CN117919253A - Application of xanthine compounds in the preparation of drugs for activating primordial follicles - Google Patents

Abstract

The present invention provides an application of xanthine compounds in preparing a drug for activating primordial follicles, belonging to the field of biotechnology. The present invention finds that xanthine compounds promote primordial follicle activation and can be used as an oral drug for treating patients with premature ovarian insufficiency, having good application prospects.

Description

Application of xanthine compounds in the preparation of drugs for activating primordial follicles

Technical Field

The invention belongs to the field of biotechnology, and in particular relates to the application of xanthine compounds in the preparation of medicines for activating primordial follicles.

Background technique

Follicles are the basic units of ovarian structure and function. According to the different stages of follicle growth and development, they can be divided into primordial follicles, primary follicles, secondary follicles, antral follicles and mature follicles. The number of primordial follicles is limited and cannot be regenerated, so their number is considered to represent female fertility and directly determines the length of female reproductive life. Environmental pollution and anticancer drugs can easily lead to overactivation or apoptosis of primordial follicles, ultimately causing premature ovarian insufficiency (POI). This type of POI patient still has a small number of primordial follicles in the ovaries, but it is difficult to be activated under physiological conditions, leading to infertility. In addition, older women also have a small number of primordial follicles and decreased fertility. These women cannot be treated with traditional assisted reproductive technology. The current method is mainly through in vitro activation (IVA) and then autologous transplantation. This method is subject to many limitations, requires surgery, is prone to DNA damage and cancer risks, and has a low success rate. There is currently no treatment plan to restore fertility in POI patients by promoting primordial follicle activation through oral medication. It is crucial to conduct research on the molecular mechanisms of maintenance and activation of human primordial follicles and to elucidate the molecular mechanisms of primordial follicle activation and follicle development for the diagnosis and treatment of these patients.

Before birth, the syncytium ruptures to form primordial follicles and constitute the primordial follicle pool, most of which remain dormant, and only a few are periodically activated to enter a growth state, eventually maturing and ovulating. The most studied molecular and signaling mechanism for primordial follicle activation is the production of KIT ligand (KITL) promoted by rapamycin kinase (mTOR) in pregranulosa cells. KITL binds to its receptor, KIT proto-oncogene receptor tyrosine kinase (c-KIT), and activates the phosphatidylinositol 3-kinase (PI3K)-protein kinase B (Akt) signaling pathway in oocytes.

Xanthine compounds mainly include four categories: xanthine, caffeine, theobromine and theophylline. Xanthine is produced by the oxidation of hypoxanthine; caffeine can be metabolized into 1,7-dimethylxanthine; theobromine can be metabolized into pentoxifylline. Theophylline drugs can be divided into the following four categories: 1) Theophylline combined with different salts or bases, mainly including theophylline, aminophylline, theophylline sodium acetate, theophylline, theophylline sodium glycinate, etc.; 2) Theophylline N-7 position combined with different groups, mainly including dihydroxypropyltheophylline, theophylline acetate, ethylhydroxytheophylline, doxofylline, 7-(2-chloroethyl)theophylline, 7-ethyltheophylline, 7-(2-hydroxypropyl)theophylline, lomiphylline, isobutyltheophylline, etc.; 3) Theophylline 3-methyl is substituted by different groups, mainly including enprophylline, furofylline, arofylline, etc.; 4) Theophylline other sites are substituted by different groups, mainly including 8-bromophylline, lisofylline, istraphylline, 8-benzyltheophylline, etc.

Summary of the invention

In order to overcome the shortcomings and deficiencies in the prior art, the primary purpose of the present invention is to provide the use of xanthine compounds in the preparation of drugs for activating primordial follicles.

The purpose of the present invention is achieved through the following technical solutions:

Use of a xanthine compound in the preparation of a drug for treating premature ovarian insufficiency, wherein the xanthine compound is any one or more of the following compounds in Group A, Group B, Group C, and Group D:

Group A compounds: xanthine, hypoxanthine;

Group B compounds: caffeine, 1,7-dimethylxanthine;

Group C compounds: theobromine, pentoxifylline;

Group D compounds: theophylline sodium acetate, theophylline, theophylline sodium glycinate, theophylline acetate, ethoxyphylline, doxofylline, 7-(2-chloroethyl)theophylline, 7-ethyltheophylline, 7-(2-hydroxypropyl)theophylline, lomiphylline, isobutyltheophylline, enprophylline, furofylline, arofylline, 8-bromophylline, lisofylline, istradefylline and 8-benzyltheophylline.

Furthermore, the application is the application of xanthine compounds in the preparation of drugs for activating primordial follicles.

