Application of FGF12 in improving intrauterine adhesion drugs

Abstract

The application discloses application of FGF12 in improving intrauterine adhesion drugs. By constructing a 95% ethanol-induced intrauterine adhesion rat model, and intrauterine injection of an adeno-associated virus carrying FGF12 gene for intervention, it is shown that FGF12 can significantly activate the Rho / ROCK signal pathway, and then promote the phosphorylation of myosin light chain subunit on myosin light chain phosphatase, inhibit the dephosphorylation of MLC, and increase the phosphorylation level of MLC in fibroblasts. The intrauterine adhesion process is inhibited, the collagen deposition is significantly reduced, the endometrial thickness and the number of glands are restored, and the expression level of TGF-β1 is increased. At the same time, the pregnancy rate is obviously improved, and the limitation of traditional treatment is broken through.

Description

Technical Field

[0001] This application relates to the field of biomedical technology, and in particular to the application of FGF12 in drugs for improving intrauterine adhesions. Background Technology

[0002] Intrauterine adhesions are a common gynecological disease among women of reproductive age. They are mainly caused by damage to the basal layer of the endometrium due to curettage after incomplete abortion, postpartum hemorrhage, or infection. They are characterized by partial or complete adhesion or occlusion of the uterine cavity. The most common symptoms of intrauterine adhesions include menstrual abnormalities, oligomenorrhea, recurrent miscarriage, premature birth, and infertility. These clinical symptoms seriously affect the fertility and physical and mental health of women of reproductive age.

[0003] Currently, cervical adhesiotomy is the preferred surgical method for patients with intrauterine adhesions, as it can separate the uterine cavity to a large extent. Common postoperative treatments include hormone therapy, intrauterine devices, uterine balloons, and anti-adhesion gels. However, these treatments are only effective for mild to moderate cases, and the recurrence rate for severe cases is as high as 40%. They are also ineffective in promoting endometrial regeneration and fertility recovery, and there is a lack of effective treatment methods.

[0004] Endometrial fibrosis is the main pathological feature of intrauterine adhesions. Promoting endometrial tissue regeneration and preventing endometrial fibrosis are key to treating intrauterine adhesions. The Rho / ROCK signaling pathway can regulate cell proliferation and participate in tissue damage repair and regeneration, such as in liver fibrosis, essential hypertension, renal fibrosis, and pulmonary fibrosis, suggesting that this signaling pathway plays an important role in the process of tissue fibrosis. Rho protein is a member of the Ras protein superfamily, including three molecules: RhoA, RhoB, and RhoC. Rho exerts various biological functions by binding to guanosine triphosphate (GTP), triggering downstream kinase cascade reactions. ROCK, also known as Rho kinase, is the most functionally detailed downstream target effector molecule of Rho, with two isoforms: ROCKI and ROCKII. ROCK receives activation signals transmitted by Rho, activating multiple amino acid sites through phosphorylation, thereby mediating a series of downstream phosphorylation / dephosphorylation reactions.

[0005] Fibroblast growth factor 12 (FGF12) is a member of the fibroblast growth factor family (FGFs). Currently, research on the function of FGF12 is limited. In tumors, FGF12 can act as an oncogene, promoting tumor cell proliferation and metastasis. Simultaneously, FGF12 can reduce radiotherapy-induced apoptosis and reduce radiotherapy-induced tissue damage. FGF12 exhibits a crucial role in the development and progression of many diseases. However, there are no reports on how FGF12 activates Rho / ROCK signaling to phosphorylate the myosin light chain (MLC) subunit on myosin light chain phosphatase (MLCP), thereby reducing MLCP activity, inhibiting MLC dephosphorylation, and increasing MLC phosphorylation levels within fibroblasts to improve intrauterine adhesions and fibrosis, and ultimately enhance fertility. Summary of the Invention

[0006] To address the problem of the difficulty in effectively promoting endometrial regeneration and restoring fertility due to intrauterine adhesions, and the lack of effective treatment methods, this application provides the application of FGF12 in drugs for improving intrauterine adhesions. By activating the Rho / ROCK signaling pathway through FGF12, it inhibits MLC dephosphorylation, increases the phosphorylation level of MLC in fibroblasts, reduces endometrial fibrosis, restores endometrial function, and improves fertility.

