Application of a mixture of collagen peptide and sodium alginate and / or NLRP3 gene knockout and / or NLRP3 inhibitor MCC950 in the prevention and / or treatment of intrauterine adhesions and inflammatory diseases.

Abstract

To provide agents for prevention and treatment of intrauterine adhesion.SOLUTION: The present invention provides a collagen peptide and sodium alginate combined mixture, comprising collagen peptide and sodium alginate. The collagen peptide and sodium alginate combined mixture has anti-inflammatory and anti-fibrosis functions.SELECTED DRAWING: Figure 4

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and specifically to a mixture combining an injectable collagen peptide and sodium alginate and / or the application of NLRP3 gene knockout and / or NLRP3 inhibitor MCC950 and drugs in the prevention and / or treatment of intrauterine adhesions and inflammatory diseases.

Background Art

[0002] In women of childbearing age, the endometrium voluntarily sheds, repairs, and regenerates due to the action of sex hormones, and scarring does not occur during this repair process. However, after damage to the basal layer of the endometrium, it is prone to impaired endometrial repair, leading to the formation of intrauterine adhesions. Intrauterine adhesions (IUA) are a common endometrial fibrosis disorder in the female reproductive system. They are mainly caused by damage to the basal layer of the endometrium due to endometrial injury or infection, which triggers an abnormal inflammatory response, leading to the deposition of large amounts of collagen fibers in the uterine cavity and causing endometrial fibrosis. The main symptoms include uterine obstruction, pelvic pain, menstrual irregularities, menopause, secondary infertility, and pregnancy complications, seriously impacting women's physical and mental health. Currently, there is still a lack of effective treatments for IUA, so effectively preventing IUA and reducing IUA complications has significant clinical importance in improving the quality of life for IUA patients. The NLRP3 inflammasome is a protein complex involved in immunomodulation and inflammatory responses, primarily composed of three different types of proteins: the NLRP3 receptor protein, an apoptosis-associated speck-like protein containing a CARD (ASC), and the aspartase precursor of the effector protein cysteine ​​(Pro-caspase-1). It plays a crucial role in the body's immune system and disease development. This invention has found that the NLRP3 inflammasome is involved in the development and progression of fibrosis in multiple organs, including the kidneys, liver, and lungs, and that inhibiting NLRP3 inflammasome activation can reduce the degree of inflammatory responses and fibrosis. However, the role of the NLRP3 inflammasome in IUA (inflammatory ulcers) has not yet been reported. While this invention demonstrates that collagen peptides (CP) and sodium alginate (SA) possess anti-inflammatory and wound-healing-promoting functions, further invention is needed to clarify the mechanism of action against IUA.

[0003] Therefore, the present invention provides novel therapeutic ideas for the clinical prevention and treatment of IUA by applying a mixture of the injectable collagen peptide and sodium alginate to damaged mouse endometrium and investigating its effect on IUA formation. [Overview of the project] [Problems that the invention aims to solve]

[0004] The present invention designs a drug for preventing and / or treating intrauterine adhesions, and the technical problem that the present invention solves is that, in the prior art, a mixture of injectable collagen peptide and sodium alginate has not been applied to the prevention and treatment of intrauterine adhesions. [Means for solving the problem]

[0005] To solve the technical problems described above, the present invention employs the following solution.

[0006] A mixture of collagen peptide and sodium alginate, characterized in that the mixture of collagen peptide and sodium alginate has anti-inflammatory and anti-fibrotic effects.

[0007] Preferably, the mixture of the collagen peptide and sodium alginate inhibits the activation of the NLRP3 inflammasome, specifically reducing the protein expression of NLRP3 and ASC, as well as the downstream inflammatory factors caspase-1, IL-1β, and IL-18.

[0008] Preferably, the mixture of the collagen peptide and sodium alginate reduces the activation and release of NLRP3 and ASC, as well as the downstream inflammatory factors caspase-1, IL-1β, and IL-18.

[0009] Preferably, the mass ratio of collagen peptide to sodium alginate is 1 to 10:1.

[0010] Preferably, the mass ratio of collagen peptide to sodium alginate is 2:1.

[0011] A mixture of collagen peptides and sodium alginate is an injectable drug used for the prevention and treatment of intrauterine adhesions.

