TGF-beta1 signal channel inhibitor, method for regulating expression of Fmod by using TGF-beta1 signal channel inhibitor and application of TGF-beta1 signal

Abstract

The invention provides a TGF-beta1 signal channel inhibitor and a method and application thereof for regulating and controlling Fmod expression, the TGF-beta1 signal channel inhibitor is decellularized amnion integrated silk fibroin / gelatin, TGF-beta1 signal transduction is antagonized by up-regulating Fmod expression, the TGF-beta1 signal channel inhibitor is suitable for being applied to a ureter porous stent, urinary tract epithelization can be increased, fibrosis can be inhibited, and the TGF-beta1 signal channel inhibitor can be used for preparing a ureter stent. Therefore, the method is used for ureteral scar-free repair treatment, ureteral defect repair treatment or ureteral reconstruction, and is of great significance to the development of ureteral tissue engineering.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and particularly relates to a TGF-β1 signal pathway inhibitor and a method and application for regulating Fmod expression. BACKGROUND

[0002] Long-segment ureteral defects pose a significant challenge to urologists, often requiring complex surgical intervention. Traditional treatment methods, such as autologous tissue transplantation (e.g., small intestine and tongue mucosa) and orthotopic kidney transplantation, are associated with significant trauma and limited success rates. Biodegradable natural polymers such as gelatin, silk fibroin (SF), and hyaluronic acid have been used to construct ureteral scaffolds, showing good results. These materials have good biocompatibility, processability, and controllable degradation rates, enhancing the regenerative capacity of the graft. Previous studies have shown that the naturally occurring RGD sequence promotes ureteral epithelial repair through the Integrin / MAPK signaling pathway. However, compared to autografts, the tissue regeneration effect of these scaffolds is still insufficient. This limitation is attributed to their biological inertness and insufficient reaction sites for the transplantation of bioactive substances, resulting in poor promotion of ureteral epithelial regeneration and anti-fibrotic effects.

[0003] Fibrosis is a complex biological process involving a delicate balance between fibroblasts and myofibroblasts, which is the core of anti-fibrotic mechanisms. The differentiation of fibroblasts into myofibroblasts plays a crucial role in tissue repair, contributing to early ECM deposition and wound contraction. However, in the later stages of repair, the sustained activation of myofibroblasts can lead to excessive fibrosis. During this process, the transforming growth factor-beta (TGF-β) related pathway plays an important role in regulating fibroblast differentiation and extracellular matrix production. Decellularized amniotic membrane (dAM) and its derived cells have shown the potential to alleviate fibrosis by downregulating TGF-β1 expression, thereby inhibiting the excessive activation of myofibroblasts. This anti-fibrotic effect of dAM is particularly important in ureteral repair, as excessive scarring can impair tissue function. However, the specific biological mechanisms of dAM's anti-fibrotic properties in ureteral tissue remain largely unknown and require further research to optimize its therapeutic potential in regenerative medicine. SUMMARY

[0004] One advantage of the present application is to provide a TGF-β1 signal pathway inhibitor and a method and application for regulating Fmod expression, wherein the TGF-β1 signal pathway inhibitor is a decellularized amniotic membrane integrated silk fibroin / gelatin, which antagonizes TGF-β1 signaling by upregulating Fmod expression, providing a new treatment strategy for ureteral defect repair treatment, and opening up a new approach for the development of urological tissue engineering and the treatment of urinary tract diseases.

[0005] Another advantage of the present application is to provide a TGF-β1 signaling pathway inhibitor and its method and application for regulating Fmod expression, wherein the elevated Fmod expression promotes fibroblast migration, crawling and collagen deposition on the scaffold surface, creating a favorable repair microenvironment, and the up-regulated Fmod further inhibits fibrosis, transforming from simple collagen deposition to proliferation and migration of ureter tissue-specific cells (uroepithelial cells and smooth muscle cells), which prevents excessive contraction of the scaffold and promotes scar-free ureter repair, which is of great significance for new ureter reconstruction therapy.

[0006] Another advantage of the present application is to provide a TGF-β1 signaling pathway inhibitor and its method and application for regulating Fmod expression, wherein the TGF-β1 signaling pathway inhibitor up-regulates Fmod gene expression, Fmod directly interacts with TGF-β1, inhibits downstream fibrosis pathways (such as p38, PI3K / AKT and MAPK), and inhibits excessive myofibroblast activation, thereby reducing ureter fibrosis and promoting scar-free repair, which has the potential to promote scar-free ureter repair.

