Application of hydrogen sulfide donor SPRC in improvement of premature ovarian insufficiency caused by chemotherapeutic drugs

Abstract

The invention provides an application of a hydrogen sulfide donor SPRC in improving early-onset ovarian insufficiency caused by chemotherapeutic drugs, relates to the technical field of biomedicine, and has the technical key point that the application of the hydrogen sulfide donor SPRC in preparing drugs for improving the early-onset ovarian insufficiency is provided. The application aims to solve the technical problems that the ovarian function and reproductive capacity of women are reduced, including the ovarian index is reduced, the number of follicles at all levels is reduced and the number of apoptotic cells in ovarian tissues is remarkably increased, caused by premature ovarian insufficiency at present. Specific verification experiments prove that by supplementing a hydrogen sulfide donor SPRC in a premature ovarian insufficiency mouse model, the weight and the ovarian index of the mouse can be increased, the normal form of the ovarian can be maintained, specifically, the SPRC can improve the number and the quality of follicles in the ovarian by reducing cell apoptosis, and finally ovarian injury caused by POI is rescued.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedical technology, and particularly relates to application of hydrogen sulfide donor SPRC in improving premature ovarian insufficiency caused by chemotherapy drugs. BACKGROUND

[0002] Premature ovarian insufficiency (POI) refers to a disease state that female experiences ovarian function decline before the age of 40, and is mainly manifested as amenorrhea, elevated level of gonadotropin and reduced level of estrogen. Potential causes of POI include iatrogenic factors such as ovarian surgery, radiotherapy and chemotherapy, environmental factors, viral infection, etc. Hormone replacement therapy (HRT) is one of the main treatment methods at present, and is regarded as a physiological replacement of estrogen, but cannot restore ovarian function, and long-term use of hormones may increase the risk of endometrial cancer, breast cancer and other diseases.

[0003] At present, there is a lack of related research on female premature ovarian insufficiency in the prior art, especially premature ovarian insufficiency induced by chemotherapy drugs cyclophosphamide and busulfan. If a method for rescuing premature ovarian insufficiency and affecting female reproductive capacity can be found, it will provide a reference for female patients with the disease in the clinic. SUMMARY

[0004] The purpose of the present application is to solve the technical problem of the limitation of the current treatment of premature ovarian insufficiency.

[0005] In order to achieve the above-mentioned purpose, the present application adopts the following technical solutions:

[0006] Application of hydrogen sulfide donor SPRC in preparation of a drug for improving premature 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 premature ovarian insufficiency by providing high ovarian index, improving ovarian morphology and inhibiting cell apoptosis.

[0010] The present application also provides a drug for improving premature ovarian insufficiency, and the active ingredient of the drug is hydrogen sulfide donor SPRC.

[0011] Preferably, the drug further comprises other excipients acceptable in pharmacy.

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

[0013] Preferably, the medicine is in one of a tablet, a capsule, a powder, a pill, a granule, a solution, a suspension, a syrup, an injection, a suppository, an inhaler or a spray.

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

[0015] 1. The present application aims at the technical problem of the decline of female ovarian function and reproductive capacity caused by early ovarian dysfunction, including the decrease of ovarian index, the decrease of the number of follicles at all levels, and the significant increase of the number of apoptotic cells in ovarian tissue, and provides a new technical solution. The in vivo supplementation of SPRC can improve the influence of POI on ovarian reproductive capacity, including reversing the significant increase of ovarian apoptotic cells and the decrease of the number of follicles in the POI group, protecting the ovary from damage by inflammation, and improving follicle quality.

[0016] 2. The hydrogen sulfide donor SPRC in the present application can provide a new solution or reference for the protection of the ovaries and fertility of patients with early ovarian dysfunction. BRIEF DESCRIPTION OF DRAWINGS

[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 the present application: a is the statistical column chart of the body weight in each group; b is the statistical column chart of the ovarian weight in each group; c is the statistical chart of the ovarian index in each group; wherein, *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 all levels in the ovaries of female mice in Example 2 of the present application: a is a representative section chart of the ovaries in each group; b is a representative picture of the primary follicle, the primary follicle, the secondary follicle and the antral follicle; c is a statistical chart of the number of follicles at all levels in each group; wherein, *P<0.05, **P<0.01, ***P<0.005, ns represents no significant difference.

[0019] Figure 3 The effects of POI and SPRC on the apoptosis of female ovarian cells in Example 3 of the present application: a is a TUNEL representative chart of ovarian sections in each group; b is a TUNEL content statistical chart of ovarian sections in each group; c is a representative chart of Western blotting of Bcl-2, BAX and GAPDH in each group; d is a column chart of the relative expression level of Bcl-2, BAX and GAPDH in each group; wherein, *P<0.05. DETAILED DESCRIPTION

[0020] The application will be further described in detail below in combination with specific examples.

[0021] The application of the hydrogen sulfide donor SPRC in the preparation of a drug for improving early ovarian dysfunction caused by a chemotherapeutic drug, specifically, the chemotherapeutic drug is cyclophosphamide and white blood cell poison.

[0022] In an embodiment, the hydrogen sulfide donor SPRC improves the ovarian damage caused by early ovarian dysfunction by providing a high ovarian index, improving ovarian morphology and inhibiting cell apoptosis.

[0023] The hydrogen sulfide donor SPRC is S-propargyl-cysteine, which is an organic compound extracted from garlic. Its chemical formula is as follows:

[0024]

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

[0026] The above content will be described in combination with specific verification experiments as follows:

[0027] Experimental materials and sources:

[0028] The materials and reagents used in the application can be obtained from commercial channels unless otherwise specified.