[0012] A method for preparing a mixture of collagen peptide and sodium alginate, comprising: step S1, taking collagen peptide powder, adding sterile water, and stirring uniformly to obtain a collagen peptide solution; and step S2, adding sodium alginate powder to the collagen peptide solution obtained in step S1, and stirring uniformly so that the mass ratio of collagen peptide to sodium alginate is 1 to 10:1 to obtain an injectable mixture of collagen peptide and sodium alginate.

[0013] NLRP3 gene knockout and / or NLRP3 inhibitor MCC950 and its application in the prevention and treatment of intrauterine adhesions. NLRP3 gene knockout and / or NLRP3 inhibitor MCC950 can be used to inhibit the NLRP3 inflammasome, reduce endometrial damage, inflammatory response, and the degree of fibrosis.

[0014] Preferably, the NLRP3 gene knockout and / or NLRP3 inhibitor MCC950 inhibits NLRP3 inflammasome activation, specifically reducing the protein expression of NLRP3 and ASC, as well as the downstream inflammatory factors caspase-1, IL-1β, and IL-18, thereby reducing the activation and release of NLRP3 and ASC, as well as the downstream inflammatory factors caspase-1, IL-1β, and IL-18. This reduces the degree of endometrial damage, inflammatory response, and fibrosis.

[0015] A drug for preventing and / or treating intrauterine adhesions, characterized in that the NLRP3 inhibitor MCC950 is applied to the prevention and / or treatment of intrauterine adhesions.

[0016] Preferably, the NLRP3 inhibitor MCC950 inhibits NLRP3 inflammasome activation, specifically by reducing the protein expression of NLRP3 and ASC, as well as the downstream inflammatory factors caspase-1, IL-1β, and IL-18, thereby reducing the activation and release of NLRP3 and ASC, as well as the downstream inflammatory factors caspase-1, IL-1β, and IL-18, and mitigating endometrial damage, inflammatory response, and fibrosis. [Effects of the Invention]

[0017] The mixture of the collagen peptide and sodium alginate, and its application in the prevention and treatment of intrauterine adhesions, have the following beneficial effects:

[0018] (1) The present invention is based on collagen peptide powder and sodium alginate powder, and the entire composition is formulated into an injectable material that is scientifically rational for use in intrauterine adhesions. Experimental results show that a mixture of injectable collagen peptide and sodium alginate can effectively inhibit the inflammatory response after endometrial injury, accelerate endometrial repair, and improve mouse reproductive capacity by inhibiting NLRP3 inflammasome expression.

[0019] (2) The present invention discloses the mechanism of action of a mixture of injectable collagen peptide and sodium alginate in the prevention and treatment of intrauterine adhesions, and provides reliable experimental evidence for the prevention and treatment of intrauterine adhesions using a mixture of injectable collagen peptide and sodium alginate.

[0020] (3) According to the research of the present invention, it has been discovered that NLRP3 gene knockout and / or the NLRP3 inhibitor MCC950 can effectively inhibit the inflammatory response after endometrial injury and accelerate endometrial repair by inhibiting NLRP3 inflammasome expression. The present invention not only discloses the anti-inflammatory and anti-fibrotic mechanism of action of NLRP3 gene knockout and / or the NLRP3 inhibitor MCC950, but also provides a new option for the prevention and treatment of intrauterine adhesions.

