A pharmaceutical composition for treating chronic pelvic inflammatory disease and a preparation method thereof

Abstract

The embodiment of the application discloses a kind of drug composition for treating chronic pelvic inflammation and preparation method thereof, belong to biological medicine technical field.A kind of drug composition for treating chronic pelvic inflammation, the final concentration of active ingredient is as follows: bone marrow mesenchymal stem cells 1×10^7 Individual / mL, Periplaneta americana extract 10-50mg / mL, Astragalus polysaccharide 50-200ug / mL, granulocyte colony-stimulating factor 5-20ng / mL, interleukin-10 10-25ug / mL, Pachyman acid 10-50mg / mL, doxycycline hydrochloride 10-40ug / mL, vitamin B6 50-100ug / mL.The drug composition provided by the application can significantly reduce uterine tissue inflammatory infiltration and tissue necrosis, improve uterine adhesion state, repair damaged uterine epithelium, and promote endometrial structure recovery.The drug composition has no obvious toxicity, and has good application prospect in the clinical treatment of chronic pelvic inflammation.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a pharmaceutical composition for treating chronic pelvic inflammatory disease and its preparation method. Background Technology

[0002] Chronic pelvic inflammatory disease (CPID) is a chronic inflammatory disease of the pelvic cavity that primarily affects women. It typically involves the reproductive organs, surrounding connective tissue, and pelvic peritoneum within the pelvic cavity, and is a common and prevalent condition in women. According to statistics, the incidence of CPID among adult women in my country exceeds 40%, and this number is increasing annually. CPID encompasses various specific conditions, such as endometritis, salpingitis, oophoritis, pelvic connective tissue inflammation, and pelvic peritonitis. Its clinical manifestations are diverse, with common symptoms including chronic lower abdominal pain, lower back discomfort or a feeling of heaviness, menstrual irregularities, and dysmenorrhea. Due to its recurrent nature, CPID significantly negatively impacts patients' daily lives. Therefore, CPID is not only a common gynecological disease but also a significant factor contributing to a decline in women's quality of life.

[0003] Treatment options for chronic pelvic inflammatory disease (PID) include medication, surgery, and other supportive therapies. Medication includes antibiotics, nonsteroidal anti-inflammatory drugs (NSAIDs), and hormones. These are convenient, readily available, and relatively inexpensive, but may lead to drug resistance and side effects, and their effectiveness is limited in some severe cases of chronic PID. Surgical treatment includes laparoscopic and open surgery. These directly target the lesions and effectively remove diseased tissue, resulting in faster recovery. However, they carry higher risks and are more expensive, and some patients may require multiple surgeries. Other supportive therapies include physical therapy, traditional Chinese medicine, and lifestyle modifications. These offer comprehensive treatment and improved quality of life, but their effects are slower and vary significantly from person to person.

[0004] Stem cell therapy for chronic pelvic inflammatory disease (PID) offers unique biological advantages: stem cells can secrete various anti-inflammatory and repair-promoting cytokines, reshaping the disordered immune microenvironment in the pelvic cavity, inhibiting excessive inflammatory responses, promoting the regeneration of damaged tissues, and improving pelvic microcirculation. However, this therapy has certain limitations: patients with chronic PID often experience pelvic fibrosis, vascular occlusion, and a hypoxic microenvironment, leading to low survival rates and poor efficacy stability after exogenous stem cell transplantation; furthermore, stem cells themselves cannot directly eliminate pathogenic microorganisms, degrade deposited inflammatory mediators, or soften old scar tissue, and their use alone is insufficient to reverse the extensive adhesions and fibrosis at the lesion site. Summary of the Invention

[0005] Therefore, embodiments of the present invention provide a pharmaceutical composition for treating chronic pelvic inflammatory disease and its application.

[0006] To achieve the above objectives, the embodiments of the present invention provide the following technical solutions:

[0007] According to a first aspect of the present invention, the present invention provides a pharmaceutical composition for treating chronic pelvic inflammatory disease, comprising an active ingredient and a pharmaceutically acceptable solvent, wherein the final concentration of the active ingredient is: 1×10^7 bone marrow mesenchymal stem cells / mL, 10-50 mg / mL of American cockroach extract, 50-200 μg / mL of Astragalus polysaccharide, 5-20 ng / mL of granulocyte colony-stimulating factor, 10-25 μg / mL of interleukin-10, 10-50 mg / mL of pachymic acid, 10-40 μg / mL of doxycycline hydrochloride, and 50-100 μg / mL of vitamin B6.

