Hirudin chitosan microsphere for repairing endometrial injury as well as preparation method and application of hirudin chitosan microsphere

Abstract

The invention belongs to the technical field of biological medicine, and discloses a hirudin chitosan microsphere for repairing endometrial injury through in-situ injection as well as a preparation method and application of the hirudin chitosan microsphere, non-toxic chitosan microspheres are adopted as a slow-release carrier, and controllable slow release of hirudin is realized by optimizing a preparation process. The chitosan microsphere loaded hirudin is locally injected into an injured uterine cavity of a mouse, experiments prove that the uterine tissue structure of an injured region of the mouse uterus is remarkably improved, cell proliferation, muscular layer reconstruction and endometrial angiogenesis of the injured region are promoted through an FOXO1-ERK1 / 2 pathway, intrauterine inflammation is inhibited, the pregnancy rate is increased, and the curative effect is improved. The traditional Chinese medicine composition has a good treatment effect and can be applied to a treatment method of severe uterine injury. The core innovation of the hirudin-chitosan microsphere delivery system is to construct the hirudin-chitosan microsphere delivery system with the targeted slow release characteristic, and a novel material is provided for treating severe uterine injury.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and particularly relates to hirudin chitosan microspheres for in situ injection to repair endometrial damage, as well as a preparation method and application thereof. Technical Background

[0002] Intrauterine adhesions (IUAs) are a common gynecological complication, primarily due to abnormal regeneration and repair of the damaged basal layer of the endometrium. Clinically, IUAs present with abnormal bleeding symptoms such as decreased menstrual volume and amenorrhea, and may lead to reproductive dysfunction such as recurrent pregnancy loss and secondary infertility. While biodegradable barrier membranes or intrauterine support balloon devices can effectively block IUAs, mechanical barrier devices alone have significant limitations in improving endometrial receptivity and restoring physiological functions such as glandular regeneration. In the adjuvant setting, while estrogen receptor-based endocrine regulation can promote endometrial angiogenesis, long-term, high-dose exogenous hormone use may increase the risk of thrombosis and drug-related complications such as breast hyperplasia. Emerging regenerative medicine technologies, including intrauterine infusion of autologous bone marrow mesenchymal stem cells or transplantation of umbilical cord-derived stem cells, have demonstrated significant improvements in endometrial thickness and glandular density in animal models. However, their clinical application remains limited by key technical bottlenecks, such as limited donor resources and low efficiency of directed differentiation. Summary of the Invention

[0003] To address the above issues, the present invention provides a method for repairing uterine damage in mice by local injection of chitosan microspheres loaded with hirudin. Specifically disclosed are hirudin-chitosan microspheres prepared by an emulsification cross-linking method and their use in endometrial repair. The present invention utilizes a sustained-release delivery system to load hirudin, which has a positive effect on promoting tissue cell proliferation and angiogenesis, into the mouse uterine cavity, locally injecting it into the damaged uterus. This promotes regeneration of the damaged uterus while restoring its function.

[0004] The present invention includes the following technical solutions:

[0005] The invention discloses use of a chitosan microsphere loaded hirudin system prepared by an emulsification cross-linking method in preparing uterine injury repair medicine and biological medicine materials.

[0006] In the present invention, the hirudin we use is a Chinese herbal monomer extracted from leeches that promotes blood circulation and removes blood stasis. Hirudin has been shown to have biological functions such as inhibiting the release of inflammatory mediators, regulating the metabolic balance of the extracellular matrix, and promoting tissue vascularization, but research on the repair of uterine damage has not yet been reported. When the uterus is damaged, the newly formed blood vessels provide nutrients and oxygen to the basal cells, supporting their proliferation and differentiation, thereby promoting the repair of the endometrium. However, patients with intrauterine adhesions have damage to the basal layer of the endometrium, which affects the proliferation and differentiation of tissue cells in the damaged area. Therefore, we invented a method that uses sustained-release chitosan microspheres loaded with hirudin for local injection into the damaged uterus. In the examples of the present invention, we observed its efficacy on uterine damage in mice in the form of sustained-release drug treatment, providing a new method and theoretical basis for the treatment of intrauterine adhesions.

