KR102434348B1 - Pharmaceutical composition for preventing or treating endometriosis comprising quercetin, luteolin, delphinidin or mixture thereof - Google Patents

Abstract

The present invention relates to a composition comprising quercetin, luteolin, delphinidin or a mixture thereof as an active ingredient, and more particularly, to prevent or treat endometriosis disease comprising quercetin, luteolin, delphinidin or a mixture thereof as an active ingredient It relates to a health functional food composition for preventing or improving endometriosis comprising a pharmaceutical composition for use and quercetin, luteolin, delphinidin or a mixture thereof as an active ingredient.
The composition according to the present invention has the effect of inhibiting the proliferation of endometriosis cells and enhancing the apoptosis effect by inhibiting phosphorylation of a sub-signal that regulates the PI3K/AKT signaling mechanism in endometriosis cells. It can be usefully used in fields related to pharmaceuticals and functional foods that can prevent and treat endometriosis caused by abnormal development of

Description

A pharmaceutical composition for preventing or treating endometriosis comprising quercetin, luteolin, delphinidin or a mixture thereof

The present invention relates to a composition comprising quercetin, luteolin, delphinidin or a mixture thereof as an active ingredient, and more particularly, to prevent or treat endometriosis disease comprising quercetin, luteolin, delphinidin or a mixture thereof as an active ingredient It relates to a health functional food composition for preventing or improving endometriosis comprising a pharmaceutical composition for use and quercetin, luteolin, delphinidin or a mixture thereof as an active ingredient.

Endometriosis is a disease that causes infertility in 35-50% of patients due to abnormal proliferation of the ovaries, fallopian tubes, and abdominal wall in women of childbearing age (Vigano et al, Best Pract Res Clin Obstet Gynaecol, 2004; Bulun, N Engl J Med, 2009). ), the pathological mechanism of this disease is being actively studied, but the exact cause of the disease is still unknown.

The basic treatment methods for endometriosis include surgical removal and hormone therapy. Since endometriosis is diagnosed based on symptoms rather than prognostic factors, the recurrence rate is within 5 years even after surgical removal. This is known to be close to 30-40% (Busacca et al. , J Obstet Gynecol, 2006: Vignali et al ., J Minim Invasive Gynecol, 2005).

Hormone treatment using a gonadotropin-free hormone agonist (GnRH agonist) or aromatase inhibitor has also been reported to have problems such as side effects and drug resistance (Gao et al. , Gynecol Oncol, 2014).

In recent years, the treatment regimen for endometriosis is changing from surgery to plant-derived treatments, but studies on the exact mechanism of action and side effects are still lacking (Johnson et al., Hum Reprod, 2013).

On the other hand, quercetin is one of the flavonols that can be consumed on average about 25-50 mg per day from various vegetables, and has been reported to cause apoptosis in gynecological cancer cells such as breast and ovarian cancer (K. Bishayee, S. Ghosh). , A. Mukherjee, R. Sadhukhan, J. Mondal, AR Khuda-Bukhsh. Cell Prolif, 46 (2013), pp. 153-163/S. Ranganathan, D. Halagowder, ND Sivasithambaram. PLoS One, 10 (2015) , Article e0141370).

In addition, luteolin is a flavone-based plant extract contained in broccoli and carrots and is known to have anti-inflammatory, anticancer, and antioxidant effects (Parhiz H, Roohbakhsh A, Soltani F, Rezaee R, Iranshahi M. Phytotherapy research: PTR) 509 2015; 29 :323-331).

In addition, delphinidin is an anthocyanin-based flavonoid extracted from pigments such as eggplant and berries, and is known to have antioxidant and anticancer effects (Hou DX, Fujii M, Terahara N, Yoshimoto M. J Biomed Biotechnol. 2004; 2004 : 321-325).

As such, various pharmacological effects of quercetin, luteolin and delphinidin have been reported, but their endometriosis therapeutic effect has not been reported so far.

Accordingly, the present inventors have made diligent efforts to develop a novel endometriosis treatment material using a natural product-derived material, and as a result, quercetin, luteolin and delphinidin inhibit the proliferation of endometriosis cell lines and have the effect of improving the cell death effect. By confirming, the present invention was completed.

