KR20220054565A - Pharmaceutical composition for preventing or treating male reproductive disease comprising butylated hydroxyanisole, butylated hydroxytoluene or mixture thereof - Google Patents

Abstract

The present invention relates to a composition comprising butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) or a mixture thereof as an active ingredient, and more specifically, to a pharmaceutical composition for preventing or treating male reproductive diseases, and a health functional food composition for preventing or alleviating male reproductive diseases, comprising BHA, BHT or a mixture thereof as an active ingredient. The composition comprising BHA, BHT or a mixture thereof according to the present invention as an active ingredient has an effect of inhibiting the proliferation of testicular cell lines and enhancing the apoptosis effect, and thus can be advantageously used in the fields related to therapeutic drugs and functional foods for preventing and treating male reproductive diseases caused by abnormal proliferation of testicular cell lines.

Description

TECHNICAL FIELD [0001] Pharmaceutical composition for preventing or treating male reproductive disease comprising butylated hydroxyanisole, butylated hydroxytoluene or mixture thereof, comprising butylhydroxyanisole, butylhydroxytoluene, or a mixture thereof

The present invention relates to a composition comprising butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) or a mixture thereof as an active ingredient, and more particularly, BHA, BHT or a composition thereof It relates to a pharmaceutical composition for preventing or treating male reproductive diseases, and a health functional food composition for preventing or improving male reproductive diseases, comprising the mixture as an active ingredient.

In the testis, there are Leydig cells, which are interstitial cells whose main function is to synthesize steroid hormones such as testosterone, and Sertoli cells, which develop reproductive cells through spermatogenesis in seminiferous tubules. The signaling pathway activated by calcium ions is reported to be related to steroidogenesis in Leedig cells and to sperm release in Sertoli cells (Lyon et al ., Biol Reprod, 2017).

It is known that when the concentration of calcium ions in the cytoplasm increases during spermatogenesis, Sertoli cell death occurs and male infertility can be induced (Liz et al. , Free Radic Biol Med, 2013). As such, the maintenance of calcium homeostasis in the testis is very important for fertilization, steroid synthesis, or spermatogenesis (Sullivan et al. , Mol Cell Endocrinol, 1984: Ogunbayo et al ., Toxicol In Vitro, 2008). ).

On the other hand, butylhydroxyanisole (BHA) and butylhydroxytoluene (BHT) are synthetic phenolic antioxidants and have been widely used as food preservatives because they have a property of preventing rancidity of fat. These substances are known to inhibit steroid synthesis at the transcriptional level, and in the case of BHT, anti-estrogenic and anti-androgen activity effects have been reported in hormone-sensitive organs (Yang et al. , Environ Sci Technol, 2018: Pop et al. , Toxicol In vitro, 2016: Pop et al. , J Appl Toxicol, 2018), a competitive inhibitor of Steroid 5 Alpha-Reductase 1 (Srd5a1) in rodents for BHA or Hydroxysteroid 11-beta in both humans and mice It has been reported that it can act as a selective inhibitor for dehydrogenase 2 (HSD11B2) (Guo et al ., Neurosci Lett, 2017: Li et al ., Pharmacology 2016).

As such, various functions and effects for BHA and BHT have been reported, but their therapeutic effect on male reproductive diseases has not been reported so far.

As a result of intensive efforts to develop a substance for treating male reproductive diseases, the present inventors have confirmed that BHA or BHT has an effect of inhibiting the proliferation of testicular cell lines and enhancing the apoptosis effect under the above-described technical background, and the present invention completed.

The present invention provides a pharmaceutical composition for preventing or treating male reproductive diseases comprising butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) or a mixture thereof as an active ingredient The purpose.

In addition, the present invention provides a health functional food composition for preventing or improving male reproductive diseases comprising butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) or a mixture thereof as an active ingredient. intended to provide

Another object of the present invention is to provide a method for inhibiting the proliferative ability of a testicular cell line using the composition.

Another object of the present invention is to provide a method for improving the killing effect of testicular cell lines using the composition.

In order to solve the above problems, the present invention is for preventing or treating male reproductive diseases comprising butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) or a mixture thereof as an active ingredient A pharmaceutical composition is provided.