[0007] Furthermore, according to the above use, the drug / biological drug material is composed of chitosan microspheres and hirudin as a sustained-release delivery system.

[0008] Furthermore, according to the above use, the drug / biological drug material upregulates the expression of the FOXO1 / ERK1 / 2 signaling pathway.

[0009] The invention also discloses hirudin chitosan microspheres prepared by an emulsification cross-linking method and use thereof in repairing endometrial damage.

[0010] Furthermore, the hirudin chitosan microspheres have a particle size of 10-200 μm, a smooth surface and a uniform particle size distribution. The in vitro sustained-release performance of the microspheres is as follows: in PBS buffer at 37° C., the cumulative release rate of hirudin is 50%-90% within 72 hours.

[0011] Furthermore, the present invention discloses the use of the hirudin chitosan microspheres in in situ injection to repair endometrial damage.

[0012] Furthermore, the present invention discloses a method for preparing the hirudin chitosan microspheres, which comprises the following steps: preparing an aqueous phase and an oil phase, emulsifying, cross-linking, washing, and freeze-drying to obtain the microspheres.

[0013] Furthermore, the above preparation method comprises the following steps:

[0014] (1) Chitosan powder was dissolved in glacial acetic acid to prepare an aqueous phase; liquid paraffin was added in a proportion of Span 80 to prepare an oil phase; the aqueous phase and the oil phase were emulsified in different proportions, washed, and freeze-dried:

[0015] (2) Add hirudin to PBS solution to a final concentration of 10 ATU / mL:

[0016] (3) Screen out chitosan microspheres with smooth and round surfaces and uniform particle size distribution, add them to the prepared hirudin solution, and store them at 4°C.

[0017] (4) Characterization of hirudin chitosan microspheres.

[0018] Furthermore, the aqueous phase in step (1) is a chitosan acetate solution with a volume fraction of 3%; the oil phase is liquid paraffin containing 2% Span80; and the cross-linking agent is a 5% glutaraldehyde solution.

[0019] Furthermore, the rotation speed of the emulsification step is 800-1200 rpm, and the emulsification time is 20-40 minutes; the temperature of the cross-linking step is 50-70° C., and the cross-linking time is 2-4 hours.

[0020] In the hirudin chitosan microspheres of the present invention, the preferred hirudin concentration is 10 ATU / mL.

[0021] The present invention has the following beneficial effects:

[0022] This invention utilizes a sustained-release chitosan microsphere system, prepared through emulsification and cross-linking, to serve as both a drug delivery vehicle and a tissue repair scaffold. This innovative application of a novel bioactive material in the repair of reproductive system injuries addresses the technical bottlenecks of traditional hirudin administration, such as its short half-life and poor targeting. By leveraging the sustained-release properties of the microsphere drug delivery system and its synergistic effects, this system simultaneously improves drug bioavailability and tissue regeneration efficiency.

[0023] Compared with existing technologies, it has the following significant advantages:

[0024] 1. Synergistic improvement of targeted sustained release and biocompatibility

[0025] The chitosan microsphere carrier prepared by emulsification cross-linking process has a particle size range of 10-200 μm (Example 1, Figure 1 AB), with a smooth and evenly distributed surface, can stably load hirudin and achieve controlled sustained release. In vitro release experiments showed that the cumulative release rate of hirudin in the microspheres in 37°C PBS buffer within 72 hours reached 50%-90% ( Figure 1 C), significantly prolonging the drug's duration of action, avoiding the short half-life of traditional drug administration and the need for frequent injections. At the same time, the inherent biocompatibility and mucosal adhesion of chitosan materials (Example 1, Figure 2 ), ensuring the long-term residence of the microspheres in the uterine cavity and providing continuous drug support for tissue repair.

[0026] 2. Promote the repair of endometrial structure and function in multiple dimensions

[0027] Animal experiments have confirmed that local injection of hirudin chitosan microspheres can significantly improve the morphology and function of damaged uterus:

[0028] Histological repair: HE staining showed that the endometrial thickness of the treatment group increased by 1.5 times compared with the natural repair group, and the glandular density increased by 2.3 times ( Figure 4 AD); Masson staining confirmed that the fibrosis area was reduced by 60% ( Figure 4 EF), indicating that microspheres can effectively inhibit abnormal collagen deposition.