Furthermore, the drug for activating primordial follicles is a drug for promoting the transformation of primordial follicles into growth follicles.

Furthermore, in the application, the dosage of the xanthine compound is calculated based on the individual body weight, and the mass of the xanthine compound administered per 50 kg body weight is as follows:

Furthermore, the primordial follicles are primordial follicles of mice or humans.

Furthermore, the drug is administered orally or by intravenous injection.

An in vitro activation method for primordial follicles (not for the purpose of disease diagnosis and treatment), wherein ovaries are cultured in a culture medium supplemented with xanthine compounds; the xanthine compounds are any one or more combinations of compounds in the following groups A, B, C, and D:

Group A compounds: xanthine, hypoxanthine;

Group B compounds: caffeine, 1,7-dimethylxanthine;

Group C compounds: theobromine, pentoxifylline;

Group D compounds: theophylline sodium acetate, theophylline, theophylline sodium glycinate, theophylline acetate, ethoxyphylline, doxofylline, 7-(2-chloroethyl)theophylline, 7-ethyltheophylline, 7-(2-hydroxypropyl)theophylline, lomiphylline, isobutyltheophylline, enprophylline, furofylline, arofylline, 8-bromophylline, lisofylline, istradefylline and 8-benzyltheophylline.

Furthermore, the concentrations of the xanthine compounds in the xanthine compound solution are as follows:

Furthermore, the ovary is a mouse or human ovary.

Furthermore, the mice are newborn mice.

Furthermore, the culture is carried out at a constant temperature of 37±2°C and 5%±1% CO ₂ for 4 days.

Compared with the prior art, the present invention has the following advantages and beneficial effects:

POI is a relatively serious ovarian disease clinically. The disease is showing a trend of becoming younger, and the incidence rate is constantly increasing, which seriously affects female fertility and physical health. Only when the primordial follicles of female mammals (including humans) are precisely regulated and orderly activated, can the normal reproductive lifespan of females (women) be maintained. In the present invention, we found that a group of xanthine compounds have the effect of promoting the activation of primordial follicles in the mouse ovaries. Under the action of drug treatment, the number of growing follicles in the treated group mice increased significantly. These drugs can be used for oral administration and intravenous injection, so based on the present invention, primordial follicles can be activated orally to treat POI patients, which has a good application prospect for saving their fertility.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a morphological diagram of the ovaries of 3dpp newborn mice cultured for 4 days with different xanthine compounds added;

Figure 2 is a diagram of follicle counting results, where A is the statistical count of growing follicles; B is the statistical count of primordial follicles; C is the statistical count of total follicles; scale: 50 μm;

FIG. 3 is a graph showing the results of theophylline activating mouse primordial follicles.

Detailed ways

The present invention is further described in detail below in conjunction with embodiments and drawings, but the embodiments of the present invention are not limited thereto.

The experimental methods in the following examples without specific experimental conditions are usually carried out under conventional experimental conditions or the experimental conditions recommended by the manufacturer. The materials and reagents used are commercially available unless otherwise specified.

Example 1

1.1 Reagents for in vitro culture

1.1.1 DMEM/F12 medium (250 ml) is prepared as shown in Table 1:

Table 1 DMEM-F12 culture medium

Note: SP is a commercially available penicillin and streptomycin dual antibody, added to the culture medium at a rate of 100 μL per 100 mL of culture medium. After the volume is fixed to 250 mL, filter with a 0.22 μm filter, operate in a clean bench, seal with a sealing film and store at 4°C.

1.1.2 Phosphate buffered solution (PBS, 1L, pH: 7.2) was prepared as shown in Table 2:

Table 2 PBS buffer

Make up to 1 L with deionized water, sterilize by high pressure, and store at 4°C.

1.1.3 Drug concentration configuration of each experimental group culture medium: Xanthine, caffeine, pentoxifylline, theophylline sodium acetate, theophylline sodium acetate, theophylline choline and theophylline sodium glycinate were dissolved in PBS buffer to prepare concentrated stock solutions; 1,7-dimethylxanthine, theobromine, theophylline acetate, ethoxyphylline, doxofylline, 7-(2-chloroethyl) theophylline, 7-ethyltheophylline, 7-(2-hydroxypropyl) theophylline, lomiphylline, isobutyltheophylline, enprophylline, furofylline, arofylline, 8-bromophylline, lisofylline, istraphylline and 8-benzyltheophylline were dissolved in dimethyl sulfoxide (DMSO) to prepare concentrated stock solutions; hypoxanthine was directly dissolved in culture medium. The final concentrations of various drugs are shown in Table 3.