[0008] American cockroach extract (Catalog No.: SNT892, Fufeng Sinote Biotechnology Co., Ltd.): Its medicinal history can be traced back to the "Shennong's Classic of Materia Medica." Ancient texts record that it is salty and neutral in nature, and enters the liver, kidney, and spleen meridians. It has the effects of breaking up blood stasis, dispersing lumps, eliminating stagnation, promoting tissue regeneration and wound healing, and clearing heat and detoxifying. In clinical applications, American cockroach extract is commonly used to treat various diseases, such as emaciation and loss of appetite caused by infantile malnutrition, swallowing discomfort caused by sore throat, local swelling and pain after insect and snake bites, skin ulceration caused by carbuncles and boils, and stomach pain caused by digestive tract ulcers.

[0009] Astragalus polysaccharide (CAS No.: 89250-26-0): is the main bioactive component of Astragalus membranaceus. Numerous in vitro and in vivo experiments and clinical studies have shown that Astragalus polysaccharide can promote the proliferation of bone marrow stromal stem cells, scavenge free radicals, inhibit lipid peroxidation to achieve antioxidant effects, regulate inflammatory signaling pathways to reduce inflammatory response, and regulate the expression of apoptosis-related proteins to inhibit abnormal cell apoptosis.

[0010] Granulocyte colony-stimulating factor (G-CSF, CAS No.: 143011-72-7): G-CSF is a bioactive substance belonging to the cytokine family. It primarily enhances the body's immune defense capabilities by stimulating the bone marrow to produce more granulocytes. Specifically, G-CSF promotes the differentiation of stem cells into granulocytes, accelerates the maturation of these cells, and improves their ability to resist microbial invasion.

[0011] Interleukin-10 (IL-10, CAS No.: 350590-73-7): IL-10 is a cytokine that plays a crucial role in the immune response, primarily secreted by immune cells such as T cells, B cells, and macrophages. It plays an important role in regulating the immune response, anti-inflammation, and suppressing excessive immune responses. IL-10 can effectively inhibit the production of pro-inflammatory cytokines and reduce the intensity of the inflammatory response.

[0012] Pachymic acid (CAS No.: 29070-92-6) is a triterpenoid compound found in the herbal medicine Poria cocos, possessing various pharmacological effects. It can directly inhibit the growth and proliferation of tumor cells, enhance the sensitivity of tumor cells to radiotherapy and chemotherapy, and exert anti-inflammatory and antioxidant effects by inhibiting inflammatory responses and scavenging free radicals. Furthermore, pachymic acid can also achieve therapeutic effects by regulating the Hippo signaling pathway and protecting the intestinal mucosal barrier.

[0013] Doxycycline hydrochloride (CAS No.: 10592-13-9): is a common tetracycline broad-spectrum antibiotic with good antibacterial effects both in vitro and in vivo. It is well absorbed orally and is mainly used for various diseases such as upper respiratory tract infections, tonsillitis, and biliary tract infections caused by sensitive Gram-positive and Gram-negative bacilli. It can also be used to treat typhus, scrub typhus, mycoplasma pneumonia, and is used to treat cholera, as well as for the prevention of malignant malaria and leptospirosis.

[0014] Vitamin B6 (CAS No.: 8059-24-3): It can inhibit sebum secretion, improve cell function, and repair the skin barrier. It can regulate sebum secretion, acne, and seborrheic dermatitis; reduce inflammatory response and assist in the treatment of eczema and contact dermatitis; participate in ceramide synthesis, repair the skin barrier, and relieve dryness and desquamation in atopic dermatitis; it can also help improve acne and eczema symptoms and reduce inflammation.