An object of the present invention is to provide a pharmaceutical composition for preventing or treating endometriosis comprising at least one selected from the group consisting of quercetin, luteolin and delphinidin as an active ingredient.

In addition, an object of the present invention is to provide a health functional food composition for preventing or improving endometriosis comprising at least one selected from the group consisting of quercetin, luteolin and delphinidin as an active ingredient.

Another object of the present invention is to provide a method for inhibiting the proliferative ability of endometriosis cells using the composition.

Another object of the present invention is to provide a method for improving the apoptosis effect of endometriosis cells using the composition.

In order to solve the above problems, the present invention provides a pharmaceutical composition for preventing or treating endometriosis comprising at least one selected from the group consisting of quercetin, luteolin and delphinidin as an active ingredient.

In addition, the present invention provides a health functional food composition for preventing or improving endometriosis comprising at least one selected from the group consisting of quercetin, luteolin and delphinidin as an active ingredient.

In addition, the present invention provides a method of inhibiting the proliferative ability of endometriosis cells using the composition.

In addition, the present invention provides a method for enhancing the apoptosis effect of endometriosis cells using the composition.

The composition according to the present invention has the effect of inhibiting the proliferation of endometriosis cells and enhancing the apoptosis effect by inhibiting phosphorylation of a sub-signal that regulates the PI3K/AKT signaling mechanism in endometriosis cells. It can be usefully used in fields related to pharmaceuticals and functional foods that can prevent and treat endometriosis caused by abnormal development of

1A to 1E show the results of analyzing the effects of quercetin, luteolin and delphinidin treatment on the proliferation of endometriosis cells.
2a to 2f show the results of analyzing the effect of quercetin, luteolin and delphinidin treatment on the death of endometriosis cells.
3a to 3f show the results of analysis of changes in mitochondrial membrane permeability in endometriosis cells, cytoplasmic calcium concentration and free radical control by quercetin, luteolin, and delphinidin.
4A to 4C show the results of analyzing the regulation of signaling mechanisms in endometriosis cells by quercetin, luteolin, and delphinidin.
Figure 5 shows the results of analyzing the effect of the combined treatment of quercetin, luteolin, delphinidin and a signaling mechanism inhibitor on the proliferation of endometriosis cells.
6a to 6c show the results of analyzing the effect of the combined treatment of quercetin, luteolin, delphinidin and a signaling mechanism inhibitor on changes in the activity of signaling proteins in cells.
7 shows the results of analyzing the lesion growth inhibitory effect in an animal model of endometriosis by quercetin and luteolin.
8A to 8I show the results of analysis of the effects of inhibiting mRNA expression of cell cycle regulatory proteins and inhibiting the growth of endometriosis cells and promoting apoptosis through combined treatment with quercetin and luteolin.
9 shows the results of analyzing the expression changes of microRNAs in endometriosis cells and lesions in animal models according to quercetin treatment.
10 shows the mechanism of inhibition of proliferation and cell death of endometriosis cells by quercetin.
11 shows the mechanism of inhibition of proliferation and cell death of endometriosis cells by luteolin.
12 shows the mechanism of suppression of proliferation and cell death of endometriosis cells by delphinidin.

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 endometriosis cells by quercetin, luteolin and delphinidin were revealed (FIGS. 10-12).

In one aspect, the present invention relates to a pharmaceutical composition for preventing or treating endometriosis comprising at least one selected from the group consisting of quercetin, luteolin and delphinidin as an active ingredient.

In another aspect, the present invention relates to a health functional food composition for preventing or improving endometriosis comprising at least one selected from the group consisting of quercetin, luteolin and delphinidin 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 and 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 weight, age, sex, administration mode, and administration time of the patient, 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 means a food manufactured and processed using raw materials or ingredients useful for the human body according to Act No. 6722 on Health Functional Foods, 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, at least one component selected from the group consisting of quercetin, luteolin and delphinidin may be characterized in that it modulates the PI3K/AKT and MAPK signaling mechanisms related to the proliferation of endometriosis cells.