In addition, the present invention provides a health functional food composition for preventing or improving male reproductive diseases comprising butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) or a mixture thereof as an active ingredient. to provide.

In addition, the present invention provides a method for inhibiting the proliferative ability of a testicular cell line using the composition.

In addition, the present invention provides a method for enhancing the killing effect of testicular cell lines using the composition.

The composition comprising BHA, BHT, or a mixture thereof according to the present invention as an active ingredient inhibits the proliferation of testicular cell lines and has an effect of enhancing the apoptosis effect, preventing male reproductive diseases caused by abnormal proliferation of testicular cell lines And it can be usefully used in fields related to therapeutic drugs and functional foods.

1 shows the results of analyzing the effect of treatment with BHA or BHT on the proliferation of testicular cell lines.
2 shows the results of analyzing the effect of treatment with BHA or BHT on the inhibition of calcium homeostasis in testicular cell lines.
3 shows the results of analyzing the effect of treatment with BHA or BHT on the amount or phosphorylation of mitochondrial proteins in testicular cell lines.
4 shows the results of analysis of the induction of mitochondrial depolarization in testicular cell lines by BHA treatment and the induction of DNA fragmentation in Leedich cells by BHT.
Figure 5 shows the results of analyzing the ER stress-inducing effect according to the treatment of BHA or BHT.
6 shows the results of analyzing mRNA expression level changes in testicular cell lines according to BHA or BHT treatment.
7 shows the results of analyzing the effects of BHA or BHT on MAPK and PI3K/AKT signaling mechanisms in testicular cell lines.
8 shows the results of analyzing the effect of the combined treatment of BHA and a signaling mechanism inhibitor on the MAPK and PI3K/AKT signaling mechanisms in testicular cell lines.
9 shows the results of analyzing the effects of BHT and signaling mechanism inhibitor combination treatment on the proliferation, MAPK and PI3K/AKT signaling mechanisms of testicular cell lines.
10 shows the results of analyzing the effects of BHA or BHT treatment on the formation of TM3 and TM4 spheroids mimicking the in vivo environment.
11 shows the mechanism of action of BHA in testicular cell lines.
12 shows the mechanism of action of BHT in testicular cell lines.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is those well known and commonly used in the art.

In the present invention, the proliferation inhibition and apoptosis mechanism of testicular cell lines by butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) were revealed ( FIGS. 11-12 ).

In one aspect, the present invention relates to a pharmaceutical composition for preventing or treating male reproductive diseases comprising BHA, BHT, or a mixture thereof as an active ingredient.

In another aspect, the present invention relates to a health functional food composition for preventing or improving male reproductive diseases comprising BHA, BHT or a mixture thereof as an active ingredient.

As used herein, the term "composition" is intended to include not only products comprising the specified ingredients, but also any products made directly or indirectly by combining the specified ingredients.

In the present invention, the pharmaceutical composition may be characterized as containing a pharmaceutically acceptable carrier, wherein the carrier is an ion exchange resin, alumina, aluminum stearate, lecithin, serum protein, buffer material, water, salt, Electrolyte, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulosic matrix, polyethylene glycol, sodium carboxymethylcellulose, polyarylate, wax, polyethylene glycol, characterized in that at least one selected from the group consisting of wool can

In the present invention, the pharmaceutical composition is formulated for intravenous, intraperitoneal, intramuscular, intraarterial, oral, intracardiac, intramedullary, intrathecal, transdermal, enteral, subcutaneous, sublingual or topical administration. It may be characterized in that it further contains any one or more adjuvants selected from the group consisting of buffers, antibacterial preservatives, surfactants, antioxidants, tonicity adjusting agents, preservatives, thickeners and viscosity modifiers, solutions, suspensions, emulsions , it may be characterized as having a formulation selected from the group consisting of gels and powders.

A suitable dosage of the pharmaceutical composition of the present invention varies depending on factors such as the severity of symptoms, the patient's weight, age, sex, administration mode, and administration time, and a skilled physician is usually effective for the desired treatment or prevention. Dosage can be readily determined.

In the present invention, health functional food refers to food manufactured and processed using raw materials or ingredients useful for the human body according to Health Functional Food Act No. 6722, and provides nutrients for the structure and function of the human body. It refers to food consumed for the purpose of controlling or obtaining useful effects for health purposes such as physiological action.