[0015] Bone marrow mesenchymal stem cells (BMSCs): In tissue regeneration, they can differentiate into various cell types such as bone, cartilage, and cardiomyocytes, migrate to damaged sites to participate in repair, and secrete growth factors to promote angiogenesis, creating conditions for tissue regeneration. In inflammatory responses, they can secrete anti-inflammatory factors, inhibit the release of pro-inflammatory cytokines, regulate immune cell activity, reduce inflammatory damage, and also prevent organ fibrosis through anti-fibrotic effects, providing new directions for the treatment of various refractory diseases.

[0016] Further, the final concentrations of the active ingredients are: 1×10^7 bone marrow mesenchymal stem cells / mL, 20 mg / mL of American cockroach extract, 100 μg / mL of astragalus polysaccharide, 8 ng / mL of granulocyte colony-stimulating factor, 15 μg / mL of interleukin-10, 25 mg / mL of pachymic acid, 20 μg / mL of doxycycline hydrochloride, and 50 μg / mL of vitamin B6.

[0017] Furthermore, the bone marrow mesenchymal stem cells are third-generation rat bone marrow mesenchymal stem cells.

[0018] Furthermore, the pharmaceutically acceptable solvent is 0.9% sodium chloride injection.

[0019] Furthermore, the dosage form of the pharmaceutical composition is an injection.

[0020] According to a second aspect of the present invention, the present invention provides a method for preparing a pharmaceutical composition for treating chronic pelvic inflammatory disease as described above, the method comprising:

[0021] (1) Bone marrow mesenchymal stem cells were prepared into a suspension using a pharmaceutically acceptable solvent;

[0022] (2) Add American cockroach extract, astragalus polysaccharide, granulocyte colony-stimulating factor, interleukin-10, pachymic acid, doxycycline hydrochloride, and vitamin B6 to the BMSCs suspension and dilute to a pharmaceutically acceptable solvent to obtain the final concentrations of bone marrow mesenchymal stem cells, American cockroach extract, astragalus polysaccharide, granulocyte colony-stimulating factor, interleukin-10, pachymic acid, doxycycline hydrochloride, and vitamin B6 as 1×10^7 cells / mL, 10-50 mg / mL, 50-200 μg / mL, 5-20 ng / mL, 10-25 μg / mL, 10-50 mg / mL, 10-40 μg / mL, and 50-100 μg / mL, respectively. Mix well to obtain the drug composition.

[0023] The embodiments of the present invention have the following advantages:

[0024] The pharmaceutical composition provided by this invention can significantly reduce the content of the pro-inflammatory factor TNF-α in uterine tissue, upregulate the expression of VEGF, PDGF, and CK19 repair factors, significantly alleviate inflammatory infiltration and tissue necrosis in uterine tissue, improve uterine adhesions, repair damaged uterine epithelium, and promote the recovery of endometrial structure. Furthermore, this pharmaceutical composition has no obvious toxicity and shows good application prospects in the clinical treatment of chronic pelvic inflammatory disease. Attached Figure Description

[0025] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely exemplary, and those skilled in the art can derive other embodiments based on the provided drawings without creative effort.

[0026] Figure 1The image shows the successful construction and verification of the rat model provided by this invention, presenting the gross observation and anatomical pathological features of the vulvar lesions. Left side: Gross morphological observation results of the vulvar region; Right side: Pathological histological observation results after dissection.

[0027] Figure 2 Comparison of bacterial colony growth in vaginal secretions from different groups of rats provided by this invention;

[0028] Figure 3 HE-stained pathological sections of rat uterine tissue provided by this invention;

[0029] Figure 4 The bar chart shows the expression levels of TNF-α protein in the uterine tissues of rats in each group, as provided in this invention.

[0030] Figure 5 The bar chart shows the VEGF protein expression levels in the uterine tissues of rats in each group provided by this invention.

[0031] Figure 6 The bar chart shows the expression levels of PDGF protein in the uterine tissues of rats in each group provided by this invention.

