CN118217291A - Application of vitamins in preparation of medicines for activating primordial follicles - Google Patents

Application of vitamins in preparation of medicines for activating primordial follicles Download PDF

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CN118217291A
CN118217291A CN202410357202.5A CN202410357202A CN118217291A CN 118217291 A CN118217291 A CN 118217291A CN 202410357202 A CN202410357202 A CN 202410357202A CN 118217291 A CN118217291 A CN 118217291A
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vitamins
primordial follicles
follicles
primordial
application
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张美佳
温建钟
彭欣宜
刘爽
张晓丹
张璐纯
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South China University of Technology SCUT
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    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
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    • A61K31/355—Tocopherols, e.g. vitamin E
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    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
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    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4415—Pyridoxine, i.e. Vitamin B6
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/455—Nicotinic acids, e.g. niacin; Derivatives thereof, e.g. esters, amides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A—HUMAN NECESSITIES
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    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
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    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/525—Isoalloxazines, e.g. riboflavins, vitamin B2
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/08—Drugs for genital or sexual disorders; Contraceptives for gonadal disorders or for enhancing fertility, e.g. inducers of ovulation or of spermatogenesis

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Abstract

The invention provides application of vitamins in preparing medicines for activating primordial follicles, and belongs to the technical field of biology. The invention discovers that vitamins promote the activation of primordial follicles, can be used as an oral medicine for treating patients with premature ovarian failure, and has good application prospect.

