Use of thyroxine and thyroxine receptor inhibitor in preparing drugs for regulating primordial follicle activation

Abstract

Provided is use of thyroxine and a thyroxine receptor inhibitor in preparing drugs for regulating primordial follicle activation, belonging to the technical field of biology. It has been discovered that the thyroxine can promote primordial follicle activation, and the thyroxine receptor inhibitor inhibits primordial follicle activation, so that they can be used as drugs for regulating primordial follicle activation in patients with premature ovarian insufficiency.

Description

Application of thyroxine and thyroxine receptor inhibitors in the preparation of drugs for regulating primordial follicle activation Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to the application of thyroid hormone and thyroid hormone receptor inhibitors in the preparation of drugs for regulating primordial follicle activation. Background Art

[0002] The female ovary contains a fixed, non-renewable number of primordial follicles, which gradually deplete with aging until they are exhausted. A primordial follicle consists of a single layer of flattened pregranulosa cells surrounding a quiescent oocyte. The activation of the primordial follicle is accompanied by oocyte growth and the differentiation of pregranulosa cells from a flattened to a cuboidal shape. During each wave of activation, a small number of primordial follicles are activated and enter the growth phase, while the majority of remaining follicles remain quiescent, maintaining a woman's fertility throughout her life. Therefore, maintaining a balance between primordial follicle dormancy and activation is crucial. The activation and development of primordial follicles is a process precisely regulated by multiple growth factors, cytokines, and protein kinases. The molecular and signaling mechanisms that have been widely studied include the production of KIT ligand (KITL, also known as SCF) by rapamycin kinase (mTOR) in pregranulosa cells. KITL binds to its receptor, KIT proto-oncogene receptor tyrosine kinase (c-KIT), and activates phosphatidylinositol 3-kinase (PI3K)-protein kinase B (Akt) signaling in the oocyte. Forkhead box O3a (FOXO3a) is then phosphorylated and transported from the nucleus to the cytoplasm, leading to primordial follicle activation. Our recent studies have shown that during primordial follicle activation, mTOR signaling in pregranulosa cells can be activated by enhanced glycolysis. Because primordial follicle activation is irreversible, orderly primordial follicle activation is crucial for female reproductive lifespan. Excessive or inactive primordial follicle activation can lead to premature ovarian insufficiency (POI).

[0003] Currently, the main causes of POI are believed to include genetic, autoimmune, iatrogenic, and environmental factors, but the etiology remains unclear in most patients. Approximately three-quarters of POI patients still have a small number of dormant primordial follicles, but these follicles are difficult to activate physiologically, making conventional IVF fertility impossible. Currently, in vitro activation (IVA) is used to reactivate these dormant primordial follicles, allowing them to be autologously transplanted to achieve conception. However, this method is only suitable for a small number of patients, and laparoscopic surgery can also cause physical damage. In addition to impaired ovarian function, POI increases the risk of cardiovascular disease, osteoporosis, cognitive decline, and mood disorders, thereby reducing quality of life. However, effective treatments for POI are currently lacking. Therefore, there is a need to explore more effective methods, such as oral medications, to regulate the activation rate of primordial follicles and provide more effective treatment strategies for POI patients.

