Application of stigmasterol in preparation of medicine for treating endometriosis

Abstract

The invention discloses application of stigmasterol in preparation of a medicine for treating endometriosis, and belongs to the technical field of biological medicine. The stigmasterol is found to be capable of effectively inhibiting migration and invasion ability of ectopic endometrial cells, improving cell senescence state and reversing macrophage M2 type polarization caused by senescence, and through the effects, the stigmasterol can regulate and control the cell senescence process and remodel the immune microenvironment, so that the effect of treating endometriosis is achieved. The invention provides application of stigmasterol in preparation of a medicine for treating endometriosis, and provides an innovative, effective and safe technical scheme for treatment of the endometriosis.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of stigmasterol in the preparation of drugs for the treatment of endometriosis. Background Technology

[0002] Endometriosis is a common benign gynecological disease characterized by the presence of functional endometrial tissue outside the uterine cavity, often accompanied by chronic inflammation, pain, and infertility. Despite being a benign condition, it exhibits high histological invasiveness and recurrence. Current treatments include drug therapy (such as progestins and GnRH agonists) and surgical intervention, but both have limitations such as high recurrence rates, significant impact on ovarian function, and substantial adverse reactions, making long-term safe and effective treatment difficult to achieve.

[0003] Recent studies have revealed that cellular senescence plays a crucial role in the development and progression of endometriosis. Senescent cells alter the local microenvironment by secreting pro-inflammatory cytokines, chemokines, and growth factors (SASP factors), promoting the migration, invasion, and immune escape of lesion cells, thereby driving disease progression. Furthermore, the senescence process exhibits significant heterogeneity in ectopic lesion tissues, with differences in the degree of senescence and immunological effects among different lesions.

[0004] Therefore, there is an urgent need to develop novel intervention strategies targeting the senescence of ectopic endometrial cells and their immune microenvironment, in order to achieve more precise and safer interventions for endometriosis.

[0005] Natural compounds, due to their multiple biological activities such as anti-inflammatory, antioxidant, and immunomodulatory effects, have become an important source for new drug development. Among them, stigmasterol is an unsaturated phytosterol widely found in plants, with the molecular formula C6H2O. 29 H 48 O3, abundant in soybeans, has been proven to possess various biological effects, including anti-inflammatory, antioxidant, and immunomodulatory properties, and is widely used in medicine, food, and cosmetics. However, to date, there is no systematic research on the role of stigmasterol in the treatment of endometriosis. This invention discovers the role of stigmasterol in regulating the senescence of endometriosis-related cells and the immune microenvironment, providing a theoretical basis and technical support for its application in the preparation of drugs for the treatment of endometriosis. Summary of the Invention

[0006] This invention reveals that stigmasterol can effectively inhibit the migration and invasion of ectopic endometrial cells, improve cellular senescence, and reverse senescence-induced M2 macrophage polarization. Through these effects, stigmasterol can regulate the cellular senescence process and reshape the immune microenvironment, thereby achieving a therapeutic effect on endometriosis.

[0007] Based on the role of stigmasterol in regulating endometriosis-related cell senescence and immune microenvironment discovered in this invention, the purpose of this invention is to provide new pharmaceutical applications for stigmasterol, specifically its use in the preparation of drugs for treating endometriosis, in order to address the problems of insufficient safety, significant side effects, and poor long-term efficacy of existing treatment regimens.

[0008] The objective of this invention is achieved through the following technical solution: This invention provides the use of stigmasterol in the preparation of a medicament for treating endometriosis. In this application, stigmasterol can be the sole active ingredient of the medicament.

[0009] The present invention also provides the use of compositions containing stigmasterol in the preparation of medicaments for treating endometriosis. In this application, the compositions containing stigmasterol may contain other substances capable of treating endometriosis, such as gestrinone, leuprolide acetate, and dinogest, in addition to stigmasterol. That is, stigmasterol can be used in combination with one or more other substances for treating endometriosis as the active ingredient of the medicament.

[0010] The drugs described above for treating endometriosis use improving cellular senescence and regulating the immune microenvironment as the core intervention strategy for treating endometriosis.

