Application of CPA4 in relieving endometrial fibrosis and hAMSCs and IUA co-culture method

Abstract

The invention discloses application of CPA4 in the aspect of relieving endometrial fibrosis and an hAMSCs and IUA co-culture method, and solves the problems that a research basis for developing other treatment methods for intrauterine adhesion is lacked at present, and limitation caused by treatment of intrauterine adhesion by stem cells cannot be overcome. The invention is realized through the following technical scheme: application of CPA4 in the aspect of relieving endometrial fibrosis and application of CPA4 in preparation of products for relieving endometrial fibrosis. The application has the advantages that the effect of CPA4 on relieving endometrial fibrosis is defined, and limitation caused by treatment of intrauterine adhesion by stem cells is overcome.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to the application of CPA4 in reducing endometrial fibrosis and a method for co-culturing hAMSCs and IUA. Background Technology

[0002] Intrauterine adhesions (IUA) are a pathological condition characterized by endometrial fibrosis and adhesion formation, often leading to amenorrhea, decreased menstrual flow, infertility, and recurrent miscarriages. It is usually caused by endometrial trauma resulting from repeated curettage, intrauterine surgery, and infection. Current treatments, such as hysteroscopic adhesiolysis combined with hormone therapy, have limited effectiveness, exhibiting high recurrence rates and insufficient endometrial regeneration. Therefore, innovative treatment strategies are urgently needed to improve the degree of endometrial fibrosis, thereby restoring endometrial structure and function, and ultimately improving women's health and fertility.

[0003] In recent years, stem cell-based therapies have emerged as a promising strategy for treating intrauterine uterine atrophy (IUA), showing significant potential to aid in endometrial repair. However, despite these encouraging findings, stem cell therapy still faces numerous challenges, such as limited stem cell sources, the possibility of immune rejection, ethical concerns, and the risk that uncontrolled proliferation may lead to tumors.

[0004] Therefore, it is necessary to study other treatment methods besides stem cell therapy, and to study cell culture systems and mechanisms of action based on these treatment methods, so as to provide a solid foundation for the final treatment method. Summary of the Invention

[0005] The technical problem to be solved by this invention is that there is a lack of research foundation for developing other treatment methods for intrauterine adhesions, and the limitations of stem cell therapy for intrauterine adhesions cannot be overcome.

[0006] This invention is achieved through the following technical solution:

[0007] Applications of CPA4 in reducing endometrial fibrosis; application of CPA4 in the preparation of products that reduce endometrial fibrosis.

[0008] Preferably, CPA4 is added directly to the cells or injected into the uterus.

[0009] Furthermore, the effective concentration of CPA4 is 10-50 ng / mL.

[0010] Furthermore, CPA4 exerts its anti-fibrotic effect by regulating FoxO1 activity.

[0011] The method for co-culturing hAMSCs with IUA cell models includes the following steps:

[0012] Step 1: Isolation and culture of hAMSCs;

[0013] Step 2: IUA cells were generated by treating THESCs with a combination of inflammatory and fibroblastic cytokines to mimic the pathological environment of IUA;

[0014] Step 3: hAMSCs and IUA cells are co-cultured in a transporous system to exchange secreted factors between the two cell types;

[0015] Step 4: After co-culture, the supernatant was collected for further analysis to determine the application of CPA4 in reducing endometrial fibrosis.

[0016] Furthermore, this study also included the use of synthetic siRNA to knock down CPA4 in hAMSCs to compare the effects of CPA4 on endometrial fibrosis.

[0017] Furthermore, the co-cultivation time is 24-72 hours.

[0018] The present invention has the following advantages and beneficial effects:

[0019] 1. This invention reveals the anti-fibrotic effect of CPA4 in the IUA model, and is based on two anti-fibrotic mechanisms: (1) CPA4 qualitatively inhibits the synthesis and secretion of fibrosis-related proteins in endometrial stromal cells, reducing the extracellular matrix components; (2) CPA4 effectively inhibits the proliferation of endometrial fibroblasts and promotes their apoptosis, thereby reducing the amount of endometrial matrix and limiting the deposition of extracellular matrix.

[0020] 2. This invention verifies the paracrine mechanism of CPA4, its safety in normal endometrial tissue, and its regulation of FOXO1 signaling. Attached Figure Description

[0021] The accompanying drawings, which are included to provide a further understanding of embodiments of the invention and form part of this application, do not constitute a limitation thereof. In the drawings:

[0022] Figure 1 The expression of ECM components was significantly altered in patients with IUA.