In the present invention, it may be characterized in that it further comprises a target inhibitor that inhibits the PI3K / AKT or MAPK signaling pathway.

In the present invention, at least one component selected from the group consisting of quercetin, luteolin and delphinidin may be characterized in that it induces oxidative stress, free radicals and lipid peroxidation in endometriosis cells.

In the present invention, the chrysin, silibinin or a mixture thereof induces depolarization of the mitochondrial membrane potential of endometriosis cells and may be characterized in that it activates the expression of apoptosis protein.

In the present invention, the quercetin and delphinidin may be characterized by reducing the mitochondrial membrane potential.

In the present invention, the quercetin and luteolin may be characterized by increasing active oxygen.

In the present invention, the luteolin and delphinidin may be characterized in that increasing the calcium ions in the cytoplasm.

In the present invention, the quercetin and luteolin may be characterized in that they reduce the size of the lesion in an autologous endometriosis animal model and regulate the expression of cell cycle regulatory proteins in the lesion.

In the present invention, the quercetin may be characterized in that it regulates the cell cycle regulatory protein Cyclin D1.

In the present invention, the quercetin may be characterized in that it modulates the Cyclin D1 target microRNA.

In the present invention, the luteolin may be characterized in that it regulates the cell cycle regulatory protein Cyclin E1.

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 proliferation ability of endometriosis cell lines using the pharmaceutical composition or health functional food composition.

In another aspect, the present invention relates to a method for improving the killing effect of endometriosis cell lines using the pharmaceutical composition or health functional food composition.

[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.

Example

<Experiment method>

Experimental animals and cell culture

VK2/E6E7, End1/E6E7 cells, which are endometriosis cell lines, were purchased from the American Type Culture Collection and used in Keratinocyte medium for monolayer culture of cells. Recombinant human epidermal growth factor (rhEGF) 0.1ng/ mL and fetal bovine serum (FBS) 0.05 mg/mL were mixed together and used.

experimental material

Quercetin and luteolin, which are candidate compositions, were purchased from Sigma-Aldrich, Inc., and delphinidin was purchased from INDOFINE Chemical Company, Inc. Cell Signaling with antibodies to phospho-AKT, ERK1/2, P70S6K, P90RSK, S6, P38 proteins and total-AKT, ERK1/2, P70S6K, P90RSK, S6, and P38 proteins to confirm the signaling mechanism by the composition It was purchased from Techonology. In addition, PCNA antibodies were purchased from Santa Cruz Biotechnology, respectively. In addition, LY294002, a PI3K inhibitor, was purchased from Cell Signaling Technology, and U0126, an ERK1/2 inhibitor, was purchased from Enzo Life Sciences to investigate the effect of inhibiting the target signaling process.

Analysis of cell proliferation ability using BrdU

To check the effect of quercetin, luteolin, and delphinidin on the proliferative ability of endometriosis cells, 100 μl of cultured ⁵ ×10 cells and 100 μl of the culture medium were dispensed into 96 wells, and quercetin, luteolin, and delphinidin were dose-dependently (0) , 5, 10, 20, 50 or 100 μM) and cultured for 48 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. VK2/E6E7 and End1/E6E7 cells were labeled with BrdU, fixed, and incubated with anti-BrdU-POD solution at room temperature for 90 minutes, and then washed 3 times. Finally, cells were reacted with 100 μl of 3,3'' substrate, and absorbance at 370 nm and 492 nm was measured using an ELISA reader to analyze cell proliferation ability.

Cell cycle analysis using propidium iodide staining

In order to check the cell cycle change of endometriosis cells by quercetin, luteolin, and delphinidin, 5×10 ⁵ cells were cultured in 6 wells until 70-80% cells were filled. Thereafter, quercetin, luteolin, and delphinidin dose-dependently (0, 5, 10, 20 μM or 0, 20, 50, 100 μM) were treated and cultured in an incubator at 37° C./5% CO ₂ for 48 hours. Then, using trypsin, the cells were removed from the culture dish, washed with PBS, and the cells were slowly mixed with 1 mL of 1× binding buffer and centrifuged to obtain a cell pellet. Next, the cells were suspended and cultured with 200 µL of 1× binding buffer, put in 100 µL in a brown 1.5 mL tube, mixed with 5 µL of RNase and 5 µL of PI, and the cells were left at room temperature for 1 hour for staining. Thereafter, 400 μL of 1× binding buffer was added, the stained solution was transferred to a 5 mL FACS tube, and the fluorescence intensity was analyzed using a flow cytometer to measure the number of cells corresponding to each cell cycle.