[0011] Furthermore, the drugs used to treat endometriosis include one or more of the following: drugs that inhibit the aging of ectopic endometrial cells, drugs that inhibit the migration and invasion of ectopic endometrial cells, drugs that reduce ectopic endometrial lesions, and drugs that reshape the immune microenvironment of endometriosis.

[0012] The preferred dosage of stigmasterol in the drug for treating endometriosis is 30-100 mg / kg.

[0013] The aforementioned drug for treating endometriosis, in addition to containing stigmasterol, or in addition to containing stigmasterol and other substances for treating endometriosis, may further contain a pharmaceutically acceptable carrier. The drug can be prepared into tablets, powders, pills, granules, capsules, solutions, suspensions, or injections using conventional pharmaceutical processes.

[0014] The present invention has the following advantages and beneficial effects: (1) Natural source and high safety: Stigmasterol is a natural active ingredient derived from plants, with few toxic side effects and good long-term safety. (2) Targeted pathogenesis: Intervene in the abnormal cell senescence and immune microenvironment in endometriosis, starting from the pathological link to improve the precision of treatment; (3) Dual regulatory effect: It can both inhibit the migration and invasion of lesion cells and improve the aging state and regulate the polarization of immune cells, thus achieving comprehensive treatment of multiple links.

[0015] In summary, this invention provides an innovative, effective, and safe technical solution for the treatment of endometriosis, which has significant theoretical value and broad application prospects. Attached Figure Description

[0016] Figure 1 Effects of stigmasterol on the function of endometriosis cells.

[0017] (A): Schematic diagram of SA-β-Gal staining in MEECs and MESCs cells of the Non-Sen. group, Sen. group, and Sen.+Stigmasterol group. This means P < 0.001.

[0018] (B): Schematic diagram of the Transwell chamber migration and invasion results of MEECs and MESCs in the Non-Sen. group, Sen. group, and Sen.+Stigmasterol group. This means P < 0.001.

[0019] Figure 2 Effects of stigmasterol on the progression of ectopic endometrial lesions in animal models.

[0020] (A): Macroscopic images and volume measurements of ectopic endometrial lesions in mice of the Non-Sen. group, Sen. group, and Sen.+Stigmasterol group. This indicates that P < 0.001; (B): Schematic diagram of SA-β-Gal staining of ectopic endometrial lesions in mice of the Non-Sen. group, Sen. group, and Sen.+Stigmasterol group. This means P < 0.001.

[0021] Figure 3 Effects of stigmasterol on ectopic endometrial lesions and splenic macrophage polarization in animal models.

[0022] (A): Immunofluorescence staining results of macrophages in ectopic endometrial tissue sections from the Non-Sen. and Sen. groups of mice. This indicates that P < 0.05. This indicates that P < 0.01.

[0023] (B): Flow cytometry results of macrophages in spleen tissue of mice in the Non-Sen. and Sen. groups. ns indicates P>0.05. This indicates that P < 0.01.

[0024] (C): Immunofluorescence staining results of macrophages in ectopic endometrial tissue sections from the Sen. group and the Sen.+Stigmasterol group mice. This indicates that P < 0.05. This indicates that P < 0.01.

[0025] (D): Flow cytometry results of macrophages in spleen tissue of mice in the Sen. group and the Sen.+Stigmasterol group. This indicates that P < 0.01. This means P < 0.001. Detailed Implementation

[0026] The core discovery of this invention is that stigmasterol can significantly inhibit the migration and invasion of ectopic endometrial cells, improve the cellular senescence state, and reverse senescence-induced macrophage M2 polarization, thereby simultaneously intervening in the cellular senescence process and the immune microenvironment, achieving effective treatment for endometriosis.

[0027] The following embodiments are used to further illustrate the present invention, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0028] Example 1 I. Experimental Methods 1. Construction of a mouse model of endometriosis Five-week-old female C57BL / 6 mice were housed in an SPF animal facility under standard conditions (22-24℃ and 60-70% relative humidity), with free access to food and water, and underwent a week of acclimatization. Untreated mice were randomly divided into two groups: a control group and an endometriosis group. The control group underwent sham surgery. In the endometriosis group, the uterine horn of the donor mouse was removed, the myometrium was dissected, and the endometrium was cut into approximately 5 mm × 5 mm pieces, which were then sutured to the peritoneum of the recipient mouse. For three consecutive days post-surgery, the recipient mice were intramuscularly injected with 30 μg / kg estradiol (HY-B0141, MedChemExpress) to promote endometrial growth. The establishment of the endometriosis model was defined as the time point four weeks after the induction of endometriosis, confirmed by histological examination. All specimens were collected during estrus.

