Medicinal preparation for repairing endometrial injury

Abstract

The invention provides a pharmaceutical preparation for repairing endometrial injury, and belongs to the technical field of biomedicine. The pharmaceutical preparation takes improved polypeptide as an active ingredient, and the amino acid sequence of the improved polypeptide is shown as SEQ ID NO.5. Experimental results show that the polypeptide can significantly promote proliferation and migration of human endometrial stromal cells, still maintains low cytotoxicity under high concentration, and shows excellent safety. Furthermore, in a rat endometrial mechanical injury model, the preparation can obviously increase the endometrial thickness, improve the Ki-67 positive cell rate, enhance the proliferation activity of endometrial cells and promote the repair of damaged tissues. Compared with the original polypeptide, the preparation disclosed by the invention shows unexpected technical effects in the aspects of repairing activity and safety, and has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to a pharmaceutical preparation for the repair of endometrial damage. Background Technology

[0002] The endometrium is a vital tissue in the female reproductive system, and its integrity and functional status are crucial for maintaining the menstrual cycle, embryo implantation, and pregnancy outcomes. Under the influence of factors such as induced abortion, curettage, hysteroscopy, infection, inflammation, and intrauterine adhesions, the endometrium can suffer varying degrees of damage, leading to endometrial thinning, reduced glandular tissue, insufficient angiogenesis, increased fibrosis, and decreased intrauterine receptivity. In severe cases, this can cause clinical problems such as decreased menstruation, amenorrhea, infertility, and recurrent implantation failure. Therefore, effectively promoting the repair of damaged endometrium and restoring its normal structure and physiological function has become a research hotspot in the fields of reproductive medicine and gynecological disease treatment.

[0003] Currently, treatment methods for endometrial injury mainly include estrogen or progesterone replacement therapy, hysteroscopic adhesiolysis, intrauterine device (IUD) or balloon placement, application of anti-adhesion materials, stem cell therapy, platelet-rich plasma (PRP) therapy, and local delivery of biomaterials. However, existing treatments still have certain limitations. For example, surgical treatment is mainly suitable for organic lesions such as intrauterine adhesions, but there is still a risk of re-adhesion and insufficient repair after surgery; although stem cell and related regenerative medicine technologies have promising applications, their preparation is complex and costly, and their safety, stability, and standardization still need further validation. Therefore, developing a pharmaceutical preparation with both good repair activity and high safety is of great significance for improving the repair effect of endometrial injury. Summary of the Invention

[0004] The purpose of this invention is to provide a pharmaceutical preparation for the repair of endometrial damage, thereby providing a safe and effective polypeptide preparation.

[0005] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides a pharmaceutical preparation for the repair of endometrial damage, the pharmaceutical preparation comprising an active ingredient and pharmaceutically acceptable excipients; The active ingredient is a modified polypeptide, and the amino acid sequence of the modified polypeptide is shown in SEQ ID NO.5.

[0006] Preferably, the concentration of the modified polypeptide in the pharmaceutical preparation is 10-160 μg / mL.

[0007] Preferably, the concentration of the modified polypeptide in the pharmaceutical preparation is 40 μg / mL.

[0008] Preferably, the endometrial damage repair refers to repairing thin endometrial tissue caused by mechanical damage or endometrial damage caused by induced abortion or curettage.

[0009] Preferably, the pharmaceutically acceptable excipient is physiological saline, sterile water, or phosphate buffer. The dosage form of the pharmaceutical preparation is selected from one of the following: solution, gel, injection, spray, sustained-release preparation, and hydrogel preparation.

[0010] Secondly, the present invention provides the application of a modified polypeptide in the preparation of a medicament for treating endometrial injury, wherein the amino acid sequence of the modified polypeptide is shown in SEQ ID NO.5.

[0011] Preferably, the endometrial injury is a thin endometrium caused by mechanical injury or endometrial injury caused by induced abortion or curettage.

[0012] Preferably, the concentration of the modified polypeptide in the drug is 10-160 μg / mL.

[0013] Preferably, the application of the modified polypeptide includes promoting the proliferation and / or migration of endometrial stromal cells.

[0014] Thirdly, the present invention provides a modified polypeptide for achieving endometrial repair, the amino acid sequence of which is shown in SEQ ID NO.5.