Analysis of apoptosis by staining with Annexin V and propidium iodide

In order to confirm the apoptosis effect of endometriosis cells by quercetin, luteolin, and delphinidin, an experiment was conducted using the FITC Annexin V Cell Death Diagnostic Kit I (BD Biosciences). First, 5×10 ⁵ cells were cultured in 6 wells. Thereafter, quercetin, luteolin, and delphinidin dose-dependently (0, 5, 10, 20 μM or 0, 20, 50, 100 μM) were treated and cultured in an incubator at 37° C./5% CO ₂ for 48 hours. Then, using trypsin, the cells were removed from the culture dish, washed with PBS, and the cells were slowly mixed with 1 mL of 1× binding buffer and centrifuged to obtain a cell pellet. Next, the cells were suspended and cultured with 200 μL of 1× binding buffer, put into a brown 1.5 mL tube, 100 μL, Annexin V 5 μL, and PI 5 μL were mixed together, and the cells were left at room temperature for 15 minutes for staining. Then, 400 μL of 1× binding buffer was added, the stained solution was transferred to a 5 mL FACS tube, and the fluorescence intensity was analyzed using a flow cytometer to measure the number of dead cells.

Immunofluorescence

3×10 ⁴ cells of VK2/E6E7, End1/E6E7 were aliquoted in a confocal dish (catalog number: 100350, SPL Life Science, Republic of Korea) and cultured, quercetin, luteolin (20 μM), delphinidin (100 μM) ) was treated for 48 hours, then the cells were fixed with methanol for 10 minutes, treated with PCNA antibody diluted to 2 μg/ml, and incubated at 4° C. for 16 hours. Then, after washing twice with PBS containing 0.1% bovine serum albumin (BSA), goat anti-mouse IgG Alexa 488 (catalog number: A-11001, Invitrogen, Carlsbad, CA, USA) was used as a secondary antibody. It was diluted 1:200 in antibody dilution buffer and incubated for 1 hour at room temperature. After washing VK2/E6E7 and End1/E6E7 cells with 0.1% BSA-PBS, DAPI staining was additionally performed to enable simultaneous observation of nuclei as well as target proteins in VK2/E6E7 and End1/E6E7 cells. After the end of the experiment, cells were observed and photographed using an LSM710 (Carl Zeiss, Thornwood, NY, USA) confocal microscope.

Cell death analysis through TUNEL reaction

3×10 ⁴ cells of VK2/E6E7, End1/E6E7 were aliquoted in a confocal dish (catalog number: 100350, SPL Life Science, Republic of Korea) and cultured, quercetin, luteolin (20 μM), delphinidin (100 μM) ) was treated for 48 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 are rinsed once with PBS and incubated on ice for 2 minutes 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), it was incubated for 1 hour at 37°C/5% CO2 incubator. Finally, after rinsing with PBS and staining with DAPI, cells were observed and photographed using an LSM710 (Carl Zeiss, Thornwood, NY, USA) confocal microscope.

Measurement of Mitochondrial Membrane Potential by JC-1 Staining

JC-1 mitochondrial membrane potential (MMP) changes were measured using a mitochondria staining kit (Cat No: CS0390, Sigma-Aldrich). 5×10 ⁵ VK2/E6E7, End1/E6E7 cells were cultured in 6 wells and cultured until 70-80% of the culture dish was filled with cells. Thereafter, quercetin, luteolin, and delphinidin dose-dependently (0, 5, 10, 20 μM or 0, 20, 50, 100 μM) were treated and cultured in an incubator at 37° C./5% CO ₂ for 48 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 an incubator at 37°C/5% CO ₂ . The stained cells were centrifuged again, washed with 1x JC-1 staining buffer, and then fluorescence intensity was analyzed using a flow cytometer.

