Use of matrine in preparation of medicine for treating endometriosis and medicine composition

Abstract

The application belongs to the technical field of traditional Chinese medicine, and discloses application of matrine in preparation of a medicine for treating endometriosis and a medicine composition. The matrine used in the application can inhibit proliferation and promote apoptosis of ectopic stromal cells of endometriosis, and can also significantly inhibit metastasis and recurrence of the ectopic stromal cells, and can be widely applied in preparation of medicines for treating endometriosis, and has important clinical value.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine technology, specifically relating to the application of matrine in the preparation of drugs for treating endometriosis and pharmaceutical compositions. Background Technology

[0002] Endometriosis refers to the ectopic growth, infiltration, and cyclical bleeding of endometrial tissue within the uterus, resulting in various clinical and pathological manifestations. Clinically, it often presents with progressively worsening dysmenorrhea, infertility, increased menstrual flow, and premenstrual spotting. Endometriosis typically affects pelvic organs such as the ovaries, fallopian tubes, and uterine ligaments, most commonly the ovaries. Endometriosis is a multifactorial, chronic, and painful disease with an incidence rate of 6%-10% in women, severely impacting their physical health and quality of life.

[0003] Currently, the clinical treatment goals for endometriosis are to reduce lesions, control pain, promote fertility, and reduce recurrence. Hormone-based drug therapy plays a crucial role in treatment, such as mifepristone, which has a relatively certain efficacy in treating endometriosis. However, it has many side effects, a high recurrence rate, is expensive, and can cause endocrine disorders. Furthermore, it requires women of childbearing age to use contraception during medication, making it difficult to maintain stable efficacy. Traditional surgical resection is often ineffective and prone to recurrence. While laparoscopic surgery has become the preferred method for lesion removal due to its widespread use, its recurrence rate is still very high, ranging from 7% to 30% over three years. It still requires combined hormone therapy, placing a significant burden on patients physiologically, psychologically, and financially.

[0004] Traditional Chinese medicine (TCM) categorizes this condition based on its symptoms into categories such as "dysmenorrhea," "abdominal masses," "menstrual disorders," and "infertility." TCM believes that coldness in the uterus, internal accumulation of phlegm and dampness, obstruction of Qi flow, blood stasis, and poor blood circulation prevent the dissipation of extravasated blood, leading to the accumulation of blood stasis, imbalance of the Chong and Ren meridians, and eventually the formation of abdominal masses. Treatment focuses on promoting blood circulation, removing blood stasis, dispersing masses, and relieving pain; while ensuring that the removal of blood stasis does not harm the body's vital energy and that the body's resistance is strengthened without retaining pathogenic factors. Modern pharmacological research shows that blood-activating and stasis-removing drugs can promote the decomposition and absorption of blood stasis, soften adhesions, shrink masses, and also dilate peripheral blood vessels, improve microcirculation, dissolve fibrin, improve the pelvic environment, regulate the Qi and blood functions of the internal organs, inhibit the growth of ectopic endometrial tissue, and regulate endocrine and immune functions, thereby eliminating lesions and reducing pelvic adhesions. In addition to treating endometriosis, blood-activating and stasis-removing drugs also have a good regulatory effect on various aspects of the body's endocrine and immune systems. Therefore, when surgery and hormone therapy are not ideal, traditional Chinese medicine offers a good alternative for treating endometriosis. Thus, identifying effective individual Chinese medicinal herbs from my country's rich resource of herbs and clarifying their roles and mechanisms in treating endometriosis is of great significance.

[0005] matrine (C 15 H 24 Matrine (N2O) is one of the main components extracted from the dried roots, plants, and fruits of the legume Sophora flavescens. It is a quinoline alkaloid compound. Studies have shown that matrine can inhibit the proliferation and metastasis of tumor cells such as lung cancer and gastric cancer, and also shows certain effects in inducing tumor cell differentiation and promoting tumor cell apoptosis; it can inhibit HeLa cell proliferation and shorten the cell life cycle, thereby reducing metastasis; it can also exert an anti-breast cancer effect by inhibiting the AKT / mTOR pathway to provide protective autophagy.

[0006] Although endometriosis is a benign disease, it exhibits malignant behaviors such as tumor-like infiltration, distant migration, and ectopic implantation. Endometrial tissue is mainly composed of glandular epithelial cells and stromal cells. Current research indicates that the abnormal proliferation, migration, and invasiveness of stromal cells are one of the main causes of endometriosis. Therefore, how to target and inhibit stromal cell proliferation, migration, and invasion, and promote stromal cell apoptosis, has become a key issue in the treatment of endometriosis. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of the prior art and provide the application of matrine in the preparation of drugs for treating endometriosis and pharmaceutical compositions thereof, specifically adopting the following technical solution:

[0008] According to a first aspect of the present invention, the use of matrine in the preparation of a drug for treating endometriosis is provided, which inhibits the proliferation rate of ectopic stromal cells by 30%-90% and promotes the apoptosis rate of ectopic stromal cells by 51%-90%.

