Pharmaceutical composition for treating uterine cavity adhesion and application thereof

Abstract

The invention relates to a pharmaceutical composition for treating uterine adhesion and application thereof. The kidney reinforcing and miscarriage preventing pills are used for improving endometrial blood perfusion and increasing the endometrial thickness, the kidney reinforcing and miscarriage preventing pills, aspirin and estrogen play a synergistic treatment role, the postoperative re-adhesion rate is remarkably reduced through combined treatment, and the fertility prognosis of a patient is improved; and the molecular mechanism of a TGF-beta1 pathway and EMT is deeply researched, and by inhibiting excessive activation of the TGF-beta1 signaling pathway, reducing endometrial fibrosis, regulating an epithelial-mesenchymal transition (EMT) process and promoting regeneration of endometrial epithelial cells, endometrial repair is further promoted, multi-target treatment is realized, and the treatment effect is remarkable. A brand-new and efficient treatment scheme is provided for treatment of intrauterine adhesion, an individualized treatment scheme can be formulated according to the specific conditions of patients, the treatment accuracy and effectiveness are improved, and important clinical significance and application value are achieved.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a pharmaceutical composition for treating intrauterine adhesions and its applications. Background Art

[0002] Intrauterine adhesions (IUA) are one of the main causes of infertility and assisted reproductive failure in women of childbearing age. It is usually caused by trauma or infection of the basal layer of the endometrium, leading to the formation of fibrotic scars and even uterine cavity occlusion. Common risk factors include pregnancy-related uterine cavity dilation and curettage, complex cesarean section, myomectomy, and pelvic inflammatory disease. Transcervical resection of adhesion (TCRA) is the main current treatment method. However, in the postoperative follow-up of patients with moderate or severe intrauterine adhesions (IUAs), the recurrence rate of adhesions after surgery is as high as 20 - 62.5%. Therefore, surgical treatment combined with postoperative adjuvant therapy has become the consensus treatment method for IUA, including estrogen-progesterone sequential therapy. However, based on current research, the exact pathogenesis of IUA is still unclear, and there is no effective treatment method for restoring fertility function and normal menstrual cycle for severe IUA.

[0003] It has been found in clinical practice that preventing recurrence of adhesions after TCRA is a key event, and extensive research has been carried out on this issue, including oral estrogen, transdermal estrogen gel, placement of intrauterine balloon or intrauterine contraceptive device, intrauterine injection of sodium hyaluronate, amniotic stem cell transplantation, and biomimetic electrical stimulation therapy. Meta-analysis found that the use of estrogen alone after TCRA can improve menstruation by 22.5 - 100.0%, while the improvement of menstruation is 63.8 - 100.0% when combined with estrogen + other adjuvant measures. According to the "Chinese Expert Consensus on Integrated Traditional Chinese and Western Medicine Diagnosis and Treatment of Intrauterine Adhesions" in 2024, in order to improve the treatment effect of patients with moderate and severe IAUs, the application of estrogen combined with other adjuvant therapies after TCRA has better curative effects. However, Bosteels et al. statistically analyzed the drug treatment data for preventing adhesion recurrence and treating infertility after TCRA before June 2017, and found that although these treatments can improve symptoms to a certain extent, the overall treatment effect is still not ideal. Because estrogen must act on the basis of sufficient residual endometrium. In the case of extensive damage to the endometrium in severe intrauterine adhesions, it is difficult for estrogen to achieve an effective therapeutic effect. At this time, the use of high-dose estrogen and an excessive estrogen environment are both ineffective, but instead promote the formation of adhesions. Therefore, new adjuvant treatment methods should be sought, hoping to obtain a more effective, economical, convenient, and less harmful way for preventing recurrence of adhesions after TCRA.

[0004] Exploring whether the combined traditional Chinese and Western medicine therapy can be more effective in treating intrauterine adhesions and preventing re-adhesions is a new direction. Traditional Chinese medicine theories such as "The Complete and Efficient Recipes for Women" and "Fu Qingzhu's Gynecology" hold that after the uterine cavity is damaged, stasis of blood accumulates in the uterine vessels, and at the same time, the kidney essence is deficient, the thoroughfare and conception vessels are damaged, resulting in the dysfunction of the reproductive gonadal axis of kidney - Tiankui - thoroughfare and conception vessels - uterine cavity. Therefore, the main basis for traditional Chinese medicine treatment is "tonifying the kidney and promoting blood circulation". Gushen Antai Pills are composed of Polygonum multiflorum, Rehmannia glutinosa, Cistanche deserticola, Dipsacus asperoides, Taxillus chinensis, Uncaria rhynchophylla, Cuscuta chinensis, Atractylodes macrocephala, Scutellaria baicalensis, and Paeonia lactiflora, and have the effects of nourishing yin and tonifying the kidney, consolidating the thoroughfare and conception vessels to prevent miscarriage, etc., and are widely used to treat early threatened abortion of kidney yin deficiency syndrome in traditional Chinese medicine.