The health functional food composition of the present invention may be formulated in the formulation of conventional health functional food known in the art, and granules, tablets, pills, suspensions, emulsions, syrups, gums, teas, jellies, various beverages, and drinks , alcoholic beverages, etc., there is no particular limitation on the type of health functional food.

The health functional food composition of the present invention may contain any herbal form suitable for administration to the body of animals including the human body, more specifically any form conventional for oral administration, for example, food or feed, additives and adjuvants of food or feed; It may be in solid form such as fortified food or feed, tablets, pills, granules, capsules and effervescent formulations, or in liquid form such as solutions, suspensions, emulsions, beverages, pastes, etc., nutrients, vitamins, electrolytes, sweeteners, colorants, organic acids, It may contain a preservative, etc., and these components may be used independently or in combination.

In the present invention, the BHA, BHT or a mixture thereof may be characterized in that it modulates the PI3K/AKT and MAPK signaling mechanisms related to the proliferation of testicular cell lines.

In the present invention, it may be characterized in that the BHA, BHT or a mixture thereof induces ER stress.

In the present invention, it may be characterized in that the BHA, BHT or a mixture thereof induces mitochondrial dysfunction and inhibition of calcium homeostasis.

In the present invention, the BHA, BHT or a mixture thereof may be characterized in that it regulates the expression of genes related to steroid biosynthesis, cell cycle, hormone receptor and spermatogenesis.

In the present invention, the male genital disease is possible as long as it is caused by abnormal proliferation of testicular cell lines, for example, Leydig cell tumor, Sertoli cell tumor, orchitis, epididymitis, testicular cancer and hydrocephalus. It can be selected from the group consisting of.

The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents.

In another aspect, the present invention relates to a method for inhibiting the proliferative ability of a testicular cell line using a composition comprising BHA, BHT, or a mixture thereof.

In another aspect, the present invention relates to a method for enhancing the killing effect of testicular cell lines using a composition comprising BHA, BHT, or a mixture thereof.

[Example]

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not to be construed as being limited by these examples. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

<Experiment method>

experimental material

BHA and BHT, which are candidate compositions, were purchased from Sigma-Aldrich, Inc., and phospho-Erk1/2, P38 MAPK, Jnk, Rps6ka1, AKT, Rps6kb1, Rps6, Gsk3β, Eif2α , Bax, Bak, Bad, Bcl-xL Cytochrome c protein and total-Erk1/2, P38 MAPK, Jnk, Rps6ka1, AKT, Rps6kb1, Rps6, Gsk3β, Eif2α antibodies were purchased from Cell Signaling Techonology. p-Perk, t-Perk, Chop, α-tubulin (Tuba) and Pcna were purchased from Santa Cruz Biotechnology Inc. In addition, as a signaling inhibitor, LY294002, a PI3K inhibitor, U0126, an ERK1/2 inhibitor, and SP600125, a JNK inhibitor, were purchased from Enzo Life Science and used.

cell culture

TM3 (immature mouse leydig cell) and TM4 (immature mouse sertoli cell) cells were purchased from the Korea Cell Line Bank (KCLB), and for monolayer culture of cells, 10% fetal calf cells were placed in high glucose Dulbecco's modified Eagle medium (DMEM). Fetal bovine serum (FBS) was mixed together and used.

BrdU Analysis of cell proliferation ability using

In order to check the effect of BHA and BHT on the proliferation ability of mouse testicular cell lines, 3×10 ³ cells cultured under FBS starvation condition and 100 μl of medium were dispensed into 96 wells and BHA and BHT were dose-dependently (0, 10 , 20, 50, 75, 100 μM) and cultured for 24 hours, and then using the BrdU kit (Cat No: 1167229001, Roche), the experiment was performed according to the manufacturer's manual. After 48 hours of incubation, 10 μM BrdU was additionally added to each well and incubated for 2 hours at 37° C./5% CO ₂ in an incubator. BrdU was labeled on Leidich cells and Sertoli cells, and cells were fixed, incubated with anti-BrdU-POD solution at room temperature for 90 minutes, and then washed three times. Finally, cells were reacted with 100 μl of 3,3',5,5'-tetramethylbenzidine substrate, and absorbance within 370 nm and 492 nm was measured using an ELISA reader to analyze cell proliferation ability.