Brief Description of the Drawings

[0021] [Figure 1] It is a comparative diagram of the results of the repair effect of the mixture of the injectable collagen peptide provided by the present invention and sodium alginate on uterine cavity adhesion tissue damage. [Figure 2] It is a comparative diagram of the results of the repair effect of the mixture of the injectable collagen peptide provided by the present invention and sodium alginate on uterine cavity adhesion tissue fibrosis. [Figure 3] It is a comparative diagram of the results of the effect of the mixture of the injectable collagen peptide provided by the present invention and sodium alginate on the activation of NLRP3 inflammasome in uterine cavity adhesion tissue. [Figure 4] It is a comparative diagram of the influence of the mixture of the injectable collagen peptide provided by the present invention and sodium alginate on the inflammatory factor levels in the serum of uterine cavity adhesion mice. [Figure 5] It is a comparative diagram of the results of the effect of the mixture of the injectable collagen peptide provided by the present invention and sodium alginate on the reproductive ability of uterine cavity adhesion. [Figure 6] It is a comparative diagram of the results of the repair effect of NLRP3 gene knockout and / or the NLRP3 inhibitor MCC950 provided by the present invention on uterine cavity adhesion tissue damage. [Figure 7]Comparison diagram of the results of the repair effect of NLRP3 gene knockout and / or NLRP3 inhibitor MCC950 provided by the present invention on the fibrosis of the adhesions in the uterine cavity. [Figure 8] Comparison diagram of the results of the action of NLRP3 gene knockout and / or NLRP3 inhibitor MCC950 provided by the present invention on NLRP3 inflammasome activation in the adhesions in the uterine cavity. [Figure 9] Comparison diagram of the influence of NLRP3 gene knockout and / or NLRP3 inhibitor MCC950 provided by the present invention on the levels of inflammatory factors in the serum of mice with uterine cavity adhesions.

Mode for Carrying Out the Invention

[0022] <Example 1> Hereinafter, referring to FIGS. 1 to 5, Example 1 of the present invention will be further described.

[0023] The composition and mixing ratio of the raw drug of the mixture combining the collagen peptide and sodium alginate of the present invention are 5 parts by weight of collagen peptide and 2.5 parts by weight of sodium alginate. The mixture combining the materials used in the present invention, namely injectable collagen peptide and sodium alginate, has the advantages of less irritation, determination of the content of active ingredients, remarkable effects, and can be used for a long time.

[0024] The preparation method of the mixture combining the injectable collagen peptide and sodium alginate of the present invention includes the following steps. Step 1: Weigh 5 g of collagen peptide powder, add 100 ml of sterile water, and stir evenly to obtain a 5% concentration collagen peptide solution. Step 2: Add 2.5 g of sodium alginate powder to the 5% collagen peptide solution obtained in Step 1, and stir evenly to obtain the mixture combining the materials, namely injectable collagen peptide and sodium alginate.

[0025] Experimental verification method: 1. Animals and reagents: All animal experiments have passed logical review and approval by Xinxiang Medical University. C57 / BL6N mice were purchased from the Animal Experiment Center of Henan SCBS Biotechnology Co., Ltd., license number SCXK(Henan)2020-0005. Collagen peptides and sodium alginate were purchased from Nantong YeMeng New Material Co., Ltd. Other materials and reagents were purchased from companies such as Beyotime, Solarbio, Proteintech, Abcam, etc., or are listed separately.

[0026] 2. Establishment of a mouse IUA model: This model is constructed to create a stable and effective mouse IUA model based on the premise that 95% ethanol acts on the endometrium to cause chemical damage, inflammatory response, and fibrosis of the endometrium. Mice with a normal estrous cycle are selected, and the model is created during the mouse's estrous period. The operating steps are as follows: Anesthesia is induced using a small animal respiratory anesthesia machine, and the mouse is given 2-3% isoflurane anesthetic to induce anesthesia. The mouse is then placed on a small animal operating table, and anesthesia is maintained by continuously inhaling 1.5-2.5% isoflurane. Subsequently, the cervix is ​​explored using a gastric tube feeding needle attached to a syringe, and 95% ethanol is injected into the cervix until ethanol is observed to flow out of the vaginal opening. After maintaining this for 1 minute, 2 ml of 0.9% saline is injected into the cervix to flush out any remaining ethanol from the uterine cavity.

[0027] 3. Grouping and processing of experimental animals: (1) Female C57 / BL6N48 mice were randomly divided into three groups: a sham surgery group, a model group, and a treatment group (CP+SA), each group consisting of 16 mice. In the model group, an IUA model was prepared by inducing endometrial damage by injecting 95% ethanol into the cervix and uterine cavity of the mice according to the method described above. In the sham surgery group, 0.9% saline was injected into the uterine cavity through the cervix of the mice. In the treatment group (CP+SA), a model was created by injecting 95% ethanol into the uterine cavity through the cervix of the mice, and then a 5% CP + 2.5% SA mixture was injected into the uterine cavity. After modeling, the mice in each group were given normal food and water.