[0009] This invention involves isolating and culturing ectopic endometrial stromal cells in vitro. The effects of the aforementioned traditional Chinese medicine monomer, matrine, on the proliferation, migration, and apoptosis of stromal cells were determined using CCK8 assays, scratch assays, and flow cytometry. The monomer showed an inhibitory effect on the proliferation of ectopic endometrial stromal cells in a concentration- and time-dependent manner; its inhibitory effect on migration also increased in a concentration-dependent manner; and its apoptosis-inducing effect increased in a concentration-dependent manner. The matrine monomer used in this invention, while inhibiting the proliferation and promoting apoptosis of ectopic endometrial stromal cells, also significantly inhibits the metastasis and recurrence of these cells.

[0010] According to a second aspect of the invention, a pharmaceutical composition for treating endometriosis is also provided, the pharmaceutical composition comprising matrine. The dosage form of the pharmaceutical composition is an injection, cream, gel, pill, or capsule.

[0011] Preferably, the concentration of matrine is 1.0 mg / mL to 1.4 mg / mL. At concentrations of 1.0 mg / mL to 1.4 mg / mL, mesenchymal cell viability is weakened, indicating that medium to high concentrations inhibit cell proliferation, and this inhibitory effect is concentration- and time-dependent. However, at concentrations below 1.0 mg / mL, mesenchymal cell viability increases after 24 hours, thereby promoting cell proliferation. At concentrations of 1.0 mg / mL to 1.4 mg / mL, the apoptosis rate of ectopic endometrial mesenchymal cells is significantly increased, while the scratch healing rate is significantly decreased, indicating enhanced ability to promote cell apoptosis and inhibit mesenchymal cell migration.

[0012] Preferably, the pharmaceutical composition further includes a pharmaceutically acceptable carrier, comprising at least one of a solvent, a dispersion medium, an isotonic regulator, and an adjuvant. The solvent is water, glycerol, or ethanol; the dispersion medium is water, ethanol, or sugar; the isotonic regulator is distilled water, glucose, a polyol, or sodium chloride; and the adjuvant is an emulsifier, preservative, solubilizer, or stabilizer. An isotonic regulator can maintain normal plasma osmotic pressure after administration, keeping the water balance inside and outside blood vessels and cells, thus maintaining normal bodily functions. Using a hypertonic solution with a higher osmotic volume than plasma can cause cell dehydration and shrinkage, affecting normal cell function.

[0013] The beneficial effects of this invention are as follows: This invention provides a new use of matrine in the preparation of drugs for treating endometriosis. The matrine used inhibits the proliferation and promotes apoptosis of ectopic stromal cells in endometriosis, while also significantly inhibiting cell metastasis and recurrence. It can be widely used in the preparation of drugs for endometriosis and has important clinical value. Attached Figure Description

[0014] Figure 1 The image shown is an identification diagram of ectopic stromal cells in endometriosis (100×); where (a) is blank; (b) is vimentin; and (c) is keratin.

[0015] Figure 2 The diagram shows the inhibition of ectopic stromal cell proliferation in endometriosis by different concentrations of matrine.

[0016] Figure 3 The image shows the inhibition of ectopic stromal cell migration in endometriosis by different concentrations of matrine.

[0017] Figure 4 The figure shows how different concentrations of matrine promote apoptosis in ectopic stromal cells of endometriosis. Detailed Implementation

[0018] The following will provide a clear and complete description of the concept and technical effects of the present invention in conjunction with the embodiments and accompanying drawings, so as to fully understand the purpose, solution and effects of the present invention. It should be noted that, unless otherwise specified, the embodiments and features in the embodiments of this application can be combined with each other.

[0019] Example 1

[0020] This embodiment describes the isolation, culture, and identification of ectopic stromal cells in endometriosis, including the following steps:

[0021] Cell isolation and culture: Ectopic endometrial tissue was collected from patients with clinical endometriosis after surgery and placed in pre-cooled sterile PBS. The tissue was then processed in a laminar flow hood within 30 minutes. After washing with PBS, the tissue was minced into pieces approximately 1 mm in size. 3 Small pieces were placed in a collagenase IV solution with a final concentration of 1 mg / mL and digested in a water bath at 37°C for 2 hours. The digestion solution was filtered through an 80-mesh and a 300-mesh sterile sieve. The filtrate was transferred into a centrifuge tube and centrifuged at 3000 rpm for 5 min. The supernatant was discarded, and the cells were resuspended in DMEM / F12 cell culture medium containing 10% fetal bovine serum and 1% penicillin-dextrin antibiotics. The cells were placed in a culture dish and incubated in a CO2 incubator for 2 hours. The medium was changed, and the cells were cultured and passaged.