[0015] The beneficial effects of this invention are as follows: This invention provides a pharmaceutical preparation for the repair of endometrial injury. Compared with existing repair methods such as hormone therapy, surgical intervention, and stem cell therapy, this invention uses a specific modified peptide as the active ingredient, exhibiting both good repair activity and safety. Experimental results show that the modified peptide can significantly promote the proliferation and migration of human endometrial stromal cells. The peptide shown in SEQ ID NO.5 exhibits a significantly better repair-promoting effect than the original Chensinin-1b and other modified peptides in in vitro experiments, while maintaining low toxicity at higher concentrations and a wider safety window. Furthermore, in a rat model of mechanical endometrial injury, this preparation can significantly increase the thickness of damaged endometrium, increase the rate of Ki-67 positive cells, enhance endometrial cell proliferation activity, and accelerate the reconstruction of damaged tissue. Therefore, the pharmaceutical preparation of this invention has significant advantages in promoting endometrial regeneration, improving the quality of endometrial repair, and enhancing its clinical application potential. Attached Figure Description

[0016] Figure 1 This is a bar chart showing the effect of each group of peptides in Example 4 of the present invention on the scratch healing rate of human endometrial stromal cells (HESCs) at 24 hours (a) and 48 hours (b). Figure 1 In the figure, *** indicates P < 0.001 compared to Chensinin-1b, and ○○○ indicates P < 0.001 compared to Chensinin-1b modified peptide 3; Figure 2 This is a statistical chart showing the Ki-67 positive cell rate in the endometrial tissue of each group of rats in Example 6 of the present invention; Figure 2 In the figure, *** indicates that P < 0.001 compared to the model group, and ●●● indicates that P < 0.001 compared to the improved Chensinin-1b group. Detailed Implementation

[0017] The present invention will be specifically described below through embodiments. It should be noted that the following embodiments are only used to further illustrate the present invention and should not be construed as limiting the scope of protection of the present invention. Those skilled in the art can make some non-essential improvements and adjustments to the present invention based on the above description.

[0018] Chensinin-1b is a derivative polypeptide (amino acid sequence: SKVWRHWRRFWHRAHRLH, SEQ ID NO.1) developed based on Chensinin-1. Its main function is antibacterial, and it also has wound repair effects. Therefore, this invention attempts to use it for endometrial repair to explore its new applications in the field of gynecological regenerative medicine.

[0019] Example 1 Effect of Chensinin-1b peptide on the proliferation of human endometrial stromal cells (HESCs) as determined by CCK-8 assay (1) HESCs cells in the logarithmic growth phase were digested with 0.25% trypsin and counted. The cell density was adjusted to 5×10⁶ cells using DMEM / F12 medium containing 10% FBS. 4 per mL.

[0020] (2) Add 100 μL of cell suspension (i.e., 5000 cells / well) to each well of a 96-well plate. Incubate at 37°C and 5% CO2 for 24 hours to allow the cells to adhere completely.

[0021] (3) Discard the original culture medium and replace it with low serum culture medium (1% FBS) for 12 hours of starvation to synchronize the cell cycle, followed by the addition of culture medium containing different concentrations of Chensinin-1b peptide: Control group: Add culture medium containing 2.5% FBS; Peptide groups: culture media containing 10, 40, 80, and 160 μg / mL of Chensinin-1b peptide, respectively; Positive control group: culture medium containing 20 ng / mL EGF was added; (4) After incubating in the incubator for 48 hours, add 10 μL of CCK-8 solution to each well and incubate in the dark for 2 hours. Use an ELISA reader to measure the optical density (OD value) of each well at a wavelength of 450 nm and calculate the cell viability.

[0022] Table 1. Effect of Chensinin-1b on HESCs cell viability (x±s, n=5)

[0023] As shown in Table 1, the experimental data indicate that Chensinin-1b exhibited some proliferative activity at a concentration of 40 μg / mL, increasing cell viability by approximately 23.5%. This demonstrates that the derived peptide possesses preliminary repair potential, but it is significantly lower than that of EGF. Furthermore, the proliferative effect disappeared at a concentration of 80 μg / mL; and at 160 μg / mL, due to the strong cationic nature of this sequence, significant cytotoxicity occurred, leading to massive cell death.

[0024] Chensinin-1b has a certain repair effect on endometrial stromal cells within a specific concentration window, but its bioactivity is limited and its safety range is narrow.

[0025] Example 2 Since the results of Example 1 showed that Chensinin-1b had limited effect when directly applied to endometrial stromal cells, this example attempts to improve it to obtain a modified peptide that can be used specifically for endometrial repair.

[0026] Modified peptide 1 Design concept: Replace some of the strongly alkaline arginine (R) with neutral glutamine (Q) or serine (S) to reduce the net positive charge and decrease electrical damage to the cell membrane.