Intracellular ROS measurement using DCFH-DA

To determine the effect of chrysophanol on ROS generation in endometriosis cell lines, 2'7'-dichlorofluorescin diacetate (DCFH-DA, Sigma), which is converted to 2'7'-dichlorofluorescin (DCF) and fluoresces in the presence of peroxide, was used. was used. VK2/E6E7 and End1/E6E7 cells were removed through trypsin and a cell pellet was obtained through centrifugation. After washing once with PBS, 10 μM of DCFH-DA was incubated in an incubator at 37° C. for 30 minutes. Thereafter, the cells were washed twice with PBS, and quercetin, luteolin, and delphinidin dose-dependently (0, 5, 10, 20 μM or 0, 20, 50, 100 μM) were incubated for 1 hour at 37° C. in an incubator. Treated cells were washed again with PBS and analyzed for DCF fluorescence intensity using flow cytometry.

Protein Expression Analysis (Western Blot)

VK2/E6E7 and End1/E6E7 cells were treated with quercetin, luteolin, delphinidin or a mixture of cell signaling inhibitors, and then total proteins were extracted from VK2/E6E7 and End1/E6E7 cells by Bradford protein assay (Bio-Rad, Hercules). , CA, USA) for protein quantification. Thereafter, the extracted protein was denatured at 95°C for 5 minutes, electrophoresed using 10% SDS/PAGE gel, transferred to a nitrocellulose membrane, incubated with primary antibody and secondary antibody sequentially, and then chemiluminescence detection (SuperSignal West Pico, Pierce, Rockford, IL, USA) reagents were used to analyze the expression of target proteins using ChemiDoc EQ system and Quantity One software (Bio-Rad) instrument.

Autologous endometriosis animal model

After anesthetizing the mouse, one side of the uterine horn was tied and cut off, and the mouse was cut into thirds and fixed in the mesenteric blood vessel of the same mouse, and then recovered for a week. After that, 100 μl of DMSO, quercetin (35mg/kg) and luteolin (40mg/kg) were each injected 10 times for one month, and the size of the transplanted endometriosis lesions was compared. RNA was extracted from three endometrial tissues from each mouse or tissues were fixed, and mRNA and protein expression levels in the tissues were checked through H&E staining, in situ hybridization, and Immunohistochemistry.

statistical analysis

For the results of this experiment, the mean and standard error 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>

Analysis of the effects of quercetin, luteolin, and delphinidin on proliferation, cell cycle inhibition and death of endometriosis cells

To analyze the changes in endometriosis cell lines VK2/E6E7 and End1/E6E7 cells by quercetin, luteolin, and delphinidin, first, quercetin, luteolin, and delphinidin were dose-dependently (0, 5, 10, 20, 50 or 100 μM). ) was cultured in the added medium for 48 hours. Then, as a result of analyzing the cell proliferation pattern, it was found that quercetin and luteolin dose-dependently decreased cell proliferation up to a concentration of 20 μM in two endometriosis cell lines ( FIGS. 1a and 1b ). Delphinidin showed its effect at a concentration of 100 μM (Fig. 1c). Then, based on the immunofluorescence technique, 20 μM quercetin, luteolin, and 100 μM delphinidin were treated for 48 hours, and PCNA, a marker of endometriosis intracellular proliferation, was fluorescently stained. As a result, it was confirmed that PCNA was overexpressed in the nucleus of each cell in the VK2/E6E7 and End1/E6E7 cell lines of the control group, but it was also confirmed that the expression was weakened according to the treatment of each extract (FIG. 1d-1f). These results show that quercetin, luteolin and delphinidin not only inhibited the proliferation of endometriosis cells, but also that the concentration-dependent treatment with quercetin, luteolin, and delphinidin stopped the cell cycle of the endometriosis cell line in subG0/G1, thereby inhibiting cell growth. was confirmed (Fig. 1g-1i).