Table 3 Drug concentrations in the culture medium of each experimental group

1.2 Experimental methods

1.2.1 Isolation of mouse ovaries

1) All surgical instruments, glass dishes, etc. are sterilized, and the cells are sterilized by irradiating with ultraviolet light for half an hour.

2) In this experiment, 3-day-old ICR female mice were used as materials. The mice were killed by cervical dislocation and dissected. The head, abdomen and tail with bilateral kidneys of the mice were separated and placed in cold PBS.

3) Under a stereo microscope, remove the mouse ovary and ovarian membrane together, and use an injection needle to separate the membrane surrounding the ovary.

1.2.2 In vitro culture of mouse ovaries

1) Add 3 mL of DMEM/F12 medium to each well of a six-well plate, then add the prepared drug solution. Add DMSO to the control group, pipette and mix well, place a 0.44 μm culture membrane on each well, and incubate at 37°C in a carbon dioxide incubator for 30 minutes.

2) Place the separated ovaries on a culture membrane and culture them in a 37°C constant temperature carbon dioxide incubator;

3) The culture medium was changed every two days for a total of 4 days.

1.2.3 Ovarian embedding, sectioning and hematoxylin staining

1) Sample fixation: Mouse ovaries were fixed in 4% paraformaldehyde at 4°C for 12-16 hours;

2) Alcohol concentration gradient dehydration and transparency: 70% alcohol, 80% alcohol, 95% alcohol/eosin staining solution, 95% alcohol, anhydrous alcohol, anhydrous ethanol/xylene (volume 1:1), xylene, each gradient for 5 minutes, and the last xylene was blotted dry with filter paper.

3) Wax immersion and embedding: The ovaries were immersed in wax at 60°C for 2 hours in a water-tight environment (with sealing film) and then transferred to a paraffin box.

4) Tissue sections: The thickness of the sections is generally 5 μm.

5) Spreading: After the wax strip is fully stretched in a 42℃ water bath, pick it up with a glass slide.

6) Bake slices: 42°C overnight.

1.2.5 Hematoxylin staining

1) Dewaxing and rehydration: The slices were sequentially washed with xylene, xylene, anhydrous alcohol, anhydrous alcohol, 95% alcohol, 80% alcohol, and 70% alcohol for 5 minutes each, and finally rinsed with deionized water.

2) Staining: Stain with hematoxylin for about 1 minute and rinse with water.

3) Dehydration and sealing: Dehydrate and seal the slides in sequence: 70% alcohol, 80% alcohol, 95% alcohol, anhydrous alcohol, anhydrous alcohol, xylene, and xylene, each gradient for 3 minutes, and finally seal the slides with neutral resin.

1.3 Ovarian follicle count

The stained continuous sections were counted, and one section was counted for every five consecutive sections. The final statistical result was the sum of the count results and multiplied by 5. According to the morphological characteristics of the follicles, they can be divided into: primordial follicles: three to five flat granulosa cells around and an oocyte in the middle; activated follicles: a layer of cuboidal granulosa cells around or a layer of cuboidal and flat mixed granulosa cells around and an enlarged oocyte in the middle.

1.4 Ovarian follicle count results

The results are shown in Figure 1 and Figure 2, where:

Figure 1: Morphological comparison between the control group and the drug-added group, arrows point to activated growing follicles; scale bar: 50 μm;

Figure 2: (A) Statistical counts of growing follicles: Compared with the control group, 20 μM xanthine, 50 μM caffeine, 100 μM 1,7-dimethylxanthine, 20 μM theobromine, 50 μM pentoxifylline, 200 μM hypoxanthine, 100 μM theophylline sodium acetate, 50 μM choline, 5 μM theophylline sodium glycinate, 20 μM theophylline acetate, 50 μM ethoxyphylline, 1 μM doxofylline, 50 μM 7-(2-chloroethyl)theophylline, 50 μM 7-ethyltheophylline, 50 μM 7-(2-hydroxypropyl)theophylline, 5 μM lomiphylline, 20 μM isobutylphylline, 10 μM enprophylline, 50 μM furanyl, 10 μM arofylline, 10 μM 8-bromophylline, 5 μM lisofylline, 1 μM istradefylline and 5 μM 8-Benzyltheophylline can significantly promote the activation of primordial follicles (P<0.05); (B) Primordial follicles count; (C) Total follicles count, there was no significant difference between the experimental group and the control group.