[0005] Epithelial mesenchymal transition (EMT) plays a crucial role in organ damage and fibrosis caused by trauma, and the same transformation process exists in the endometrial tissue of IUAs. Research shows that the expression of mesenchymal cell markers such as α - SMA and Vimentin increases in the endometrial tissue of IUAs, while the expression of epithelial marker cells such as E - cadherin decreases, and these phenotypes are consistent with type II EMT. In addition, the expression of transforming growth factor β1 (TGF - β1) is significantly increased in the endometrial tissue of IUA, and it may mediate the activation of fibroblasts and the abnormal synthesis of extracellular matrix by activating the downstream Smad signaling pathway, thus affecting endometrial fibrosis, and it has been proved that this process is closely related to EMT. Therefore, current research mainly focuses on the connection between TGF - β1 and intrauterine adhesions. However, TGF - β1 is regulated by multiple cytokines, hormones, enzymes, and microRNAs, and can mediate fibrosis through the activation of part of the classical Wnt signal. At the same time, considering that TGF - β1 is involved in other systems including the immune system, directly targeting TGF - β1 is unlikely to be feasible in treatment. Therefore, finding the key molecules and signaling pathways between the TGF - β1 pathway and EMT for in - depth research is a potential new direction, and it has very important clinical significance to clarify the etiology and pathogenesis of IUA. Summary of the Invention

[0006] The object of the present invention is to solve the problems existing in uterine adhesions in the prior art, and aims to provide a new method for treating intrauterine adhesions and preventing postoperative re-adhesions. The present invention combines the traditional Chinese medicine for tonifying the kidney and promoting blood circulation, Gushen Antai Pills, with aspirin, and at the same time applies cyclic hormone therapy. By deeply studying the differential expression changes of downstream proteins in the TGF-β1 pathway, key genes and proteins are found, and it is explored whether they have an impact on the EMT process; the endometrium is repaired through the EMT pathway to improve the endometrial repair effect of patients with intrauterine adhesions, reduce the postoperative re-adhesion rate, and restore the reproductive function and normal menstrual cycle of patients, so as to lay a theoretical foundation for selecting appropriate clinical treatment measures and effective combined drug regimens.

[0007] To solve the above technical problems, the present invention is achieved through the following technical solutions.

[0008] In a first aspect of the present invention, there is provided a pharmaceutical composition for treating intrauterine adhesions and / or improving physiological indices related to intrauterine adhesions, comprising Gushen Antai Pills and aspirin.

[0009] Preferably, the physiological indices related to intrauterine adhesions are selected from uterine receptivity and endometrial morphology.

[0010] Preferably, the Gushen Antai Pills are composed of Polygonum multiflorum Thunb., Rehmannia glutinosa Libosch., Cistanche deserticola Ma, Dipsacus asper Wall., Taxillus sutchuenensis (Lecomte) Danser, Uncaria rhynchophylla (Miq.) Jacks., Cuscuta chinensis Lam., Atractylodes macrocephala Koidz., Scutellaria baicalensis Georgi, and Paeonia lactiflora Pall.

[0011] Preferably, the Gushen Antai Pills can be prepared by conventional methods in the art or obtained commercially; unless otherwise specified, the Gushen Antai Pills used in the context of the present invention are all purchased from Beijing Boran Pharmaceutical Co., Ltd. (including commercial preparations of different batches with batch numbers including 203918-240405). It should be understood that the different batches of drugs only reflect the difference in their production time, and according to the Good Manufacturing Practice (GMP), each batch of drugs needs to pass unified quality inspections, including ingredient content, purity, microbial limit, etc., to ensure compliance with national standards. Therefore, for different batches of drugs, their efficacy and safety are consistent.

[0012] Preferably, the pharmaceutical composition optionally further comprises one or more of estrogen and estrogen derivatives.

[0013] Preferably, the estrogen is selected from one or more of estradiol, estrone, and estriol.

[0014] Preferably, the estrogen derivative is selected from one or more of estradiol benzoate, estradiol valerate, ethinylestradiol, quinestrol, and conjugated estrogens.

[0015] In the second aspect of the present invention, there is provided a pharmaceutical preparation for treating intrauterine adhesions and / or improving physiological indicators related to intrauterine adhesions, comprising Gushen Antai Pills, aspirin, and a pharmaceutically acceptable carrier.

[0016] Preferably, the physiological indicators related to intrauterine adhesions are selected from uterine receptivity and endometrial morphology.

[0017] Preferably, the Gushen Antai Pills are composed of Polygonum multiflorum Thunb., Rehmannia glutinosa Libosch., Cistanche deserticola Ma, Dipsacus asperoides C. Y. Cheng et T. M. Ai, Taxillus sutchuenensis (Lecomte) Danser, Uncaria rhynchophylla (Miq.) Jacks, Cuscuta chinensis Lam., Atractylodes macrocephala Koidz., Scutellaria baicalensis Georgi, and Paeonia lactiflora Pall.