[0028] 4. Sample collection and processing: Seven days after modeling, eight mice were randomly selected from each group, anesthetized with 2-3% isoflurane inhalation, blood was collected from the eyes, centrifuged, and the upper serum was collected and cryopreserved for use in ELISA testing. Uterine tissue was collected, and a portion of the tissue was stored at -80°C for use in measuring protein content. A portion of the tissue was fixed with a freshly prepared 4% paraformaldehyde solution, and conventional paraffin-embedded sections were used for subsequent histomorphological staining and modified Masson trichrome staining.

[0029] 5. Hematoxylin-eosin (HE) staining for observing morphological changes in the endometrium: Uterine tissue sections are regularly dewaxed with water, stained with hematoxylin for 5 minutes, rinsed with running tap water for about 10 minutes, stained with eosin for 1 minute, dehydrated to clear, mounted on neutral gum, and then the pathological morphological changes of the endometrium are observed under a microscope, photographs are taken under 10×4x and 10×20x microscopes, and the number of endometrial glands is counted using ImageJ software.

[0030] 6. Modified Masson trichrome staining to observe the degree of endometrial fibrosis: Uterine tissue sections were regularly dewaxed with water, mordanted with Bouin's solution for 2 hours, stained with celestite blue for 2 minutes, stained with Mayer hematoxylin for 2 minutes, separated with acidic ethanol fractionation solution for 10 seconds, treated with phosphomolybdic acid solution for 10 minutes, stained with aniline blue for 5 minutes, treated with weak acid for 2 minutes, dehydrated to clear, sealed with neutral gum, observed the degree of endometrial fibrosis under a microscope, photographs were taken under 10×4x and 10×20x microscopes, and the degree of endometrial fibrosis in each group was analyzed and calculated using ImageJ software.

[0031] 7. Western blot isonoassay to detect the expression status of NLRP3, ASC, caspase-1, IL-1β, and IL-18 proteins in the uterine tissue of mice from each group: After homogenizing the uterine tissue of mice from each group, proteins were extracted and their concentrations measured. Gel preparation, sample loading, electrophoresis, electroporation, and blocking were then performed in sequence. The corresponding primary and secondary antibodies were incubated, the membranes were washed, and the samples were developed using the Azure electrochemiluminescence imaging system. The gray values ​​of the target and reference proteins were then analyzed and calculated using Image-Pro Plus software.

[0032] 8. Enzyme-linked immunosorbent assay (ELISA) to detect serum IL-1β and IL-18 content in mice from each group: Serum samples were collected from each group of mice, and the IL-1β and IL-18 content in each group's mouse serum was detected strictly according to the operating procedure of the ELISA detection kit. A standard curve was created with the standard concentration on the vertical axis and the OD value on the horizontal axis, and the levels of the indicators measured in each serum sample were calculated.

[0033] 9. Detection of fertility: After 7 days of modeling, the remaining mice in each group were placed in cages at 8am every night according to a male-to-female ratio of 1:2. Vaginal smears were examined at 8am the following day, and the first day of pregnancy was marked when sperm were observed under a microscope. The pregnancy status of the mice was observed, the number of pregnant mice in each group was recorded, and the pregnant mice were sacrificed on day 14 of pregnancy, with the number of embryos recorded.

[0034] 10.Statistical analysis: The data of this invention is mean number ± standard deviation (x - The results are expressed as ±s, quantitatively analyzed using Image J and Image Pro-Plus, and statistically analyzed and graphed using GraphPad Prism 8.0. Data comparisons between multiple groups are analyzed using one-way ANOVA and multiple comparison tests.

[0035] result: Figure 1 shows the reparative effect of a mixture of injectable collagen peptide and sodium alginate on intrauterine adhesion tissue.

[0036] In Figure 1, mouse uteruses were treated with a mixture of injectable collagen peptide and sodium alginate (CP+SA treatment group) after intrauterine adhesion modeling. The reparative effect of the mixture of injectable collagen peptide and sodium alginate on the endometrium was detected by HE staining.

[0037] Compared to the model group, the results show a significant improvement in the degree of endometrial damage in the CP+SA treatment group, with increased endometrial thickness, reduced inflammatory cell infiltration, and a corresponding increase in the number of endometrial glands compared to before.

[0038] Figure 2 shows the anti-fibrotic effect of a mixture of injectable collagen peptide and sodium alginate in intrauterine adhesion tissue.