[0027] The specific amino acid sequence is as follows: SKVWRHWQS FWHQAHQLH, SEQ ID NO.2; Modified peptide 2 Design concept: Tryptophan (W) and phenylalanine (F) in the original sequence are prone to hydrophobic aggregation on the membrane surface. Therefore, some W is replaced with alanine (A) or tyrosine (Y) which has a similar structure but lower hydrophobicity.

[0028] The specific amino acid sequence is as follows: SKAYRHARRFAHRAHRLH, SEQ ID NO.3; Modified Peptide 3 Design concept: Modify the sites attacked by proteases. Use N-terminal acetylation (Ac-) and C-terminal amidation (-NH2) for terminal shielding.

[0029] The specific amino acid sequence is as follows: Ac-SKVWRHWRRFWHRAHRLH-NH2, SEQ ID NO.4; Modified Peptide 4 Design concept: The strategy of "charge distribution optimization + flexible linker" is adopted. The core active group (WR group) is retained, but glycine (G) is introduced in the middle as a flexible hinge, so that the peptide can bind to the cell receptor more flexibly, rather than forcibly inserting into the membrane.

[0030] The specific amino acid sequence is as follows: SKVWRGWRRFGHRAHRLH, SEQ ID NO.5; The above-mentioned polypeptides were synthesized by Jier Biochemical (Shanghai) Co., Ltd., with a purity of ≥95%.

[0031] Example 3 Detection of the effect of modified peptides on the proliferation of human endometrial stromal cells (1) HESCs cells in the logarithmic growth phase were digested with 0.25% trypsin and counted. The cell density was adjusted to 5×10⁶ cells using DMEM / F12 medium containing 10% FBS. 4 per mL.

[0032] (2) Add 100 μL of cell suspension (i.e., 5000 cells / well) to each well of a 96-well plate. Incubate at 37°C and 5% CO2 for 24 hours to allow the cells to adhere completely.

[0033] (3) Discard the original culture medium and replace it with low serum medium (1% FBS) for 12 hours of starvation to synchronize the cell cycle, followed by the addition of a culture medium containing 40 μg / mL and 160 μg / mL of modified peptides: Control group: Add culture medium containing 2.5% FBS; Low-dose Chensinin-1b group: culture medium containing 40 μg / mL Chensinin-1b peptide; High-dose Chensinin-1b group: culture medium containing 160 μg / mL Chensinin-1b peptide; Low-dose Chensinin-1b modified peptide group 1: culture medium containing 40 μg / mL Chensinin-1b modified peptide 1; High-dose Chensinin-1b modified peptide group 1: culture medium containing 160 μg / mL Chensinin-1b modified peptide 1; Low-dose Chensinin-1b modified peptide group 2: culture medium containing 40 μg / mL Chensinin-1b modified peptide 2; High-dose Chensinin-1b modified peptide group 2: culture medium containing 160 μg / mL Chensinin-1b modified peptide 2; Low-dose Chensinin-1b modified peptide group 3: culture medium containing 40 μg / mL Chensinin-1b modified peptide 3; High-dose Chensinin-1b modified peptide group 3: culture medium containing 160 μg / mL Chensinin-1b modified peptide 3; Low-dose Chensinin-1b modified peptide group 4: culture medium containing 40 μg / mL Chensinin-1b modified peptide 4; High-dose Chensinin-1b modified peptide group 4: culture medium containing 160 μg / mL of Chensinin-1b modified peptide 4.

[0034] (4) After incubating in the incubator for 48 hours, add 10 μL of CCK-8 solution to each well and incubate in the dark for 2 hours. Use an ELISA reader to measure the optical density (OD value) of each well at a wavelength of 450 nm and calculate the cell viability.

[0035] Table 2. Effects of each modified peptide on the viability of HESCs (x±s, n=5)

[0036] This indicates that the cell viability was significantly higher than that of Chensinin-1b (P < 0.01). This indicates that the cell viability was significantly lower than that of Chensinin-1b (P < 0.001).

[0037] As shown in Table 2, while modified Chensinin-1b peptides 1 and 2 exhibited lower toxicity at high concentrations, their promoting effects were poor. Modified Chensinin-1b peptide 3 showed significantly improved promoting effects at low concentrations, but its promoting effect was poor at high concentrations. In contrast, modified Chensinin-1b peptide 4 not only demonstrated significantly better proliferative effects than other modified peptides at low concentrations but also maintained excellent promoting effects at high concentrations.