[0007] Another advantage of the present application is to provide a TGF-β1 signaling pathway inhibitor and its method and application for regulating Fmod expression, 218 genes up-regulated by TGF-β1 but down-regulated by SGA scaffold treatment are determined, which may represent fibrosis markers; on the contrary, 554 genes down-regulated by TGF-β1 but up-regulated by SGA scaffold treatment may be protective factors, and fibromodulin regulates TGF-β1-related biological processes, which provides a new ureter reconstruction therapy strategy.

[0008] Another advantage of the present application is to provide a TGF-β1 signaling pathway inhibitor and its method and application for regulating Fmod expression, TGF-β1 is determined as one of the primary interaction proteins of Fmod, and Fmod always interacts with TGF-β1 under various conditions, and experimental results show that Fmod can inhibit the pro-fibrotic activity of TGF-β1 by direct binding, which is of great significance for tissue repair and remodeling development.

[0009] According to one aspect of the present application, the present application provides a TGF-β1 signaling pathway inhibitor, wherein the TGF-β1 signaling pathway inhibitor is a decellularized amniotic membrane integrated silk fibroin / gelatin.

[0010] The decellularized amniotic membrane integrated silk fibroin / gelatin antagonizes TGF-β1 signaling by up-regulating Fmod expression.

[0011] The decellularized amniotic membrane integrated silk fibroin / gelatin antagonizes TGF-β1 signaling by up-regulating Fmod expression.

[0012] According to another aspect of the present application, the present application also provides a method for regulating Fmod expression by a TGF-β1 signaling pathway inhibitor, wherein the method for regulating Fmod expression is up-regulating Fmod expression.

[0013] wherein TGF-β1 is a primary interaction protein of Fmod, and the Fmod inhibits the activity of fibroblasts by interacting with TGF-β1.

[0014] wherein the TGF-β1 signaling pathway inhibitor reduces ureteral fibrosis by inhibiting downstream fibrosis pathways p38, PI3K / AKT and MAPK.

[0015] wherein after the treatment of the TGF-β1 signaling pathway inhibitor, the expression of the phosphorylation levels of downstream pathway proteins α-SMA and p38, JNK, ERK, Smad2 / 3, PI3K, AKT are reduced.

[0016] wherein the 554 genes down-regulated by TGF-β1 but up-regulated by the treatment of the TGF-β1 signaling pathway inhibitor are protective factors.

[0017] wherein the 218 genes up-regulated by TGF-β1 but down-regulated by the treatment of the TGF-β1 signaling pathway inhibitor represent fibrosis markers, and the Fmod-TGF-β1 interaction can reverse fibrosis.

[0018] According to another aspect of the present application, the present application also provides a TGF-β1 signaling pathway inhibitor, wherein the TGF-β1 signaling pathway inhibitor is suitable for being applied in a ureteral stent to promote scarless tissue repair and remodeling of the ureter. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 、 Figure 2 and Figure 3 are schematic diagrams of bioinformatics analysis of FBs gene expression under different conditions.

[0020] Figures 4 to 6 is a schematic diagram of PPI network construction analysis results of Fmod-related genes.

[0021] Figure 7 and Figure 8 are schematic diagrams of results analysis of Fmod expression in BFs under different culture conditions.

[0022] Figure 9 and Figure 10 are schematic diagrams of identification of Fmod and TGF-β1 interaction.

[0023] Figure 11is a schematic diagram of the pathological examination results of ureter regeneration in vivo 3 months after the operation of the Ctrl group.

[0024] Figure 12 and Figure 13 is a schematic diagram of the histological analysis results of the tubular stent 2 weeks after implantation in rabbits.

[0025] Figure 14 is a schematic diagram of the medical image analysis results of the animal experiment of the Ctrl group.

[0026] Figure 15 and Figure 16 is a schematic diagram of the results of the urinary tract patency after ureter defect repair for 3 months.

[0027] Figure 17 and Figure 18 is a schematic diagram of the analysis of the degree of fibrosis of the ureter graft segment 3 months after the operation.