[0039] Mouse uteruses were treated with a mixture of injectable collagen peptide and sodium alginate (CP+SA treatment group) after modeling intrauterine adhesions. The improvement effect of the mixture on endometrial fibrosis was detected by modified Masson trichrome staining.

[0040] Compared to the model group, the results show that the CP+SA treatment group showed a significant improvement in the degree of endometrial damage and a reduction in the degree of endometrial fibrosis.

[0041] Figure 3 shows the effect of a mixture of injectable collagen peptide and sodium alginate on NLRP3 inflammasome activation in intrauterine adhesion tissue.

[0042] Mouse uteruses were treated with a mixture of injectable collagen peptide and sodium alginate (CP+SA treatment group) after modeling intrauterine adhesions. Seven days after modeling, uterine tissue was collected, proteins were extracted, and Western blotting was performed.

[0043] Part A of Figure 3 shows the effect of a mixture of injectable collagen peptide and sodium alginate on the NLRP3 content in intrauterine adhesion tissue.

[0044] Part B of Figure 3 shows the effect of a mixture of injectable collagen peptide and sodium alginate on the ASC content in intrauterine adhesion tissue.

[0045] Part C in Figure 3 shows the effect of a mixture of injectable collagen peptide and sodium alginate on the caspase-1 content in intrauterine adhesion tissue.

[0046] Part D in Figure 3 shows the effect of a mixture of injectable collagen peptide and sodium alginate on the IL-1β content in intrauterine adhesion tissue.

[0047] Section E of Figure 3 shows the effect of a mixture of injectable collagen peptide and sodium alginate on the IL-18 content in intrauterine adhesion tissue.

[0048] Compared to the model group, the results show that CP+SA combination therapy can reduce the expression of NLRP3, ASC, caspase-1, IL-1β, and IL-18 proteins in the uterine tissue of mice with intrauterine adhesions, suggesting that CP+SA combination therapy may alleviate uterine inflammation and fibrosis by inhibiting the NLRP3 inflammasome pathway.

[0049] Figure 4 shows the effect of a mixture of injectable collagen peptide and sodium alginate on inflammatory factor levels in the serum of mice with intrauterine adhesions.

[0050] Mouse uteruses were treated with a mixture of injectable collagen peptide and sodium alginate (CP+SA treatment group) after modeling for intrauterine adhesions. Seven days after modeling, mouse serum was collected and ELISA detection was performed.

[0051] Part A of Figure 4 shows the effect of a mixture of injectable collagen peptide and sodium alginate on the IL-1β content in the serum of mice with intrauterine adhesions.

[0052] Part B of Figure 4 shows the effect of a mixture of injectable collagen peptide and sodium alginate on the IL-18 content in the serum of mice with intrauterine adhesions.

[0053] Compared to the model group, the results show a significant decrease in serum levels of IL-1β and IL-18 in the CP+SA treatment group.

[0054] Figure 5 shows the effect of a mixture of injectable collagen peptide and sodium alginate on the reproductive capacity of mice with intrauterine adhesions.

[0055] Mouse uteruses were treated with a mixture of injectable collagen peptide and sodium alginate (CP+SA treatment group) after intrauterine adhesion modeling. Seven days after modeling, male and female mice were placed in cages at 8am every night according to a 1:2 male-to-female ratio. Vaginal smears were examined at 8am the following day, and the first day of pregnancy was marked when sperm were observed under a microscope. The pregnancy status of the mice was observed, the number of pregnant mice in each group was recorded, and the pregnant mice were sacrificed on day 14 of pregnancy, with the number of embryos recorded.

[0056] Part A of Figure 4 shows the effect of a mixture of injectable collagen peptide and sodium alginate on the number of pregnancies in mice with intrauterine adhesions.

[0057] Part B of Figure 4 shows the effect of a mixture of injectable collagen peptide and sodium alginate on the number of embryos after pregnancy in mice with intrauterine adhesions.

[0058] Compared to the model group, the results showed a significant increase in the number of pregnancies and embryos after pregnancy in the CP+SA treatment group, suggesting that the combination of CP and SA can effectively improve the reproductive capacity of mice.

[0059] <Example 2> The present invention will be further described below with reference to Figures 6 to 9.