[0023] A) Levels of FN and type I, III, IV, and V collagen were detected by IHC, scale bar =

[0024] 100 μm. B) The matrix metalloproteinases MMP2 and MMP9 and their natural inhibitors were evaluated by IHC.

[0025] Formulation TIMP1

[0026] Expression levels of TIMP2, scale bar = 100 μm.

[0027] C) The expression level of Ki67 in endometrial tissue of IUA patients was detected by immunofluorescence (IF), scale bar = 100 μm. Data are expressed as mean ± standard deviation, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, NS indicates no statistical significance.

[0028] Figure 2 The image shows the proteomic analysis of three groups of supernatants, among which...

[0029] A heatmap shows the range of differentially expressed proteins among the groups. The Venn plot shows the number of overlapping proteins (16) and unique proteins in the differentially expressed protein datasets for each group. The top 10 proteins with the highest expression levels were identified by PCR (n=3).

[0030] B) Both PCR and WB analyses confirmed the presence of the highest levels of CPA4 mRNA and protein in hAMSCs and their supernatant (n=3). Data represent mean ± standard deviation.

[0031] *p<0.05,**p<0.01,***p<0.001,****p<0.0001, NS indicates no statistical significance.

[0032] Figure 3 The results showed that CPA4 significantly reduced the expression of fibrosis-related genes in IUA-THESCs.

[0033] A) The effective concentration and time of CPA4 treatment were determined by PCR and WB (n=3);

[0034] B) A CPA4 knockdown hAMSCs model (hAMSCssi-CPA4) was created using specific siRNA, and the changes in antifibrotic effect before and after CPA4 knockdown were evaluated by PCR and WB (n=3).

[0035] C) CPA4 was knocked down in the IUA-THESCs cell model to eliminate the effect of endogenous CPA4 (n=3). Data are expressed as mean ± standard deviation. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, NS indicates no statistical significance.

[0036] Figure 4 The results show that the effective concentration of CPA4 does not affect the biological function of normal THESCs.

[0037] A) The expression levels of molecules related to proliferation (PCNA), cell cycle (CDK2, Cyclin D1) and apoptosis (Bcl2, Bax) in THESCs were detected by qPCR and WB (n=3);

[0038] B) The effects of CPA4 on the proliferation and cell cycle distribution of THESCs were evaluated using Edu assays and flow cytometry, scale bar = 200 μm, (n = 3);

[0039] C) The effect of CPA4 on apoptosis in THESCs was analyzed by flow cytometry (n=3). Data are expressed as mean ± standard deviation. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, and NS indicates no statistical significance.

[0040] Figure 5 The CPA4 protein secreted by hAMSCs is internalized by IUA-THESCs via endocytosis to exert its effects.

[0041] A) Western blot analysis was performed to assess the expression level of the Flag-marked CPA4 in hAMSCs and the expression level in co-cultured IUA-THESCs (n=3).

[0042] B) The expression of Flag-CPA4 in IUA-THESCs was tracked by IF before and after co-culturing with hAMSCs, scale bar = 100 μm, (n = 3);

[0043] C) Use 488 marked CPA4 and tracked its trajectory into IUA-THESCs via IF, scale bar = 200 μm, (n = 3);

[0044] D) The uptake of CPA4 was inhibited using the endocytosis inhibitor Pitstop to assess changes in protein effects (n=3). Data are presented as mean ± standard deviation. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, NS indicates no statistical significance.

[0045] Figure 6 The results showed that CPA4 inhibited the proliferation of IUA-THESCs and promoted their apoptosis.

[0046] A) The changes in gene expression related to proliferation (PCNA), cell cycle distribution (CDK2, Cyclin D1) and apoptosis (Bcl2, Bax, cleaved caspase 3) in IUA-THESCs before and after CPA4 intervention were detected by qPCR and WB (n=3).

[0047] B) Edu assay and flow cytometry were used to assess the proliferation capacity and cell cycle distribution of IUA-THESCs before and after CPA4 intervention, scale bar = 50 μm, (n = 3).

[0048] C) Flow cytometry is also used to assess the apoptosis of IUA-THESCs before and after CPA4 intervention.

[0049] (n=3). Data are expressed as mean ± standard deviation, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, NS indicates no statistical significance.

[0050] Figure 7 The results showed that CPA4 enhanced FOXO1 expression in IUA-THESCs and promoted the dephosphorylation of p-Smad protein.