[0007] This application provides the use of FGF12 in drugs for improving intrauterine adhesions. The drug application activates Rho / ROCK signaling by injecting FGF12, which phosphorylates the MLC subunits on MLCPs, reduces MLCP activity, thereby inhibiting MLC dephosphorylation, increasing the phosphorylation level of MLC in fibroblasts, and improving intrauterine adhesion fibrosis.

[0008] Preferably, the protein expression of the improved intrauterine adhesion fibers is detected by immunohistochemical SP method.

[0009] Preferably, the injection is an intraperitoneal injection.

[0010] Preferably, the immunohistochemical SP method for detecting protein expression is: the expression of Rho / ROCK signaling pathway-related proteins and TGF-β1 in rat endometrial tissue.

[0011] Preferably, the intrauterine adhesions are a rat model of intrauterine adhesions induced by 95% ethanol.

[0012] Preferably, the method for preparing the 95% ethanol-induced intrauterine adhesion rat model is as follows: (1) Select female rats in estrus, fast and deprive them of water before the operation, inject an anesthetic into the abdominal cavity, and expose the uterus; (2) Clamp the uterus near the ovary and near the vagina with hemostatic forceps to form a closed cavity; (3) Infuse the uterine cavity with 95% ethanol and keep it full for 3 minutes; (4) Aspirate the ethanol and rinse the uterine cavity with physiological saline; (5) Suture the abdominal cavity and feed the patient for at least 14 days postoperatively to allow for the formation of intrauterine adhesions.

[0013] Preferably, the Rho / ROCK signaling pathway-related proteins are RhoA, RhoB, RhoC, ROCKI, and ROCKI.

[0014] Preferably, the anesthetic in step (1) is 10% chloral hydrate, and the injection dose is 3 mL / kg.

[0015] Preferably, in step (3), the 95% ethanol perfusion volume is 1 mL / side of uterus.

[0016] Preferably, the application is also used to improve pregnancy rates.

[0017] The beneficial effects of the embodiments in this application are as follows: (1) Target innovation: FGF12 can improve intrauterine adhesion fibrosis by regulating the Rho / ROCK signaling pathway, providing a new molecular target and mechanism of action for the treatment of intrauterine adhesion. FGF12 is a brand-new therapeutic target for improving intrauterine adhesion.

[0018] (2) FGF12 treatment increased the pregnancy rate of rats with intrauterine adhesions from 0% to 40%, which is the "gold standard" indicator for evaluating endometrial function repair.

[0019] (3) It breaks through the limitations of traditional treatment methods (such as surgical separation, hormone therapy, etc.) and expands the biological treatment ideas for intrauterine adhesions; by activating Rho / ROCK signal transduction, it affects the phosphorylation process of MLC subunits of MLCP, and ultimately increases the phosphorylation level of MLC in fibroblasts, inhibiting the fibrosis process at the cellular and molecular level. The mechanism of action is clear and highly targeted, providing a clear theoretical basis for subsequent drug development. Attached Figure Description

[0020] Figure 1 HE staining image of the endometrium of the control group in Experiment 1 of this application; Figure 2 Masson staining image of intrauterine adhesions in Experiment Example 1 of this application. Detailed Implementation

[0021] The technical solutions of this application will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments in this application, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of this application.

[0022] The reagents and equipment used in the embodiments of this disclosure are all conventional and commercially available.

[0023] Example 1 Establishment of intrauterine adhesion model (1) Animal preparation: Eighty healthy female Sprague Dawley rats (250g-280g, 6-8 weeks old) were purchased from Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). All SD rats were housed in an SPF-grade environment at the Experimental Animal Center of Wenzhou Medical University, with a constant temperature of 23±2℃, a 12h light-dark cycle, and a relative humidity of 50%. All rats had free access to food and water. Before the start of the experiment, all experimental rats were required to be acclimatized for more than a week to ensure their health for the subsequent construction of the intrauterine adhesion model. The rats were fasted and deprived of water one day before the operation.