[0032] Figure 7 The bar chart shows the expression levels of CK-19 protein in the uterine tissues of rats in each group, as provided in this invention. Detailed Implementation

[0033] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0034] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0035] The following content details the extraction and identification of bone marrow mesenchymal stem cells (BMSCs):

[0036] 1. Bone marrow sample collection: Four-week-old SD rats were selected, euthanized by cervical dislocation, and disinfected by immersion in 75% medical alcohol for 5-10 minutes. The femur or tibia of the rat was then removed, and the femur and tibia were dissected bilaterally under manipulation, removing surrounding muscle tissue while preserving the bone integrity. The purified bone was transferred to a new culture dish containing PBS, and both ends of the bone shaft were cut off to expose the medullary cavity. The medullary cavity was aspirated using a syringe and rinsed with PBS solution containing penicillin and streptomycin to collect the sample.

[0037] 2. Isolation of BMSCs: Centrifuge tubes containing bone marrow fluid at 1500 rpm for 5 minutes, discard the supernatant, and resuspend the pellet in complete culture medium. Slowly add the resuspended cell suspension dropwise onto PERcoll separation medium to create a density gradient. Centrifuge at 2000-2500 rpm for 25 minutes, aspirate the middle layer (white mist-like cell layer), wash the cells with PBS, centrifuge at 1800 rpm for 5 minutes, discard the supernatant, and retain the cell pellet.

[0038] 3. Culturing BMSCs: The precipitate was resuspended in DMEM medium containing 10% fetal bovine serum, with the addition of penicillin and streptomycin (final concentration 500 U / mL), and seeded into T25 flasks. The flasks were incubated at 37°C in a 5% CO2 incubator. After 24 hours of culture, the culture medium was discarded, and the cells were washed 2-3 times with PBS to remove non-adherent cells. Fresh culture medium was then added, and the cells were cultured again. Half of the culture medium was replaced every 3 days. The cells typically reached confluence in about 10 days. At this point, the cells were digested with 0.25% trypsin and passaged at a 1:2 ratio every 3-5 days. Well-grown third-generation cells were selected for subsequent experiments.

[0039] 4. Cell Morphology and Identification: Microscopic observation revealed that the cells were uniform in morphology, appearing spindle-shaped or flattened. BMSCs positively expressed CD90 and CD44, and negatively expressed CD34, consistent with the surface marker characteristics of BMSCs. This confirms that the cells we isolated and cultured in vitro were purified BMSCs.

[0040] Example 1

[0041] This embodiment provides a pharmaceutical composition for treating chronic pelvic inflammatory disease, with the following final concentrations of active ingredients: 1×10^7 bone marrow mesenchymal stem cells / mL, 20 mg / mL of American cockroach extract, 100 μg / mL of astragalus polysaccharide, 8 ng / mL of granulocyte colony-stimulating factor, 15 μg / mL of interleukin-10, 25 mg / mL of pachymic acid, 20 μg / mL of doxycycline hydrochloride, 50 μg / mL of vitamin B6, and the balance being 0.9% sodium chloride injection.

[0042] Its preparation method includes the following steps:

[0043] (1) Take 1×10^8 bone marrow mesenchymal stem cells and prepare about 5 mL of BMSCs suspension using 0.9% sodium chloride injection;

[0044] (2) Add 200mg of American cockroach extract, 1mg of astragalus polysaccharide, 80ng of granulocyte colony-stimulating factor, 150μg of interleukin-10, 250mg of pachymic acid, 200μg of doxycycline hydrochloride and 500μg of vitamin B6 to the BMSCs suspension, add 0.9% sodium chloride injection to make up to 10ml, mix well and the drug composition is obtained.

[0045] Example 2

[0046] This embodiment provides a pharmaceutical composition for treating chronic pelvic inflammatory disease, with the following final concentrations of active ingredients: 1×10^7 bone marrow mesenchymal stem cells / mL, 10 mg / mL of American cockroach extract, 50 μg / mL of astragalus polysaccharide, 10 ng / mL of granulocyte colony-stimulating factor, 20 μg / mL of interleukin-10, 10 mg / mL of pachymic acid, 10 μg / mL of doxycycline hydrochloride, 50 μg / mL of vitamin B6, and the balance being 0.9% sodium chloride injection.

[0047] The preparation method is the same as in Example 1.