quercetin, luteolin, delphinidin Analysis of the killing effect of endometriosis cell lines according to application

In order to measure the number of cells killed in endometriosis cells by quercetin, luteolin, and delphinidin, each substance was dose-dependently treated and incubated for 48 hours, stained with Annexin V and propidium iodide (PI), and then using a flow cytometer. to measure the number of dead cells (FIGS. 2a-2c). As a result, it was found that quercetin, luteolin, and delphinidin act in VK2/E6E7 and End1/E6E7 cells to increase the number of apoptotic cells in a dose-dependent manner. In addition, TUNEL assay was performed through tetramethyl-rhodamine-dUTP labeling to determine whether DNA fragmentation occurred in endometriosis cell lines killed by each plant-derived extract. As a result, quercetin, luteolin, and delphinidin all induced red fluorescence according to DNA fragmentation in the nucleus of VK2/E6E7 and End1/E6E7 cell lines ( FIGS. 2d-2f ). Through these results, it was confirmed that quercetin, luteolin, and delphinidin effectively induce apoptosis through DNA fragmentation in endometriosis cell lines.

quercetin, luteolin, to delphinidin Analysis of changes in mitochondrial membrane permeability and cytoplasmic calcium concentration and free radical regulation in cells with endometriosis

In order to check the mitochondrial membrane potential change by quercetin and delphinidin, each corresponding substance was treated in an endometriosis cell line and incubated for 48 hours, followed by JC-1 staining and FACS analysis. As a result, it was confirmed that the mitochondrial membrane potential in endometriosis cells was dose-dependently decreased by quercetin and delphinidin ( FIGS. 3a and 3b ). In addition, luteolin and delphinidin significantly increased intracytoplasmic calcium ions (FIGS. 3c, 3d). Also, DCF fluorescence to determine whether oxidative stress in VK2/E6E7, End1/E6E7 cells is induced by quercetin and luteolin. As a result of confirming the level of intracellular reactive oxygen species (ROS) production through observation, DCF fluorescence in the cell line treated with quercetin (Fig. 3e) and luteolin (Fig. 3f) increases in a dose-dependent manner, and the increased reactive oxygen species is lipid in the cytoplasm. It was found to cause lipid peroxidation.

quercetin, luteolin, to delphinidin Analysis of the regulation of signaling mechanisms in cells of endometriosis by

Phosphorylation patterns of PI3K and ERK1/2 signaling pathways associated with proliferation were investigated in order to identify signaling mechanisms affecting cell death and inhibition of cell proliferation induced by quercetin, luteolin, and delphinidin in endometriosis cells. It was confirmed to be dependent (FIG. 4). As a result, it was shown that quercetin decreased phosphorylation of ERK1/2-P90RSK, P38MAPK, and AKT-P70S6K-S6 proteins in a dose-dependent manner in endometriosis cell lines VK2/E6E7 and End1/E6E7 cells (FIG. 4a). It was confirmed that luteolin killed endometriosis cells by increasing phosphorylation of P38MAPK protein while decreasing phosphorylation of ERK1/2, JNK, and AKT-P70S6K-S6 proteins ( FIG. 4b ). Delphinidin inhibited the activity of ERK1/2 and AKT-P70S6K-S6 signaling proteins and at the same time increased phosphorylation of P90RSK and P38 MAPK proteins in a dose-dependent manner ( FIG. 4c ). These results indicate that quercetin, luteolin, and delphinidin regulate PI3K/AKT and MAPK signaling mechanisms in endometriosis cells.

quercetin, luteolin, with delphinidin Analysis of cell proliferation change pattern through combination treatment with signal transduction mechanism inhibitor