[0051] A) Heatmaps were used to assess changes in gene expression in IUA-THESCs before and after intervention. KEGG enrichment analysis showed that CPA4 mainly affects specific signaling pathways.

[0052] B) and C) The expression changes of FOXO1 and FoxO3 related molecules in the FOXO signaling pathway and Smad2 / 3 / 4 related molecules in the Samd signaling pathway were detected by qPCR and WB (n=3);

[0053] D) The cytoplasmic and nuclear localization of FOXO1 cells in IUA-THESCs after CPA4 intervention was detected by IF. Scale bar = 100 μm, (n = 3). Data are expressed as mean ± standard deviation. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, NS indicates no statistical significance.

[0054] Figure 8 CPA4 was shown to reduce endometrial fibrosis by regulating the FoxO1 / Smad axis.

[0055] A) Use Western blotting (WB) to assess the effective concentration of the FoxO1 inhibitor (AS1842856) (n=3);

[0056] B) The expression levels of fibrosis-related molecules such as FN, type I collagen, type III collagen and p-Smad2 / 3 in IUA-THESCs cells before and after AS1842856 intervention were analyzed by qPCR and WB (n=3).

[0057] C) The changes in the expression levels of proliferation, cell cycle and apoptosis-related molecules in IUA-THESCs cells were assessed by qPCR and WB (n=3);

[0058] D) Co-immunoprecipitation (Co-IP) was performed to confirm the interaction between Smad2 / 3 and FoxO1 after CPA4 treatment (n=3). Data are expressed as mean ± standard deviation, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. NS indicates no statistical significance.

[0059] Figure 9 CPA4 demonstrated effectiveness in treating IUA rats.

[0060] A) After injection of CPA4, the level of fibrotic molecules in the endometrial tissue of IUA rats was detected by Western blotting (n=3);

[0061] B) Hematoxylin and eosin (HE) staining and Masson staining were used to assess changes in endometrial thickness, glandular number, and fibrotic area after CPA4 treatment; changes in the expression of endometrial epithelial marker CK19 and fibrotic molecules type I collagen and FN were detected by immunofluorescence, scale bar = 100 μm, (n = 3).

[0062] C) CPA4 knockdown human amniotic mesenchymal stem cells were injected into the uterus of IUA rats. Changes in endometrial thickness, glandular number, and fibrotic area were assessed using HE and Masson staining. Simultaneously, changes in the expression of CK19 and the fibrotic molecules type I collagen and FN were detected by immunofluorescence. Scale bar = 100 μm, (n = 3). Data are expressed as mean ± standard deviation. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. NS indicates no statistical significance.

[0063] Figure 10 The study showed that CPA4 reduced endometrial fibrosis in IUA rats by regulating FoxO1.

[0064] A) After injecting AS1842856 into the uterus of healthy rats, the effect of AS1842856 on normal endometrial tissue was assessed by HE staining and Masson staining. At the same time, IHC was used to detect the expression level of fibrosis markers in endometrial tissue. Scale bar = 200 μm, (n = 3).

[0065] B) CPA4 and AS1842856 were simultaneously injected into the uterus of IUA rats, and the changes in the expression of fibrosis molecules and epithelial markers were analyzed by IF. Scale bar = 100 μm, (n = 3). Data are expressed as mean ± standard deviation. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, NS indicates no statistical significance.

[0066] Figure 11This is a schematic diagram of the co-culture chamber of the present invention. Detailed Implementation

[0067] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the embodiments and accompanying drawings. The illustrative embodiments and descriptions of the present invention are only used to explain the present invention and are not intended to limit the present invention.

[0068] The following description is provided for the embodiments:

[0069] Antibodies and reagents

[0070] (1) Purchased from Wuhan Sanying Biotechnology Co., Ltd. (Proteintech): CPA4 (26824-1-AP), cytokeratin 19 (10712-1-AP), fibronectin (15613-1-AP), FoxO1 (18592-1-AP), phosphorylated FoxO1 (28757-1-AP), MMP2 (10373-2-AP), MMP9 (10375-2-AP), TIMP1 (16644-1-AP), TIMP2 (17353-1-AP), Ki67 (27309-1-AP), type I collagen (67288-1-Ig), type III collagen (22734-1-AP), CDK2 (10122-1-AP).

[0071] (2) Purchased from Cell Signaling Technology: SMAD2 / 3 (#3102), phosphorylated SMAD2 (Ser465 / 467) / SMAD3 (Ser423 / 425) (#8828) and type IV collagen (#44204).