[0060] This invention provides the application of NLRP3 gene knockout and / or NLRP3 inhibitor MCC950 in the prevention and / or treatment of intrauterine adhesions mediated by the NLRP3 inflammasome.

[0061] In specific examples, prevention and / or treatment are targeted prevention and / or targeted therapy.

[0062] Experimental method: 1. Animals and reagents: All animal experiments have passed logical review and approval by Xinxiang Medical University. C57 / BL6N mice were purchased from the Animal Experiment Center of Henan SCBS Biotechnology Co., Ltd., license number SCXK(Henan)2020-0005. Gene knockout mice were provided by Professor Wang Xianwei's team at Xinxiang Medical University, and the NLRP3 inhibitor MCC950 was purchased from GLPBIO. Other materials and reagents were purchased from companies such as Beyotime, Solarbio, Proteintech, Abcam, or are listed separately.

[0063] 2. Establishment of a mouse IUA model: This model is constructed to create a stable and effective mouse IUA model based on the premise that 95% ethanol acts on the endometrium to cause chemical damage, inflammatory response, and fibrosis of the endometrium. Mice with a normal estrous cycle are selected, and the model is created during the mouse's estrous period. The operating steps are as follows: Anesthesia is induced using a small animal respiratory anesthesia machine, and the mouse is given 2-3% isoflurane anesthetic to induce anesthesia. The mouse is then placed on a small animal operating table, and anesthesia is maintained by continuously inhaling 1.5-2.5% isoflurane. Subsequently, the cervix is ​​explored using a gastric tube feeding needle attached to a syringe, and 95% ethanol is injected into the cervix until ethanol is observed to flow out of the vaginal opening. After maintaining this for 1 minute, 2 ml of 0.9% saline is injected into the cervix to flush out any remaining ethanol from the uterine cavity.

[0064] 3. Grouping and processing of experimental animals: (1) 32 female C57 / BL6N mice were randomly divided into four groups: a sham surgery group, an IUA model group, and an NLRP3 inhibitor MCC950 group, each containing 8 mice, as well as an NLRP3 gene knockout (NLRP3-KO) group, also containing 8 mice. In the model group, an IUA model was prepared by inducing endometrial damage by injecting 95% ethanol into the cervix and uterine cavity of the mice according to the method described above. In the sham surgery group, 0.9% saline was injected into the uterine cavity through the cervix of the mice. In the NLRP3 inhibitor MCC950 group, 95% alcohol was injected into the cervix of the mice and held for 1 minute to damage the endometrium, after which the remaining alcohol in the uterine cavity was rinsed with saline, and the mice were reared normally. On the day of modeling, the NLRP3 inhibitor MCC950 was injected intraperitoneally at a dose of 10 mg / kg mouse body weight, and samples were collected after 7 days of continuous injection. In the NLRP3-KO group, 95% alcohol was injected into the cervix of the mice and held for 1 minute to damage the endometrium. After that, the remaining alcohol in the uterine cavity was rinsed with saline, and the mice were simply reared normally. Samples were collected 7 days after modeling.

[0065] 4. Sample collection and processing: Seven days after modeling, mice were collected from each group, anesthetized with 2-3% isoflurane inhalation, blood was collected from the eyes, centrifuged, and the upper serum was collected and cryopreserved for use in ELISA testing. Uterine tissue was collected, and a portion of the tissue was stored at -80°C for use in measuring protein content. A portion of the tissue was fixed with a freshly prepared 4% paraformaldehyde solution, and conventional paraffin-embedded sections were used for subsequent histomorphological staining and modified Masson trichrome staining.

[0066] 5. Hematoxylin-eosin (HE) staining for observing morphological changes in the endometrium: Uterine tissue sections are regularly dewaxed with water, stained with hematoxylin for 5 minutes, rinsed with running tap water for about 10 minutes, stained with eosin for 1 minute, dehydrated to clear, mounted on neutral gum, and then the pathological morphological changes of the endometrium are observed under a microscope, photographs are taken under 10×4x and 10×20x microscopes, and the number of endometrial glands is counted using ImageJ software.