Immunofluorescence

3×10 ⁴ Leidich cells and Sertoli cells were dispensed and cultured in a confocal dish (catalog number: 100350, SPL Life Science, Republic of Korea) with 300 μl of medium containing 10% FBS, followed by 24 hours FBS After further culturing under starvation, BHA and BHT were treated with 100 μM for 24 hours, cells were fixed with methanol for 10 minutes, and PCNA antibody purchased from Santa Cruz Biothecnology and diluted to 2 μg/ml was treated. Treated with mouse IgG and incubated for 16 hours at 4 ℃. After that, it was washed twice with PBS containing 0.1% BSA (bovine serum albumin), and as a secondary antibody, goat anti-mouse IgG Alexa 488 (catalog number: A-11001, Invitrogen, Carlsbad, CA, USA) was diluted 1:200 in antibody dilution buffer and incubated at room temperature for 1 hour, then washed with 0.1% BSA-PBS, and then DAPI staining was additionally performed to enable simultaneous observation of the nucleus as well as the target protein in the cell. . After the end of the experiment, cells were observed and photographed using an LSM710 (Carl Zeiss, Thornwood, NY, USA) confocal microscope.

Propidium Cell cycle analysis with iodide (PI)

In order to confirm the cell cycle arresting effect of BHA and BHT in mouse testicular cell lines, an experiment was conducted using BD Biosicences' Propidium iodide (PI) reagent. First, 1×10 ⁵ cells were cultured in 6 wells, and when the cells were filled in a 60% culture dish, they were further cultured in FBS starvation state for 24 hours. Thereafter, BHA and BHT were treated in a dose-dependent manner (0, 10, 20, 50, 75, 100 μM) and cultured in an incubator at 37° C./5% CO ₂ for 24 hours. Thereafter, cells were removed from the culture dish using trypsin, washed twice with PBS, fixed with 70% ethanol, and stored at 4° C. for one day. Next, washing was performed twice with PBS, and 5 μL of 10 mg/ml RNase A and 5 μL of 50 mg/ml PI were mixed together in PBS and stained at room temperature for 30 minutes while blocking light. Thereafter, 400 μL of PBS was added to transfer the stained solution to a 5 mL FACS tube, and the fluorescence intensity was analyzed using a flow cytometer to measure changes in the cell cycle.

Fluo -4 In the cytoplasm through AM staining calcium concentration change measurement

In order to measure the intracytoplasmic calcium concentration, it was measured using Fluo-4 AM. 1×10 ⁵ Leidich and Sertoli cells were cultured in 6 wells, and when the cells were filled in a 60% culture dish, they were further cultured in FBS starvation state for 24 hours. Thereafter, BHA or BHT was dose-dependently treated and cultured in an incubator at 37° C./5% CO ₂ for 24 hours. Then, using trypsin, the cells were removed from the culture dish and centrifuged to obtain a cell pellet. After dissolving the cells in Fluo-4 staining solution, they were incubated for 20 minutes at 37°C/5% CO ₂ in an incubator. The stained cells were centrifuged again, washed with PBS, and then centrifuged again to obtain a cell pellet, and the fluorescence intensity was analyzed using a flow cytometer.

Rhod -2 Measurement of calcium concentration in mitochondrial matrix through AM staining

To measure the intracytoplasmic calcium concentration, it was measured using Rhod2-AM. 1×10 ⁵ mouse testicular cell lines were cultured in 6 wells, and when the cells were filled in a 60% culture dish, they were further cultured in FBS starvation state for 24 hours. Thereafter, BHA or BHT was dose-dependently treated and cultured in an incubator at 37° C./5% CO ₂ for 24 hours. Then, using trypsin, the cells were removed from the culture dish and centrifuged to obtain a cell pellet. After dissolving the cells in Rhod2 staining solution, light was blocked and incubated for 30 minutes in a refrigerator at 4°C. The stained cells were centrifuged again to release the cell pellet with 500 μl of HBSS (w/o Ca ^{2 +} , Mg ^{2 +} ), and then incubated for 10 minutes in an incubator at 37° C./5% CO ₂ , using a flow cytometer. Fluorescence intensity was analyzed.