[0048] Example 3

[0049] This embodiment provides a pharmaceutical composition for treating chronic pelvic inflammatory disease, with the following final concentrations of active ingredients: 1×10^7 bone marrow mesenchymal stem cells / mL, 50 mg / mL of American cockroach extract, 180 μg / mL of astragalus polysaccharide, 20 ng / mL of granulocyte colony-stimulating factor, 15 μg / mL of interleukin-10, 50 mg / mL of pachymic acid, 40 μg / mL of doxycycline hydrochloride, 100 μg / mL of vitamin B6, and the balance being 0.9% sodium chloride injection.

[0050] The preparation method is the same as in Example 1.

[0051] Comparative Example 1

[0052] This comparative example provides a pharmaceutical composition for treating chronic pelvic inflammatory disease, with the following final concentrations of active ingredients: 1×10^7 bone marrow mesenchymal stem cells / mL, 80 mg / mL of American cockroach extract, 8 ng / mL of granulocyte colony-stimulating factor, 15 μg / mL of interleukin-10, 25 mg / mL of pachymic acid, 20 μg / mL of doxycycline hydrochloride, 50 μg / mL of vitamin B6, and the balance being 0.9% sodium chloride injection.

[0053] The preparation method is the same as in Example 1.

[0054] Comparative Example 2

[0055] This comparative example provides a pharmaceutical composition for treating chronic pelvic inflammatory disease, with the following final concentrations of active ingredients: 1×10^7 bone marrow mesenchymal stem cells / mL, 300 μg / mL astragalus polysaccharide, 5 ng / mL granulocyte colony-stimulating factor, 105 μg / mL interleukin-1, 80 mg / mL pachymic acid, 50 μg / mL doxycycline hydrochloride, and the balance being 0.9% sodium chloride injection.

[0056] The preparation method is the same as in Example 1.

[0057] Comparative Example 3

[0058] This comparative example provides a pharmaceutical composition for treating chronic pelvic inflammatory disease, wherein the final concentration of the active ingredient is as follows:

[0059] The formula contained 1×10^7 bone marrow mesenchymal stem cells / mL, 20 mg / mL of American cockroach extract, 20 μg / mL of astragalus polysaccharide, 50 ng / mL of granulocyte colony-stimulating factor, 2 μg / mL of interleukin-10, 25 mg / mL of pachymic acid, 60 μg / mL of doxycycline hydrochloride, 200 μg / mL of vitamin B6, with the remainder being 0.9% sodium chloride injection.

[0060] The preparation method is the same as in Example 1.

[0061] Experimental Example 1

[0062] (I) Experimental Methods

[0063] 1. Control group: A total of 51 female SD rats, weighing between 200-230 grams, were selected as the control group, and no treatment was given.

[0064] 2. CPID Model Establishment: The remaining 45 rats were used to establish the CPID model. Rats were anesthetized via intraperitoneal injection of 2% sodium pentobarbital (40 mg / kg). A 1 cm incision was made in the lower abdomen, surrounding tissues were freed, and both uteri were exposed. A bacterial suspension of Staphylococcus aureus and Escherichia coli (concentration approximately 1 × 10^8 CFU / mL) was injected into each uterine cavity, with a volume of 0.1 mL per side. After model establishment, the rats underwent 14 days of acclimatization feeding.

[0065] 3. Model Validation: Three rats were randomly selected 14 days after model establishment for dissection to verify successful model establishment. Typical inflammatory pathological changes were observed, including pelvic congestion, inflammatory swelling, and significant fibrous tissue adhesions; the uterus was congested, with some areas showing uterine fluid accumulation, decreased uterine elasticity, and hardening; simultaneously, the vulva was open, with slight redness, congestion, moisture, and discharge in the vulvar area. The combined presence of these symptoms was considered a successful model establishment. The remaining 42 modeled rats were used for subsequent experiments.

[0066] 4. Experimental grouping: 42 successfully modeled rats were randomly divided into 7 groups of 6 rats each: model group (CPID group), experimental group 1, experimental group 2, experimental group 3, comparative group 1, comparative group 2, and comparative group 3.

[0067] 5. Drug intervention: Experimental groups 1-3 and comparative group-3 were given the drug compositions of Examples 1-3 and Comparative Groups 1-3, respectively. The control group and the model group were injected with the same dose of physiological saline. The injections were given three times on the 14th day (day 1 of treatment), the 21st day (day 7 of treatment), and the 28th day (day 14 of treatment) after modeling.