[0029] Cell proliferation and myometrial reconstruction: Immunohistochemistry results showed that the number of PCNA-positive cells increased by 80% compared with the control group ( Figure 5 AD), the α-SMA positive area expanded 1.8 times ( Figure 6 AD), suggesting that microspheres accelerate injury repair by activating cell proliferation and muscle fiber regeneration.

[0030] Promoting angiogenesis and inhibiting inflammation: vWF staining showed that microvessel density increased 2-fold ( Figure 7 AD), the number of CD45 positive cells decreased by 70% ( Figure 8 AD), indicating that hirudin synergistically with chitosan microspheres can promote angiogenesis and inhibit local inflammatory response.

[0031] 3. Significant potential for clinical translation

[0032] Fertility function experiments showed that the pregnancy rate of mice treated with hirudin chitosan microspheres was 3 times higher than that of the natural repair group, and the embryo survival rate reached 85% ( Figure 9 AB), proving that this technical solution can not only restore the endometrial structure, but also significantly improve reproductive function, providing an innovative strategy for the clinical treatment of intrauterine adhesions and secondary infertility. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 -- Electron micrograph of hirudin chitosan microspheres, schematic diagram of particle size distribution, and schematic diagram of hirudin sustained release from hirudin chitosan microspheres at different time points;

[0034] Figure 2 ——Schematic diagram of in vitro biocompatibility of hirudin chitosan microspheres;

[0035] Figure 3 ——Schematic diagram of hirudin chitosan microspheres implanted in the mouse uterus after in situ injection;

[0036] Figure 4 ——Image of the results of repairing damaged uterus in mice by in situ injection of hirudin-chitosan microspheres;

[0037] Figure 5 ——Results of in situ injection of hirudin-chitosan microspheres promoting proliferation of uterine stromal cells in damaged mice;

[0038] Figure 6——Image of the results of in situ injection of hirudin-chitosan microspheres to restore the damaged uterine myometrium of mice;

[0039] Figure 7 ——Results of in situ injection of hirudin-chitosan microspheres promoting angiogenesis in damaged endometrium of mice;

[0040] Figure 8 ——Image showing the results of in situ injection of hirudin-chitosan microspheres inhibiting lipopolysaccharide-induced uterine inflammation in mice;

[0041] Figure 9 ——Results of in situ injection of hirudin-chitosan microspheres improving the pregnancy rate of model mice;

[0042] Figure 10 ——Transcriptome sequencing analysis and verification of the hirudin chitosan microsphere group and the natural repair group. DETAILED DESCRIPTION

[0043] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0044] The reagents or instruments used in the examples of the present invention without indicating the manufacturer are all conventional reagent products that can be obtained through commercial purchase.

[0045] Example 1

[0046] Preparation and Characterization of Hirudin-containing Chitosan Microspheres

[0047] (1) Preparation of hirudin chitosan microspheres:

[0048] 1) Preparation of aqueous phase: Weigh 1.5 g of chitosan powder and dissolve it in 1 mL of glacial acetic acid. Add 49 mL of distilled water to obtain a 3% chitosan acetic acid solution. Stir continuously until the chitosan is dissolved. Allow to stand to remove bubbles and store at 4°C.

[0049] 2) Oil phase preparation: 49 mL of liquid paraffin was mixed with 1 mL of Span 80 surfactant to form a stable oil phase medium and stored at room temperature.

[0050] 3) Emulsification: Mix the two phases at a volume ratio of 8:1, rotate at 1000 rpm, and emulsify for 30 minutes.

[0051] 4) Cross-linking: 5% glutaraldehyde cross-linking agent was slowly added to the emulsified system and placed in a constant temperature water bath at 60° C. for 3 hours for cross-linking.

[0052] 5) Washing: The solution was centrifuged at 3000 rpm, the supernatant discarded, and the precipitate washed with petroleum ether to remove residual surfactant. The microspheres were placed in water and the surface of the microspheres was transparent and smooth, indicating no oil spots. The microspheres were dehydrated three times with anhydrous ethanol and washed several times with deionized water. The purified microspheres were immersed in a 10 ATU / mL hirudin solution in PBS (pH = 7.4) and stored in a dark place at 4°C for 72 hours to achieve drug loading.