[0018] Preferably, the pharmaceutical composition optionally further comprises one or more of estrogen and estrogen derivatives.

[0019] Preferably, the estrogen is selected from one or more of estradiol, estrone, and estriol.

[0020] Preferably, the estrogen derivatives are selected from one or more of estradiol benzoate, estradiol valerate, ethinylestradiol, quinestrol, and conjugated estrogens.

[0021] Preferably, the pharmaceutically acceptable carrier is selected from one or more of fillers, binders, disintegrants, lubricants, antioxidants, bacteriostatic agents, colorants, flavoring agents, fragrances, chelating agents, stabilizers, solvents, emulsifiers, and solubilizers.

[0022] Preferably, the dosage form of the pharmaceutical preparation is selected from one of powders, granules, tablets, capsules, dripping pills, films, suppositories, emulsions, gels, solutions, suspensions, aerosols, and sprays.

[0023] In the third aspect of the present invention, there is provided the use of a composition in the preparation of a drug for treating intrauterine adhesions and / or improving physiological indicators related to intrauterine adhesions and / or preventing intrauterine re-adhesion after separation of intrauterine adhesions, wherein the composition comprises Gushen Antai Pills and aspirin.

[0024] Preferably, the physiological indicators related to intrauterine adhesions are selected from uterine receptivity and endometrial morphology.

[0025] Preferably, the Gushen Antai Pills are composed of Polygonum multiflorum Thunb., Rehmannia glutinosa Libosch., Cistanche deserticola Ma, Dipsacus asperoides C. Y. Cheng et T. M. Ai, Taxillus sutchuenensis (Lecomte) Danser, Uncaria rhynchophylla (Miq.) Jacks, Cuscuta chinensis Lam., Atractylodes macrocephala Koidz., Scutellaria baicalensis Georgi, and Paeonia lactiflora Pall.

[0026] Preferably, the Gushen Antai Pills can be prepared by conventional methods in the art or obtained commercially. Unless otherwise specified, the Gushen Antai Pills used in the context of the present invention are all purchased from Beijing Boran Pharmaceutical Co., Ltd. (including commercial preparations of different batches with batch number 203918 - 240405). It should be understood that different batches of drugs only reflect the difference in their production time. According to the Good Manufacturing Practice (GMP), each batch of drugs needs to pass unified quality inspections, including ingredient content, purity, microbial limit, etc., to ensure compliance with national standards. Therefore, for different batches of drugs, their efficacy and safety are consistent.

[0027] Preferably, the composition optionally further comprises one or more of estrogen and estrogen derivatives.

[0028] Preferably, the estrogen is selected from one or more of estradiol, estrone, and estriol.

[0029] Preferably, the estrogen derivative is selected from one or more of estradiol benzoate, estradiol valerate, ethinylestradiol, quinestrol, and conjugated estrogens.

[0030] Based on traditional Chinese medicine theory, the pathogenesis of intrauterine adhesion is "kidney deficiency and blood stasis", and the treatment should focus on "tonifying the kidney and promoting blood circulation". The present invention selects Gushen Antai Pills (composed of Polygonum multiflorum Thunb., Rehmannia glutinosa Libosch., Cistanche deserticola Ma, Dipsacus asper Wall., Taxillus chinensis (DC.) Danser, Uncaria rhynchophylla (Miq.) Jacks., Cuscuta chinensis Lam., Atractylodes macrocephala Koidz., Scutellaria baicalensis Georgi, and Paeonia lactiflora Pall.) as the representative of traditional Chinese medicine for tonifying the kidney and promoting blood circulation, and plays a role through the following mechanisms: improving endometrial blood supply: traditional Chinese medicine for tonifying the kidney and promoting blood circulation can promote the generation of endometrial microvessels, increase the blood perfusion of the endometrium, and provide nutritional support for endometrial repair; inhibiting fibrosis: active substances in traditional Chinese medicine components (such as baicalin and paeoniflorin) can inhibit the over - activation of the TGF - β1 / Smad pathway, reduce the activation of fibroblasts and the deposition of ECM; regulating the EMT process: traditional Chinese medicine for tonifying the kidney and promoting blood circulation can inhibit the EMT process by regulating the expression of transcription factors such as Snail and Slug, and promote the regeneration and functional recovery of endometrial epithelial cells.

[0031] Aspirin is a commonly used non-steroidal anti-inflammatory drug in clinical practice, with the functions of anti-platelet aggregation and inhibition of platelet adhesion. Initially, aspirin was found to have an anti-hepatic fibrosis effect in patients with chronic liver disease. In subsequent studies, it was found that aspirin could reduce fibrosis markers in endometrial tissue, including TGF-β, CTGF, type I collagen, and type III collagen. The present invention further found that the combined use of aspirin on the basis of Gushen Antai Pills can inhibit the inflammatory response, reduce the formation of postoperative adhesions, significantly treat intrauterine adhesions, improve endometrial receptivity and its morphology. Among them, aspirin can inhibit the expression of TGF-β1, p-Smad2, and p-Smad3 in endometrial tissue through the TGF-β1 / Smad1-based signaling pathway, and can inhibit endometrial fibrosis. At the same time, estrogen is used in combination on this basis, combining cycle hormone therapy with traditional Chinese medicine for tonifying the kidney and promoting blood circulation. Through the mechanism of action of multiple targets and multiple pathways, it synergistically promotes the repair of the endometrium, reduces fibrosis and the occurrence of re-adhesions, generates obvious synergistic therapeutic activity, and also helps to avoid the risk of re-adhesion caused by a single high-dose estrogen.