In order to analyze the effect of signaling molecules regulated by quercetin, luteolin, and delphinidin selected through a dose-dependent analysis on the proliferation of endometriosis cells, 20 μM of LY294002 (PI3K inhibitor) or U0126 (ERK1/2 inhibitor) in endometriosis cells ) 10 μM was co-treated with quercetin, luteolin 20 μM and delphinidin 100 μM (Fig. 5). As a result, the proliferation of endometriosis cells decreased by quercetin was additionally decreased by the combined treatment of LY294002, U0126, and SB203580. In particular, when each cell line was treated with LY294002 and SB20358 alone Contrary to this, it was shown that the cell proliferation inhibitory effect was efficiently improved (FIG. 5a). The proliferation of endometriosis cells decreased by luteolin was shown to be additionally decreased by the combined treatment with LY294002, U0126, SP600125, and SB203580. In particular, when each cell line was treated with a signaling inhibitor alone, each cell Contrary to this, it was shown that the cell proliferation inhibitory effect was efficiently improved (Fig. 5b). The proliferation of endometriosis cells decreased by delphinidin was shown to be additionally decreased due to the combined treatment of LY294002, U0126, and SB203580. In particular, the combination treatment compared to when each cell line was treated with a signaling inhibitor alone. It was shown that the cell proliferation inhibitory effect was improved efficiently (FIG. 5c).

quercetin, luteolin, with delphinidin AKT , MAPK Analysis of Signal Transmitter Phosphorylation Pattern in Endometriosis Cells Using a Signal Transduction Mechanism Inhibitor Mixture

LY294002 20 μM, U0126 10 μM, SP600125 20 μM, and SB203580 20 μM in endometriosis cells were added in a 37°C/CO ₂ incubator to elucidate the exact molecular mechanism regulated by quercetin, luteolin, and delphinidin in endometriosis cells. After incubation for 1 hour, 20 μM of quercetin, luteolin, and 100 μM of delphinidin were each treated for 30 minutes to extract proteins, and phosphorylation of target molecules was analyzed through Western blot. As a result, the phosphorylation of ERK1/2 and P90RSK, which were decreased by quercetin, was further inhibited when both VK2/E6E7 and End1/E6E7 were treated with U0126, and P38 phosphorylation was further inhibited by the combination treatment of quercetin and SB203580. was shown. Phosphorylation of AKT reduced by quercetin was further inhibited by the combined treatment of apigenin and LY294002, and the phosphorylation of P70S6K and S6 was further weakened when combined with LY294002 than when quercetin was treated alone. was confirmed (Fig. 6a). In addition, phosphorylation of ERK1/2 and JNK, which were decreased by luteolin, was further inhibited when U0126 or SP600125 was treated together, and phosphorylation of P38 increased by luteolin was inhibited by the combination treatment of SB203580. . Phosphorylation of AKT, P70S6K and S6 reduced by luteolin was further inhibited by co-treatment with luteolin and LY294002 ( FIG. 6b ). Upon co-treatment with delphinidin and a signaling inhibitor, ERK1/2 was further decreased by LY294002 and U0126, and phosphorylation of P90RSK increased by treatment with delphinidin was also decreased by LY294002 and U0126. The increased phosphorylation of P38 MAPK was decreased by all signaling inhibitors in VK2/E6E7 cells, but decreased by U0126 and SB203580 in End1/E6E7 cells. LY294002 inhibited all phosphorylation of AKT-P70S6K-S6 decreased by delphinidin, AKT phosphorylation was decreased by SB203580, and phosphorylation of P70S6K was decreased by U0126 (Fig. 6c). Through these results, the MAPK and AKT signaling pathways regulated by quercetin, luteolin, and delphinidin interact with each other, and the antiproliferative effect of endometriosis cells was confirmed.

Confirmation of endometriosis lesion growth inhibitory effect of quercetin and luteolin in autologous animal model

When quercetin (35mg/kg) and luteolin (40mg/kg) were injected intraperitoneally 10 times in an animal model of endometriosis transplanted into the mesentery by dividing the uterine horn into thirds, the size of the lesion was significantly larger than that of the control group injected with solvent alone. was decreased ( FIGS. 7a and 7d ). In the lesions of mice injected with quercetin, the expression of mRNA ( FIG. 7b ) and protein ( FIG. 7c ) of Ccnd1, a cell cycle regulatory protein, was decreased compared to mice injected with solvent alone. In addition, the mRNA expression of the cell cycle regulatory proteins Ccne1, Cdk2, and Cdk4 was decreased in the lesions of the mice injected with luteolin compared to the lesions of the mice injected with only the solvent (FIG. 7e).