[0028] Figure 19 、 Figure 20 and Figure 21 is a schematic diagram of the analysis of the pathological examination results of ureter regeneration in vivo 3 months after the operation. DETAILED DESCRIPTION

[0029] The following description is provided to enable any person skilled in the art to practice the present application. The preferred embodiments in the following description are only examples of the present application and various modifications can be made by those skilled in the art without departing from the spirit and scope of the present application. The basic principles defined in the following description can be applied to other embodiments, modifications, improvements, equivalents and other technical solutions without departing from the spirit and scope of the present application.

[0030] The present application provides a TGF-β1 signal pathway inhibitor and its method and application for regulating Fmod expression, wherein the TGF-β1 signal pathway inhibitor is a decellularized amniotic membrane integrated silk fibroin / gelatin, a tubular porous scaffold (SGA, i.e. SF / gelatin / pepsin / dAM) is prepared by integrating silk fibroin / gelatin with decellularized amniotic membrane (dAM) for ureter repair and reconstruction, and then the role of decellularized amniotic membrane integrated silk fibroin / gelatin in regulating Fmod expression and inhibiting TGF-β1 signal transduction is studied. That is, silk fibroin and pigskin gelatin are mixed in proportion, and a decellularized amniotic membrane integrated silk fibroin / gelatin tubular porous scaffold is prepared by integrating silk fibroin and gelatin solution with decellularized amniotic membrane (dAM) through freeze-drying, compression and cross-linking method. The decellularized amniotic membrane integrated silk fibroin / gelatin scaffold retains many functional properties of dAM, can enhance ureteral epithelialization, and at the same time inhibit fibrosis. Experiments show that it has the potential to promote scar-free ureter repair, and is suitable for application in ureter reconstruction treatment.

[0031] Fibroblast induction and transcriptomic analysis: 20000 fibroblasts (FBs) were transferred to each well of a 24-well plate and cultured overnight. TGF-β1 20 ng / mL was used to induce the phenotypic change of fibroblasts to myofibroblasts and overexpress ECM. At the same time, SGA scaffolds were added to observe their anti-fibrosis effect. After 72 hours of culture, cells were collected for large-scale RNA sequencing (performed by Suzhou Qiantang Biotechnology Co., Ltd.). The results were screened for relevant targets by GO and KEGG analysis. Protein-protein interaction (PPI) data were obtained from STRING (http: / / string-db.org / ) or Genemania (http: / / genemania.org / ) and visualized using Cytoscape software.

[0032] Each group was named according to the culture conditions: Ctrl; TGF-β1 (TGF-β1); TAM (TGF-β1 + SGA).

[0033] According to the manufacturer's protocol, siRNA and GP-transfect-Mate were used to transfect fibroblasts (FBs). Briefly, cells were seeded in a 24-well plate (20000 cells / well) and allowed to adhere overnight. Then, cells were transfected with 20 nM siRNA and the medium was replaced with complete high glucose medium overnight and maintained for 72 hours. Finally, qRT-PCR was used to detect the expression of fibromodulin (Fmod). The primer sequences of Fmod and the corresponding siRNA are shown in Table 1.

[0034]

[0035] Western blotting (WB) and co-immunoprecipitation (Co-IP) experiments: WB experiments were used to verify the levels of relevant proteins and phosphorylation. For Co-IP, whole cell lysates were immunoblotted with the corresponding antibodies to detect protein expression. Co-IP was performed using Protein A / G PLUS-Agarose Immunoprecipitation Reagent (Santa Cruz) according to the manufacturer's instructions. Experiments were performed using anti-Fmod antibodies (Santa Cruz). The interacting proteins were detected by WB using anti-TGF-β1 antibodies (Abcam).

[0036] Specific steps: The cells treated with different treatments were collected and the protein was extracted using lysis buffer. The cell lysate was incubated on ice for 1 hour, and then the supernatant was collected by centrifugation. 40 μg of total protein was loaded into each lane and electrophoresis was performed using a 10% SDS-PAGE gel. The target protein on the SDS-PAGE gel was transferred to a polyvinylidene fluoride membrane (PVDF, 0.45 μm), and then blocked with 5% blocking buffer at 37°C for 1 hour. The PVDF membrane was treated with primary antibodies for GAPDH (proteintech), a-SMA (Abcam), p38, p-p38, JNK, p-JNK, ERK, p-ERK (CST), Smad2 / 3 (Affinity, China), p-smad2, p-smad3 (Abcam), PI3K, p-PI3K, AKT and p-AKT (CST) at 4°C overnight, respectively. Subsequently, the membrane was washed with TBST three times and incubated with secondary antibodies for 1 hour. The membrane was scanned using Imagelab software and the gray value was measured to show the expression of differential proteins.