[0053] 6) Freeze-drying: The drug-loaded microspheres are quickly frozen in liquid nitrogen and then transferred to a freeze dryer for freeze-drying.

[0054] The electron microscope scanning and particle size distribution analysis of hirudin chitosan microspheres showed that the chitosan microspheres prepared with an oil-water ratio of 8:1 were smooth and round, and their particle size distribution was uniform ( Figure 1 AB).

[0055] (2) Sustained release curve of hirudin chitosan microspheres

[0056] For in vitro release studies, hirudin-encapsulated chitosan microspheres were placed in 10 mL of PBS buffer solution and placed on a thermostatic shaker (37°C, 100 rpm). 1 mL of the suspension was extracted at various time intervals and centrifuged (3000 rpm, 5 min). The amount of free hirudin in the supernatant was determined by the BCA assay, and the cumulative percentage of hirudin released was calculated from the standard curve. An equal volume of fresh PBS buffer was added to the release medium to return it to the original volume.

[0057] The results showed that when the chitosan microspheres with an oil-water ratio of 8:1 were used to encapsulate hirudin, the drug release rate gradually slowed down over time ( Figure 1 C).

[0058] (3) Effects of hirudin chitosan microspheres on the ring formation and proliferation of HUVECs in vitro

[0059] Chitosan microspheres and extracts of hirudin-loaded chitosan microspheres were prepared for use. The effects of hirudin-loaded chitosan microspheres and chitosan microspheres on HUVECs proliferation were detected using a CCK-8 kit. Cells in the logarithmic growth phase were plated at 5×10 cells per well. 3 The cells were seeded at a density of 500 μL in a 96-well plate and cultured for 24 hours. 100 μL of extract medium and 10 ATU / mL hirudin culture medium were added to each well. The control group was added with culture medium containing 10% FBS and incubated for 24 hours. CCK-8 solution was added to measure cell viability.

[0060] Liquid matrix gel was inoculated into 96-well plates and incubated in a 37°C incubator for 30 min to solidify. HUVECs were digested with trypsin and resuspended in serum-free DMEM medium at a density of 1×10 per well. 4A volume of 100 μL was added to each well. The HUVECs group served as a blank control. The 10% FBS complete medium group served as a positive control. HUVECs were resuspended in hirudin medium (hirudin stock solution was resuspended in serum-free DMEM medium to a concentration of 10 ATU / mL), chitosan microsphere extract, and hirudin-chitosan microsphere extract. 100 μL of each group was added to the Matrigel per well, incubated at 37°C for 24 hours, and observed under an inverted microscope. ImageJ software was used to count the number of tubes, length, and branching nodes in the three imaged areas.

[0061] The results showed that the proliferation capacity of HUVECs cultured in the extract of chitosan microspheres encapsulated with hirudin was significantly increased compared with the control group ( Figure 2 B). The number of rings, branch points and length of HUVECs treated with hirudin chitosan microspheres in vitro were significantly higher than those in the control group, indicating that hirudin chitosan microspheres have a positive effect on promoting angiogenesis ( Figure 2 AB).

[0062] (4) Local injection of hirudin chitosan microspheres into the mouse uterus

[0063] Before local injection into the uterine cavity, hirudin-containing chitosan microspheres were labeled with rhodamine B. Uterine tissue was collected from mice on days 3, 6, 9, and 12 after injection. Fluorescent images were obtained using an in vivo imaging system on days 3, 6, 9, and 12 post-injection to detect the colonization of the hirudin-containing chitosan microspheres in the uterus.

[0064] The results showed that on the 3rd day after injection, obvious fluorescence could be observed in the damaged uterine area of ​​mice. On the 6th day after injection, the fluorescence intensity was similar to that on the 3rd day. On the 9th and 12th day after local injection, the fluorescence intensity gradually weakened, but there was no statistical significance compared with the fluorescence intensity on the 3rd day. Chitosan microspheres can be adsorbed in the damaged mouse uterine cavity ( Figure 3 AB).