[0004] Thyroxine is a hormone secreted by the thyroid gland that acts on almost all cells in the human body. Thyroxine includes triiodothyronine (T3) and tetraiodothyronine (T4). The formation of thyroxine goes through six processes: synthesis, storage, iodination, reabsorption, decomposition and release: 1. Follicular epithelial cells take up amino acids from the blood, synthesize the precursor of thyroglobulin in the rough endoplasmic reticulum, and then add sugar and concentrate in the Golgi complex to form secretory granules, which are then discharged into the follicular cavity for storage by exocytosis. 2. Follicular epithelial cells can take up iodide ions (I - ), I - Activated by peroxidase. -4. Under the influence of thyroid-stimulating hormone (TSH) secreted by the pituitary gland, follicular epithelial cells endocytose the iodinated thyroglobulin within the follicular cavity, forming glial vesicles. 5. The glial vesicles fuse with lysosomes, where iodinated thyroglobulin is broken down by hydrolases to form a small amount of T3 and a large amount of T4. 6. T3 and T4 are released into the bloodstream at the cell base. 100% of plasma T4 originates from the thyroid gland, 20% of plasma T3 comes directly from the thyroid gland, and 80% comes from the peripheral conversion of plasma T4. T3 is the primary biologically active thyroid hormone, while T4 is the primary hormone secreted by the thyroid gland and is considered a precursor of T3. T4 requires conversion to T3 by deiodinases in peripheral tissues to function. Thyroid hormone receptors are a very important family of nuclear receptor proteins, consisting of two main groups: α receptors and β receptors. Based on structural differences, α receptors are divided into two subfamilies, α1 and α2, and β receptors are also divided into two subfamilies, β1 and β2. These receptors are important carriers of thyroid hormone signals, both peripherally and internally. They function at the cellular level and participate in the regulation of body metabolism. Furthermore, thyroid hormone receptors can interact with non-nuclear receptor proteins, such as cytokines and transcription factors, to regulate cellular function. T3 and T4 enter target cells via diffusion or carrier-mediated transport, involving membrane transporters such as MCT8, MCT10, and Oatp1a2. Within target cells, thyroid hormones exert their functions directly by activating their nuclear receptors, stimulating or inhibiting the expression of transcriptional genes that depend on dimerization of the retinoic acid X receptor (RXR) and / or coactivators, such as steroid receptor coactivator (SRC). In addition to nuclear receptors, thyroid hormones can also act indirectly by binding to the membrane protein αvβ3 integrin, activating signal transduction cascades through MAPK and ERK1 / 2, and regulating the transcription and phosphorylation of their nuclear receptors. The biological effects of thyroxine are primarily manifested in three aspects: 1. It promotes metabolism of substances and energy in the body, primarily by promoting the oxidative breakdown of energy sources such as carbohydrates, proteins, and fats, thereby increasing oxygen consumption and releasing energy. 2. It promotes physical and intellectual development, significantly impacting the skeletal, nervous, and reproductive systems. 3. It enhances the excitability of the nervous system, particularly the sympathetic nervous system. Thyroxine is crucial for normal reproductive function in humans and animals. T3 and T4 can directly act on the ovaries, uterus, and placenta through specific nuclear receptors that regulate the development and metabolism of these organs. Furthermore, T3 and T4 can exert their effects indirectly through various interactions with other hormones and growth factors, such as estrogen, prolactin (PRL), and insulin-like growth factor (IGF), and by influencing the release of gonadotropin-releasing hormone (GnRH) within the hypothalamic-pituitary-gonadal axis. Therefore, both high and low blood thyroxine levels can lead to a decrease in the number of large-antral follicles in rats.In vitro experiments have demonstrated that thyroxine promotes the growth and ovulation rate of rat secondary follicles. Furthermore, when used in combination with FSH, T3 can enhance the proliferation of granulosa cells of antral follicles and reduce apoptosis. The interaction between T3 and gonadotropins also inhibits excessive androgen production by antral theca cells and stimulates estrogen production by granulosa cells. Thyroxine not only participates in the maturation of preovulatory follicles and mouse cumulus cells through ERK1 / 2 signaling but also participates in the meiotic maturation of oocytes of large antral follicles in cattle and pigs. These findings suggest an important role for thyroxine in the female reproductive system, but there are no reports on its role in regulating primordial follicle activation.

[0005] Summary of the Invention

[0006] 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 thyroid hormone and / or thyroid hormone receptor inhibitors in the preparation of drugs for regulating primordial follicle activation.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] Use of thyroid hormone and / or thyroid hormone receptor inhibitors in the preparation of medicines for treating primary ovarian insufficiency.

[0009] Furthermore, the primary ovarian insufficiency includes primary ovarian insufficiency caused by overactivation or inactivation of primordial follicles.

[0010] Furthermore, the application is the application of thyroxine in the preparation of a drug for promoting the activation of primordial follicles, or the application of a thyroxine receptor inhibitor in the preparation of a drug for inhibiting the activation of primordial follicles.