Protein expression analysis ( western blot )

Mouse testicular cells were treated with BHA, BHT, or a mixture with a cell signaling inhibitor, and then total proteins were extracted from testicular cell lines, and proteins were quantified by Bradford protein assay (Bio-Rad, Hercules, CA, USA). Thereafter, the extracted protein was denatured at 95°C for 5 minutes, electrophoresis was performed using 10% SDS/PAGE gel, transferred to a nitrocellulose membrane, and the primary antibody and secondary antibody were incubated sequentially, and then chemiluminescence detection (SuperSignal West Pico, Pierce, Rockford, IL, USA) reagent, ChemiDoc EQ system, and Quantity One software (Bio-Rad) were used to analyze the expression of the target protein.

JC -1 Mitochondria through staining membrane potential Measure

JC-1 mitochondrial membrane potential (MMP) changes were measured using a mitochondria staining kit (Cat No: CS0390, Sigma-Aldrich). 1×10 ⁵ mouse testicular cells were cultured in 6 wells, and when the cells were filled in a 60% culture dish, they were further cultured in FBS starvation state for 24 hours. Thereafter, BHA was treated in a dose-dependent manner and cultured in an incubator at 37° C./5% CO 2 for 24 hours. Then, using trypsin, the cells were removed from the culture dish and centrifuged to obtain a cell pellet. Cells were dissolved in JC-1 staining solution and incubated for 20 minutes in a 37°C/5% CO2 incubator. The stained cells were centrifuged again, washed with 1x JC-1 staining buffer, and then fluorescence intensity was analyzed using a flow cytometer.

TUNEL Analysis of apoptosis through reaction

3×10 ⁴ mouse Leedig cells were aliquoted and cultured in a confocal dish (catalog number: 100350, SPL Life Science, Republic of Korea) with 300 μl of medium containing 10% FBS, followed by FBS starvation for 24 hours. After further incubation, 100 μM of BHT was treated for 24 hours. Thereafter, the cells were air-dried and incubated with 4% paraformaldehyde at room temperature for 1 hour to fix the cells. The fixed cells were washed once with PBS and incubated for 2 minutes on ice using a solution containing 0.1% Triton X-100 in 0.1% sodium citrate. Next, using the TUNEL staining mixture included in In Situ Cell Death Detection Kit, TMR red (Roche), 37° C./5% CO ₂ Incubated in an incubator for 1 hour. Finally, after rinsing with PBS and staining with DAPI, cells were observed and photographed using an LSM710 (Carl Zeiss, Thornwood, NY, USA) confocal microscope.

quantitative reverse transcription polymerase chain reaction

BHA and BHT-treated Leidich and Sertoli cells were subjected to RNA extraction using Trizol reagent according to the manufacturer's manual. cDNA was synthesized with 1 μg of total RNA and AccuPower premix, and gene expression was measured with SYBR Green (Sigma-Aldrich) and StepOnePlus real-time PCR system (Applied Biosystems, Foster City, CA). The expression of the target gene was calculated as the C _T value, and normalized based on the GAPDH expression. PCR conditions were carried out at 95°C for 3 minutes, and then the 95°C 30 seconds, 60°C 30 seconds, and 72°C 30 seconds conditions were amplified 40 times.

Formation of Spheroids

After dissolving Leedig and Sertoli cells in a medium prepared by adding 10% fetal bovine serum (FBS) to high glucose Dulbecco's modified Eagle medium (DMEM) to make 10 ml of a single-cell suspension, , 25 μl was dispensed at a concentration of 6000 cells per cell drop on the lid of a 100-mm tissue culture dish. To prevent drying, 10 ml of PBS was dispensed in the culture dish, and the lid of the culture dish in which the cells were dispensed was turned upside down. Cell droplets were cultured in an incubator at 37° C./5% CO ₂ for 2-3 days. After taking a spheroid formed with Leica DM2500, image analysis was performed. For statistical processing, three different images were used.

statistical analysis

For the results of this experiment, the mean and standard deviation were calculated using a statistical analysis system (SAS), and one-way ANOVA was performed. Significance tests were performed at the P < 0.05 level.

