chemotherapy drugs cyclophosphamide and busulfan. Specific verification experiments have demonstrated that hydrogen sulfide donor SPRC can improve ovarian damage caused by premature ovarian insufficiency in females. Specifically, the experimental results of this application demonstrate that supplementing with hydrogen sulfide donor SPRC in a mouse model of premature ovarian insufficiency can increase mouse body weight and ovarian index, maintain normal ovarian morphology, including increasing the number of follicles at each stage and reducing apoptotic ovarian cells.

Claims

1. The application of hydrogen sulfide donor SPRC in the preparation of drugs to improve early-onset ovarian insufficiency induced by chemotherapy, characterized in that: The chemical formula of the hydrogen sulfide donor SPRC is shown below: 。 2. The application of the hydrogen sulfide donor SPRC according to claim 1 in the preparation of a drug to improve early-onset ovarian insufficiency induced by chemotherapy drugs, characterized in that: The chemotherapy drugs are cyclophosphamide and busulfan.

Images