[0024] (2) Grouping: Eighty 6-8 week old female SD rats were randomly divided into four groups: control group, intrauterine adhesion model group, AAV (Adeno-Associated Virus) empty vector group, and FGF12 treatment group. Except for the control group, which was injected with saline into the uterine cavity, the other three groups were used to establish an intrauterine adhesion model. The AAV empty vector group was injected with empty vector virus (without FGF12 gene) into the uterine cavity. The FGF12 treatment group was injected with AAV virus carrying the FGF12 gene into the uterine cavity. The AAV virus carrying the FGF12 gene was purchased from Genechem Co., Ltd (Shanghai, China). The viral titer was diluted with saline to 2×10⁻⁶. 11 The dosage was 100 μL per dose, and intrauterine adhesion modeling was performed 3 weeks after injection.

[0025] (3) Establishment of intrauterine adhesion model: After acclimatization, vaginal smears were obtained at 9:00 am every day and Gram staining was performed to ensure that all rats used to establish intrauterine adhesion model were in estrus. The rat intrauterine adhesion model was established by chemical damage method: 95% ethanol was perfused into both uterine cavities, and 10% chloral hydrate was injected into the peritoneum (3 mL / kg) to anesthetize the rats and put them in a supine position. After removing the hair from the lower abdomen of the rats, the abdominal cavity was opened and the uterus was exposed by longitudinal incision along the midline of the abdomen. After locating the left uterus, the uterus near the ovary end and near the vagina end was clamped with a miniature hemostat to form a closed cavity to prevent ethanol from damaging the ovary and vagina. Then, 95% ethanol was slowly perfused into the uterine cavity with a 1 ml syringe and kept the uterine cavity full and white for 3 min to induce severe uterine adhesion. The ethanol in the uterine cavity was aspirated, and the uterine cavity was flushed 3 times with the same volume of physiological saline and the vascular clamp was released. The contralateral uterus was treated in the same way.

[0026] (4) The injured uterus was returned to the abdominal cavity and repositioned. The abdominal cavity was rinsed with normal saline and gently dried with sterile gauze. The abdominal incision was disinfected with iodine solution. Each layer of the abdominal cavity was sutured layer by layer. Penicillin powder was applied to the muscle layer after suturing. After the skin layer was sutured, it was disinfected with povidone-iodine. The control group underwent the same procedure, but only normal saline was injected.

[0027] Example 2 Tissue morphology assessment using HE staining and Masson staining The steps for HE staining are as follows: (1) Tissue fixation: Fresh uterine tissue was collected from rats after euthanasia and fixed in 4% paraformaldehyde for 24 hours.

[0028] (2) Dehydration: Dehydrated the tissue with gradient ethanol (70%, 80%, 90%, 95%, 100%, 1h each) using a dehydrator, and then impregnated with transparent paraffin using xylene.

[0029] (3) Embedding: Place the tissue into the embedding mold, pour in the molten paraffin, quickly adjust the position of the tissue (keep the longitudinal or transverse section of the uterus flat), and form a paraffin block after cooling.

[0030] (4) Slicing and baking: The paraffin block was sliced ​​using a microtome to a thickness of 5 μm. The slices were collected on glass slides and baked at 60°C for 1 to 2 hours.

[0031] (5) HE staining: The above-mentioned dried slides were placed in an HE staining machine for dewaxing with xylene and staining with hematoxylin and eosin to obtain HE-stained slides.

[0032] (6) Mounting: Use neutral resin for mounting.

[0033] (7) Microscopic examination: At 100× magnification, three fields of view were randomly selected from each slice to measure the number of endometrial glands and the thickness of the endometrium (from the basement membrane to the epithelial apex surface), and the average value of the endometrium was calculated using Image Pro software.

[0034] The steps for B. Masson staining are as follows: (1) Tissue fixation: Same as HE staining procedure.

[0035] (2) Dehydration: Same as HE staining procedure.

[0036] (3) Embedding: Same as HE staining procedure.