<Results and considerations>

BHA and BHT's Mouse TM3 (Leedig cells) and TM4 ( Sertoli proliferation inhibitory effect on cells)

To find out whether BHA and BHT have antiproliferative effects on mouse testicular cells, TM3 and TM4 cells, cell proliferation analysis using BrdU was performed ( FIG. 1 ). In the case of BHA, both mouse Leedig cells (TM3) and Sertoli cells (TM4) showed an antiproliferative effect, and when treated up to 100 μM, the cell proliferation rate decreased to 50% (TM3) and 35% (TM4). results (Fig. 1A). In the case of BHT, only TM3 showed a specific anti-proliferative effect, and it was also shown that the cell proliferation ability was reduced by 48% at a concentration of 100 μM ( FIG. 1D ). The green fluorescence of PCNA protein is a marker of cell proliferation, and the relative expression levels were compared through immunofluorescence. As with the previous results, PCNA expression was decreased by about 62% (TM3) and 80% (TM4) when treated with BHA compared to the control group ( p < 0.001), and expression was significantly increased by about 79% in the group treated with BHT. It was confirmed that the decrease ( p < 0.01) (Fig. 1B, 1E). Finally, as a result of analyzing the cell cycle pattern through PI staining, it was confirmed that the number of cells in sub-G1 increased by about 1.6 to 2-fold or the number of cells in G0/G1 decreased when BHA or BHT dose-dependent treatment was performed (Fig. 1C, 1F). ). Through these results, it was confirmed that BHA inhibits cell proliferation and causes cell cycle arrest in mouse testicular cell lines, and that BHT inhibits cell proliferation specifically TM3.

BHA or BHT Analysis of mitochondrial dysfunction and mitochondrial protein expression change in TM3 and TM4 cells induced by

In order to investigate the effect of BHA or BHT on calcium homeostasis, the relative calcium ion concentration in the cytoplasm or mitochondria was measured through Fluo4-AM and Rhod2-AM staining (FIG. 2). When BHA or BHT was treated in a dose-dependent manner (0, 10, 20, 50, 75, 100 μM) in mouse Leedig cells (TM3), the cytoplasmic calcium concentration was 48.5% (BHA) compared to the control (100%). , decreased to 74.5% (BHT), and the calcium ion concentration in the mitochondrial matrix also showed a tendency to decrease significantly to 76.4% (BHA) and 68.1% (BHT) ( p < 0.001) ( FIGS. 2A, 2C, 2E, 2F). In contrast, when BHA was dose-dependently treated with mouse Sertoli cells (TM4), the relative calcium ion concentrations in the cytoplasm and mitochondria were significantly increased by 1.85 and 3.93 times, respectively ( FIGS. 2B and 2D ). In addition, changes in the expression of several mitochondrial proteins involved in mitochondrial apoptosis were confirmed through Western blot. First, upon BHA treatment, the pro-apoptotic proteins Bax, Bak, and cytochrome c were significantly increased up to 1.4-fold in both Leidich cells and Sertoli cells, and the amount of anti-apoptotic protein Bcl-xL and Bad Phosphorylation was reduced ( FIGS. 3A-3D ). Subsequently, even when TM3 was dose-dependently treated with BHT (0, 20, 50, 100 μM), except for Bak, the expression levels of pro-apoptotic proteins (Bax, cytochrome c ) increased 1.5-1.6 times, and anti-apoptotic Proteins (Bad, Bcl-xL) showed a tendency to decrease by about 20-30% (FIG. 3E). In addition, mitochondrial depolarization was induced in both testicular cell lines upon BHA treatment, and DNA fragmentation induced in Leedich cells upon BHT treatment was confirmed by immunofluorescence labeling with red fluorescence ( FIG. 4 ). Through these results, it was confirmed that BHA and BHT inhibit calcium homeostasis and induce mitochondrial apoptosis in mouse testicular cell lines.