[0011] Furthermore, the thyroxine is any one of T3 (triiodothyronine) and T4 (thyroxine) or a combination of both; the thyroid receptor inhibitor is any one of KB130015 and Debutyldronedarone hydrochloride (SR35021 hydrochloride) or a combination of both.

[0012] Furthermore, in the application, the dosage of thyroxine is calculated based on individual body weight, and the dosage range per 50 kg body weight is: T3: 0.00005-0.5 mg / day, T4: 0.00009-0.9 mg / day; the dosage of thyroid receptor inhibitors is calculated based on individual body weight, and the dosage range per 50 kg body weight is: KB130015: 0.222-2220 mg / day, Debutyldronedarone hydrochloride: 2.75-27500 mg / day.

[0013] Furthermore, the primordial follicles are mammalian primordial follicles.

[0014] Furthermore, the mammal is a mouse or a human.

[0015] Furthermore, the drug is administered orally or by intravenous injection.

[0016] Application of thyroxine in the preparation of drugs for primordial follicle activation in vitro.

[0017] Furthermore, the thyroxine is T3 or T4.

[0018] Furthermore, the primordial follicle in vitro activation drug is a drug that promotes the transformation of primordial follicles into growth follicles.

[0019] An in vitro activation method for primordial follicles for non-disease diagnosis and treatment purposes, comprising culturing ovaries in an in vitro activation reagent; the in vitro activation reagent contains thyroxine; the thyroxine is T3 or T4.

[0020] Furthermore, the concentrations of T3 and T4 used are 1±0.5 nM and 1.5±0.5 nM, respectively.

[0021] Furthermore, the ovary is a mouse ovary.

[0022] Furthermore, the mice are newborn mice.

[0023] Furthermore, the culture is carried out at a constant temperature of 37±2°C and 5%±1% CO2 for 4 days.

[0024] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0025] POI is a relatively serious ovarian disease clinically. The disease is showing a trend of becoming younger, and the incidence rate is constantly increasing, seriously affecting women's fertility and physical and mental health. In order to ensure that female mammals (including humans) have mature eggs available throughout their reproductive cycle, the activation of primordial follicles in their primordial follicle reservoir must be precisely regulated. Excessive activation of primordial follicles or primordial follicle activation disorders under physiological conditions can lead to POI. In the present invention, we found that thyroxine can promote the activation of primordial follicles, and the thyroxine receptor inhibitors KB130015 and Debutyldronedarone hydrochloride have the effect of inhibiting the activation of primordial follicles in the ovaries of newborn mice. Thyroid tablets, as a common drug, can be administered orally and have good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 shows the morphological observation and follicle counting results of the ovaries of 3 dpp newborn mice cultured for 4 days after supplementation with the thyroid hormones T3 and T4 of the present invention, the thyroid hormone receptor inhibitor KB130015, and Debutyldronedarone hydrochloride; wherein (a) is a comparison of the ovarian histological morphology of the control group and each group (scale: 50 μm); (b) is a statistical count of primordial follicles and growing follicles in each group. DETAILED DESCRIPTION

[0027] The present invention will be described in further detail below with reference to the embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0028] In the following examples, experimental methods without specific experimental conditions were generally performed under conventional experimental conditions or those recommended by the manufacturer. Materials and reagents used were commercially available unless otherwise specified.

[0029] Example 1

[0030] 1. Experimental Materials

[0031] 1. Preparation of Reagents Related to In Vitro Culture

[0032] 1.1 Preparation of drug solution

[0033] Weigh appropriate amounts of thyroxine T3, T4, thyroxine receptor inhibitor KB130015, and debutyldronedarone hydrochloride powder, prepare a storage solution using DMSO as solvent, wait until the solids are fully dissolved, filter with a 0.2 μm filter, seal with parafilm, and store at -20°C.

[0034] 1.2 DMEM / F12 medium preparation (Table 1)

[0035] Table 1. DMEM / F12 culture medium

[0036] After all the solids are dissolved, filter with a 0.22 μm filter, operate in a clean bench, seal with sealing film, and store at 4°C.

[0037] 1.3 Preparation of phosphate buffered solution (PBS) (Table 2)

[0038] Table 2. PBS buffer

[0039] Autoclave and store at 4°C.