[0037] (4) Sectioning and drying: Same as HE staining procedure.

[0038] (5) Masson staining: 1) Nuclear staining: Solution A (2% iron alum solution): Weigh 2g of iron alum, dissolve in 100mL of distilled water, and store in the dark; Solution B (0.5% hematoxylin solution): Weigh 0.5g of hematoxylin, dissolve in 100mL of 95% ethanol, and store in the dark; Working solution: Mix equal volumes of solution A and solution B before use; Immerse the sections in the working solution for 10-15 minutes until the cell nuclei turn blue-black, then rinse quickly once with distilled water to remove excess staining solution; 2) Differentiation and Blueing: Differentiation: Differentiate with 1% hydrochloric acid ethanol (hydrochloric acid: ethanol = 1:100) for a few seconds until the cytoplasmic background is clear and the cell nucleus is of appropriate color (avoid over-differentiation which will cause the nuclear staining to become light); Blueing: Rinse with tap water for 5-10 minutes to make the cell nucleus appear a stable blue-black color. 3) Collagen fiber staining: Masson's blue staining solution (0.5g brilliant green + 1mL glacial acetic acid + 100mL distilled water). Immerse the sections in the blue staining solution for 5-10 minutes until the collagen fibers turn a clear blue. Rinse quickly with distilled water 1-2 times to remove excess staining solution. 4) Cytoplasmic staining: Acid fuchsin solution (0.5% acid fuchsin + 1 mL glacial acetic acid + 100 mL distilled water). Immerse the sections in the acid fuchsin solution for 3-5 minutes to make the cytoplasm and red blood cells turn red. Rinse once with distilled water.

[0039] (6) Dehydration, clearing and mounting: Dehydration with graded ethanol, clearing with xylene, and mounting with neutral resin.

[0040] (7) Microscopic examination: Under a 200× magnification microscope, three fields of view were randomly selected for each slice, and the fibrosis rate was calculated using ImageJ software (National Institutes of Health): the area of ​​endometrial stroma fibrosis / the area of ​​endometrial stroma and glands (excluding cavities).

[0041] Example 3 Immunohistochemical SP method (1) Dewaxing and dehydration: The paraffin slices were baked in an oven at 60℃ for 30 minutes, and then immersed in xylene I for 10 minutes → xylene II for 10 minutes for dewaxing; anhydrous ethanol I for 5 minutes → anhydrous ethanol II for 5 minutes → 95% ethanol for 5 minutes → 80% ethanol for gradient dehydration; and rinsed twice with distilled water for 5 minutes each time.

[0042] (2) Antigen retrieval and blocking endogenous peroxidase: Add 3% H2O2 (prepared with PBS) to cover the tissue, incubate at room temperature in the dark for 10 min, and rinse with PBS 3 times, 5 min each time.

[0043] (3) Blocking non-specific binding: Add 10% normal goat serum to cover the tissue, incubate at room temperature for 1 hour, and then discard the serum.

[0044] (4) Primary antibody incubation: Add diluted primary antibody to cover the tissue, TGF-β1, RhoA, RhoC, ROCKI and ROCKII antibodies (Abcam, UK) and RhoB (Beijing Bio-Sens), with 1% BSA as the dilution buffer. Incubate overnight in a humidified chamber at 4°C. The next day, wash three times with PBS for 5 minutes each time.

[0045] (5) Add streptomycin-avidin-peroxidase solution, incubate at room temperature for 30 min, wash with PBS 3 times, 5 min each time. (6) Add biotin-labeled goat anti-rabbit IgG polymer to cover the tissue, incubate at room temperature in the dark for 30 min, and wash with PBS 3 times, 5 min each time.

[0046] (7) Add DAB (3,3'-Diaminobenzidine, 3,3'-diaminobenzidine) solution to cover the tissue, and terminate the reaction with distilled water in 1-2 minutes.

[0047] (8) Counterstaining and mounting: Counterstain cell nuclei with hematoxylin for 30s, rinse with tap water to return to blue, and perform gradient dehydration: 80% ethanol for 1min → 95% ethanol for 1min → anhydrous ethanol I for 1min → anhydrous ethanol II for 1min, xylene I for 2min → xylene II for 2min for clearing, and mount with neutral resin.