[0065] Example 2

[0066] Application of hirudin chitosan microspheres in mouse uterine injury model

[0067] A model of intrauterine adhesions was established in proestrus mice by mechanical curettage combined with lipopolysaccharide (LPS)-induced double injury. Mice were anesthetized with isoflurane, secured on an operating table, and the lower abdominal hair was shaved. The skin was disinfected with 75% ethanol. A 1.2 cm longitudinal incision was made along the midline of the abdomen, and the subcutaneous tissue and peritoneum were gently separated to expose the bilateral uterine horns. In the sham group, only the laparotomy and suture were performed without intrauterine intervention. In the natural repair group (SR), a 0.2 cm longitudinal incision was made in the bilateral uterine horns using micro-ophthalmic scissors. Micro-toothed forceps were inserted through the incisions and mechanical curettage was performed until the endometrium showed diffuse bleeding and the myometrium was exposed. Subsequently, a 3-0 sterile cotton thread soaked in 6 mg / L LPS solution was placed in the uterine cavity, with the end of the thread positioned 1 mm externally as a marker. After curettage, the chitosan microsphere group (CS) received a 100 μL chitosan microsphere suspension injection. In the hirudin group (Hirudin), after curettage, 100 μL of PBS buffer containing 10 ATU / mL hirudin was injected into the uterus, and a cotton thread was placed in place. In the hirudin chitosan microsphere group (CS / Hirudin), after curettage, 100 μL of a hirudin-loaded chitosan microsphere suspension was injected. Penicillin 200,000 IU was injected continuously for three days postoperatively. The intrauterine lipopolysaccharide cotton thread was removed 48 hours after surgery. Uterine samples were collected 30 and 60 days after surgery for HE staining and immunohistochemistry to assess uterine recovery. Masson staining was used to observe the degree of fibrosis. A fertility study was performed on rats 60 days after surgery to assess uterine function recovery.

[0068] (1) HE staining

[0069] Paraffin-embedded sections were placed in a 65°C oven for 30 minutes, then dewaxed using xylene I / II / III (15 minutes, 10 minutes, and 10 minutes, respectively), hydrated with 100% ethanol for 10 minutes, and then with 95%, 80%, 70%, and 50% graded ethanol for 1 minute each. Hematoxylin was applied for 1 minute, followed by a 1-minute rinse in deionized water to remove excess color. Sections were then differentiated in 1% hydrochloric acid-ethanol solution for 15 seconds and rinsed with running water for 1 minute. Sections were stained with eosin for 2 minutes, dehydrated with graded ethanol (80%, 95%, 100%) for 5 minutes each, and cleared twice with xylene (5 minutes each). Sections were mounted with neutral gum, and the staining results were observed and photographed under a standard light microscope.

[0070] HE staining results showed that the thickness and gland number of the endometrium repaired by hirudin chitosan microspheres were higher than those in other treatment groups. This shows that hirudin chitosan microspheres can repair damaged endometrium ( Figure 4 AD).

[0071] (2) Masson collagen staining

[0072] After baking, dewax the sections and hydrate them. Mix Weiger's Iron Hematoxylin Solution A and Solution B in a 1:1 ratio (prepared immediately before use) and drop onto the circled tissue. Stain for 5 minutes and rinse with distilled water. Differentiate the tissue with acidic ethanol solution for 10 seconds and rinse with distilled water. Debluing with Masson's bluing solution for 5 minutes and rinse with distilled water. Stain with Ponceau red solution for 5 minutes and then with weak acid working solution for 1 minute. Stain with phosphomolybdic acid for 1 minute and then with weak acid working solution for 1 minute. Stain with aniline blue for 1 minute and then with weak acid working solution for 1 minute. Dehydrate rapidly with 5% ethanol for 2-3 seconds, then with anhydrous ethanol three times for 10 seconds each. Clear with xylene three times for 2 minutes each. Mount the sections with neutral gum.

[0073] Masson collagen staining was used to detect the collagen deposition and fibrosis degree of the endometrium in each group. The experimental results showed that treatment with hirudin chitosan microspheres could reduce the fibrosis degree of the damaged area of ​​the uterus and improve the damaged structure of the uterus ( Figure 4 E,F).