[0040] 2. Isolation and in vitro culture of mouse ovaries

[0041] 1. Isolation of Mouse Ovaries

[0042] 1.1 Surgical instruments (ophthalmic straight scissors, 10 cm toothed forceps, pointed forceps, 1 mL syringe), 90 mm culture dishes, six-well plates, etc. were placed in a clean bench and irradiated with UV light for 30 minutes. Intercellular UV light was used for 30 minutes.

[0043] 1.2 In this experiment, 3-day-old ICR female mice were sacrificed by cervical dislocation. The abdomen was disinfected with 70% alcohol, and the abdominal cavity was opened through a V-shaped incision at the umbilicus. The mice were divided into the head and tail along the upper edge of the kidneys, and the tail was placed in pre-cooled PBS.

[0044] 1.3 Under a stereomicroscope, remove the mouse ovary along with the surrounding connective tissue and use a 1 mL syringe needle to peel off the connective tissue surrounding the ovary.

[0045] 2. In vitro culture of mouse ovaries

[0046] 2.1 Add 3 mL of DMEM / F12 medium to each well of a six-well plate, then add various drugs to make the final concentrations of T3: 1 nM, T4: 1.5 nM, thyroid receptor inhibitor KB130015: 5 μM, and Debutyldronedarone hydrochloride: 60 μM, respectively. Mix well by pipetting, place a 0.44 μm culture membrane on each well, and incubate in a 37°C incubator for 30 minutes.

[0047] 2.2 Place the isolated ovaries on a culture membrane and culture in a 37°C, 5% CO2 incubator;

[0048] The medium was changed every two days and cultured for 4 days.

[0049] 3. Hematoxylin staining

[0050] 1. Paraffin Embedding of Ovaries

[0051] 1.1 Sample fixation: Mouse ovaries cultured for 4 days were fixed in 4% paraformaldehyde at 4°C for 12-16 hours;

[0052] 1.2 Alcohol concentration gradient dehydration and transparency: 70% ethanol, 80% ethanol, 95% ethanol / eosin staining solution, 95% ethanol, anhydrous ethanol, anhydrous ethanol / xylene (volume 1:1), xylene, each gradient for 5 minutes, and the xylene in the last step was dried with filter paper.

[0053] 1.3 Wax immersion and embedding: Wax the tissue in a 60℃ water bath for 3 hours, transfer the tissue to an iron paraffin box, cover the box, take it out and let it solidify at room temperature.

[0054] 2. Slicing

[0055] 2.1 Turn on the microtome, adjust the slice thickness to 5 μm, and start continuous sectioning after fixing the wax block.

[0056] 2.2 Spreading: After the wax strip is fully stretched in a 42℃ water bath, pick it up with a glass slide.

[0057] 2.3 Bake the slices at 42℃ overnight.

[0058] 3. Hematoxylin staining

[0059] 3.1 Dewaxing and Rehydration: Place the slides with tissue attached in a series of xylene, xylene, absolute alcohol, absolute alcohol, 95% ethanol, 80% ethanol, and 70% ethanol steps. Each step lasts for 5 minutes, and the slides are then rinsed with deionized water.

[0060] 3.2 Staining: Stain with hematoxylin solution for 1 minute and 30 seconds, then rinse with deionized water.

[0061] 3.3 Dehydration and mounting: The slides were sequentially dehydrated with 70% ethanol, 80% ethanol, 95% ethanol, anhydrous ethanol, anhydrous ethanol, xylene, and xylene, each gradient for 3 minutes, and then mounted with neutral resin.

[0062] 4. Ovarian follicle count

[0063] 1. Follicle Counting by Hematoxylin Staining

[0064] Based on their morphological characteristics, follicles can be divided into the following categories: primordial follicles: small oocytes surrounded by three to five flat granulosa cells; growing follicles: larger oocytes surrounded by one or more layers of cuboidal granulosa cells, or a mixture of cuboidal and flat granulosa cells; and atretic follicles: pyknotic oocytes and / or one or more pyknotic granulosa cells. Counts were performed on hematoxylin-stained serial sections, with one count taken every five sections. The final result was the sum of the counts multiplied by five.