[0032] In summary, the present invention combines the cycle hormone therapy of modern medicine with the theory of tonifying the kidney and promoting blood circulation in traditional Chinese medicine. Using Gushen Antai Pills to improve endometrial blood perfusion and increase endometrial thickness, it plays a synergistic therapeutic role with aspirin and estrogen. Through combined treatment, the postoperative re-adhesion rate is significantly reduced, and the fertility prognosis of patients is improved; and it deeply studies the molecular mechanism of the TGF-β1 pathway and EMT. By inhibiting the excessive activation of the TGF-β1 signaling pathway, reducing endometrial fibrosis, regulating the epithelial-mesenchymal transition (EMT) process, and promoting the regeneration of endometrial epithelial cells, thereby promoting endometrial repair, achieving multi-target treatment, providing a new and efficient treatment plan for the treatment of intrauterine adhesions, and also helping to formulate an individualized treatment plan according to the specific situation of patients, which has important clinical significance and application value for improving the accuracy and effectiveness of treatment. Brief Description of the Drawings

[0033] Figure 1 It is a schematic diagram of the results of immunohistochemical HE staining analysis of the uterus of IUA rats.

[0034] Figure 2 It is a schematic diagram of the results of Masson staining analysis of the uterus of IUA rats.

[0035] Figure 3 It is a schematic diagram of the results of immunohistochemical analysis of the uterus of IUA rats.

[0036] Figure 4 It is a schematic diagram of the results of real-time quantitative polymerase chain reaction analysis of endometrial receptivity markers in the uterus of IUA rats.

[0037] Figure 5 It is a schematic diagram of the analysis results of the expression of other biological targets related to endometrial receptivity in IUA rats in vivo.

[0038] Figure 6 It is a schematic diagram of the analysis results of the EMT process in IUA rats. Detailed implementation manners

[0039] To make the objectives, technical solutions and effects of the present invention clearer and more definite, the present invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0040] Unless otherwise specified, the reagents used in the present invention are all commercially available. The experimental methods used in the present invention, such as molecular experiments, cell experiments, animal experiments, etc. are all conventional methods and techniques in the art. For the conduct of animal experiments, the experimental protocols have been approved by the Ethics Committee of Wuhan University and follow its guidelines.

[0041] Representative results in biological experiment replicates are presented in the context figures, and the data are shown as mean±SD or mean±SEM as specified in the figures. All experiments were repeated at least three times. The data were analyzed using GraphPad Prism 9.0 or SPSS 22.0 software. Conventional medical statistical methods such as t-test, chi-square test, analysis of variance, rank sum test, etc. were used to compare the mean differences between two or more groups. p<0.05 was considered a significant difference.

[0042] Example 1 Construction of IUA rat model

[0043] (1) Take ten-week-old sexually mature female S / D rats (purchased from the Animal Experiment Center of Wuhan University). Under the same controlled conditions (20°C, 12 hours: 12-hour light / dark cycle, lights on at 6:00 am), provide food and fresh water for each rat.

[0044] (2) All S / D rats were anesthetized by pentobarbital injection, placed in the supine position, the abdomen was shaved, and disinfected with povidone iodine. After making a vertical incision in the midline of the lower abdomen, the uterine horns were identified. Take a 20-cm long No. 10 sterile surgical thread and soak it in a lipopolysaccharide saline solution (6 mg / mL) for 24 h to prepare a modeling cotton thread. In the IUA group, a 2.5-cm longitudinal incision was made on the left uterus, and the upper and middle endometrium was scraped with a curette until the four walls of the uterus were rough; the lipopolysaccharide cotton thread was placed in the uterine cavity, and about 3 cm of the stump was left to protrude from the abdominal incision; the uterus was sutured, and the abdominal cavity was rinsed with normal saline and then closed; after 2 days, the stump of the cotton thread was pulled to remove the cotton thread in the uterine cavity; in the sham operation group, the rats only received the incision wound and no treatment was performed on the endometrium. The S / D rats were sacrificed 7 days after modeling.