of cell cycle regulatory proteins mRNA Confirmation of the effect of suppressing the growth of endometriosis cells and promoting apoptosis through combination treatment with expression suppression and quercetin and luteolin

In endometriosis cells, VK2/E6E7, End1/E6E7, CCND1 siRNA dose-dependent (10, 20, 40 nM) treatment effectively reduced intracellular CCND1 mRNA expression compared to Control siRNA-treated cells (Fig. 8a), among them, intracellular CCND1 And it was confirmed that PCNA mRNA expression was similar when 40 nM CCND1 siRNA treatment and 20 μM quercetin were treated ( FIG. 8b ). Cell growth was also decreased by the dose-dependent treatment of CCND1 siRNA, and in both cells, 40 nM of siRNA showed a similar degree of cell growth inhibition to that of 20 μM of quercetin ( FIG. 8c ). In addition, apoptosis of endometriosis cells was further increased when CCND1 siRNA and quercetin were co-treated than when control siRNA and quercetin were co-treated ( FIG. 8D ). Cell cycle inhibition also showed a tendency to further increase cells in sub G0/G1 phase when CCND1 siRNA and quercetin were co-treated than when control siRNA and quercetin were co-treated (FIG. 10e). Luteolin also confirmed that CCNE1 and PCNA mRNA expression was further reduced when 40 nM CCNE1 siRNA and 20 μM luteolin were combined treatment than when control siRNA and luteolin were combined ( FIG. 8f ). It was also confirmed that cell growth was further decreased by co-treatment of CCNE1 siRNA with luteolin in a dose-dependent manner rather than the combination treatment with control siRNA and tureolin (FIG. 8g). In addition, apoptosis of endometriosis cells was further increased when CCNE1 siRNA and luteolin were co-treated than when control siRNA and luteolin were co-treated (FIG. 8h). Cell cycle inhibition also showed that when CCNE1 siRNA and luteolin were co-treated compared to when control siRNA and luteolin were co-treated, sub G0/G1 cells increased more and G0/G1 cells decreased in VK2/E6E7. In End1/E6E7, G2/M phase cells increased and G0/G1 decreased ( FIG. 8i ).

Treatment of endometriosis cells and lesions in animal models following quercetin treatment microRNAs Check expression changes

MicroRNAs having a high binding probability with the VK2/E6E7, End1/E6E7, and CCNE1 genes in endometriosis cells were selected ( FIGS. 9A and 9B ). In VK2/E6E7 cells, miR-503-5p, miR-1283, miR-3203, miR-3714 and miR-6867-5p increased according to quercetin treatment. In addition, End1/E6E7 also increased miR-503-5p, miR-1283, miR-3714 and miR-6867-5p according to quercetin treatment (FIGS. 9c-9g). Selected miR-503-5p and miR-546 in mice also decreased within the lesion following quercetin injection ( FIGS. 9H and 9I ).

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 (5)

Including any one selected from the group consisting of quercetin, luteolin and delphinidin as an active ingredient,
Further comprising a target inhibitor that inhibits the PI3K / AKT signaling pathway,
The target inhibitor for inhibiting the PI3K / AKT signaling pathway is a pharmaceutical composition for preventing or treating endometriosis, characterized in that LY294002. According to claim 1,
Further comprising a target inhibitor that inhibits the MAPK signaling pathway,
The target inhibitor for inhibiting the MAPK signaling pathway is a pharmaceutical composition for preventing or treating endometriosis, characterized in that U0126 or SB203580. Including any one selected from the group consisting of quercetin, luteolin and delphinidin as an active ingredient,
Further comprising a target inhibitor that inhibits the PI3K / AKT signaling pathway,
The target inhibitor for inhibiting the PI3K / AKT signaling pathway is a health functional food composition for preventing or improving endometriosis, characterized in that LY294002. A method of inhibiting the proliferation ability of a mammalian non-human endometriosis cell line using the composition according to claim 1 . A method for enhancing the killing effect of endometriosis cell lines derived from mammals other than humans by using the composition according to claim 1 .

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