[0074] (3) Immunohistochemical PCNA staining

[0075] Uterine tissues from each group of mice were collected 30 and 60 days after surgery. The tissues were immersed in 4% paraformaldehyde overnight, dehydrated in graded ethanol, and embedded in paraffin. Transverse sections were cut at a thickness of 5 μm. After dewaxing and hydration, the sections were placed in sodium citrate buffer and exposed to antigenic epitopes using high-pressure heat repair. After repair, the sections were cooled to room temperature and washed three times with PBS (5 minutes each). The tissues were covered with 3% hydrogen peroxide blocking solution and incubated at room temperature for 10 minutes to quench endogenous peroxidase activity. After washing with PBS, the sections were blocked with 10% normal goat serum for 30 minutes at room temperature to block nonspecific binding sites. The blocking solution was discarded, and the primary antibody against PCNA (1:200, Proteintech) was added dropwise, and the sections were incubated overnight at 4°C in a humidified chamber. For a negative control, the primary antibody was replaced with PBS. The sections were warmed the next day and washed three times with PBS (5 minutes each). An HRP-conjugated goat anti-rabbit IgG secondary antibody was added dropwise, and the sections were incubated at 37°C for 60 minutes. Develop with DAB colorimetric solution in the dark. Monitor under a microscope until a clear positive signal and no background staining are observed. Terminate the reaction with deionized water. After sections are dehydrated and transparent, mount with neutral gum and observe the staining results under an optical microscope and photograph them.

[0076] Immunohistochemical PCNA staining can detect cell proliferation in the damaged uterine area. 30 and 60 days after surgery, the PCNA expression in the uterine damaged area in the group injected with hirudin chitosan microspheres was significantly higher than that in the other treatment groups. This result suggests that hirudin chitosan microspheres can repair damaged endometrium by promoting cell proliferation. Figure 5 AD).

[0077] (4) Immunohistochemical α-SMA staining

[0078] The method was the same as that of PCNA staining, and the primary antibody was α-SMA (1:1000, Abcam).

[0079] Immunohistochemical α-SMA staining was used to evaluate the regeneration of the uterine myometrium. The experimental results showed that after treatment with hirudin chitosan microspheres, the regeneration of myometrial muscle fibers was significantly promoted, and the positive area was higher than that of other treatment groups, indicating that hirudin chitosan microspheres can promote the regeneration of damaged uterine myometrium in mice and promote uterine repair ( Figure 6 AD).

[0080] (5) Immunohistochemical vWF staining

[0081] The method was the same as that of PCNA staining, and the primary antibody was vWF (1:1000, Abcam).

[0082] Immunohistochemical vWF staining was used to evaluate uterine angiogenesis. The experimental results showed that the hirudin chitosan local injection group significantly increased the number of blood vessels in the damaged uterine area, and the distribution was even, and the microvascular density was significantly higher than that of the other treatment groups. This shows that hirudin chitosan microspheres can promote the angiogenesis of damaged uterus and repair damaged uterus ( Figure 7 AD).

[0083] (6) Immunohistochemical CD45 staining

[0084] The method was the same as that for PCNA staining, and the primary antibody was CD45 (1:300, Abcam).

[0085] The mouse model of intrauterine adhesions was induced by mechanical curettage combined with lipopolysaccharide to induce inflammation. The leukocyte count was evaluated by immunohistochemical staining for CD45. The results showed that the local injection of hirudin chitosan significantly reduced the leukocyte count in the damaged uterine area, suggesting that local injection of hirudin chitosan inhibited the inflammation of the damaged endometrium. Figure 8 AD).

[0086] (7) Recovery of pregnancy function

[0087] Thirty days after surgery, 8-week-old fertile male Balb / c mice were co-housing with female mice to assess the recovery of postoperative uterine pregnancy function. The day after co-housing, the presence of a vaginal plug was checked. If present, the gestational age was recorded as 0.5 days. Female mice were euthanized at 17.5 days of gestation, and the embryo status was examined and recorded.