[0037] Molecular dynamics simulation: Fmod and TGF-β1 were selected for molecular docking simulation to explore their interaction mechanism. The amino acid sequence was obtained from the UniProt database (https: / / www.uniprot.org / ) and the corresponding 3D structure model was retrieved from the AlphaFold database (https: / / alphafold.com / ). Ramachandran plot analysis was performed using MOE 2015 software to evaluate the effectiveness of the protein structure. Molecular docking analysis was performed using the docking module in the Schrödinger 2018 software suite. A blind docking strategy was adopted, with the entire protein surface set as a potential binding site. The maximum number of conformations searched was set to 10000, and the final retained conformations were limited to 30.

[0038] Biological evaluation of ureter repair: Biological evaluation of ureter repair includes HE staining and Masson trichrome staining to evaluate inflammation and collagen deposition. The level of fibrosis was evaluated by qRT-PCR (primer sequences are shown in Table 2), and the level of uroepithelialization and fibrosis was further evaluated by AE1 / AE3, Fmod and a-SMA multiplex fluorescent staining.

[0039]

[0040] Data analysis: All measurements were performed at least in triplicate, and data are presented as mean ± standard deviation (SD). Comparisons between two groups were made using the t-test, while multiple comparisons were made using one-way ANOVA followed by Tukey's post hoc analysis using SPSS 20 software. A p-value < 0.05 was considered statistically significant.

[0041] 1. Anti-fibrotic and transcriptomic analysis While early myofibroblast differentiation can promote wound contraction and collagen deposition, thus facilitating tissue repair, sustained contraction and ECM protein secretion by myofibroblasts are the main causes of ureteral fibrosis. WB showed that a-SMA protein expression was low in untreated BFs (Ctrl) and significantly increased after TGF-β1 induction, mimicking ureteral fibrosis. Figures 1 to 6 Bioinformatics analysis of FBs gene expression under different conditions: Figure 1 (A), (B) of FIG. 1 are schematic diagrams of the results of detecting the protein and mRNA expression of a-SMA in FBs under different culture conditions using Western blotting and qRT-PCR techniques. Figure 2 (A) of FIG. 2 is a Venn diagram of the number of differentially expressed genes in the Ctrl, TGF-β1, and TAM groups; (B), (C) are volcano plots of transcriptomic analysis, where (B) is the differentially expressed genes of Ctrl vs TGF-β1 group, (C) is the differentially expressed genes of TGF-β1 vs TAM group. Figure 3 are extracellular matrix-related genes that are inversely regulated in GO and KEGG pathways in TAM relative to Ctrl vs TGF-β1 group. Figure 4 and Figure 5 PPI network analysis of Fmod-related genes in FIG. 3, Figure 6 is the interaction network of Fmod and its possible related genes constructed in the GeneMANIA database. Data are presented as mean ± standard deviation (*p < 0.05, ****p < 0.0001).

[0042] Figure 7 and Figure 8 are analyses of Fmod expression in BFs under different culture conditions. Figure 7 is immunofluorescence of Fmod (green) and a-SMA (red) in BFs under different culture conditions on day 5, with DAPI used to stain the cell nuclei. Figure 8 is a semi-quantitative analysis of Fmod expression. Wherein (a) is Figure 7(a) Semi-quantitative analysis of immunofluorescence in the middle. (b) qRT-PCR analysis of Fmod relative mRNA expression level. Data are expressed as mean ± standard deviation (*p<0.05, **p<0.01, ****p<0.0001).