Description

Application of vitamins in preparation of medicines for activating primordial follicles
Technical Field
The invention belongs to the technical field of biology, and particularly relates to application of vitamins in preparation of medicines for activating primordial follicles.
Background
Follicles are fundamental units of ovarian structure and function, and can be classified into primordial follicles, primary follicles, secondary follicles, luminal follicles, and mature follicles, according to the different stages of follicular growth and development. Primordial follicles are limited in number and non-renewable, so their number is considered to represent female fertility, directly determining the length of female reproductive life. Genetic factors, environmental pollution, anticancer drugs, etc. easily cause excessive activation or apoptosis of primordial follicles, and ultimately lead to premature ovarian failure (POI). There are often small numbers of profound primordial follicles in the ovaries of such POI patients, but they are difficult to activate under physiological conditions, resulting in infertility. Furthermore, women of advanced age also have reduced fertility due to reduced numbers of primordial follicles. These women cannot be treated with traditional assisted reproductive techniques. The method adopted at present is mainly through an In Vitro Activation (IVA) mode and then autograft. The method is subject to a number of limitations, requires surgery, is prone to DNA damage and risk of cancer, and has a low success rate. There is currently no therapeutic regimen for restoring fertility in POI patients by promoting primordial follicular activation via oral medications. Initial regulator research on the maintenance and activation of human primordial follicles is carried out, and the molecular mechanism of primordial follicle activation and follicular development is clarified, so that the research is important for diagnosis and treatment of patients.
The syncytia of female mammals breaks down to form primordial follicles and constitutes a pool of primordial follicles, with most primordial follicles remaining dormant at each periodic recruitment, with only a few primordial follicles activated into growth, eventually maturing and ovulating. The most studied molecule and signaling mechanism for primordial follicle activation is that rapamycin kinase (mTOR) in pre-granulosa cells promotes the production of KIT ligands (KIT l). KIT l binds to its receptor KIT proto-oncogene receptor tyrosine kinase (c-KIT), activating the phosphatidylinositol 3-kinase (PI 3K) -protein kinase B (Akt) signaling pathway in oocytes.
Vitamins mainly include Vitamins A (VA), B (VB), C (VC), D (VD), E (VE) and K (VK). VA can regulate cell proliferation and differentiation, and has important effects on reproductive tissues and embryogenesis in mammals. VB1, also known as thiamine, has the effect of maintaining normal sugar metabolism. VB2, also known as riboflavin, promotes development and cell regeneration. VB3, also known as niacin, is converted in our body tissues into the major metabolically active form: the coenzymes Nicotinamide Adenine Dinucleotide (NAD) and Nicotinamide Adenine Dinucleotide Phosphate (NADP). NAD is also involved in energy metabolism and plays a key role in cellular functions, such as controlling gene expression. NADP is able to promote anabolic reactions such as synthesis of cholesterol and fatty acids and plays an important role in maintaining the antioxidant function of cells. VB4, also known as adenine, is a constituent of nucleic acids and coenzymes, involved in the synthesis of DNA and RNA in the body, and is an essential component for maintaining the metabolism of the organism. VB5, also known as pantothenic acid, is converted in vivo into CoA or acyl carrier proteins, involved in sugar, fat, protein and energy metabolism. VB6, also called pyridoxine, is a component of some coenzymes in the body, and participates in various metabolic reactions, especially has close relation with amino acid metabolism, and can be clinically used for preventing and treating vomiting of pregnancy and vomiting of radiation sickness. VB7, also called biotin, is an indispensable substance for normal metabolism of fat and protein, and is a necessary nutrient for maintaining natural growth and development of human body and normal human body function and health. VB9, also known as folic acid, is involved in the metabolism of genetic material and proteins; influence animal reproductive performance; affecting secretion of animal pancreas; promoting the growth of animals; improving immunity. VB12, also called cobalamin, has the functions of activating amino acid and promoting nucleic acid biosynthesis, and can promote protein synthesis, and has important effect on infant growth and development. VC can delay cell aging and apoptosis, and promote repair of partial tissues. VD has the main functions of regulating calcium and phosphorus metabolism, promoting intestinal calcium and phosphorus absorption and bone calcification, and maintaining the balance of blood calcium and blood phosphorus. VE can maintain normal reproductive function of organism and delay aging of organism. VK mainly promotes blood coagulation and may also be involved in skeletal metabolism.
Disclosure of Invention
In order to overcome the defects and shortcomings in the prior art, the primary purpose of the invention is to provide application of vitamins in preparing medicines for activating primordial follicles.
The aim of the invention is realized by the following technical scheme:
Use of vitamins in the manufacture of a medicament for the treatment of premature ovarian failure, said vitamins being one or more of the following combinations: VB1, VB2, VB3, VB4, VB5, VB6, VB9, VB12, VC and VE.
Further, the application is the application of vitamins in preparing medicines for activating primordial follicles.
Further, the drug for activating the primordial follicles is a drug for promoting the conversion of the primordial follicles to the growth follicles.
Further, in said application, the doses of said vitamins are calculated on the basis of the body weight of the individual, the amounts of vitamins administered per 50kg of body weight being as follows:
further, the primordial follicle is a mouse or human primordial follicle.
Further, the administration mode of the medicine is oral administration or intravenous injection administration.
An in vitro activation method of primordial follicle (for non-disease diagnosis and treatment purpose) comprises culturing ovary in vitamin solution; the vitamins are one or more of the following combinations: VB1, VB2, VB3, VB4, VB5, VB6, VB9, VB12, VC and VE.
Further, the vitamin concentrations in the vitamin solution are shown below:
Further, the ovary is a mouse or human ovary.
Further, the mice are neonatal mice.
Further, the culture was carried out at 37.+ -. 2 ℃ and 5.+ -. 1% CO 2 at constant temperature for 4 days.
Compared with the prior art, the invention has the following advantages:
POI is a clinically severe ovarian disease that presents a trend toward younger age, with increasing morbidity, severely affecting female fertility and physical well-being. Only when the primordial follicles of female mammals, including humans, are carefully regulated and orderly activated, it is possible to maintain the normal reproductive life of the female (female). In the present invention we have found that vitamins have the effect of promoting the activation of primordial follicles in the ovaries of mice, and that the number of follicles grown in treated mice is significantly increased under the action of drug treatment. The medicines can be used for oral administration and intravenous injection, so that the medicine can be used for treating POI patients by activating primordial follicles through oral administration, and has good application prospect for saving fertility.
Drawings
FIG. 1 is a morphology of 3dpp neonatal mice ovaries cultured for 4 days with different vitamins;
FIG. 2 is a graph of follicular count results, wherein (A) growth follicular count is counted; (B) raw follicular statistics count;
(C) Counting total follicles; ruler: 50 μm.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but embodiments of the present invention are not limited thereto.
The test methods for specific experimental conditions are not noted in the examples below, and are generally performed under conventional experimental conditions or under experimental conditions recommended by the manufacturer. The materials, reagents and the like used, unless otherwise specified, are those obtained commercially.
Example 1
1.1 In vitro culture related reagent configuration
1.1.1DMEM/F12 Medium (250 ml) configuration as shown in Table 1:
TABLE 1DMEM-F12 Medium
SP was a commercial penicillin and streptomycin diabody and was added to 100mL of medium per 100. Mu.L. After volume was set to 250mL, it was filtered through a 0.22 μm filter, handled in a super clean bench, sealed with a sealing film and stored at 4 ℃.
1.1.2 Phosphate buffered saline (Phosphate buffered solution, PBS,1L, pH: 7.2) as shown in Table 2:
TABLE 2PBS buffer
Deionized water was used to determine the volume to 1L, autoclaved and stored at 4 ℃.
1.1.3 Configuration of the drug concentration in the culture medium of each experimental group: dissolving VB3, VB4, VB5, VB6 and VC with ultrapure water to prepare a concentrated stock solution; VB1, VB2, VB9, VB12 and VE were dissolved in dimethyl sulfoxide (DMSO) to prepare a concentrated stock solution. The final concentrations of each drug are shown in Table 3.
TABLE 3 drug concentration in the Medium of each experimental group
1.2 Experimental methods
1.2.1 Isolation of mouse ovaries
1) All surgical instruments, glass plates and the like are sterilized, and ultraviolet lamps are turned on among cells to irradiate for half an hour for sterilization.
2) In this experiment, 3 days of large ICR females were used as material, the dissection of the mice was sacrificed by cervical dislocation, the head and abdomen of the mice were separated from the tail with bilateral kidneys, and the tail was placed in cold PBS.
3) The ovaries and ovarian membranes of the mice were removed together and the periovaries-wrapped membranes were separated with a syringe needle under stereomicroscope procedures.
1.2.2 In vitro culture of mouse ovaries
1) 3ML of DMEM/F12 culture medium is added into each hole of a six-hole plate, then a prepared drug solution is added, DMSO is added into a control group, the mixture is blown and evenly mixed, a 0.44 mu m culture film is placed on each hole, and the mixture is placed in a carbon dioxide incubator for incubation for 30 minutes at 37 ℃.
2) Placing the separated ovary on a culture membrane, and culturing in a constant-temperature carbon dioxide incubator at 37 ℃;
3) The medium was changed every two days and co-cultured for 4 days.
1.2.3 Ovarian embedding, section and hematoxylin staining
1) Fixing the sample: the ovaries of mice were fixed in 4% paraformaldehyde for 12-16 hours at 4 ℃;
2) Alcohol concentration gradient dehydration and transparency: 70% alcohol, 80% alcohol, 95% alcohol/eosin dye solution, 95% alcohol, absolute alcohol/xylene (volume 1:1), xylene, 5 minutes per gradient, and finally xylene was blotted dry with filter paper.
3) Wax dipping and embedding: ovaries were waxed in a water-insulated (sealing film) environment at 60 ℃ for 2 hours and transferred to paraffin boxes.
4) Tissue section: the slices are typically 5 μm thick.
5) Spreading: the wax belt is fully stretched in a water bath kettle at 42 ℃ and then fished out by a glass slide.
6) Baking slices: overnight at 42 ℃.
1.2.5 Hematoxylin staining
1) Dewaxing and rehydrating: sequentially passing the sliced pieces through xylene, absolute alcohol, 95% alcohol, 80% alcohol and 70% alcohol. Each gradient was run for 5 minutes and finally rinsed with deionized water.
2) Dyeing: the cells were stained with hematoxylin dye for about 1 minute and rinsed with water.
3) And (3) removing the water sealing piece: sequentially passing through 70% alcohol, 80% alcohol, 95% alcohol, anhydrous alcohol, xylene, and xylene for 3 min each gradient, and sealing with neutral resin.
1.3 Ovarian follicle count
The stained serial sections are counted, one for every 5 consecutive sections, and the final statistical result is to multiply the counting result by 5 after adding. The morphological characteristics of follicles can be distinguished into: primordial follicles: three to five flattened granulosa cells around and one oocyte in the middle; activating follicles: a layer of granulosa cells surrounding it or a layer of granulosa cells mixed around it and flattened and a growing oocyte in between.
1.4 Ovarian follicle count results
The results are shown in fig. 1 and 2, wherein:
fig. 1: morphological comparisons between control and drug-added groups, arrows point to activated growth follicles; ruler: 50 μm;
Fig. 2: (a) growth follicle statistics count: compared with the control group, 1 mu MVB1, 10 mu MVB2, 1 mu MVB3, 1 mu MVB4, 20 mu MVB5, 10 mu MVB6, 40 mu MVB9, 10 mu MVB12, 250 mu MVC, 1 mu MVE can significantly promote the activation of primordial follicles (P < 0.05); (B) raw follicular statistics count; (C) Total follicular statistics were counted with no significant difference between the experimental and control groups.
The invention cultures 3dpp newborn mice ovaries with or without vitamins for 4 days, and observes the ovaries structure. Serial section statistical analysis showed that the number of primordial follicular activations ),1μMVB1(521.67±30.14)、10μMVB2(651.67±32.15)、1μMVB3(628.33±29.30)、1μMVB4(631.67±36.86)20μM VB5(670±36.05)、10μMVB6(718.33±32.53)、40μMVB9(683.33±60.07)、10μMVB12(566.67±25.66)、250μMVC(653.33±30.14)、1μMVE(745±40) per ovary was significantly enhanced compared to the control group (423.33 ±15.28, data indicating activation of primordial follicles).
In addition, the increase of the growth follicles caused by the action of vitamins slightly reduced the number of primordial follicles, but the total number of ovaries of mice was not affected, which indicates that vitamins did not affect primordial follicles and apoptosis of the growth follicles.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