[0038] Example 4 Detection of the effect of modified peptides on the migration ability of human endometrial stromal cells (1) HESCs cells in the logarithmic growth phase were digested with 0.25% trypsin and counted, using 2×10⁻⁶ cells per cell. 5 The cells were seeded at a density of 1 cell per well in 6-well plates and cultured in complete medium containing 10% FBS until the cells reached 100% confluence (forming a dense monolayer). (2) Discard the original culture medium and replace it with serum-free DMEM / F12 culture medium for starvation treatment for 12 hours to eliminate the interference of cell proliferation on the migration results; (3) Using a 200μL sterile pipette tip, draw a uniform straight line vertically in the center of the cell monolayer in each well, and gently rinse 3 times with PBS to remove the detached cells; (4) Replace the culture medium with one containing 1% FBS, and add 40 μg / mL of each group of polypeptides respectively: Control group: only 1% FBS medium was added; Chensinin-1b group; Chensinin-1b modified peptides groups 1, 2, 3, and 4.

[0039] (5) Place it in a cell culture incubator and continue to culture for 24 hours. After taking a picture, use ImageJ software to measure the scratch width at 0 hours, 12 hours and 24 hours respectively, and calculate the scratch healing rate.

[0040] from Figure 1The results showed that, regardless of whether it was at 12 hours or 24 hours, the effect of Chensinin-1b modified peptide 4 on promoting the migration ability of HESCs cells was significantly better than that of Chensinin-1b, and also significantly better than that of Chensinin-1b modified peptide 3. Specifically, the healing rate at 24 hours was 16.63±1.72% in the control group, 26.31±1.71% in the Chensinin-1b group, 34.88±2.77% in the Chensinin-1b modified peptide 3 group, and 57.69±2.97% in the Chensinin-1b modified peptide 4 group at 48 hours, which was approximately 1.65 times that of Chensinin-1b modified peptide 3, demonstrating a very excellent effect.

[0041] Example 5 To investigate the repair effects of different peptides on mechanical damage to the endometrium. 1. Animal pretreatment Healthy adult female Sprague-Dawley (SD) rats, 8–10 weeks old and weighing 220–250g, were selected. Environmental adaptation: SD rats were cultured for 7 days under a 12-hour light / dark cycle, with free access to food and water, at a temperature of 22±2°C and a humidity of 50±10%.

[0042] 2. Experimental groups (6 animals per group) Sham surgery group: only exploratory laparotomy was performed, without uterine curettage or medication; Model group: Mechanical scraping + physiological saline; Chensinin-1b peptide group: mechanical scraping + Chensinin-1b treatment; Chensinin-1b modified peptide group 4: mechanical scraping + Chensinin-1b modified peptide 4 treatment; One-way ANOVA pretest showed no statistically significant difference in body weight among the groups.

[0043] 4. Mechanical injury modeling and drug administration (1) The rats in the model group and the treatment group were fasted for 12 hours before the operation and anesthetized with 1% sodium pentobarbital on the day of estrus; (2) Fix the rat in a supine position on a 37°C heated operating table and disinfect it three times with iodine solution and 75% alcohol; (3) Make a 2cm incision in the midline of the abdomen, gently pull out the bilateral uterine horns with toothless forceps, and make a longitudinal micro-incision of about 2mm at the end of each uterine horn near the ovary (about 0.5cm from the ovary); (4) Insert the hook-shaped needle into the uterine cavity and rotate it along the longitudinal axis of the uterus and scrape the endometrial layer 10 times. In the sham surgery group, only a 2mm incision is made and no scraper is inserted.

[0044] (5) Use a microsyringe to inject the corresponding polypeptide solution or physiological saline into the uterine cavity of the rat. The polypeptide solution concentration is 160 μg / mL, and the sterile physiological saline is prepared in a volume of 50 μL (this concentration is safe and effective due to the dilution of the fluid in the uterine cavity). (6) Immediately ligate the distal incision (near the cervix) twice with sterile silk thread to prevent leakage of the medication.

[0045] (7) Gently return the uterus to the abdominal cavity, check for bleeding and torsion, suture layer by layer, and apply erythromycin ointment to the skin to prevent infection.

[0046] (8) During the anesthesia recovery period, the patient was placed in a 37°C incubator until fully awake. Immediately after the operation, ibuprofen 5mg / kg was injected subcutaneously for analgesia, and penicillin G sodium 20000U / kg was injected intramuscularly for 3 consecutive days.

[0047] 5. Endometrial thickness measurement Seven days after modeling, the rats were anesthetized, sacrificed, and the bilateral uteruses were quickly removed, fixed with 4% paraformaldehyde for 24 hours, dehydrated in a gradient, embedded in paraffin, sectioned, and stained with hematoxylin and eosin.

[0048] The endometrium was photographed under a microscope, and the endometrial thickness was measured using ImageJ software. The results are shown in Table 3.