[0072] (3) Purchased from ABmart: Bcl2 (T40056), bax (T40051), and cleaved Caspase 3 (TA7022).

[0073] (4) Purchased from ABclonal: PCNA (A12427) and Cyclin D1 (A1301).

[0074] (5) Purchased from Solarbio: Recombinant human CPA4 protein (P05406).

[0075] (6) Purchased from Selleck: AS1842856 (S8222) and Pitstop2 (S9670).

[0076] (7) Purchased from Homeland Bio: FBS, model GL1001.

[0077] Example 1

[0078] Construction of a co-culture system for hAMSCs and IUA cells

[0079] Step 1: hAMSCs were isolated and cultured according to the previously established method;

[0080] Specifically, the experiment used DMEM / F12 medium containing fetal bovine serum, purchased from Homeland Bio, catalog number GL1001, Nanjing, China.

[0081] Step 2: IUA cells were generated by treating THESCs with a combination of inflammatory and fibroblastic cytokines to mimic the pathological environment of IUA.

[0082] Step 3: hAMSCs and IUA cells are co-cultured in a transpore system to exchange secretory factors between the two cell types while maintaining physical separation;

[0083] After the co-culture system was maintained for 48 hours, the supernatant was collected for proteomics analysis to identify key bioactive proteins secreted by hAMSCs.

[0084] The specific methods for co-cultivation are as follows:

[0085] (1) Item preparation

[0086] Two 50ml centrifuge tubes (containing 20-30ml PBS and 0.5ml-1ml of penicillin-dextrose antibody); gloves; a styrofoam box filled with ice.

[0087] (2) Specific steps

[0088] a. Go to the obstetrics operating room to find a suitable case (excluding hypertension, diabetes, and infectious diseases), obtain the placenta, peel off the amnion from the fetal side, remove blood and mucus, put the tissue into a pre-prepared centrifuge tube, tighten the cap, put it into a foam box filled with ice, and bring it back to the laboratory.

[0089] b. Pack the ice cubes in a new foam box for easy handling on the ice.

[0090] Pour the tissue into a kidney dish, wash repeatedly with low-temperature PBS to remove blood and mucus, then cut the amnion into small pieces, aliquot into 15ml centrifuge tubes, add 1:1 trypsin and digest for 15 minutes (incubate at 37 degrees Celsius), shaking well during the process, centrifuge for 5-10 minutes at 1200-1500 RPM, discard the supernatant, add trypsin again, centrifuge, and repeat this process 4 times.

[0091] Add DMEM / F12 medium with a serum concentration of 10% in volume equal to the added trypsin to stop the trypsin reaction. Shake well, centrifuge again, discard the supernatant, transfer the tissue to a 50ml centrifuge tube, add 1:1.5 collagenase for 1 hour (shake well every 15 minutes), filter once through a sieve, collect the filtrate, centrifuge again as above, discard the supernatant, resuspend the cells in 10% DMEM / F12 medium, and complete the plate culture.

[0092] c. Stem cell culture conditions: sterile incubator at 37 degrees Celsius, 5% CO2 concentration.

[0093] d. Stem cell culture medium formula: DMEM / F12 basal medium, 5%-10% fetal bovine serum, 1% streptomycin / penicillin.

[0094] e. Co-culture procedure: Using a co-culture chamber, add amniotic mesenchymal stem cells to the upper chamber and IUA cells to the lower culture dish, such as... Figure 11 As shown, all cells were cultured in a sterile incubator at 37 degrees Celsius in DMEM / F12 medium containing 5% CO2 and 10% serum. Because the bottom of the upper chamber has a water-permeable membrane, small molecules can pass through, but cells cannot, thus achieving molecular exchange between the two cell types while maintaining physical cell isolation. However, sufficient cytokine exchange cannot be guaranteed, but the opportunity for exchange can be increased by extending the co-culture time.