[0088] The experimental results suggest that the number and development of embryos in mice treated with hirudin chitosan microspheres are better than those in other treatment groups, indicating that hirudin chitosan microspheres can improve the damaged uterine function of rats ( Figure 9 AB).

[0089] Example 3

[0090] Molecular mechanism of hirudin-chitosan microspheres in repairing uterine damage

[0091] (1) Transcriptome sequencing

[0092] Thirty days after surgery, uterine tissue samples from the natural repair group and the hirudin-chitosan microsphere-treated group were subjected to RNA sequencing analysis (three biological replicates per group). Total RNA was extracted using TRIzol reagent, and RNA purity was assessed using a NanoDrop 2000 spectrophotometer, and RNA integrity was assessed using an Agilent 2100 Bioanalyzer. RNA sequencing was performed by Shanghai Ouyi Biotechnology Co., Ltd. using an Illumina NovaSeq 6000 platform, employing 150-bp paired-end sequencing to generate approximately 40 million raw reads per sample. cDNA libraries were prepared using the NEBNext Ultra RNA Library Preparation Kit, followed by ribosomal RNA removal using the Ribo-Zero Gold rRNA Removal Kit. Raw sequencing data were quality controlled, adapter trimmed, and aligned to the mouse reference genome GRCm39. Gene expression was quantified using the htseq-count joint counting mode. Differential expression between groups was analyzed using the DESeq2 package in R, with multiplexed analysis. Gene ontology (GO) enrichment analysis of differentially expressed genes (DEGs) was performed using the clusterProfiler software package and Fisher's exact test, while KEGG pathway analysis was performed using KOBAS-i and hypergeometric test and false discovery rate correction.

[0093] This study used transcriptome sequencing technology to observe the molecular level changes after treatment with hirudin chitosan microspheres. The sequencing results showed that after treatment with hirudin chitosan microspheres, 1618 genes were upregulated and 1380 genes were downregulated. GO enrichment and KEGG enrichment analysis were performed based on these differentially expressed genes. GO analysis results showed that the differentially expressed genes were mainly enriched in cell proliferation regulation. KEGG enrichment analysis showed that hirudin chitosan microspheres may be involved in the changes in signaling pathways such as FOXO and PI3K-AKT ( Figure 10 AC).

[0094] (2) Expression of proliferation genes in mice after local injection of hirudin chitosan microspheres

[0095] Uterine tissue was collected 30 days after surgery and stored at -80°C for RT-qPCR. Uterine tissue was removed from -80°C cryopreservation, thawed on ice until suitable for manipulation, and then rapidly transferred to a centrifuge tube containing Trizol reagent for tissue RNA extraction. RNA concentration was measured using a NanoDrop 1000 spectrophotometer. Tissue RNA was reverse transcribed into cDNA. Target gene expression was assessed by RT-qPCR. mRNA levels were calculated using the 2-ΔΔCt method, using GAPDH as an endogenous control.

[0096] The present invention uses RT-qPCR to detect changes in the expression levels of genes related to damaged uterus in each group. The ERK1 / 2 expression levels of the natural repair group, chitosan group, and hirudin group were compared with the hirudin chitosan microsphere local injection group, and it was found that the expression level of ERK1 / 2 in the hirudin chitosan microsphere local injection group was significantly increased. In addition, when the FOXO1 expression levels in each group were statistically analyzed, it was found that the FOXO1 expression levels in the natural repair group, chitosan group, and hirudin group were significantly reduced, while the FOXO1 expression level in the hirudin chitosan microsphere local injection group was significantly increased ( Figure 10 DE).

[0097] (3) Changes in protein levels in mice after local injection of hirudin chitosan microspheres

[0098] The uterine tissues of each group of mice were taken out at -80℃ and placed on ice. PMSF and RIPA lysate were added to the tissue to extract tissue protein. BCA protein assay kit was used for quantification. A sample of 40 μg of protein was loaded into the lane of 10% SDS-PAGE electrophoresis for separation. After electrophoresis, the protein was transferred to a PVDF membrane. In order to block non-specific binding sites, the membrane was soaked in 5% skim milk for 1 hour. The membrane was incubated with primary antibodies FOXO1 (1:1000), ERK1 / 2 (1:1000), and GAPDH (1:10000) at 4℃ overnight. The membrane was washed with TBST 3 times, 5 minutes each time. The membrane was incubated with goat anti-rabbit IgG at room temperature for 1 hour. The blot signal was captured using an enhanced chemiluminescence (ECL) kit.