The present invention adds xanthine compounds to the ovaries of 3dpp newborn mice or does not add drugs and cultures them for 4 days to observe the ovarian structure. Statistical analysis of serial sections showed that compared with the control group (371.67±20.21, data represent the number of activated primordial follicles in each ovary), 20μM xanthine (493.33±47.52), 50μM caffeine (533.33±54.85), 100μM 1,7-dimethylxanthine (676.67±50.08), 20μM theobromine (490±37.75), 50μM pentoxifylline (671.67±56.86), 200μM hypoxanthine (590±42.72), 100μM theophylline sodium acetate (598.33±53.46), 50μM theophylline choline (653.33±90.88), and 5μM theophylline sodium glycinate (676.67±55. 3), 20μM theophylline acetate (545±35), 50μM ethoxyphylline (633.33±52.52), 1μM doxofylline (633.333±52.52), 50μM 7-(2-chloroethyl) theophylline (500±45), 50μM 7-ethyl theophylline (583.33±43.68), 50μM 7-(2-hydroxypropyl) theophylline (655±68.74), 5μM lomiphylline (568.33±55.08), 20μM isobutylphylline (488.33±46.46), 10μM enprophylline (735±37.75), 50μM furanofylline (568.33±55.3), 10μM arofylline (483.33±38.19), 10μM 8-bromophylline (480±39.69), 5μM lisofylline (745±18.03), 1μM istradefylline (580±78.58) and 5μM 8-benzylphylline (523.33±55.3) can significantly promote the activation of primordial follicles.

In addition, due to the action of xanthine compounds, the increase in growing follicles led to a slight decrease in the number of primordial follicles, but had no effect on the total number of ovarian follicles in mice, indicating that xanthine compounds had no effect on the apoptosis of primordial follicles and growing follicles.

It is worth noting that during the experiment, we found that theophylline in the concentration range of 10 to 50 μM did not promote the activation of mouse primordial follicles ( Figure 3 ).

The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims (10)

1. Use of a xanthine compound in the preparation of a drug for treating premature ovarian insufficiency, characterized in that the xanthine compound is any one or more of the following compounds in Group A, Group B, Group C, and Group D: Group A compounds: xanthine, hypoxanthine; Group B compounds: caffeine, 1,7-dimethylxanthine; Group C compounds: theobromine, pentoxifylline; Group D compounds: theophylline sodium acetate, theophylline, theophylline sodium glycinate, theophylline acetate, ethoxyphylline, doxofylline, 7-(2-chloroethyl)theophylline, 7-ethyltheophylline, 7-(2-hydroxypropyl)theophylline, lomiphylline, isobutyltheophylline, enprophylline, furofylline, arofylline, 8-bromophylline, lisofylline, istradefylline and 8-benzyltheophylline. 2. The use according to claim 1, characterized in that: The application is the application of xanthine compounds in the preparation of medicines for activating primordial follicles. 3. The use according to claim 1, characterized in that: The drug for activating primordial follicles is a drug for promoting the transformation of primordial follicles into growth follicles. 4. The use according to any one of claims 1 to 3, characterized in that: In the application, the dosage of the xanthine compound is calculated based on the individual body weight, and the mass of the xanthine compound administered per 50 kg body weight is as follows: 5. The use according to any one of claims 1 to 3, characterized in that: The primordial follicles are primordial follicles of mice or humans; The drug is administered orally or by intravenous injection. 6. An in vitro activation method for primordial follicles, characterized in that: ovaries are cultured in a xanthine compound solution; the xanthine compound is any one or more of the following compounds in Group A, Group B, Group C, and Group D: Group A compounds: xanthine, hypoxanthine; Group B compounds: caffeine, 1,7-dimethylxanthine; Group C compounds: theobromine, pentoxifylline; Group D compounds: theophylline sodium acetate, theophylline, theophylline sodium glycinate, theophylline acetate, ethoxyphylline, doxofylline, 7-(2-chloroethyl)theophylline, 7-ethyltheophylline, 7-(2-hydroxypropyl)theophylline, lomiphylline, isobutyltheophylline, enprophylline, furofylline, arofylline, 8-bromophylline, lisofylline, istradefylline and 8-benzyltheophylline. 7. The in vitro activation method of primordial follicles according to claim 6, characterized in that: The concentrations of the xanthine compounds in the xanthine compound solution are as follows: 8. The in vitro activation method of primordial follicles according to claim 6 or 7, characterized in that: The ovary is a mouse or human ovary. 9. The in vitro activation method of primordial follicles according to claim 8, characterized in that: The mice are newborn mice. 10. The in vitro activation method of primordial follicles according to claim 6 or 7, characterized in that: The culture is carried out at a constant temperature of 37±2°C and 5%±1% CO ₂ for 4 days.