[0095] Comparative Example 1

[0096] The supernatant of hAMSCs was collected as control experimental group 1;

[0097] Comparative Example 2

[0098] Collect the supernatant of individual IUA-THESCs as control experimental group 2;

[0099] Example 2

[0100] The methods for determining the use of CPA4 in reducing endometrial fibrosis include:

[0101] 1. Analysis of major components accumulated in the ECM of IUA patients

[0102] The extracellular matrix (ECM) provides structural support and biochemical signaling for cells, normally maintaining homeostasis. However, in the presence of fibrosis, this balance is disrupted, leading to excessive ECM deposition. To identify key components of ECM accumulation in patients with intrauterine fibrosis (IUA), this invention collected endometrial samples from six IUA patients at the University City Hospital Affiliated to Chongqing Medical University. Immunohistochemistry (IHC) was used to detect fibronectin (FN), type I collagen, type III collagen, type IV collagen, and type V collagen, which are the main components of typical endometrial ECM. The results showed no statistically significant difference in type IV collagen between the healthy control group (NC) and the IUA group. However, significant differences were observed in FN, type I collagen, and type III collagen, with FN and type I collagen showing significant variation. Furthermore, this invention evaluated Ki67, a biomarker indicating endometrial proliferation. The results showed that, compared with normal subjects, IUA patients had significantly reduced endometrial epithelial cell proliferation, suggesting that fibrosis may severely hinder endometrial regeneration and repair, such as… Figure 1 As shown in A and C.

[0103] To further clarify whether ECM accumulation in IUA patients is due to excessive ECM production or reduced degradation, this invention evaluated the expression levels of matrix-degrading enzymes MMP2 and MMP9, as well as their natural inhibitors TIMP1 and TIMP2. The results showed no significant difference between IUA patients and healthy controls. This suggests that excessive ECM production, rather than reduced degradation, is the cause of endometrial fibrosis. In subsequent experiments, we focused on strategies to reduce the production of extracellular matrix components, such as… Figure 1 As shown in B.

[0104] 2. Proteomics analysis

[0105] Proteomics analysis of the supernatant collected from the co-culture system includes the following steps:

[0106] Proteins were extracted and digested with trypsin, followed by peptide labeling. Proteins in the samples were identified and quantified using liquid chromatography-tandem mass spectrometry (LC-MS / MS). Data were analyzed using bioinformatics tools to identify differentially expressed proteins of different secretory factors that may be involved in IUA treatment. The results are shown below. Figure 2 As shown.

[0107] The results showed that a series of secreted proteins were found, exhibiting significant differences among the three analyzed groups (p < 0.05 and |log2(fold change)| > 1), such as... Figure 2 The heatmap for A(a) is shown. To further elucidate the characteristics and commonalities of these intergroup secreted proteins, this invention performed a Wien analysis on the intergroup differential protein dataset, as shown... Figure 2The Venn diagram in A(b) shows the number of shared proteins (16) and unique proteins in each group of differentially expressed protein datasets, revealing 16 distinct proteins. Among these 16 representative candidates, the top 10 factors were identified by quantitative PCR (qPCR) and Western blot (WB): CPA4, XYLT1, VCAN, DPP4, LAMC2, KRT8, PAPPA, BGN, RAP1B, and SERPINA3. Figure 2 The PCR identification results of A(c) are shown in the figure.

[0108] The results show that, Figure 2 As shown in Figure B, the differences in CPA4 expression at both the mRNA and protein levels were most pronounced among the groups. Notably, CPA4 levels were significantly higher in hAMSCs compared to IUA-THESCs. Although CPA4 levels increased further in co-cultured hAMSCs, they remained stable in co-cultured IUA-THESCs. Furthermore, Western blot analysis revealed significantly enhanced CPA4 expression in both hAMSCs conditioned medium and co-culture conditioned medium compared to IUA-THESCs conditioned medium. These findings suggest that CPA4 may play a crucial role in the treatment of IUA with hAMSCs, requiring further confirmation.

[0109] 3. CPA4 inhibits the expression of fibrosis-related genes in IUA-THESCs.

[0110] This invention introduced three different concentrations of exogenous recombinant CPA4 protein into an IUA cell model. qPCR and Western blotting were used to monitor the accumulation of FN and type I collagen, the main components in the extracellular matrix of IUA patients. Figure 3 As shown in Figure A, compared to the IUA group, the expression of FN and type I collagen in IUA-THESCs treated with three different concentrations of CPA4 did not decrease after 24 hours. Therefore, the CPA4 exposure time was prolonged. After 48 hours, when treated with a concentration of 50 ng / mL, there was a significant reduction in both FN and type I collagen at the mRNA and protein levels. Therefore, this concentration and exposure time were selected for subsequent experiments.

[0111] This invention uses synthetic siRNA to explicitly knock down CPA4 in hAMSCs. Subsequently, the CPA4-knockdown hAMSCs were co-cultured with IUA-THESCs. The results are as follows... Figure 3 As shown in Figure B, the expression levels of FN and type I collagen were increased in the hAMSCsi-CPA4 group compared to the hAMSCs group. These results indicate that the ability of hAMSCs to alleviate endometrial fibrosis was partially weakened after CPA4 knockdown.