[0045] Example 2 Endometrial Receptivity Assessment

[0046] Prepare the IUA rat model according to Example 1. Subsequently, the rats are randomly divided into 6 groups, denoted as Group 1 - Group 6, with 6 rats in each group. Among them, Group 1 is the control group (Control group, C group), and each rat is injected with the same volume of sterile PBS. During the experiment, the health, behavior, and bleeding status of the rats are observed every other day; Group 2 is the IUA model group (Intrauterine adhesion group, IUA group), and each rat is injected with the same volume of sterile PBS. During the experiment, the health, behavior, and bleeding status of the rats are observed every other day; Group 3 is the estrogen treatment group (estrogen group (estradiol valerate (Progynova, 1mg), the same below), E2 group). After successful IUA modeling, 10.8 mg·kg -1 of estrogen is administered by gavage once a day for 2 weeks; Group 4 is the estrogen + aspirin treatment group (IUA + estrogen + aspirin group, E2 + APC group). After successful IUA modeling, 10.8 mg·kg -1 of estrogen is administered by gavage, and at the same time, the corresponding dose of aspirin is administered by gavage, once a day, with a dose of 54 mg·kg -1 , for 4 weeks; Group 5 is the estrogen + Gushen Antai Pills treatment group (IUA + estrogen + Gushen Antai Pills group, E2 + GAP group). After successful IUA modeling, 10.8 mg·kg -1 of estrogen is administered by gavage, and at the same time, the corresponding dose of Gushen Antai Pills is administered by gavage, three times a day, with a dose of 64.8 g·kg -1 / time, for 4 weeks; Group 6 is the estrogen + aspirin + Gushen Antai Pills treatment group (IUA + estrogen + aspirin + Gushen Antai Pills group, E2 + APC + GAP group). After successful IUA modeling, 10.8 mg·kg -1 of estrogen is administered by gavage, and at the same time, the corresponding dose of aspirin is administered by gavage, once a day, with a dose of 54 mg·kg -1 , and Gushen Antai Pills is administered by gavage, three times a day, with a dose of 64.8 g·kg -1 / time, for a total of 4 weeks.

[0047] After the experiment, hematoxylin-eosin (HE) staining and Masson staining were used to evaluate uterine morphological changes such as endometrial thickness, fibrosis degree, and gland number in the two groups. Immunohistochemical detection, RT-qPCR, Western blot assays, etc. were used to evaluate the protein expression changes of endometrial receptivity markers: estrogen receptor ESR1, integrin alpha-V / beta-3, and vascular endothelial growth factor (VEGFA).

[0048] The specific steps of HE staining were as follows: The rat uterus was fixed in 4% paraformaldehyde for 24 hours and embedded in paraffin. The slides were first dewaxed and rehydrated, and then stained with hematoxylin and eosin. The whole image of the pathological uterus was visually observed and the gland number was calculated to predict intrauterine adhesion (IUA).

[0049] The specific steps of Masson staining were as follows: The dewaxed and rehydrated slides were incubated with the Masson staining mixture for 5 minutes, and then stained with phosphomolybdic acid-aniline blue solution for 6.5 minutes. The area of collagen fibers stained blue relative to the total view was calculated by Image Pro-Plus 6.0.

[0050] The experimental results of HE staining and Masson staining were as shown in Figure 1-2 respectively. Among them, the experimental results of HE staining showed that under the HE staining microscope in the control group, the endometrial layer, serosa layer, and muscular layer were normal. The endometrial mucosal layer was composed of single-layer columnar epithelial cells, and the glands in the submucosal layer were arranged neatly with a large number, and no inflammatory cell infiltration was seen; in the IUA model group, the endometrium was damaged, the endometrial stromal structure was loose, the number of endometrial glands was significantly reduced, the arrangement was sparse, and a large number of inflammatory cells infiltrated at the endometrial injury site; compared with the IUA model group, the endometrium was thickened in each drug group, the gland number increased, and a small amount of inflammatory cell infiltration was seen; compared with the control group, the endometrial gland counts in the IUA model group, estrogen group, aspirin group, and Gushen Antai Pills treatment group of rats were all reduced (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001), indicating that the model was successfully established; compared with the IUA model group, the endometrial gland counts in each drug group increased, indicating that drug treatment could promote the regeneration of endometrial glands after injury, and the estrogen + aspirin + Gushen Antai Pills group was particularly obvious ( # p < 0.05, ## p < 0.01, ### p < 0.001, #### p < 0.001) (see Figure 1 ).

[0051] The results of Masson staining showed that under the microscope, collagen fibers were blue, and mucosa, muscle, and blood vessels were dark red; in the control group, there were a small amount of blue collagen fibers in the endometrial stroma, which were wavy and arranged neatly; in the IUA model group, a large area of fibrous tissue hyperplasia was seen, which was in the form of cords and arranged tightly; in each drug administration group, the distribution of collagen fibers in the uterine tissue was sparse and arranged relatively regularly, and partial gland regeneration was visible; compared with the control group, the fibrotic area in the IUA model group, estrogen group, aspirin and Gushen Antai Pills treatment group increased significantly (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.001), indicating that mechanical injury + lipopolysaccharide infection could successfully induce an increase in collagen fibers in the rat endometrium and form intrauterine adhesions; compared with the IUA model group, the fibrotic area in the estrogen + Gushen Antai Pills group decreased significantly ( # p < 0.05, ## p < 0.01, ### p < 0.001, #### p < 0.001), indicating that after treatment with estrogen and Gushen Antai Pills, the formation of collagen fibers could be inhibited and the process of intrauterine adhesions could be delayed (see Figure 2 ).