[0043] like Figure 1 As shown in (A), when induced myofibroblasts were co-cultured with the SGA scaffold, α-SMA protein expression was significantly reduced, suggesting that the SGA scaffold may antagonize the pro-fibrotic effect of TGF-β1 through a paracrine mechanism. Figure 1 As shown in (B) above, qRT-PCR results confirmed these findings at the mRNA level. Figure 2 As shown in (A), transcriptome analysis revealed significant gene expression changes induced by TGF-β1 and SGA scaffold treatments. Venn plots showed 1300, 3160, and 2237 differentially expressed genes in Ctrl vs TGF-β1, TGF-β1 vs TAM, and Ctrl vs TAM comparisons, respectively. Figure 2 As shown in (B) and (C), the volcano plots reveal 615 upregulated genes and 665 downregulated genes in Ctrl vs TGF-β1, while 2240 upregulated genes and 871 downregulated genes are found in TGF-β1 vs TAM. Figure 3 As shown, further analysis identified 218 genes upregulated by TGF-β1 but downregulated by SGA scaffold treatment, potentially representing fibrosis biomarkers. Conversely, 554 genes downregulated by TGF-β1 but upregulated by SGA scaffold treatment may be protective factors. GO and KEGG analyses of ECM-related genes were visualized in a heatmap, showing opposite expression patterns between the TGF-β1 and TAM groups, indicating that these genes are key regulators of SGA scaffold-mediated fibrosis reversal. Figure 3 In this context, BP represents biological processes, MF represents molecular functions, and CC represents cellular components. For example... Figure 4 As shown, PPI network analysis of these genes revealed extensive interactions, suggesting the potential formation of specific protein complexes or signaling pathways in fibrosis regulation. Figure 5 As shown, in this network, Fmod acts as a key regulatory node, participating in the interactions of multiple ECM-related proteins. It is noteworthy that, as... Figure 6 As shown, TGF-β1 was identified as one of the primary interacting proteins of Fmod, suggesting that Fmod may inhibit the profibrotic activity of TGF-β1 by direct binding.

[0044] like Figure 7 and Figure 8As shown, expression analysis of Fmod under different conditions showed significant group differences. Fmod expression was significantly elevated in the SGA group compared to the Ctrl group, decreased in the TGF-β1 group, and restored to higher levels when TGF-β1 -induced myofibroblasts were co-cultured with SGA scaffolds. qRT-PCR results confirmed these protein expression patterns at the mRNA level. These findings collectively indicate that SGA scaffolds have the potential to reverse fibrosis by modulating key ECM-associated genes and proteins, particularly through the Fmod-TGF-β1 interaction.

[0045] Fmod is a small leucine-rich proteoglycan that plays a key role in extracellular matrix organization and collagen fiber generation. In the fetal wound healing model, Fmod expression rapidly decreased during the transition from scarless to scar repair. Loss-of-function and gain-of-function experiments confirmed the importance of Fmod in fetal-type scarless repair and its impact on TGF-β ligand and receptor levels in fetal and adult skin. Notably, Fmod deficiency in adult mice resulted in increased scar area and impaired dermal fibrosis, manifested as severely impaired fibroblast migration and delayed granulation tissue formation. These phenotypes are very similar to those observed in fibroblast-specific TGF-β receptor knockout mice, indicating that Fmod is crucial for the correct expression and function of TGF-β during wound repair. Moreover, Fmod has been shown to elicit a more "fetal-like" pro-migratory and pro-contractile phenotype in adult dermal fibroblasts.

[0046] 2. Protein interactions and characterization The results of the molecular dynamics simulation of the interaction between Fmod and TGF-β1 were analyzed, and the results are shown in Figure 9 and Figure 10 , which are schematic diagrams of the identification results of the interaction between Fmod and TGF-β1. Among them, in Figure 9 (A) is the docking modeling of Fmod and TGF-β1; where (a) is a surface plot, with Fmod in cyan and TGF-β1 in green. (b) is a ribbon plot; (c) is a ribbon plot showing key amino acid residues. Figure 9 (B) in Figure 9 (C) is an ELISA analysis of TGF-β1 secreted by FBs pretreated with TGF-β1 and stimulated with SGA scaffold extracts and Fmod siRNA. Figure 10Figures (A) and (B) show the Western blot bands and semi-quantitative analysis. In TGF-β1-stimulated FBs, Fmod knockdown resulted in high expression of TGF-β1-related downstream pathway proteins (α-SMA, p38, p-p38, JNK, p-JNK, ERK, p-ERK, Smad2 / 3, p-Smad2, p-Smad3, PI3K, p-PI3K, AKT, and p-AKT), while SGA scaffold treatment resulted in low expression. Data are presented as mean ± standard deviation (*p<0.05, ****p<0.0001).