[0112] CPA4 has been confirmed to reduce the expression of genes associated with fibrosis. However, the effect of endogenous CPA4 in IUA-THESCs must be considered. Exogenous CPA4 protein was administered to IUA-THESCs in both the si-CPA4 group and the IUA-THESCs group. Figure 3 As shown in Figure C, compared with the IUA-THESCs group, there were no significant differences in the expression levels of FN and type I collagen in the IUA-THESCs si-CPA4 group at both the protein and mRNA levels. This indicates that endogenous CPA4 in IUA-THESCs has a relatively small effect on reducing fibrosis.

[0113] 4. Safety verification of CPA therapy

[0114] CPA4 protein was added to cells to assess whether exogenous CPA4 protein at a concentration of 50 ng / mL affected the proliferation and apoptosis of normal THECSs. Results are as follows: Figure 4 As shown in Figure A, the mRNA and protein expression levels of key biomarkers related to proliferation, cell cycle regulation, and apoptosis, such as PCNA, Cyclin D1, CDK2, Bcl-2, and Bax, did not show significant differences among the experimental groups.

[0115] The EdU incorporation experiment further confirmed the above results, such as Figure 4 As shown in Figure B, there was no significant difference in the percentage of EdU-positive cells among the groups (p>0.05), indicating that cell proliferation was not affected by CPA4 treatment. Flow cytometry analysis of cell cycle distribution after PI staining showed no significant difference in the percentages of cells in G0 / G1, S, and G2 / M phases among the groups (p>0.05), indicating that CPA4 treatment did not affect the overall cell cycle progression. Further assessment of apoptosis was performed using Annexin V-FITC / PI staining by flow cytometry. Figure 4 As shown in Figure C, the percentages of early (Annexin V+ / PI-) and late (Annexin V+ / PI+) apoptotic cells in the treatment group were almost not increased compared to the control group (p>0.05), suggesting that CPA4 treatment did not induce apoptosis. These results indicate that a concentration of 50 ng / ml of CPA4 can exert an anti-fibrotic effect without causing functional impairment in THESCs.

[0116] 5. The source and direction verification of CPA4

[0117] hAMSCs OE-CPA4, overexpressing CPA4 with adenovirus, were co-cultured with IUA-THESCs. Figure 5A showed that Flag-CPA4 was present in both hAMSCs and their conditioned medium. These findings confirm that Flag-CPA4 originates from hAMSCs and is secreted into the extracellular space. Furthermore, Flag-CPA4 was also detected in IUA-THESCs after co-culture.

[0118] To further verify the expression of Flag in target cells, an immunofluorescence experiment was performed. The results are as follows: Figure 5 As shown in Figure B, CPA4 expression was positive in IUA-THESCs after co-culture, while no expression was detected in IUA-THESCs cultured alone.

[0119] Pre-use CPA4, marked with 488, handles IUA-THESCs. For example... Figure 5 As shown in C and 5D, positive expression was observed in IUA-THESCs after CPA4 treatment. Furthermore, to determine the key role of endocytosis in promoting CPA4 entry into IUA-THESCs, the endocytosis inhibitor Pitstop2 was used to disrupt the endocytic process in IUA-THESCs. The study found that Pitstop2 significantly counteracted the expected antifibrotic effect of CPA4. Therefore, it can be concluded that CPA4 is secreted by hAMSCs and endocytosed by IUA-THESCs to exert its biological effects.

[0120] 6. Effects of CPA4 on endometrial fibroblasts (IUA-THESCs)

[0121] Endometrial fibroblasts originate from fibroblasts and produce abundant extracellular matrix. Therefore, excessive proliferation of endometrial fibroblasts leads to extracellular matrix accumulation, ultimately causing endometrial fibrosis. Compared to normal THESCs, the expression of PCNA, Cyclin D, CDK2, and Bcl2 was upregulated in the IUA-THESCs group, while the expression of cleaved caspase 3 was significantly reduced. Notably, the levels of these markers partially recovered after CPA4 treatment, such as... Figure 6 As shown in Figure A.