[0068] 6. Administration: The rats were placed in a supine position and anesthetized using a 2% isoflurane machine, with the pelvic region elevated 3 cm. 200 μL of the drug composition was drawn using a sterile microsyringe. The needle was then slowly inserted into the vagina to a depth of approximately 2-3 cm and manually injected into the rat's cervical canal at a rate of 20 μL / min. After injection, the rats were kept in place for 10 minutes to minimize disturbance caused by fluid flow.

[0069] 7. Observation Indicators and Methods: Rats were dissected 14 days after modeling to observe for congestion, fluid accumulation, masses, adhesions, etc., in both uteri, verifying the success of the modeling. On day 14 post-treatment, uterine tissue pathology was performed: uterine tissue preserved in 4% paraformaldehyde solution was removed and dehydrated using graded alcohol treatments; then, the tissue was cleared with xylene; the cleared tissue blocks were then embedded in paraffin, sectioned, stained with hematoxylin and eosin (HE), and the pathological changes in the uterine tissue were observed under a microscope. On day 14 post-treatment, rat uterine tissue homogenate was collected and analyzed using ELISA.

[0070] 8. Safety testing: On day 14 after drug administration, albumin, alkaline phosphatase, transaminase, renal function and protein metabolism-related parameters of rats in experimental groups 1-3 and the control group were tested.

[0071] (II) Results

[0072] 1. Verification of successful rat model establishment

[0073] Fourteen days after modeling, the vulva was visibly red and congested, accompanied by vulvar moisture, discharge, and vulvar laxity. Dissection revealed significant congestion and redness in the rat uterus, thickened uterine walls, blurred and disordered uterine anatomy, adhesions between the uterus and surrounding pelvic tissues, decreased uterine elasticity, and hardening—all indicative of inflammatory changes. Figure 1 ).

[0074] 2. Culture of uterine and vaginal irrigation fluid

[0075] On day 14 post-treatment, rats in the control group, model group (CPID group), experimental groups 1, 2, and 3, and comparative groups 1, 2, and 3 were anesthetized. A sterile, moistened cotton swab was gently inserted into the rat's vagina approximately 2-3 cm and rotated counterclockwise 5 times to ensure full contact with the vaginal wall. The swab was removed, and the tip was placed in a sterile test tube containing an appropriate amount of MS culture medium. The test tube was gently shaken to thoroughly mix the sample on the swab with the culture medium. The swab was removed, and the remaining culture medium was poured into a sterile petri dish, labeled with the group information. The petri dishes containing the samples were incubated at 37°C for 24 hours. The morphological characteristics of the colonies were observed and recorded under a microscope, including size, number, turbidity, and type. Microscopic results showed that the control group solution was clear and transparent, and no colonies were detected. Figure 2 A); The model group solution was significantly turbid and showed a variety of different colony morphologies ( Figure 2 B); The solution in experimental group 1 was clear, with only a small number of colonies appearing ( Figure 2 C); The solutions in experimental groups 2 and 3 were slightly yellow, and the number of colonies increased significantly, but the turbidity was still lower than that of the model group (C). Figure 2 D and Figure 2 E); The solutions in comparative groups 1 and 2 were light yellow with high turbidity, and the number of colonies was significantly higher than that in experimental group 1 ( Figure 2 F and Figure 2 G); The solution in control group 3 was yellow and highly turbid, with an increased number of colonies (G); Figure 2 H).