[0022] Cell identification: Cells (ectopic mesenchymal cells) were digested with trypsin to prepare a cell suspension, seeded in 6-well plates, and cultured overnight. The next day, the supernatant was discarded, and the cells were washed three times with PBS and fixed with 4% paraformaldehyde for 30 minutes. Residual liquid was aspirated, and one drop of 3% H2O2 was added to each well to block endogenous peroxidase. The cells were incubated at room temperature for 10 minutes. The cells were then washed three times with PBS, and primary antibodies (violin diluted 1:70, keratin diluted 1:100) were added, enough to cover the cells, and incubated for 1 hour. The cells were washed three times with PBS, and secondary antibody was added, and incubated for 30 minutes. The cells were washed three times with PBS, and DAB staining was performed. After thorough staining under a microscope, the staining was stopped with tap water. Results are as follows: Figure 1 As shown, the isolated and cultured cells stained brownish-yellow after incubation with vimentin, but not stained after incubation with cytokeratin, indicating that primary endometrial stromal cells were successfully isolated and cultured with a purity of over 90%.

[0023] Example 2

[0024] This embodiment is an experiment on the proliferation of ectopic stromal cells in endometriosis, including the following steps:

[0025] The cells (ectopic mesenchymal cells) were digested with pancreatic enzymes to prepare a cell suspension, and 3×10 4CFU cells were seeded in 96-well plates and incubated in a CO2 incubator for 24 hours. After 24 hours, different concentrations of the drug were added according to the experimental groups. The normal culture medium was DMEM / F12 cell culture medium containing 10% fetal bovine serum and 1% penicillin-dextrose antibody. Each group had 5 replicates, and the groups were as follows:

[0026] ① Blank group (normal culture medium, no cells);

[0027] ② Experimental control group (normal culture medium + cells, group 0);

[0028] ③ Solvent group (normal culture medium + cells + 2% PBS, PBS group);

[0029] ④ 0.8 mg / mL experimental group (normal culture medium + cells + 0.8 mg / mL matrine (purchased from Sigma-Aldrich, CAS No.: 519-02-8), 0.8 mg / mL group);

[0030] ⑤ 1.0 mg / mL experimental group (normal culture medium + cells + 1.0 mg / mL matrine, 1.0 mg / mL group).

[0031] ⑥ 1.2 mg / mL experimental group (normal culture medium + cells + 1.2 mg / mL matrine, 1.2 mg / mL group);

[0032] ⑦ 1.4 mg / mL experimental group (normal culture medium + cells + 1.4 mg / mL matrine, 1.4 mg / mL group).

[0033] Drug treatment was performed at 0, 24, 48, 72, and 96 hours, respectively. The culture medium was filtered off, CCK8 solution was added, and the cells were incubated in a CO2 incubator for 2 hours. The absorbance (OD) was then measured at 450 nm using a microplate reader. Cell viability was calculated as: Cell viability = (Experimental group OD - Blank group OD) / (Control group OD - Blank group OD) × 100%. Figure 2As shown (from top to bottom: 0.8 mg / mL group, PBS group, 1.0 mg / mL group, 1.2 mg / mL group, 1.4 mg / mL group), compared with the PBS group, the low drug concentration group (0.8 mg / mL) showed enhanced mesenchymal cell viability after 24 hours, indicating that low concentration promotes cell proliferation; while the medium- and high drug concentration groups (1.0 mg / mL, 1.2 mg / mL, 1.4 mg / mL) showed weakened mesenchymal cell viability, indicating that medium- and high concentrations inhibited cell proliferation, and the inhibitory effect was concentration- and time-dependent. At 96 h, the 1.0 mg / mL group inhibited mesenchymal cell proliferation by 30%; the 1.2 mg / mL group inhibited mesenchymal cell proliferation by 61%; and the 1.4 mg / mL group inhibited mesenchymal cell proliferation by 90%.