[0029] 2. Establishment of an aging mouse model: Mice with endometriosis were randomly divided into three groups: non-sen., D-galactose-induced aging group (Sen.), and aging + stigmasterol intervention group (Sen. + Stigmasterol). Mice in the Non-Sen. group received intraperitoneal injections of 500 mg / kg PBS daily for 4 weeks after the establishment of the endometriosis model. Mice in the Sen. group received intraperitoneal injections of 500 mg / kg D-galactose (D-gal, HY-N0210, MedChemExpress) daily for 4 weeks after the establishment of the endometriosis model. Mice in the Sen. + Stigmasterol group were fed 50 mg / kg stigmasterol (HY-N0131, MedChemExpress) once daily for 3 weeks. For the control treatment regimen, corn oil (HY-Y1888, MCE) was administered as a placebo. Ectopic endometrial tissue was collected during the first estrus cycle after all treatments were completed. The experimental procedures, data collection, and processing were performed using a double-blind method. All protocols and experiments were approved by the Laboratory Animal Ethics Committee of Renmin Hospital of Wuhan University (Ethics Approval No.: WDRM-20230410D).

[0030] 3. Culture and treatment of endometrial epithelial cells (MEECs) and endometrial stromal cells (MESCs) (1) MEECs and MESCs cell origin: obtained by peeling ectopic endometrium from mice with endometriosis under a dissecting microscope.

[0031] (2) Extraction of MEECs and MESCs: The obtained ectopic endometrial tissue was digested at 37°C with 0.1% type II collagenase (C2-BIOC, Sigma) for 30 minutes. Then, it was centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, and the precipitate was seeded into a complete medium consisting of 20% fetal bovine serum (16000-044, Gibco), 1% penicillin-streptomycin (15140122, Gibco), and 79% DMEM medium (8121728, Gibco), and incubated at 37°C and 5% CO2. After the formation of confluent monolayers, mouse endometrial epithelial cells (MEECs) and mouse endometrial stromal cells (MESCs) were separated and purified by pipetting and differential adhesion.

[0032] (3) Culture of MEECs and MESCs: The obtained MEECs and MESCs were cultured in DMEM medium supplemented with 10% FBS, penicillin / streptomycin sulfate (100 U / mL and 100 μg / mL, respectively) in a cell culture incubator at 37°C and supplemented with 5% CO2.

[0033] (4) Treatment of MEECs and MESCs: D-gal was dissolved in sterile PBS, and stigmasterol was dissolved in ethanol to prepare a drug solution. MEECs and MESCs were induced with D-gal and stigmasterol and divided into Non-Sen. group, Sen. group, and Sen.+Stigmasterol group. The Non-Sen. group was treated with PBS for 48 hours. The Sen. group was treated with 20 mg / mL D-gal for 48 hours, and the Sen.+Stigmasterol group was treated with 20 mg / mL D-gal for 24 hours, and then treated with 8 μg / mL stigmasterol and 20 mg / mL D-gal for 24 hours.

[0034] 4. Detection of cell senescence: (1) For MEECs and MESCs: The cells to be tested were seeded in six-well culture plates and cultured under suitable conditions until the cells had good morphology and no obvious contact inhibition was observed. Subsequently, the culture medium was removed, the cells were gently washed with PBS, and stained according to the instructions of the SA-β-gal staining kit. During the staining process, the cells were incubated overnight at 37°C in a CO2-free environment. The next day, the staining results were observed and photographed under an inverted microscope to record the staining results. The proportion of positive cells or the staining intensity were then analyzed.

[0035] (2) For ectopic endometrial tissue: The target tissue was prepared into frozen sections and stained according to the instructions of the SA-β-gal staining kit. During the staining process, the cells were incubated overnight at 37°C in a CO2-free environment. The next day, the staining results were recorded by observing and photographing the tissue under an inverted microscope. The proportion of positive cells was then analyzed.