[0029]

[0030]

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

[0032] Example 1: Effect of hydrogen sulfide donor SPRC on the body weight and ovarian index of early ovarian dysfunction mice

[0033] 1. Model establishment

[0034] Eight-week-old ICR female mice were selected and 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 2HE staining of the ovarian sections of each group of mice shown in Figure a, it is obvious from the figure that the ovaries of the SPRC group are larger and the number of follicles is increased compared to the POI group. Figure 2 b The typical images of the original follicle, primary follicle, secondary follicle, antral follicle, and atretic follicle are presented in sequence to facilitate the counting of each type of follicle in each group.

[0046] 2. Follicle count

[0047] To avoid repeated counting, follicles were counted every 2 tissue sections, which could well show the number of follicles at each stage in the ovarian tissue.

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

[0049] Example 3: Effect of hydrogen sulfide donor SPRC on ovarian cell apoptosis in premature ovarian insufficiency mice

[0050] 1. Immunofluorescence of ovarian paraffin sections

[0051] To further explore the apoptosis mechanism caused by POI, in this example, the ovarian tissues of each group were taken, and the TUNEL staining of the mouse ovarian paraffin sections was performed. The paraffin sections were prepared according to the above method. After deparaffinization and hydration with gradient alcohol, 20 μg / ml of proteinase K was added to each section, and the reaction was carried out at room temperature for 20 minutes. PBS was washed 3 times to completely wash away the proteinase K. 50 μL of TUNEL detection solution was added to each sample, and a suitable size of sealing film was cut and covered on the sample to prevent evaporation of the TUNEL detection solution. The sample was incubated at 37°C in the dark for 60 minutes. Finally, the sample was mounted with DAPI-containing anti-fluorescence quencher, and then observed under an immunofluorescence microscope and scanned. The number of apoptotic cells was counted using Image J software.

[0052] The results are shown in Figure 3 As shown in Figures a and b, the number of apoptotic cells after TUNEL staining in the POI group was significantly increased compared with the CON group, but the number of apoptotic cells in the SPRC group was significantly reduced.

[0053] 2. Ovarian immunoblotting

[0054] Take the mouse ovary, grind in 200 μL RIPA lysis buffer and 2 μL protease inhibitor to homogenate, take the supernatant after centrifugation. Use BCA protein kit to detect protein concentration and required lysis buffer volume, and add protein and lysis buffer according to the measured data respectively. Add 7 μL of LDS loading buffer and 0.5 μL of β-mercaptoethanol to each tube, then heat the proteins in each group to 100°C for 5 minutes and store in a -20°C refrigerator. Prepare the electrophoresis solution and transfer membrane solution. Put the electrophoresis tank into the ice bath prepared in advance. Use constant current voltage 120V, work for 1 hour and 10 minutes. Cut the PVDF membrane to the appropriate size for transfer, set the current to 0.2A, and work for 1.5 hours. After transfer, block with blocking solution. Dilute BAX, Bcl-2, GAPDH antibodies (1:1000) with Western primary antibody diluent, and incubate the membrane overnight (4°C). The next day, incubate the secondary antibody in the dark for 1 hour. Finally, place the membrane in the immunoprotein imaging system for scanning, develop and expose, and detect the protein expression levels of apoptosis-related factors BAX and Bcl-2.

[0055] The results are shown in Figure 3 c, d, compared with the CON group, the POI group mouse apoptosis factor Bcl-2 / Bax protein expression level ratio was significantly reduced. But compared with the POI group, the Bcl-2 / Bax protein expression level ratio of the SPRC middle dose and high dose groups in the ovary was significantly increased.

[0056] Based on the analysis of the above verification test, the hydrogen sulfide donor SPRC can improve the number and quality of ovarian follicles in the ovary by reducing apoptosis, and ultimately rescue the ovarian damage caused by POI.

[0057] In summary, the present application provides the application of hydrogen sulfide donor SPRC in improving early ovarian dysfunction induced by chemotherapy drugs cyclophosphamide and busulfan, and the specific verification test proves that hydrogen sulfide donor SPRC can improve the ovarian damage caused by early ovarian dysfunction in females. Specifically, the experimental results of the present application prove that: supplementing hydrogen sulfide donor SPRC in the early ovarian dysfunction mouse model can improve the body weight and ovarian index of mice, maintain the normal morphology of the ovary, including increasing the number of follicles at all levels and reducing the number of apoptotic cells in the ovary.

Claims

1. Use of hydrogen sulfide donor SPRC in the preparation of a drug for improving premature ovarian dysfunction.

2. The use of hydrogen sulfide donor SPRC according to claim 1 in the preparation of a drug for improving early ovarian dysfunction, characterized in that: The premature ovarian dysfunction is premature ovarian dysfunction caused by a chemotherapeutic drug.

3. The use of hydrogen sulfide donor SPRC according to claim 2 in the preparation of a drug for improving early ovarian dysfunction, characterized in that: The chemotherapeutic drug is cyclophosphamide and busulfan.

4. The use of hydrogen sulfide donor SPRC according to claim 3 for the preparation of a medicament for improving early ovarian insufficiency, characterized in that: The hydrogen sulfide donor SPRC improves the ovarian damage caused by premature ovarian dysfunction by providing a high ovarian index, improving ovarian morphology, and inhibiting cell apoptosis.

5. A medicament for improving early ovarian insufficiency, characterized by: The active ingredient of the drug is the hydrogen sulfide donor SPRC.

6. The medicament for improving early ovarian dysfunction according to claim 5, characterized by: The drug also includes other excipients acceptable in pharmacy.

7. The medicament for improving early ovarian dysfunction according to claim 5, characterized by: The drug is in oral or non-oral dosage form. 8.The drug for improving early ovarian dysfunction according to claim 5, characterized in that: The drug is one of tablets, capsules, powders, pills, granules, solutions, suspensions, syrups, injections, suppositories, inhalers, or sprays.

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