[0067] 6. Modified Masson trichrome staining to observe the degree of endometrial fibrosis: Uterine tissue sections were regularly dewaxed with water, mordanted with Bouin's solution for 2 hours, stained with celestite blue for 2 minutes, stained with Meyer hematoxylin for 2 minutes, separated with acidic ethanol fractionation solution for 10 seconds, treated with phosphomolybdic acid solution for 10 minutes, stained with aniline blue for 5 minutes, treated with weak acid for 2 minutes, dehydrated to clear, sealed with neutral gum, observed the degree of endometrial fibrosis under a microscope, photographs were taken under 10×4x and 10×20x microscopes, and the degree of endometrial fibrosis in each group was analyzed and calculated using ImageJ software.

[0068] 7. Western blot isonoassay to detect the expression status of NLRP3, ASC, caspase-1, IL-1β, and IL-18 proteins in the uterine tissue of mice from each group: After homogenizing the uterine tissue of mice from each group, proteins were extracted and their concentrations measured. Gel preparation, sample loading, electrophoresis, electroporation, and blocking were then performed in sequence. The corresponding primary and secondary antibodies were incubated, the membranes were washed, and the samples were developed using the Azure electrochemiluminescence imaging system. The gray values ​​of the target and reference proteins were then analyzed and calculated using Image-Pro Plus software.

[0069] 8. Enzyme-linked immunosorbent assay (ELISA) to detect serum IL-1β and IL-18 content in mice from each group: Serum samples were collected from each group of mice, and the IL-1β and IL-18 content in each group's mouse serum was detected strictly according to the operating procedure of the ELISA detection kit. A standard curve was created with the standard concentration on the vertical axis and the OD value on the horizontal axis, and the levels of the indicators measured in each serum sample were calculated.

[0070] 9.Statistical analysis: The data in this study are expressed as mean number ± standard error, quantitatively analyzed using Image J and Image Pro-Plus, and statistically analyzed and graphed using GraphPad Prism 8.0. Data comparisons between multiple groups are analyzed using one-way ANOVA and multiple comparison tests.

[0071] 10.Results: Figure 6 shows the repair effect of NLRP3 gene knockout and / or NLRP3 inhibitor MCC950 on intrauterine adhesion tissue. In Figure 6, mouse uteruses were treated with NLRP3 gene knockout and / or NLRP3 inhibitor MCC950 after intrauterine adhesion modeling, and the repair effect of a mixture of injectable collagen peptide and sodium alginate on the endometrium was detected by HE staining.

[0072] The results show that, compared to the sham surgery group, the endometrium in the intrauterine adhesion model group was thinned and destroyed, the intrauterine epithelium disappeared, and the endometrial glands decreased. Compared to the model group, after the application of NLRP3 gene knockout and / or the NLRP3 inhibitor MCC950, the degree of endometrial damage was significantly improved, the thickness of the endometrium increased compared to before, inflammatory cell infiltration decreased, and the number of endometrial glands also increased correspondingly.

[0073] Figure 7 shows the anti-fibrotic effects of NLRP3 gene knockout and / or the NLRP3 inhibitor MCC950 in intrauterine adhesion tissue. After treating mouse intrauterine adhesions with NLRP3 gene knockout and / or the NLRP3 inhibitor MCC950, the ameliorative effect of a mixture of injectable collagen peptide and sodium alginate on endometrial fibrosis was detected by modified Masson trichrome staining.

[0074] The results show that, compared to the model group, the NLRP3 gene knockout and / or NLRP3 inhibitor MCC950 groups exhibited significantly improved endometrial damage and reduced endometrial fibrosis.

[0075] Figure 8 shows the effect of NLRP3 gene knockout and / or NLRP3 inhibitor MCC950 on NLRP3 inflammasome activation in intrauterine adhesion tissue. After treating mouse intrauterine adhesions with NLRP3 gene knockout and / or NLRP3 inhibitor MCC950, uterine tissue was collected, proteins were extracted, and Western blotting was performed 7 days after modeling.

[0076] Part A of Figure 8 shows the effect of NLRP3 gene knockout and / or the NLRP3 inhibitor MCC950 on NLRP3 content in intrauterine adhesion tissue.

[0077] Part B of Figure 8 shows the effect of NLRP3 gene knockout and / or the NLRP3 inhibitor MCC950 on ASC content in intrauterine adhesion tissue.