[0034] Example 3

[0035] This embodiment is a scratch assay for ectopic stromal cells in endometriosis, including the following steps:

[0036] Sterile scratch chambers were placed in 24-well plates, and cells (ectopic mesenchymal cells) were digested with trypsin to prepare a cell suspension. The suspension was then incubated at 3 × 10⁻⁶ cells / well. 5 CFU cells were seeded at a density in microcells, and 24-well plates were incubated in a CO2 incubator for 24 hours. Culture medium containing different concentrations of the drug was added according to the experimental groups. The scratch chambers were then removed, and cell healing was recorded by photographing at 0 and 24 hours. The scratch healing rate was calculated using ImageJ software. The normal culture medium was DMEM / F12 cell medium containing 10% fetal bovine serum and 1% penicillin-dextrin antibiotics. Grouping was as follows:

[0037] ① Experimental control group (normal culture medium + cells, group 0);

[0038] ② Solvent group (normal culture medium + cells + 2% PBS, PBS group);

[0039] ③ 0.8 mg / mL experimental group (normal culture medium + cells + 0.8 mg / mL matrine, 0.8 mg / mL group);

[0040] ④ 1.0 mg / mL experimental group (normal culture medium + cells + 1.0 mg / mL matrine, 1.0 mg / mL group).

[0041] ⑤ 1.2 mg / mL experimental group (normal culture medium + cells + 1.2 mg / mL matrine, 1.2 mg / mL group);

[0042] ⑥ 1.4 mg / mL experimental group (normal culture medium + cells + 1.4 mg / mL matrine, 1.4 mg / mL group).

[0043] like Figure 3 As shown, compared with the PBS group, the scratch healing rate of the drug concentration groups (0.8 mg / mL, 1.0 mg / mL, 1.2 mg / mL, and 1.4 mg / mL) was significantly reduced, with the 1.2 mg / mL group showing the most significant inhibition of mesenchymal cell migration (P<0.01).

[0044] Example 4

[0045] This embodiment is an experiment on apoptosis of ectopic stromal cells in endometriosis, including the following steps:

[0046] Cells (ectopic mesenchymal cells) were digested with pancreatic enzymes to prepare a cell suspension, at a concentration of 3 × 10⁻⁶. 4 CFU cells were seeded at a density suitable for 6-well plates and incubated in a CO2 incubator for 24 hours. The culture medium was then removed, and different concentrations of the drug were added to the culture medium according to the experimental groups. After 48 hours of incubation, the cells were digested with trypsin without EDTA, centrifuged, and stained with Annexin V (FITC) and propidine iodide (PI). The cells were incubated in the dark for 15 minutes, centrifuged, washed with PBS, resuspended in binding buffer, and analyzed. The normal culture medium was DMEM / F12 cell culture medium containing 10% fetal bovine serum and 1% penicillin-dextrin antibiotics. Grouping was as follows:

[0047] ① Experimental control group (normal culture medium + cells, group 0);

[0048] ② Solvent group (normal culture medium + cells + 2% PBS, PBS group);

[0049] ③ 0.8 mg / mL experimental group (normal culture medium + cells + 0.8 mg / mL matrine, 0.8 mg / mL group);

[0050] ④ 1.0 mg / mL experimental group (normal culture medium + cells + 1.0 mg / mL matrine, 1.0 mg / mL group).

[0051] ⑤ 1.2 mg / mL experimental group (normal culture medium + cells + 1.2 mg / mL matrine, 1.2 mg / mL group);

[0052] ⑥ 1.4 mg / mL experimental group (normal culture medium + cells + 1.4 mg / mL matrine, 1.4 mg / mL group).

[0053] like Figure 4As shown (with annexin V-fluorescein isothiocyanate on the x-axis and propidium iodide on the y-axis; the first row from left to right represents group 0, PBS, and 0.8 mg / mL; the second row from left to right represents group 1.0 mg / mL, 1.2 mg / mL, and 1.4 mg / mL), compared with the PBS group, the apoptosis rates of the drug concentration groups (1.0 mg / mL, 1.2 mg / mL, and 1.4 mg / mL) were significantly increased, and the apoptosis-promoting effect was concentration-dependent. The 1.0 mg / mL group promoted apoptosis rates of up to 51% in mesenchymal cells; the 1.2 mg / mL group promoted apoptosis rates of up to 81.8% in mesenchymal cells; and the 1.4 mg / mL group promoted apoptosis rates of up to 91.4% in mesenchymal cells.

[0054] Although the description of the invention has been quite detailed and particularly of several described embodiments, it is not intended to limit it to any of these details or embodiments or any particular embodiment, but should be considered as providing a broad possible interpretation of the claims by referring to the appended claims and taking into account the prior art, thereby effectively covering the intended scope of the invention. Furthermore, the invention has been described above with respect to embodiments foreseeable by the inventors in order to provide a useful description, and non-substantial modifications to the invention that have not yet been foreseen may still represent equivalent modifications.

Claims

1. The application of matrine as the sole active ingredient in the preparation of drugs for treating endometriosis, characterized in that, The concentration of matrine is 1.0 mg / mL-1.4 mg / mL. The matrine inhibits the proliferation rate of ectopic mesenchymal cells by 30%-90% and promotes the apoptosis rate of ectopic mesenchymal cells by 51%-90%.

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