[0122] The IUA group showed a higher proportion of Edu-positive cells (p<0.05), which returned to baseline levels after CPA4 treatment. Figure 6As shown in B and 6C, flow cytometry results revealed a significant decrease in the percentage of cells in the G0 / G1 phase in the IUA group. The percentages of cells in the S and G2 / M phases increased (p<0.05), indicating that more cells were arrested in these phases. In the CPA4 group, the number of cells in the S and G2 / M phases was significantly reduced, while the number of cells in the G0 / G1 phase showed an increasing trend, although this trend was not statistically significant. The proportions of early (Annexin V+ / PI-) and late apoptotic cells (Annexin V+ / PI+) were significantly lower in the IUA group than in the control group (p<0.05), and this was corrected in the CPA4 group. These results collectively suggest that CPA4 can inhibit the excessive proliferation of endometrial fibroblasts and promote their apoptosis in IUA.

[0123] 7. Mechanism of action of CPA4 in IUA-THESCs

[0124] To further investigate the downstream role of CPA4 in IUA treatment, RNA sequencing was performed to analyze changes in mRNA in IUA-THESCs before and after CPA4 treatment. Based on KEGG enrichment, such as... Figure 7 As shown in Figure A, the FoxO1 pathway is the primary pathway. FoxO1 and FoxO3 are key factors in the FoxO1 pathway and may be potential downstream components of CPA4. IUA-THESCs were treated with exogenous CPA4 protein, hAMSCs, and hAMSCssi-CPA4, respectively. Results showed that FoxO1 expression was downregulated in the IUA-THESCs group, but upregulated after treatment with exogenous CPA4 and hAMSCs, partially offsetting the CPA4 knockdown effect. In contrast, FoxO3 showed no significant difference between the THESCs group and IUA-THESCs (p>0.05), but exhibited a consistent trend after CPA4 or hAMSC intervention, such as... Figure 7 As shown in B.

[0125] The TGFβ / Smad pathway has attracted attention due to its induction of endometrial fibrosis in IUA cases and has also shown enrichment in KEGG analysis. To investigate whether CPA4 regulates the TGFβ / Smad pathway at the mRNA transcriptional level, this invention used qPCR to detect Smad2, Smad3, and Smad4. Analysis showed no statistically significant difference in the levels of Smad2, Smad3, or Smad4 between the CPA4 group and the IUA group. Conversely, a significant increase in Smad2 and Smad3 phosphorylation was observed in the IUA group, while this phenomenon significantly decreased after CPA4 intervention. These findings suggest that CPA4 exerts its anti-fibrotic effect primarily by regulating Smad protein phosphorylation rather than changes at the transcriptional level. Figure 7 As shown in A and 7C.

[0126] FoxO1 is a multifunctional transcription factor that primarily functions in the cell nucleus, while also existing in the cytoplasm as an inactive phosphorylated form (p-FoxO1). This invention uses immunofluorescence to assess its distribution and investigate whether FoxO1 is concentrated in the nucleus to achieve its functional purpose. Results showed that, compared to the standard group, nuclear expression of FoxO1 was reduced in the IUA group, while cytoplasmic expression was increased. Conversely, CPA4 treatment increased nuclear expression of FoxO1, while cytoplasmic levels decreased. In summary, CPA4 treatment of IUA-THESCs led to upregulation of FoxO1 levels, correspondingly concentrated in the nucleus, while active p-Smad2 / 3 protein was downregulated. Figure 7 As shown in D.

[0127] 8. Mechanism of CPA4 in reversing endometrial fibrosis

[0128] As an inhibitor of Foxo1, AS1842856 enhanced Foxo1 phosphorylation, which is unusual in atypical cases because phosphorylation usually indicates the active state. The effective doses of AS at three concentrations (50 nM, 100 nM, and 200 nM) in normal THESCs were determined using Western blotting. Results showed that the p-Foxo1 / Foxo1 ratio was significantly increased at 100 nM and 200 nM. Simultaneously, the expression levels of FN and type I collagen were significantly increased at these concentrations, indicating that Foxo1 inhibition begins at 100 nM and promotes fibroblast formation. Further Western blotting analysis showed significantly enhanced Smad2 / 3 phosphorylation in the AS1842856 group compared to the THESCs group. This finding suggests that AS1842856, as a Foxo1 inhibitor, effectively promotes Smad2 / 3 phosphorylation, such as… Figure 8 As shown in Figure A.