[0076] 3. Pathological morphological analysis of rat uterine tissue

[0077] On day 14 of treatment, tissues preserved in 4% paraformaldehyde solution were removed and dehydrated sequentially using different concentrations of ethanol (70% ethanol for 1 hour, 80% ethanol for 1 hour, 90% ethanol for 40 minutes, 95% ethanol twice for 30 minutes, and 100% ethanol twice for 30 minutes). Subsequently, xylene was used for clearing. The cleared tissue blocks were then embedded in paraffin, prepared into sections, and finally stained with hematoxylin and eosin (HE) for microscopic observation of pathological changes. Results showed that the uterine tissue of the control group rats had clear and intact structures in all layers, with no glandular hyperplasia or destruction, no inflammatory cell infiltration, and no significant changes in uterine cell morphology. Figure 3 A); In the model group rats, the boundaries between the layers of the uterine wall were blurred, the epithelial cells and mucosal tissue were severely sloughed off, the cells were arranged in a disordered manner, accompanied by significant inflammatory cell infiltration, and the cell staining was uneven, with a large number of vacuoles present. Figure 3 B); In experimental group 1, the uterine tissue layers were relatively intact, with slight necrosis and shedding of epithelial cells, accompanied by a small amount of inflammatory cell infiltration. The cells were evenly stained, orderly arranged, and significantly reduced in vacuoles. Although the epithelial mucosa layer was thinner than that of the control group rats, the treatment effect was more obvious compared with other groups. Figure 3 C); In experimental groups 2 and 3, the endometrial epithelial cell structure was partially missing, and inflammatory cell infiltration was observed. Compared with experimental group 1, the irregularity of the epithelial mucosa structure was more severe, the mucosal cells were swollen, the cell staining was uneven, and there were many irregularly arranged cells with a significantly increased number of vacuoles. Figure 3 D and Figure 3 E). In the uterine tissue of comparative group 1, the tissue structure of each layer was uneven, the cell staining was uneven, the mucosal tissue was thinner, and the treatment effect was significantly worse than that of experimental group 1. Figure 3 F); In comparative groups 2 and 3, some cells in the uterine tissue were shed, the structure was disordered, inflammatory cells were infiltrated in all layers, cell staining was uneven, most cells were arranged randomly, the number of vacuoles was much greater than in experimental group 1, and the epithelial mucosa tissue of comparative groups 2 and 3 was severely damaged. Figure 3 G and Figure 3 H).

[0078] 4. Changes in relevant factors in the uterine mucosa

[0079] In the pathological process of CPID, changes in TNF-α, VEGF, PDGF, and CK-19 collectively influence the microenvironment at the lesion site, including inflammatory responses, angiogenesis, tissue repair, and cell activity. The interactions among these factors are crucial for disease progression and treatment.

[0080] TNF-α (tumor necrosis factor-α) is an important pro-inflammatory cytokine that plays a crucial role in the immune system. It can activate immune cells, such as macrophages and T lymphocytes, thereby promoting the release of inflammatory mediators and leading to enhanced local inflammatory responses. The role of TNF-α in the inflammatory response is complex; it can induce apoptosis of target cells, an effect that may negatively impact the repair of damaged tissues. In CPID, increased TNF-α levels exacerbate the local inflammatory response, thus worsening tissue damage and pain. Excessive inflammatory responses can activate the immune system, further damaging surrounding healthy tissues. Fourteen days after treatment, uterine tissue from each group of animals was collected and homogenized, and TNF-α expression was subsequently detected. The results showed that compared with the control group, the TNF-α content in experimental groups 1-3 was slightly higher, but significantly lower than that in the model group and comparative groups 1-3. Among them, experimental group 1 had the lowest TNF-α expression level. Figure 4 ).

[0081] VEGF (vascular endothelial growth factor) is primarily responsible for promoting angiogenesis, which is crucial for wound healing and tissue repair. VEGF accelerates angiogenesis by promoting endothelial cell proliferation and migration, providing more blood supply to damaged tissues, thereby promoting the transport of nutrients and oxygen and accelerating the removal of metabolic waste. In CPID, increased VEGF contributes to the repair of damaged tissues and cell proliferation, alleviating tissue hypoxia and promoting tissue regeneration by improving local blood circulation. Fourteen days after treatment, uterine tissue from each group of animals was collected and homogenized, and VEGF expression was subsequently detected. The results showed that compared with the control group, the VEGF levels in experimental groups 1-3 were decreased, but still higher than those in control groups 1-3, and significantly higher than those in the model group. Among them, experimental group 1 had the highest VEGF content. Figure 5 ).