BHA or BHT mouse Leedig cells according to treatment and Sertoli Analysis of the effect of endoplasmic reticulum stress induction in cells

Western blot was performed to determine whether ER stress was induced in Leidich cells and Sertoli cells during treatment with BHA and BHT ( FIG. 5 ). First, when BHA was dose-dependently (0, 20, 50, 100 μM) treated in TM3 and TM4 cells, phosphorylation of Perk protein, an initial signal of ER stress, in both cells was 1.6-fold (TM3), 1.8-fold (TM3) and 1.8-fold ( TM4) was confirmed to increase gradually (FIG. 5A). Subsequently, it was confirmed that the phosphorylation of Eif2α, known as a lower signal, and the amount of protein in Chop also increased significantly from 1.5 to 2.8 times compared to the control ( FIGS. 5B-C ). When BHT was treated in TM3 cells, phosphorylation and Chop of Perk and Eif2α as above, as well as protein expression of Grp78, active Atf6α, and Ire1α, known as other early signals, were also significantly increased ( FIGS. 5D-E ). Through these results, it was confirmed that BHA and BHT induce endoplasmic reticulum stress in mouse testis cell lines.

BHA and BHT Leedich cells and Sertoli intracellular mRNA Expression regulation effect analysis

Reverse transcription polymerase chain reaction was performed to confirm the change in mRNA expression level upon treatment with BHA and BHT in mouse Leedig cells and Sertoli cells. First, it was confirmed that when 100 μM of BHA was treated, the expression of Star, Hsd17b1, Srd5a1, and Srd5a3, which are genes encoding enzymes involved in steroid neogenesis, was significantly reduced compared to the control group (FIG. 6A). In addition, the expression of Jun, Junb, and Jund genes, which are known to be involved in the development of germ cells or hormone regulation as transcription factors, were also significantly reduced except for Jund expression in Leedig cells. In addition, the gene expression of Insl3, Inbha, and Ar showed different patterns for each tissue.

When 100 μM of BHT was treated in mouse Leydig cells, genes related to cell cycle progression such as Ccnd1, Cdk4, and Ccne1 decreased by 58%, 21%, and 54%, respectively, and P21, which blocks it, increased by 9 times. , showed results corresponding to the previous cell cycle analysis experiments. However, the genes related to steroid neogenesis, Star, Cyp19a1, and Hsd17b1, showed a tendency to increase during BHT treatment compared to the control group, but Srd5a1 and Srd5a3 to decrease, each showing a different trend ( FIG. 6B ). In addition, the expression of Lhr slightly increased by about 1.2 times, while the expression of Egfr and Jud decreased by 50% and 20%, respectively, compared to the control group.

BHA and BHT In TM3, TM4 cells upon treatment MAPK and PI3K / AKT Analysis of changes in signaling mechanisms

Western blotting was performed to investigate the signaling mechanisms of MAPK and PI3K/AKT pathways in mouse testis cell lines following BHA and BHT treatment. First, when BHA was dose-dependently (0, 20, 50, 100 μM) treatment to mouse Leidhig and Sertoli cells, both cell lines, except for Rps6ka1, showed MAPK signaling pathways (Erk1/2, P38, Jnk). It was confirmed that phosphorylation was increased (FIG. 7A). Phosphorylation of Rps6ka1 was increased up to 1.49 fold in Leedig cells, but decreased by 0.53 fold in Sertoli cells. In the case of the Pi3k/Akt signaling pathway, in Leedich cells, the sub-signals Rps6kb1 and Rps6 were significantly decreased by 29%, 19%, and 69%, respectively, from the decrease in Akt according to BHA treatment ( FIG. 7B ). Conversely, in Sertoli cells, only Akt and Rps6kb1 were slightly increased according to BHA treatment, and there was no significant change in Rps6 ( FIG. 7B ). Contrary to BHA, it was confirmed that phosphorylation of all proteins of the Pi3k/Akt signaling pathway in Leedich cells was increased during BHT treatment ( FIG. 7C ). It was confirmed that the phosphorylation tendency of Erk1/2, Jnk, and P38 increased by 1.4 to 3.2 times, except that the expression level of Rps6ka1 showed a slight decrease by about 19% compared to the control group (FIG. 7D). Through these results, it was confirmed that BHA or BHT activates the MAPK signaling pathway and regulates the PI3K/AKT signaling pathway.

signal transduction inhibitors and BHA or BHT Analysis of changes in TM3 and TM4 cell proliferation and MAPK and PI3K/AKT signaling mechanisms during combination treatment