[0129] To investigate whether CPA4 reverses endometrial fibrosis by influencing Foxo1, four experimental groups were established: THESCs, IUA-THESCs, CPA4 (with IUA-THESCs treated with CPA4), and AS1842856 (with IUA-THESCs treated with both CPA4 and AS1842856). Results showed that CPA4 effectively reduced fibrosis-related IUA expression, proliferation, cell cycle, and p-Smad / Smad levels, while enhancing apoptosis-related IUA expression. Furthermore, administration of AS1842856 partially counteracted the effects of CPA4, suggesting that CPA4 may reverse endometrial fibrosis through its interaction with Foxo1. Figure 8 As shown in B and 8C.

[0130] To investigate the interaction between FoxO1 and Smad2 / 3 under different conditions, co-immunoprecipitation (Co-IP) experiments were performed on IUA-THESCs before and after CPA4 treatment. In each group, FoxO1 was immunoprecipitated using a specific FoxO1 antibody, and the presence of Smad2 / 3 in the immunoprecipitate was assessed. Similarly, Smad2 / 3 was also immunoprecipitated to confirm the presence of FoxO1. The results showed that the interaction between FoxO1 and Smad2 / 3 was significantly increased after CPA4 treatment compared to the untreated IUA-THESCs group. CPA4 appeared to enhance FoxO1 expression, promote its binding to Smad2 / 3, and further inhibit the phosphorylation level of Smad2 / 3, thereby inhibiting the fibrotic process, such as... Figure 8 As shown in D.

[0131] 9. Efficacy and optimal concentration of exogenous CPA4 in vivo for the treatment of IUA

[0132] This invention conducted an IUA rat model experiment according to previously reported methods. Changes in fibrotic areas, endometrial thickness, and glandular number before and after CPA4 treatment were assessed using Masson and HE staining. Results showed that, as Figure 9 As shown in A and 9B, compared with the IUA group, the 1 μg / mL concentration group showed a significant reduction in fibrosis and improved endometrial thickness, approaching normal levels. Although the number of glands increased in this group, it remained significantly lower than in the sham-operated group, and further increases in concentration did not produce any additional significant effect. Furthermore, the positive expression of type I collagen and FN was reduced. Simultaneously, the CK19 level in the 1 μg / mL group was increased compared to the sham-operated group, but no obvious dose-dependent relationship was observed.

[0133] hAMSCs and hAMSCssi-CPA4 were injected into the uterine cavity of patients with intrauterine fibrosis (IUA). In the hAMSCssi-CPA4 group, the positive immunofluorescence expression of type I collagen and fibroblasts (FN) increased, while the level of cytokeratin 19 decreased. Compared with the hAMSCs group, CPA4 effectively alleviated endometrial fibrosis. Figure 9 As shown in C.

[0134] 10. Research on the mechanism by which CPA4 reduces endometrial fibrosis

[0135] The effect of AS1842856 on promoting endometrial fibrosis was evaluated using the same method. Figure 10The results show that AS can induce fibrosis, which was confirmed by MASSON and IHC analyses, and the effect was only observed at a concentration of 10 μM. When AS1842856 was added to CPA4, fibrosis-related expression was significantly increased compared to CPA4 alone. Conversely, CK19 expression was reduced, indicating that CPA4 effectively treats IUA by modulating Foxo1 in vivo.

[0136] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. The application of CPA4 in reducing endometrial fibrosis, characterized in that, Application of CPA4 in the preparation of products that reduce endometrial fibrosis.

2. The application of CPA4 according to claim 1 in reducing endometrial fibrosis, characterized in that, The effective concentration of CPA4 is 10-50 ng / mL.

3. The application of CPA4 according to claim 1 in reducing endometrial fibrosis, characterized in that, CPA4 exerts its anti-fibrotic effect by regulating FoxO1 activity.

4. A method for co-culturing hAMSCs with an IUA cell model, characterized by: Includes the following steps: Step 1: Isolation and culture of hAMSCs; Step 2: IUA cells were generated by treating THESCs with a combination of inflammatory and fibroblastic cytokines to mimic the pathological environment of IUA; Step 3: hAMSCs and IUA cells are co-cultured in a transporous system to exchange secreted factors between the two cell types; Step 4: After co-culture, collect the supernatant for further analysis to determine the application of CPA4 as described in any one of claims 1-3 in reducing endometrial fibrosis.

5. The method for co-culturing hAMSCs and IUA cell model according to claim 4, characterized in that: It also includes using synthetic siRNA to knock down CPA4 in hAMSCs to compare the effects of CPA4 on endometrial fibrosis.

6. The method for co-culturing hAMSCs and IUA cell model according to claim 4 or 5, characterized in that: The co-culture time is 24-72 hours.

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