[0082] PDGF (platelet-derived growth factor) is an important growth factor that promotes the proliferation and migration of fibroblasts and endothelial cells, contributing to tissue repair. In CPID, increased PDGF levels aid in the repair of damaged tissue. PDGF promotes wound healing by stimulating the synthesis and remodeling of the extracellular matrix. Furthermore, PDGF can regulate the inflammatory response, inhibiting excessive inflammation and thus protecting tissues from further damage. Fourteen days after treatment, uterine tissue from each group of animals was collected and homogenized, followed by detection of PDGF expression. Results showed that compared to the control group, PDGF levels in experimental groups 1-3 were significantly lower, but still higher than in control groups 1-3, and significantly higher than in the model group. Experimental group 1 had the highest PDGF content. Figure 6 ).

[0083] CK-19 (keratin 19), as a marker of epithelial cells, reflects the activity and state of these cells through changes in its expression level. In CPID, increased CK-19 reflects the active state of epithelial cells, indicating their active participation in the repair process of damaged tissue. Upregulation of CK-19 is generally associated with tissue regeneration and repair, suggesting that the body is actively responding to inflammation and damage. Fourteen days after treatment, uterine tissue was collected from each group of animals and homogenized, followed by detection of CK-19 expression. The results showed that compared with the control group, the CK-19 level in experimental groups 1-3 was significantly decreased, but still significantly higher than that in control groups 1-3 and the model group. Among them, experimental group 1 had the highest CK-19 content. Figure 7 ).

[0084] 5. After drug intervention, there were no significant differences in various indicators between the experimental groups 1-3 and the control group. All indicators were within the normal reference range, indicating that the drug composition provided in this embodiment of the invention did not produce significant toxic reactions, and its metabolic safety was preliminarily verified.

[0085] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A pharmaceutical composition for treating chronic pelvic inflammatory disease, characterized in that, The product contains active ingredients and pharmaceutically acceptable solvents, wherein the final concentrations of the active ingredients are: 1×10^7 bone marrow mesenchymal stem cells / mL, 10-50 mg / mL of American cockroach extract, 50-200 μg / mL of astragalus polysaccharide, 5-20 ng / mL of granulocyte colony-stimulating factor, 10-25 μg / mL of interleukin-10, 10-50 mg / mL of pachymic acid, 10-40 μg / mL of doxycycline hydrochloride, and 50-100 μg / mL of vitamin B6.

2. The pharmaceutical composition for treating chronic pelvic inflammatory disease according to claim 1, characterized in that, The final concentrations of the active ingredients are as follows: 1×10^7 bone marrow mesenchymal stem cells / mL, 20 mg / mL American cockroach extract, 100 μg / mL astragalus polysaccharide, 8 ng / mL granulocyte colony-stimulating factor, 15 μg / mL interleukin-10, 25 mg / mL pachymic acid, 20 μg / mL doxycycline hydrochloride, and 50 μg / mL vitamin B6.

3. The pharmaceutical composition for treating chronic pelvic inflammatory disease according to claim 1, characterized in that, The bone marrow mesenchymal stem cells were third-generation rat bone marrow mesenchymal stem cells.

4. The pharmaceutical composition for treating chronic pelvic inflammatory disease according to claim 1, characterized in that, The pharmaceutically acceptable solvent is 0.9% sodium chloride injection.

5. The pharmaceutical composition for treating chronic pelvic inflammatory disease according to claim 1, characterized in that, The dosage form of the pharmaceutical composition is an injection.

6. The method for preparing the pharmaceutical composition for treating chronic pelvic inflammatory disease according to claim 1, characterized in that, The method includes: (1) Bone marrow mesenchymal stem cells were prepared into a suspension using a pharmaceutically acceptable solvent; (2) Add American cockroach extract, astragalus polysaccharide, granulocyte colony-stimulating factor, interleukin-10, pachymic acid, doxycycline hydrochloride, and vitamin B6 to the BMSCs suspension and dilute to a pharmaceutically acceptable solvent to obtain the final concentrations of bone marrow mesenchymal stem cells, American cockroach extract, astragalus polysaccharide, granulocyte colony-stimulating factor, interleukin-10, pachymic acid, doxycycline hydrochloride, and vitamin B6 as 1×10^7 cells / mL, 10-50 mg / mL, 50-200 μg / mL, 5-20 ng / mL, 10-25 μg / mL, 10-50 mg / mL, 10-40 μg / mL, and 50-100 μg / mL, respectively. Mix well to obtain the drug composition.

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