[0052] With the continuous in-depth study of steroid hormones and their receptor systems, researchers have gradually realized that normal sex hormone levels in the blood do not necessarily mean normal physiological functions of sex hormones. If target cells lack corresponding receptors or receptor functions are abnormal, the hormone still cannot exert normal physiological effects. Estrogen receptor α (ERα) is a member of the ligand-dependent transcriptional activity factor superfamily and is a subtype of ER, which is expressed in the cell nucleus and cytoplasm. The results of immunohistochemistry are as Figure 3 shown. The results showed that estrogen receptor ESR1 was expressed in both endometrial glandular epithelial cells and stromal cells, mainly with nuclear staining. The expression of ESR1 in glandular epithelium was particularly significant. After the establishment of the IUA rat model, the expression of estrogen receptor ESR1 in glandular epithelial cells and stromal cells increased significantly, showing diffuse strong positivity. Compared with the IUA model group, the estrogen group, aspirin and Gushen Antai Pills treatment group could rescue the overexpression of ESR1, indicating that the estrogen group, aspirin and Gushen Antai Pills treatment group could restore the functional abnormality of estrogen receptor ESR1 after the establishment of the IUA model and promote estrogen to exert physiological effects more effectively.

[0053] Vascular endothelial growth factor VEGFA and integrin β3 (αVβ3) are used as indicators of endometrial receptivity. Vascular endothelial growth factor A (VEGFA), as a pro-angiogenic endothelial cell growth factor that induces angiogenesis in vivo, immunohistochemical results show that the positive expression of VEGF protein is mainly located in the cytoplasm of endometrial glandular epithelial cells and stromal cells, presenting brown-yellow granules. It is more dispersed in the surrounding stroma and shows discontinuous expression. Some endometrial vascular endothelial cells are stained positively. After the establishment of the IUA rat model, the expression of vascular endothelial growth factor VEGFA in endometrial glandular epithelial cells and stromal cells is significantly inhibited. Compared with the IUA model group, the estrogen group and the treatment groups with aspirin and Gushen Antai Pills can rescue the expression of VEGFA.

[0054] Immunohistochemical results show that integrin αvβ3 mainly appears in the cytoplasm, cell membrane of the free edge of endometrial glandular epithelial and luminal epithelial cells, and the cytoplasm of stromal cells. The staining of glandular epithelial and luminal epithelial cells is more obvious than that of stromal cells. After the establishment of the IUA rat model, the expression of integrin αvβ3 in endometrial glandular epithelial cells and stromal cells is significantly enhanced. Compared with the IUA model group, the estrogen group and the treatment groups with aspirin and Gushen Antai Pills can rescue the overexpression of integrin αvβ3. In summary, the above results indicate that the estrogen group and the treatment groups with aspirin and Gushen Antai Pills can significantly rescue endometrial receptivity and improve the prognosis of intrauterine adhesion.

[0055] The specific steps for real-time quantitative polymerase chain reaction (RT-qPCR) analysis of endometrial receptivity markers and fibrosis-related indicators are as follows: After extracting mRNA from endometrial tissue, qRT-PCR is used for mRNA quantitative detection. The experimental steps are as follows: (1) RNA extraction; (2) RNA agarose gel electrophoresis; (3) Reverse transcription of RNA into cDNA; (4) Quality detection of cDNA and primers; (5) Quantitative PCR detection.

[0056] The specific steps of the Western blot experiment are as follows: After extracting total protein from endometrial tissue, add the corresponding primary antibody for incubation respectively, and select a suitable secondary antibody. Use the hypersensitive ECL luminescent solution to luminesce, and analyze the band gray value with Quantity one, with GAPDH as the internal reference.

[0057] The experimental results are shown in Table 1 below and Figure 4As shown in the figure. The results showed that the expressions of estrogen receptor ESR1 and integrin αVβ3 increased after the establishment of the IUA rat model. By comparing the estrogen group with the estrogen + Gushen Antai Pills group, it was found that the expressions of estrogen receptor ESR1 and integrin αVβ3 were significantly inhibited; compared the estrogen + aspirin group with the estrogen + Gushen Antai Pills group, although there was no difference in the expressions of estrogen receptor ESR1 and integrin αVβ3, the expressions of estrogen receptor ESR1 and integrin αVβ3 in the estrogen + Gushen Antai Pills group were lower; compared the estrogen + aspirin group / estrogen + Gushen Antai Pills group with the estrogen + aspirin + Gushen Antai Pills group, the expressions of estrogen receptor ESR1 and integrin αVβ3 were significantly inhibited. It indicated that the combined application of estrogen + aspirin + Gushen Antai Pills could better rescue the endometrial receptivity of IUA rats.