[0036] 5. Detection of cell migration and invasion capabilities: (1) Cell migration detection: MEECs or MESCs in logarithmic growth phase were digested into single cells and the density was adjusted to 4 × 10⁻⁶ cells. 4 Cells were seeded per well in 3 μm Transwell chambers. The upper chamber contained 1% FBS, and the lower chamber contained 10% FBS. After culturing at 37°C for 48 hours, cells were fixed with 4% paraformaldehyde for 15 minutes, followed by staining with 0.1% crystal violet for 15 minutes. Residual cells in the upper chamber were gently wiped away with a medical swab. Cells were then photographed and counted under an upright microscope.

[0037] (2) Cell invasion detection: The upper chamber side of the 3 μm pore size Transwell chamber was pre-coated with Matrigel (354237, Corning), and the other steps were the same as those for cell migration detection.

[0038] 6. Detection of macrophage polarization: (1) For ectopic endometrial lesions: The target tissue was prepared into frozen sections, blocked with 3% BSA, and M1 and M2 macrophages were labeled with anti-CD86 and anti-CD206 antibodies, respectively, to distinguish different phenotypes. Subsequently, CoraLite594-labeled goat anti-rabbit IgG (H+L) secondary antibody and CoraLite488-labeled goat anti-mouse IgG (H+L) secondary antibody were used for fluorescent labeling to achieve visualization detection of different macrophage subsets. Finally, the cell nuclei were counterstained with DAPI. The images were observed and photographed under an upright microscope, and the proportion of positive cells was subsequently analyzed.

[0039] (2) For spleen tissue: Mouse spleens were obtained and mechanically ground to obtain a cell suspension. The obtained cell suspension was centrifuged, the supernatant was discarded, and the cells were resuspended in erythrocyte lysis buffer. The suspension was allowed to stand for a certain period of time to remove erythrocyte components. The suspension was then centrifuged again to obtain a mixed cell pellet containing various immune cells. The cell surface was blocked using an Fc receptor blocker. Cell phenotypic labeling was performed using different fluorescently labeled antibodies: FITC-CD11b antibody and PerCP / Cyanine5.5-F4 / 80 antibody were used to label all macrophage populations; PE-CD86 antibody was used to label M1 macrophages; and APC-CD206 antibody was used to label M2 macrophages. The cell samples were then detected and analyzed by flow cytometry.

[0040] II. Experimental Results 1. Stigmasterol inhibits the aging state and invasion / metastasis of MEECs and MESCs. (1) Detection of senescence in endometriosis cells MEECs and MESCs from the Non-Sen. group, Sen. group, and Sen.+Stigmasterol group were subjected to SA-β-Gal staining. Results are as follows: Figure 1 As shown in (A), compared with the Non-Sen. group, MEECs and MESCs cells in the Sen. group showed obvious senescence, while MEECs and MESCs cells in the Sen.+Stigmasterol group did not show obvious senescence.

[0041] (2) Detect the migration and invasion ability of endometriotic cells. MEECs and MESCs from the Non-Sen. group, Sen. group, and Sen.+Stigmasterol group were used for Transwell cell migration and invasion experiments. Results are as follows: Figure 1 As shown in (B), compared with the Non-Sen. group, the migration and invasion abilities of MEECs and MESCs in the Sen. group were increased, while the migration and invasion abilities of MEECs and MESCs decreased after treatment with Stigmasterol.

[0042] 2. Stigmasterol inhibits the size and aging of ectopic endometrial lesions in animal models. (1) Detection of the size of ectopic endometrial lesions in mice Ectopic endometrial lesions were collected from mice in the Non-Sen. group, Sen. group, and Sen.+Stigmasterol group, and the size of the lesions was measured. Results are as follows: Figure 2 As shown in (A), compared with the Non-Sen. group, the ectopic endometrial lesions in the Sen. group mice were larger, while compared with the Sen. group, the ectopic endometrial lesions in the Sen.+Stigmasterol group mice were smaller.