[0049] Table 3 Comparison of endometrial thickness and relative recovery rate in rats of different groups

[0050] As can be seen from the results in Table 3, on the 7th day after mechanical curettage modeling, the endometrial thickness of the rats in the model group was only 192.3±25.8μm, which was significantly reduced compared with the sham surgery group, indicating that mechanical injury successfully induced the pathological characteristics of thin endometrium.

[0051] Further analysis revealed that the intima thickness increased in all treatment groups after intervention with different peptides. The intima thickness in the Chensinin-1b group was 265.4 ± 28.1 mm, with a relative recovery rate of only 24.9%, indicating limited repair efficacy. In contrast, the modified Chensinin-1b peptide 4 group exhibited extremely strong repair activity, with a recovery rate approximately 3.15 times that of the original peptide group, demonstrating a very significant repair effect on damaged intima tissue.

[0052] 6. Ki-67 immunohistochemical staining The paraffin-embedded sections were dewaxed and hydrated, subjected to high-pressure heat repair with citrate buffer (pH 6.0), blocked with endogenous peroxidase by 3% H2O2, and blocked with 5–10% normal goat serum. Add Ki-67 primary antibody (1:200 dilution, overnight at 4°C); incubate sequentially with biotin-labeled secondary antibody, streptavidin-HRP complex, DAB staining, hematoxylin counterstaining, gradient dehydration, clearing, and then mount with neutral resin.

[0053] The endometrial repair area was photographed under a microscope (×400x). Hotspot areas with the highest concentration of Ki-67 positive cells were selected. ImageJ software was used to randomly count the total number of cells and the number of Ki-67 positive cells in each slice, calculating the Ki-67 positive cell rate. The results are shown below. Figure 2 As shown.

[0054] from Figure 2 It can be seen that the Ki-67 positive cell rate in the sham surgery group was 35.4±3.2%, the positive cell rate in the model group was 8.2±1.3%, the Ki-67 positive cell rate in the peptide Chensinin-1b group was 15.6±1.8%, and the Ki-67 positive cell rate in the Chensinin-1b modified peptide 4 group was 31.28±2.6%.

[0055] In contrast, the model group showed a significant decrease in Ki-67 positive cell rate after mechanical scraping injury, indicating that severe mechanical damage destroyed the germinal layer of the endometrium, resulting in a severe lack of local cell proliferation dynamism and difficulty in initiating effective self-repair.

[0056] Although the Chensinin-1b peptide group was superior to the model group, it was still far below the normal physiological level, indicating that the original peptide had limited ability to mobilize the division of damaged endometrial cells. The Ki-67 positive cell rate of the Chensinin-1b modified peptide group 4 was close to that of the sham-operated group, indicating that it significantly enhanced the cell proliferation dynamics during the endometrial repair process, thereby accelerating re-epithelialization and tissue reconstruction.

Claims

1. A pharmaceutical preparation for repairing endometrial damage, characterized in that, The pharmaceutical preparation contains an active ingredient and pharmaceutically acceptable excipients; The active ingredient is a modified polypeptide, and the amino acid sequence of the modified polypeptide is shown in SEQ ID NO.

5.

2. The pharmaceutical preparation according to claim 1, characterized in that, The concentration of the modified polypeptide in the pharmaceutical preparation is 10-160 μg / mL.

3. The pharmaceutical preparation according to claim 2, characterized in that, The concentration of the modified polypeptide in the pharmaceutical preparation is 40 μg / mL.

4. The pharmaceutical preparation according to claim 1, characterized in that, The endometrial damage repair refers to repairing thin endometrial tissue caused by mechanical damage or endometrial damage caused by induced abortion or curettage.

5. The pharmaceutical preparation according to claim 1, characterized in that, The pharmaceutically acceptable excipients are physiological saline, sterile water or phosphate buffer; The dosage form of the pharmaceutical preparation is selected from one of the following: solution, gel, injection, spray, sustained-release preparation, and hydrogel preparation.

6. The use of a modified polypeptide in the preparation of a medicament for treating endometrial injury, characterized in that, The amino acid sequence of the modified polypeptide is shown in SEQ ID NO.

5.

7. The application according to claim 6, characterized in that, The aforementioned endometrial injury refers to thin endometrium caused by mechanical injury or endometrial damage caused by induced abortion or curettage.

8. The application according to claim 7, characterized in that, The concentration of the modified polypeptide in the drug is 10-160 μg / mL.

9. The application according to claim 8, characterized in that, The modified peptide is used to promote the proliferation and / or migration of endometrial stromal cells.

10. A modified polypeptide for achieving endometrial repair, characterized in that, The amino acid sequence of the modified polypeptide is shown in SEQ ID NO.5.

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