Claims (10)

1. The application of vitamins in preparing medicines for treating premature ovarian failure is characterized in that: the vitamins are any one or more compound combinations: VB1, VB2, VB3, VB4, VB5, VB6, VB9, VB12, VC and VE.
2. The use according to claim 1, characterized in that:
the application is the application of vitamins in preparing medicines for activating primordial follicles.
3. The use according to claim 1, characterized in that:
the medicine for activating the primordial follicles is a medicine for promoting the conversion of the primordial follicles to the growth follicles.
4. A use according to any one of claims 1-3, characterized in that:
in said application, the doses of said vitamins are calculated on the basis of the body weight of the individual, the mass of vitamin administered per 50kg of body weight being as follows:
5. A use according to any one of claims 1-3, characterized in that:
the original follicle is of a mouse or a human;
the medicine is orally taken or intravenous injection.
6. An in vitro activation method of primordial follicles, characterized by: culturing ovary in vitamin solution; the vitamins are any one or more compound combinations: VB1, VB2, VB3, VB4, VB5, VB6, VB9, VB12, VC and VE.
7. The method of in vitro activation of primordial follicles according to claim 6, wherein:
the vitamin concentration in the vitamin solution is shown as follows:
8. the method of in vitro activation of primordial follicles according to claim 6 or 7, characterized in that:
The ovary is mouse or human ovary.
9. The method of in vitro activation of primordial follicles according to claim 8, wherein:
The mice are neonatal mice.
10. The method of in vitro activation of primordial follicles according to claim 6 or 7, characterized in that:
the culture is carried out for 4 days at the constant temperature of 37+/-2 ℃ and 5+/-1% CO 2.
CN202410357202.5A 2024-03-27 2024-03-27 Application of vitamins in preparation of medicines for activating primordial follicles Pending CN118217291A (en)

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