In order to further clarify the mechanism of the effects of intracellular BHA and BHT, additional experiments were performed by co-treatment with a signaling mechanism inhibitor. For the antiproliferative effect of BHA on mouse testicular cells, synergistic effects were shown when U0126 (Erk1/2 inhibitor) and SP600125 (Jnk inhibitor) were treated ( FIG. 8A ). BHA-induced phosphorylation of Erk1/2 was inhibited by co-treatment with U0126 and SP600125, and phosphorylation of JNK was inhibited by co-treatment with three inhibitors ( FIG. 8B ). The inactivation of Akt and Rps6 proteins reduced by BHA was restored upon treatment with U0126 and SP600125 ( FIG. 8C ). In addition, phosphorylation of Rps6kb1 was completely inhibited by LY294002 (Akt inhibitor), and it was restored in TM3 cells by SP600125 combination treatment ( FIG. 8C ). In TM4 cells, the expression level of Akt pathway signaling proteins was restored when U0126 and SP600125 were treated.

In the case of BHT, co-treatment with U0126 and SP600125 showed a synergistic effect in inhibiting the proliferation of mouse Leedig cells, and when treated with SB203580 (P38 inhibitor), cell proliferation was slightly restored ( FIG. 9A ). In the case of the signaling pathway, phosphorylation was inhibited by LY294002 and SB203580 in the PI3K/AKT pathway, and in the case of Rps6, the original level was restored upon treatment with U0126 and SP600125 ( FIG. 9B ). Conversely, the activation of JNK induced by BHT was more activated when treated with U0126 and SB203580, and recovered to the original level when treated with LY294002 (Fig. 9C). Phosphorylation of P38 by BHT did not show a pattern of recovery to the original phosphorylation level even when treated in combination with any inhibitor except SB203580 ( FIG. 9C ).

BHA and BHT's processing in in vivo TM3, TM4 that mimics the environment spheroid Analysis of impact on formation

Using the spheroid culture method, which is a system that mimics the 3D in vivo environment, the effect of the spheroid formation of BHA and BHT was confirmed. The formation of spheroids of TM3 and TM4 in the medium treated with BHA (100 μM) was 30% (TM3) and 19% (TM4) reduced in size, respectively, than the spheroids formed in the medium treated with the control, and the density was also 45 each. % (TM3), decreased by 73% (TM4) (Fig. 10A-B). In the case of BHT (100 μM), there was no significant difference in TM4 between the control group and the treatment group, whereas the size of the spheroids made of TM3 decreased by about 10% and the density decreased by 21% (Fig. 10C-D). Through this, it was indirectly proved that BHA and BHT have cell proliferation inhibitory and apoptotic effects on TM3 or TM4 cells even in an environment that mimics in vivo as the results performed in vitro.

As described above in detail a specific part of the content of the present invention, for those of ordinary skill in the art, this specific description is only a preferred embodiment, and it is clear that the scope of the present invention is not limited thereby. something to do. Accordingly, it is intended that the substantial scope of the present invention be defined by the appended claims and their equivalents.

Claims (8)

butylated hydroxytoluene (BHT) as an active ingredient,
A pharmaceutical composition for the prevention or treatment of male reproductive diseases caused by overproliferation of Leydig cells, and selected from the group consisting of Leydig cell tumors, orchitis, epididymitis, testicular cancer and hydrocephalus. According to claim 1,
The BHT is a pharmaceutical composition for the prevention or treatment of male reproductive diseases, characterized in that it activates the MAPK signaling mechanism. According to claim 1,
The BHT is a pharmaceutical composition for the prevention or treatment of male reproductive diseases, characterized in that inducing endoplasmic reticulum stress. butylated hydroxytoluene (BHT) as an active ingredient,
A health functional food composition for preventing or improving male reproductive diseases, which is caused by overproliferation of Leydig cells, and is selected from the group consisting of Leydig cell tumors, orchitis, epididymitis, testicular cancer and hydrocephalus. 5. The method of claim 4,
The BHT is a health functional food composition for the prevention or improvement of male reproductive diseases, characterized in that it activates the MAPK signaling mechanism. 5. The method of claim 4,
The BHT is a health functional food composition for the prevention or improvement of male reproductive diseases, characterized in that inducing endoplasmic reticulum stress. A method of inhibiting the proliferative ability of Leedich cells derived from mammals other than humans using the composition of claim 1 . A method of improving the killing effect of Leidich cells derived from mammals other than humans using the composition of claim 1 .

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