[0047] .like Figure 9 As shown in (A), (a) and (b) demonstrate that molecular docking reveals a unique "key-lock" complementary binding mode between Fmod and TGF-β1. Analysis of the interfacial residues in (c) indicates that the interaction is primarily driven by hydrophobic contacts, with additional polar interactions providing specificity and stability.

[0048] Protein-protein interactions were experimentally verified using Co-IP and Western blotting. The results are as follows: Figure 9 As shown in (B), Fmod consistently interacts with TGF-β1 under various conditions. RNA interference combined with ELISA revealed that TGF-β1 induction significantly increased TGF-β1 secretion in FBs, and Fmod knockdown further exacerbated this effect (p<0.0001). Figure 9 As shown in (C), SGA scaffold treatment reduced the secretion of TGF-β1 in myofibroblasts (p<0.05), and this effect was attenuated when Fmod was silenced.

[0049] like Figure 10 As shown in (A) and (B), Western blotting analysis revealed that TGF-β1 induced upregulation of α-SMA expression and activated both canonical (p-Smad2 / 3) and non-canonical (p-p38, p-JNK, p-ERK, p-PI3K, p-AKT) TGF-β1 signaling pathways, which play crucial roles in fibrosis. Fmod knockdown enhanced this activation, while SGA scaffold treatment significantly inhibited it. This inhibitory effect was attenuated when Fmod was silenced. These findings suggest that the SGA scaffold may antagonize TGF-β1 signaling by upregulating Fmod expression and thus inhibiting fibroblast activation.

[0050] 3. Results of ureteral repair experiment Histological analysis of the Ctrl group showed that the ureter had a well-preserved tissue structure, including the epithelial layer, lamina propria, smooth muscle layer, and connective tissue layer. The stellate lumen was lined by a folded urothelial lining. Repair experimental results were as follows... Figures 11 to 21 As shown.

[0051] Figure 11 For pathological examination of ureteral regeneration in vivo 3 months after operation in the Ctrl group. (A) H&E staining. (B) Masson staining.

[0052] Figure 12 and Figure 13 Histological analysis of tubular stents in rabbits in vivo 2 weeks after implantation. Figure 12 Macroscopic photographs, HE staining and Masson staining optical microscope images of the defect site, and IF images 2 weeks after implantation of different stents. The triangle indicates the stent position. In the IF images, Fmod, a-SMA and nuclei are shown in red, green and blue, respectively (black scale bar = 100 μm; white scale bar = 50 μm). Figure 13 (A) Quantitative analysis of Masson trichrome staining to show the collagen area around the stent; (B) Quantitative analysis of Fmod and a-SMA expression levels in A; (C) q-PCR results showing that Fmod and a-SMA were significantly increased in the SGA group. The agarose gel electrophoresis of RT-PCR amplification products is shown in the figure. Data are expressed as mean ± standard deviation (*p < 0.05, **p < 0.01, ***p < 0.001).

[0053] Figure 14 Medical image analysis of the Ctrl group animals. (A) Gross and CT / CTU images of the urinary tract. (B) Micro-CT scanning of the transplanted segment. The red-dotted area represents the patency of the urinary tract.

[0054] Figure 15 and Figure 16 Urinary tract patency 3 months after ureteral defect repair. Figure 15 In (A), gross and CT images are shown, and in (B) and (C), 3D reconstruction images of the urinary tract in the SGA and Ctrl groups are shown, respectively. In (D), Micro-CT was used to assess the patency of the graft. The red-dotted area represents the patency of the urinary tract. Figure 16 Results of renal function analysis including the degree of hydronephrosis, the degree of ureteral dilation, and the serum BUN and Cr concentrations in each group. Data are expressed as mean ± standard deviation (*p < 0.05, **p < 0.01).

[0055] Figure 17 and Figure 18 Analysis of the degree of fibrosis in the ureteral graft segment 3 months after operation. Figure 17 RT-PCR amplification products of CTGF, Col1a1 and Fmod in the ureteral graft segment were analyzed by agarose gel electrophoresis. GAPDH was used as an internal control. Figure 18qRT-PCR analysis for CTGF, Col1a1 and Fmod expression in the grafted ureteral segment. Results were normalized to GAPDH expression. Data are expressed as mean ± standard deviation (**p < 0.01, ***p < 0.001, ****p < 0.0001).