[0048] (9) Microscopic examination: Under a 200× microscope, three fields of view were randomly selected from each slide to observe the DAB-positive cell signal.

[0049] Example 4 Fertility test The degree of endometrial repair was assessed by assessing whether the endometrium could implant a fertilized egg and support embryonic development to late pregnancy. On the 15th day after modeling, at 6 pm that day, rats were caged together at a female-to-male ratio of 1:2 for one week. At 9 am the next morning, the presence of vaginal plugs or the presence of sperm in vaginal smears were checked. If sperm was present, it was recorded as day 0.5 of pregnancy. On day 12.5 of pregnancy, the pregnant rats were euthanized. The number of embryos implanted in both uteri and the pregnancy rate of each group were recorded and compared.

[0050] Example 5 Statistical processing The results of Examples 1-5 were statistically analyzed using Graphpad Prism 9.0. Data are expressed as mean ± standard deviation (SD) of at least three independent experiments. Student's t-test was used to perform statistical analysis on variables between two groups. One-way ANOVA was used for comparison of variables among multiple groups. p < 0.05 was considered statistically significant.

[0051] Experimental Example 1 Rat uterine morphology test Ten rats from each group were sacrificed on day 15 after the establishment of the intrauterine adhesion rat model, and the remaining 10 rats from each group were used for breeding experiments.

[0052] On day 15 after modeling, the Y-shaped uterus of rats in the model group became uneven in thickness, less smooth, and less resilient. In contrast, the uterine cavity of rats in the control group was regular in shape and structurally intact, consisting of the endometrium, myometrium, and adventitia. The endometrium was a single layer of columnar epithelium covering the uterine cavity surface, with abundant, oval or round glands. HE staining and Masson staining were performed on paraffin sections of the uterus from both the control and model groups. HE staining of the endometrium in the control group was as follows. Figure 1 As shown, HE staining revealed that the control group had an intact endometrium (798.66±87.81 μm) and a complete lumen covered by columnar epithelium. In the model group, the endometrium was atrophied and its thickness decreased (350.65±20.10 μm, p<0.05). Extensive collagen deposition led to fibrosis, and Masson staining showed collagen fibers in the blue area. Figure 2 As shown, compared with the control group (28.07±5.41%), the model group showed severe proliferation of collagen fibers (55.09±4.04%, p<0.05); these results demonstrate the success of the rat intrauterine adhesion model.

[0053] Experimental Example 2 Expression of TGF-β1 in rat endometrial tissue The expression data of TGF-β1 in the four experimental groups are shown in Table 1: Table 1. Expression of TGF-β1

[0054] The p < 0.01 was statistically significant. The TGF-β1 expression level in the FGF12 treatment group was significantly higher than that in the model group and close to that in the control group. FGF12 can increase the TGF-β1 expression level.

[0055] Experimental Example 3 Expression of Rho / ROCK signaling pathway-related proteins in rat endometrial tissue The expression data of RhoA, RhoB, RhoC, ROCKI, and ROCKII in the four experimental groups are shown in Table 2: Table 2 Expression Data

[0056] Test Example 4 Effects of FGF12 on fertility in rats Fertility is considered the gold standard for assessing the recovery of endometrial function. Female mice were euthanized on day 12.5 of gestation, and their uteruses were collected. Pregnancy data are shown in Table 3. Table 3 Comparison of pregnancy rates among experimental groups

[0057] The above results indicate that FGF12 has a therapeutic effect on damaged endometrium, effectively restoring endometrial function, improving endometrial receptivity, and providing a good foundation for subsequent embryo implantation and development.

[0058] In summary, this application systematically proposes the application of FGF12 in drugs for improving intrauterine adhesions. Through animal experiments and molecular mechanism studies, it has been confirmed that FGF12 activates the Rho / ROCK signaling pathway, regulates MLC phosphorylation levels, thereby inhibiting endometrial fibrosis, promoting tissue repair and functional recovery, and significantly improving pregnancy rates.