[0058] Vascular endothelial growth factor A (VEGFA), as a pro-angiogenic factor that induces angiogenesis in vivo, the experimental results showed that the expression of VEGFA was inhibited after the establishment of the IUA rat model. By comparing the estrogen group with the estrogen + Gushen Antai Pills group, the expression of VEGFA was rescued; compared the estrogen + aspirin group / estrogen + Gushen Antai Pills group with the estrogen + aspirin + Gushen Antai Pills group, the expression of VEGFA was more effectively rescued (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

[0059] The Bliss model was used to evaluate the synergistic effects of estrogen + aspirin + Gushen Antai Pills on the three genes of ESR1, αVβ3 and VEGFA. The specific calculation formula was as follows:

[0060] For ESR1 and αVβ3, E 预测 =E IUA+雌激素+阿司匹林 +E IUA+雌激素+固肾安胎丸 -E IUA+雌激素+阿司匹林 ×E IUA+雌激素+固肾安胎丸 ;

[0061] When E IUA+雌激素+阿司匹林+固肾安胎丸 >E 预测 ,it indicated that the combined use of several drugs produced a synergistic effect.

[0062] Among them, E i =1-M i / M IUA+雌激素 ,M i was the 2 -△△CT value of each group, and M IUA was the 2 -△△CT value of the IUA + estrogen group.

[0063] For VEGFA, E 预测 =E IUA+雌激素+阿司匹林 +EIUA+雌激素+固肾安胎丸 -E IUA+雌激素+阿司匹林 ×E IUA+雌激素+固肾安胎丸 ;

[0064] When E IUA+雌激素+阿司匹林+固肾安胎丸 > E 预测 it indicates that the combination of several drugs has produced a synergistic effect.

[0065] Where E i = M i / M IUA+雌激素 -1, M i is the 2 -△△CT value of each group, and M IUA is the 2 -△△CT value of the IUA + estrogen group.

[0066] It can be calculated that for ESR1, E 预测 is 0.523, and E IUA+雌激素+阿司匹林+固肾安胎丸 is 0.713; for αVβ3, E 预测 is 0.598, and E IUA+雌激素+阿司匹林+固肾安胎丸 is 0.639; for VEGFA, E 预测 is 0.803, and E IUA+雌激素+阿司匹林+固肾安胎丸 is 1.013. Obviously, the combined use of estrogen, aspirin, and Gushen Antai Pills has produced a significant synergistic effect compared with the use of single drugs or two drugs.

[0067] Table 1 Effects of different treatment methods on ESR1, VEGFA, and αVβ3 (2 -△△CT )

[0068]

[0069] Furthermore, Western blot experiments were used to detect the expression levels of other biological targets related to endometrial receptivity (AKT1, CXCR4, EGF, RUNX2) and fibrosis-related indicators (MMP9, TGFβ-1) in the bodies of IUA rats. The results are as Figure 5As shown. TGF-β1 is a member of the TGF superfamily and is the main endogenous growth factor for fibrous scar formation. It participates in the entire process of wound repair and healing. Its sustained high concentration and overexpression may be the main cause of fibrous scar formation. It is currently recognized as the strongest pro-fibrotic factor and a key mediator in the pathway that causes tissue fibrous scar formation. The results showed that the TGFβ-1 signaling pathway would be overactivated in the pro-fibrotic effect. After the IUA rat model was established, the expression of TGFβ-1 was overactivated. Compared with the estrogen group and the estrogen + Gushen Antai pill group, the expression of TGFβ-1 was inhibited; compared with the estrogen + aspirin group / estrogen + Gushen Antai pill group and the estrogen + aspirin + Gushen Antai pill group, the expression of TGFβ-1 was more effectively inhibited. This indicates that estrogen + aspirin + Gushen Antai Pills achieves anti-fibrosis effects through the TGFβ-1 signaling pathway (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