[0043] (2) Detection of senescence in ectopic endometrial lesions in mice The ectopic endometrial lesions obtained above were prepared into frozen sections, stained with β-galactosidase, and images were acquired and analyzed under a microscope. The results are as follows: Figure 2 As shown in (B), compared with the Non-Sen. group, the ectopic endometrial lesions in the Sen. group mice showed a higher degree of aging, while compared with the Sen. group, the ectopic endometrial lesions in the Sen.+Stigmasterol group mice showed a significantly lower degree of aging.

[0044] 3. Stigmasterol inhibits aging-induced ectopic endometrial lesions and M2 polarization of splenic macrophages in animal models. (1) Detection of macrophage polarization in ectopic endometrial lesions in mice Ectopic endometrial tissue was sectioned from mice in the Non-Sen. and Sen. groups, and CD86+M1 and CD206+M2 macrophages were detected by immunofluorescence staining. Results are as follows: Figure 3 As shown in (A), compared with the Non-Sen. group, the number of CD86+M1 macrophages in the ectopic endometrial tissue of the Sen. group mice was significantly reduced, while the number of CD206+M2 macrophages was significantly increased.

[0045] Ectopic endometrial tissue was sectioned from mice in the Sen. group and the Sen.+Stigmasterol group, and CD86+M1 and CD206+M2 macrophages were detected by immunofluorescence staining. Results are as follows: Figure 3As shown in (C), compared with the Sen. group, the number of CD86+M1 macrophages in the ectopic endometrial tissue of the Sen.+Stigmasterol group mice was significantly increased, while the number of CD206+M2 macrophages was significantly decreased.

[0046] (2) Detection of macrophage polarization in mouse spleen tissue Spleen tissues were collected from mice in the Non-Sen. and Sen. groups, and CD86+ (M1) and CD206+ (M2) macrophages were detected by flow cytometry. Results are as follows: Figure 3 As shown in (B), compared with the Non-Sen. group, there was no significant difference in CD86+M1 macrophages in the spleen tissue of the Sen. group mice, while CD206+M2 macrophages were significantly increased.

[0047] Spleen tissues were collected from mice in the Sen. group and the Sen.+Stigmasterol group, and CD86+ (M1) and CD206+ (M2) macrophages were detected by flow cytometry. Results are as follows: Figure 3 As shown in (D), compared with the Sen. group, the number of CD86+(M)1 macrophages in the spleen tissue of the Sen.+Stigmasterol group mice was significantly increased, while the number of CD206+(M2) macrophages was significantly decreased.

[0048] The preferred embodiments of the present invention have been described in detail above. The implementation of the present invention is not limited to the above embodiments. Those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of the present invention. All such equivalent modifications or substitutions are included within the scope defined by the claims of this application.

Claims

1. The application of stigmasterol in the preparation of drugs for the treatment of endometriosis.

2. The application according to claim 1, characterized in that: Stigmasterol is the only active ingredient in drugs used to treat endometriosis.

3. Use of compositions containing stigmasterol in the preparation of medicaments for the treatment of endometriosis.

4. The application according to claim 3, characterized in that: Stigmasterol is used in combination with one or more other substances used to treat endometriosis as an active ingredient in drugs for treating endometriosis.

5. The application according to claim 4, characterized in that: Other substances used to treat endometriosis include gestrinone, leuprolide acetate, and dinogest.

6. The application according to any one of claims 1-5, characterized in that: The drugs described for treating endometriosis focus on improving cellular senescence and regulating the immune microenvironment as the core of their treatment.

7. The application according to any one of claims 1-5, characterized in that: The drugs used to treat endometriosis include one or more of the following: drugs that inhibit the aging of ectopic endometrial cells, drugs that inhibit the migration and invasion of ectopic endometrial cells, drugs that reduce ectopic endometrial lesions, and drugs that reshape the immune microenvironment of endometriosis.

8. The application according to any one of claims 1-5, characterized in that: The dosage of stigmasterol is 30-100 mg / kg.

9. The application according to any one of claims 1-5, characterized in that: The medication for treating endometriosis also contains a pharmaceutically acceptable carrier.

10. The application according to any one of claims 1-5, characterized in that: The dosage forms of the drugs used to treat endometriosis include tablets, powders, pills, granules, capsules, solutions, suspensions, or injections.

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