[0059] FGF12 activates the Rho / ROCK signaling pathway, promoting MLC subunit phosphorylation on MLCPs, reducing MLCP activity, inhibiting MLC dephosphorylation, and increasing MLC phosphorylation levels in fibroblasts, thereby inhibiting fibrosis and improving endometrial structure and function. Using a rat model of intrauterine adhesions induced by 95% ethanol, HE staining showed significant recovery of endometrial thickness in the FGF12 treatment group. Masson staining showed reduced collagen fiber deposition and significantly improved fibrosis. Immunohistochemical SP method detection of TGF-β1, RhoA, RhoB, RhoC, ROCKI, and ROCKI protein expression showed that TGF-β1 and Rho / ROCK pathway-related protein expression were significantly upregulated in the FGF12 treatment group, approaching the levels of the control group.

[0060] Fertility experiments showed that the pregnancy rate in the FGF12 treatment group reached 40%, which was significantly higher than that in the model group and the empty vector group, proving that it effectively restores endometrial receptivity and fertility.

[0061] Although embodiments of this application have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of this application. All equivalent changes and improvements made within the scope of this application shall still fall within the patent coverage of this application.

Claims

1. The application of FGF12 in the preparation of drugs for improving intrauterine adhesions, characterized in that, The drug application activates Rho / ROCK signaling through FGF12 injection, causing phosphorylation of MLC subunits on MLCPs, reducing MLCP activity, thereby inhibiting MLC dephosphorylation, increasing the phosphorylation level of MLC in fibroblasts, and improving intrauterine adhesions and fibrosis.

2. The application of FGF12 as described in claim 1 in the preparation of drugs for improving intrauterine adhesions, characterized in that, The injection was administered intraperitoneally.

3. The application of FGF12 as described in claim 1 in the preparation of drugs for improving intrauterine adhesions, characterized in that, The model of intrauterine adhesions was a rat model of intrauterine adhesions induced by 95% ethanol.

4. The application of FGF12 as described in claim 1 in the preparation of drugs for improving intrauterine adhesions, characterized in that, The assessment of improvement in intrauterine adhesions and fibrosis was performed using immunohistochemical SP method to detect protein expression.

5. The application of FGF12 as described in claim 4 in the preparation of drugs for improving intrauterine adhesions, characterized in that, The immunohistochemical SP method for detecting protein expression was used to detect the expression of Rho / ROCK signaling pathway-related proteins in rat endometrial tissue.

6. The application of FGF12 as described in claim 3 in the preparation of drugs for improving intrauterine adhesions, characterized in that, The method for preparing the 95% ethanol-induced intrauterine adhesion rat model is as follows: (1) Select female rats in estrus, fast and deprive them of water before the operation, inject an anesthetic into the abdominal cavity, and expose the uterus; (2) Clamp the uterus near the ovary and near the vagina with hemostatic forceps to form a closed cavity; (3) Infuse the uterine cavity with 95% ethanol and keep it full for 3 minutes; (4) Aspirate the ethanol and rinse the uterine cavity with physiological saline; (5) Suture the abdominal cavity and feed the patient for at least 14 days postoperatively to allow for the formation of intrauterine adhesions.

7. The application of FGF12 as described in claim 5 in the preparation of drugs for improving intrauterine adhesions, characterized in that, The proteins associated with the Rho / ROCK signaling pathway are RhoA, RhoB, RhoC, ROCKI, and ROCKII.

8. The application of FGF12 as described in claim 6 in the preparation of drugs for improving intrauterine adhesions, characterized in that, The anesthetic used in step (1) is 10% chloral hydrate, and the injection dose is 3 mL / kg.

9. The application of FGF12 as described in claim 6 in the preparation of drugs for improving intrauterine adhesions, characterized in that, In step (3), the 95% ethanol perfusion volume is 1 mL per uterus.

10. The use of FGF12 as described in any one of claims 1-9 in the preparation of a drug for improving intrauterine adhesions, characterized in that, The application is also used to improve pregnancy rates.

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