[0070] Epithelial-mesenchymal transition (EMT) is considered to be one of the potential pathogenesis of IUAs. After the endometrial basal layer is damaged, epithelial cells regenerate impaired, and they undergo a morphological transformation to a mesenchymal (stromal) cell phenotype and acquire migration ability, leading to fibroblast proliferation and excessive deposition of extracellular matrix, and adhesion scar formation. E-cadherin (E-cad), as an epithelial marker, is a member of the cadherin family and is commonly found in epithelial cells, placenta and mesoderm cells. It mainly mediates adhesion between cells of the same type and is mostly expressed at cell-cell junctions. It plays an important role in maintaining the polarity, integrity and cell differentiation of epithelial cells. The decreased expression of the epithelial marker E-cad is the most significant cell phenotypic change in the EMT process. Furthermore, the EMT process of IUA rats was analyzed experimentally. After the IUA rat model was established, the expression of E-cad was inhibited. The epithelial marker E-cad was rescued to a certain extent in the estrogen group and the estrogen + Gushen Antai Wan group. The epithelial marker E-cad was more effectively increased in the estrogen + aspirin group / estrogen + Gushen Antai Wan group compared with the estrogen + aspirin + Gushen Antai Wan group (see Figure 6 )(*p<0.05, **p<0.01, ***p<0.001), further clarified the synergistic effect of estrogen + aspirin + Gushen Antai Pills; and based on the grayscale ratio (relative content ratio) of the internal reference group (GAPDH) for quantitative analysis (see Table 2), the above-mentioned Bliss model was used to evaluate the synergistic effect of estrogen + aspirin + Gushen Antai Pills on E-cad. It was calculated that for E-cad, E 预测 is 0.865, E IUA+雌激素+阿司匹林+固肾安胎丸was 1.978, further clarifying the synergistic effect of estrogen + aspirin + Gushen Antai Pills. In addition, vimentin (Vimentin) and N-cadherin (N-cad) are used as mesenchymal markers. After the establishment of the IUA rat model, the expressions of Vim and N-cad increased. Comparing the estrogen group with the estrogen + Gushen Antai Pills group, the mesenchymal markers Vim and N-cad were inhibited to a certain extent; compared with the estrogen + aspirin group / estrogen + Gushen Antai Pills group, the estrogen + aspirin + Gushen Antai Pills group more effectively inhibited the mesenchymal markers Vim and N-cad (see Figure 6 )(*p < 0.05, **p < 0.01, ***p < 0.001).

[0071] In summary, the combined use of estrogen + aspirin + Gushen Antai Pills exerts a more effective synergistic antifibrotic effect by acting on the EMT process through the TGFβ-1 signaling pathway.

[0072] Table 2 Effects of different treatment methods on Vimentin, N-cad and E-cad

[0073]

[0074]

[0075] Based on the above, the present invention combines the cyclic hormone therapy of modern medicine with the theory of tonifying the kidney and promoting blood circulation of traditional Chinese medicine, uses Gushen Antai Pills to improve endometrial blood perfusion and increase endometrial thickness, plays a synergistic therapeutic role, significantly reduces the postoperative re-adhesion rate through combined treatment, and improves the fertility prognosis of patients; and deeply studies the molecular mechanism of the TGF-β1 pathway and EMT, reduces endometrial fibrosis by inhibiting the overactivation of the TGF-β1 signaling pathway, inhibits the epithelial-mesenchymal transition (EMT) process, promotes the regeneration of endometrial epithelial cells, and then promotes endometrial repair, achieving multi-target treatment, providing a new and efficient treatment plan for intrauterine adhesions, and also helping to formulate individualized treatment plans according to the specific conditions of patients, which has important clinical significance and application value for improving the accuracy and effectiveness of treatment.

[0076] The above specific implementation part specifically introduces the analysis methods involved in the present invention. It should be noted that the above introduction is only to help those skilled in the art better understand the methods and ideas of the present invention, rather than a limitation on the relevant content. Without departing from the principle of the present invention, those skilled in the art can also make appropriate adjustments or modifications to the present invention, and the above adjustments and modifications should also fall within the protection scope of the present invention.

Claims

1. A pharmaceutical composition for treating intrauterine adhesions and / or improving physiological indices related to intrauterine adhesions, characterized in that, It includes Gushen Antai Pills and aspirin.

2. The pharmaceutical composition according to claim 1, wherein The physiological indicators related to intrauterine adhesion are selected from uterine receptivity and endometrial morphology.

3. The pharmaceutical composition according to claim 1, wherein, Optionally, the pharmaceutical composition further includes one or more of estrogen and estrogen derivatives.

4. A pharmaceutical preparation for treating intrauterine adhesions and / or improving physiological indices related to intrauterine adhesions, characterized in that, It includes Gushen Antai Pills, aspirin, and a pharmaceutically acceptable carrier.

5. The pharmaceutical preparation according to claim 4, characterized in that, The physiological indicators related to intrauterine adhesion are selected from uterine receptivity and endometrial morphology.

6. The pharmaceutical preparation according to claim 4, characterized in that, Optionally, the pharmaceutical composition further includes one or more of estrogen and estrogen derivatives.

7. The pharmaceutical preparation according to claim 4, characterized in that, The pharmaceutically acceptable carrier is selected from one or more of fillers, binders, disintegrants, lubricants, antioxidants, bacteriostatic agents, colorants, flavoring agents, fragrances, chelating agents, stabilizers, solvents, emulsifiers, and solubilizers.

8. Use of a composition in the preparation of a medicament for treating intrauterine adhesions and / or improving physiological indices related to intrauterine adhesions and / or preventing intrauterine re-adhesions after separation of intrauterine adhesions, characterized in that, The composition includes Gushen Antai Pills and aspirin.

9. The application according to claim 8, characterized in that, The physiological indicators related to intrauterine adhesion are selected from uterine receptivity and endometrial morphology.

10. The application according to claim 8, characterized in that Optionally, the composition further includes one or more of estrogen and estrogen derivatives.

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