[0056] Figures 19 to 21 Pathological examination of ureteral regeneration in vivo 3 months after surgery. Figure 19 (A) HE and Masson staining of engineered ureteral tissues after different post-processing, (B) IF images of ureteral conduits to assess epithelialization (AE1 / AE3, green) and Fmod levels (red). Figure 20 (A), (B) are the quantitative analysis of Masson trichrome staining, showing collagen area and smooth muscle area, respectively. (C), (D) are the quantitative analysis of AE1 / AE3 and Fmod expression levels. Figure 21 Schematic diagram of fibroblast regulation mechanism on dAM-containing scaffolds. Data are expressed as mean ± standard deviation (*p < 0.05).

[0057] As shown in Figure 11 , collagen fibers were confined to the lamina propria and almost no penetration to other layers, similar to the normal ureteral structure. For other groups, the results of SGA scaffolds in the in vivo ureteral repair experiment were encouraging. As shown in Figure 12 and Figure 13 (A), macroscopic observation showed that both SG and SGA scaffolds were not completely degraded and were covered by a large amount of connective tissue after two weeks of implantation. Histological examination showed that both SG and SGA scaffolds had good biocompatibility with less inflammatory cell infiltration, and Masson staining showed that the collagen layer on the surface of SGA scaffolds was significantly thicker than that of SG scaffolds (p < 0.01), indicating enhanced early ECM deposition. As shown in Figure 12 and Figure 13 (B), IF staining showed positive expression of α-SMA (green) and Fmod (red) on the surface of both scaffolds, and the positive area of SGA was significantly higher (p < 0.001). As shown in Figure 13 (C), qRT-PCR results confirmed that the expression levels of Fmod and α-SMA in the SGA group were significantly higher (p < 0.05), indicating that SGA scaffolds may better promote tissue repair and remodeling.

[0058] As shown in Figure 14 , the ureter of the Ctrl group showed normal size without dilation or granuloma. The kidney was healthy without hydronephrosis. In the imaging examination, CT and CTU scans showed no hydronephrosis or ureteral dilation, normal urine flow, and no significant stenosis as Figure 14The lumen was confirmed to be patent by Micro-CT, without tissue overgrowth as shown in Fig. 1A. Figure 14 The lumen was confirmed to be patent by Micro-CT, without tissue overgrowth as shown in Fig. 1A.

[0059] As shown in Fig. 1B, Fig. 1C, and Fig. 1D, the SGA group showed less ureteral dilation and hydronephrosis compared to the PP46-gel and SG groups, with ureteral diameter and hydronephrosis levels similar to the control group. Figure 15 and Figure 16 As shown in Fig. 1B, Fig. 1C, and Fig. 1D, the SGA group showed less ureteral dilation and hydronephrosis compared to the PP46-gel and SG groups, with ureteral diameter and hydronephrosis levels similar to the control group. Figure 15 and Figure 16 As shown in Fig. 1B, Fig. 1C, and Fig. 1D, the SGA group showed less ureteral dilation and hydronephrosis compared to the PP46-gel and SG groups, with ureteral diameter and hydronephrosis levels similar to the control group. Figure 15 As shown in Fig. 1B, Fig. 1C, and Fig. 1D, the SGA group showed less ureteral dilation and hydronephrosis compared to the PP46-gel and SG groups, with ureteral diameter and hydronephrosis levels similar to the control group. Figure 15 As shown in Fig. 1B, Fig. 1C, and Fig. 1D, the SGA group showed less ureteral dilation and hydronephrosis compared to the PP46-gel and SG groups, with ureteral diameter and hydronephrosis levels similar to the control group.

[0060] Histological examination at 3 months showed epithelial cell growth in the lumen of the graft in all experimental groups. As shown in Fig. 2A and Fig. 2B, the SGA group exhibited a pseudostratified epithelial growth pattern with less collagen deposition and increased smooth muscle tissue compared to the PP46-gel and SG groups. Figure 19 and Figure 20 As shown in Fig. 2A and Fig. 2B, the SGA group exhibited a pseudostratified epithelial growth pattern with less collagen deposition and increased smooth muscle tissue compared to the PP46-gel and SG groups. Figure 19 and Figure 20 IF staining in Fig. 2B and Fig. 2C showed that the epithelium in the SGA group was more extensive with folding structures, indicating partial recovery of ureteral contractile function for urine transport. Figure 20 As shown in Fig. 2D, Fmod expression was mainly localized below the epithelial layer, which was significantly higher in the SGA group.