[0078] Part C of Figure 8 shows the effect of NLRP3 gene knockout and / or the NLRP3 inhibitor MCC950 on caspase-1 content in intrauterine adhesion tissue.

[0079] Part D of Figure 8 shows the effect of NLRP3 gene knockout and / or the NLRP3 inhibitor MCC950 on IL-1β content in intrauterine adhesion tissue.

[0080] Part E of Figure 8 shows the effect of NLRP3 gene knockout and / or the NLRP3 inhibitor MCC950 on IL-18 content in intrauterine adhesion tissue.

[0081] The results show that, compared to the sham surgery group, the expression levels of NLRP3, ASC, caspase-1, IL-1β, and IL-18 in mouse uterine tissue were significantly increased in the intrauterine adhesion group. Compared to the model group, treatment with NLRP3 gene knockout and / or the NLRP3 inhibitor MCC950 can reduce the protein expression of NLRP3, ASC, caspase-1, IL-1β, and IL-18 in the uterine tissue of mice with intrauterine adhesions. This suggests that NLRP3 gene knockout and / or the NLRP3 inhibitor MCC950 may reduce uterine inflammation and fibrosis by inhibiting the NLRP3 inflammasome pathway.

[0082] Figure 9 shows the effects of NLRP3 gene knockout and / or NLRP3 inhibitor MCC950 on inflammatory factor levels in the serum of mice with intrauterine adhesions. After modeling intrauterine adhesions in mouse uteruses, mice were treated with NLRP3 gene knockout and / or the NLRP3 inhibitor MCC950. Seven days after modeling, mouse serum was collected and ELISA detection was performed.

[0083] Part A of Figure 9 shows the effect of NLRP3 gene knockout and / or the NLRP3 inhibitor MCC950 on IL-1β content in the serum of mice with intrauterine adhesions.

[0084] Part B of Figure 9 shows the effect of NLRP3 gene knockout and / or the NLRP3 inhibitor MCC950 on IL-18 content in serum from mice with intrauterine adhesions.

[0085] Part C of Figure 9 shows the effect of NLRP3 gene knockout and / or the NLRP3 inhibitor MCC950 on TNF-α content in serum from mice with intrauterine adhesions.

[0086] The results show that, compared to the sham surgery group, levels of inflammatory factors IL-1β, IL-18, and TNF-α were significantly increased in the intrauterine adhesion model group, and compared to the model group, serum levels of IL-1β, IL-18, and TNF-α were significantly decreased in the NLRP3 gene knockout and / or NLRP3 inhibitor MCC950 group.

[0087] Although the present invention has been described illustratively with reference to the attached drawings, it is clear that the implementation of the present invention is not limited to the above-described methods. As long as various improvements are made to the method concept and technical solutions of the present invention, or as long as the concept and technical solutions of the present invention are directly applied to other situations without improvement, they all fall within the scope of the protection of the present invention.

Claims

1. A composition for preventing and / or treating intrauterine adhesions, the composition comprising a collagen peptide and sodium alginate, The composition is used to prevent and / or treat intrauterine adhesions by inhibiting the activation of the NLRP3 inflammasome associated with intrauterine adhesions, thereby reducing the protein expression of NLRP3 and ASC, as well as the downstream inflammatory factors caspase-1, IL-1β, and IL-18, and by reducing the activation and release of NLRP3 and ASC, as well as the downstream inflammatory factors caspase-1, IL-1β, and IL-18. A composition characterized by the following features.

2. The composition according to claim 1, characterized in that the mass ratio of collagen peptide to sodium alginate is 1 to 10:

1.

3. The composition is characterized in that the mass ratio of collagen peptide to sodium alginate is 2:

1. The composition according to claim 2.

4. An injectable drug for preventing and / or treating intrauterine adhesions, characterized in that it contains the composition described in any one of claims 1 to 3 as an active ingredient.

5. A method for preparing a composition used to prevent and / or treat intrauterine adhesions, Step S1 involves adding sterile water to collagen peptide powder and stirring uniformly to obtain a collagen peptide solution, Step S2 involves adding sodium alginate powder to the collagen peptide solution and uniformly stirring so that the mass ratio of collagen peptide to sodium alginate is 1 to 10:1 to obtain an injectable composition. A preparation method characterized by including the following.

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