[0099] The present invention detected the expression levels of ERK1 / 2 and FOXO1 proteins in the damaged uterus of each group by Western blot. Compared with the natural repair group (SR), chitosan group (CS), and hirudin group (Hirudin), the expression levels of ERK1 / 2 and FOXO1 proteins in the hirudin chitosan microsphere group (CS / Hirudin) were significantly increased, suggesting that hirudin chitosan microspheres may promote the repair of damaged mouse uterus by regulating ERK1 / FOXO1. Figure 10 FG).

[0100] The above experimental results demonstrate that local injection of hirudin-chitosan microspheres significantly improves the repair of endometrial damage and the restoration of physiological function in mice. Histological observations revealed more complete glandular structure and more active cell proliferation in the treatment group compared to the control group. Functional recovery assessments confirmed its ability to effectively improve pregnancy outcomes in mice. Molecular mechanistic studies revealed an association with ERK1 / 2 and FOXO1. The chitosan microspheres prepared by the emulsification and cross-linking method in this invention deliver hirudin for a localized sustained-release effect, providing a new therapeutic approach for patients with severe uterine damage.

[0101] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Therefore, based on the innovative concept of the present invention, changes and modifications to the embodiments described herein, or equivalent process transformations made using the contents of the present invention specification, directly or indirectly applying the above technical solutions to other related technical fields are all included in the scope of protection of the patent of the present invention.

Claims

1. The use of hirudin in the preparation of drugs / biological medicine materials for repairing uterine damage.

2. The use according to claim 1, characterized in that The drug / biological drug material is chitosan microspheres loaded with hirudin.

3. The use according to claim 1, characterized in that The drug / biological drug material is used to upregulate the expression of the FOXO1 / ERK1 / 2 signaling pathway.

4. A hirudin chitosan microsphere for in situ injection to repair endometrial damage, characterized in that: The hirudin chitosan microspheres are composed of chitosan microspheres loaded with hirudin.

5. The hirudin chitosan microspheres according to claim 4, characterized in that The chitosan microspheres have a particle size of 10-200 μm, a smooth surface and uniform particle size distribution. The in vitro sustained-release performance of the microspheres is as follows: in a PBS buffer solution at 37° C., the cumulative release rate of hirudin within 72 hours is 50%-90%.

6. Use of the hirudin chitosan microspheres according to claim 4 or 5 in a drug for in situ injection to repair endometrial damage.

7. The method for preparing hirudin chitosan microspheres according to claim 4 or 5, characterized in that: The following steps are involved: (1) Chitosan powder is dissolved in glacial acetic acid to prepare an aqueous phase; Span 80 is added to liquid paraffin in proportion to prepare an oil phase; the aqueous phase and the oil phase are emulsified, cross-linked, washed, and freeze-dried in different proportions: (2) Add hirudin to PBS solution to a final concentration of 10 ATU / mL: (3) Screen out chitosan microspheres with smooth and round surfaces and uniform particle size distribution, add them to the prepared hirudin solution, and store them at 4°C. (4) Characterization of hirudin chitosan microspheres.

8. The method for preparing hirudin chitosan microspheres according to claim 6, characterized in that: The aqueous phase in the step (1) is a chitosan acetate solution with a volume fraction of 3%; the oil phase is liquid paraffin containing 2% Span80; and the cross-linking agent is a 5% glutaraldehyde solution.

9. The method for preparing hirudin chitosan microspheres according to claim 6, characterized in that: The rotation speed of the emulsification step in step (1) is 800-1200 rpm, and the emulsification time is 20-40 minutes; the temperature of the cross-linking step is 50-70° C., and the cross-linking time is 2-4 hours.

10. The method for preparing hirudin chitosan microspheres according to claim 6, characterized in that: The hirudin concentration in the hirudin chitosan microspheres is 10 ATU / mL.

Images