[0065] 5. Results

[0066] Figure 1 (a) shows the comparison of ovarian morphology between the control group and the groups supplemented with T3, T4, the thyroxine receptor inhibitor KB130015, and debutyldronedarone hydrochloride. Figure 1 (b) shows the counts of primordial and growing follicles. As can be seen, compared with the control group, the number of growing follicles in the thyroxine-added groups increased significantly, including the control group (358.33±32.15), the T3 group (598.33±27.54), and the T4 group (696.97±61.10). The number of growing follicles in the thyroxine receptor inhibitor-added groups decreased significantly, including the KB130015 group (240.00±22.91) and the debutyldronedarone hydrochloride group (235.00±15.00). There was no significant difference in the number of primordial follicles between the groups and the control group. Scale bar: 50 μm. ***P<0.005.

[0067] The above results indicate that thyroxine promotes the activation of primordial follicles, while thyroxine receptor inhibitors KB130015 and Debutyldronedarone hydrochloride inhibit the activation of primordial follicles, and have a regulatory effect on the activation of primordial follicles.

[0068] 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 considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. Use of thyroxine and / or thyroxine receptor inhibitors in the preparation of drugs for the treatment of primary ovarian insufficiency.

2. Use of the thyroid hormone and / or thyroid hormone receptor inhibitor according to claim 1 in the preparation of a medicament for treating primary ovarian insufficiency, characterized in that: The primary ovarian insufficiency includes primary ovarian insufficiency caused by overactivation or inactivation of primordial follicles.

3. Use of the thyroid hormone and / or thyroid hormone receptor inhibitor according to claim 2 in the preparation of a medicament for treating primary ovarian insufficiency, characterized in that: The application is the application of thyroxine in the preparation of a drug for promoting the activation of primordial follicles, or the application of a thyroxine receptor inhibitor in the preparation of a drug for inhibiting the activation of primordial follicles.

4. Use of the thyroid hormone and / or thyroid hormone receptor inhibitor according to any one of claims 1 to 3 in the preparation of a medicament for treating primary ovarian insufficiency, characterized in that: The thyroxine is any one of T3 and T4 or a combination of two thereof; the thyroxine receptor inhibitor is any one of KB130015 and Debutyldronedarone hydrochloride or a combination of two thereof.

5. Use of the thyroid hormone and / or thyroid hormone receptor inhibitor according to any one of claims 1 to 3 in the preparation of a medicament for treating primary ovarian insufficiency, characterized in that: In the application, the dosage of thyroxine is calculated according to individual body weight, and the dosage range per 50 kg body weight is: T3: 0.00005-0.5 mg / day, T4: 0.00009-0.9 mg / day; the dosage of thyroid receptor inhibitor is calculated according to individual body weight, and the dosage range per 50 kg body weight is: KB130015: 0.222-2220 mg / day, Debutyldronedarone hydrochloride: 2.75-27500 mg / day. The primordial follicles are primordial follicles of mammals; the mammals are mice or humans; The drug is administered orally or by intravenous injection.

6. The use of thyroxine in the preparation of a drug for activating primordial follicles in vitro, characterized in that: The thyroxine is T3 and T4.

7. The use of thyroxine in the preparation of a drug for activating primordial follicles in vitro according to claim 6, characterized in that: The primordial follicle in vitro activation drug is a drug that promotes the transformation of primordial follicles into growth follicles.

8. An in vitro method for activating primordial follicles for non-disease diagnosis and treatment purposes, characterized in that: The ovaries are taken and cultured in an in vitro activation reagent; the in vitro activation reagent contains thyroxine; the thyroxine is T3 and T4.

9. The in vitro activation method of primordial follicles for non-disease diagnosis and treatment purposes according to claim 8, characterized in that: The concentrations of thyroxine T3 and T4 used are 1±0.5 nM and 1.5±0.5 nM respectively.

10. The in vitro activation method of primordial follicles for non-disease diagnosis and treatment purposes according to claim 8 or 9, characterized in that: The ovary is a mouse ovary; The culture is carried out at a constant temperature of 37±2°C and 5%±1% CO2 for 4 days.

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