[0061] As shown in Fig. 2A and Fig. 2B, the SGA group exhibited a pseudostratified epithelial growth pattern with less collagen deposition and increased smooth muscle tissue compared to the PP46-gel and SG groups. Figure 17 and Figure 18As shown, gene expression analysis showed that the SGA group had lower levels of fibrosis-related markers (connective tissue growth factor, CTGF and Col1a1) and higher Fmod expression compared to the PP46-gel and SG groups. Specifically, the SGA group had significantly lower levels of CTGF and Col1a1 than the PP46-gel (p < 0.001 and p < 0.0001, respectively) and SG groups (p < 0.01), but higher than the control group (p < 0.01). The SGA group had significantly higher Fmod expression than the PP46-gel (p < 0.001) and SG groups (p < 0.01), consistent with the IF results, but lower than the control group (p < 0.01). These findings suggest that elevated Fmod expression during the proliferation phase promotes fibroblast migration, crawling, and collagen deposition on the scaffold surface, creating a favorable repair microenvironment. During the remodeling phase, the upregulated Fmod further inhibits fibrosis, shifting from simple collagen deposition to the proliferation and migration of ureter-specific cells (uroepithelial cells and smooth muscle cells). This process prevents excessive contraction of the scaffold and promotes scar-free ureteral repair.

[0062] In summary, the present application successfully developed a dAM-integrated SGA scaffold suitable for ureteral defect repair through a simple freeze-drying, compression, and cross-linking method. The scaffold effectively promotes uroepithelial regeneration and anti-fibrosis properties, promoting scar-free ureteral healing. Mechanistically, the SGA upregulates Fmod gene expression, which directly interacts with TGF-β1 to inhibit downstream fibrosis pathways (such as p38, PI3K / AKT, and MAPK), inhibiting excessive myofibroblast activation, thereby reducing ureteral fibrosis and promoting scar-free repair, as shown. Figure 21 The present application provides a new ureteral defect repair treatment strategy and provides theoretical support for the translational application of dAMs in regenerative medicine, which is of great significance for promoting the development of urological tissue engineering and opening up new avenues for the treatment of urological diseases.

[0063] It should be understood by those skilled in the art that the embodiments of the application described above and shown in the drawings are only examples and do not limit the application. The purpose of the application has been fully and effectively achieved. The function and structural principles of the application have been demonstrated and described in the embodiments, and the implementation of the application can be modified or changed in any way without departing from the principles described.

Claims

1. A TGF-β1 signal pathway inhibitor characterized by, The TGF-β1 signaling pathway inhibitor is a decellularized amniotic membrane integrated silk fibroin / gelatin. 2.The TGF-β1 signaling pathway inhibitor of claim 1, wherein the decellularized amniotic membrane integrated silk fibroin / gelatin antagonizes TGF-β1 signaling by upregulating Fmod expression. 3.The TGF-β1 signaling pathway inhibitor of claim 2, wherein the decellularized amniotic membrane integrated silk fibroin / gelatin is prepared by integrating enzymatically decellularized amniotic membrane with silk fibroin and gelatin solution, using freeze-drying, compression and cross-linking methods.

4. The method of claim 1, wherein the TGF-β1 signaling pathway inhibitor is a TGF-β1 neutralizing antibody. The method for regulating Fmod expression is upregulating Fmod expression. 5.The method for regulating Fmod expression of the TGF-β1 signaling pathway inhibitor of claim 4, wherein TGF-β1 is a primary interaction protein of Fmod, and the Fmod inhibits the activity of fibroblasts by interacting with TGF-β1. 6.The method for regulating Fmod expression of the TGF-β1 signaling pathway inhibitor of claim 5, wherein the TGF-β1 signaling pathway inhibitor alleviates ureteral fibrosis by inhibiting downstream fibrosis pathways p38, PI3K / AKT and MAPK. 7.The method for regulating Fmod expression of the TGF-β1 signaling pathway inhibitor of claim 4, wherein after the TGF-β1 signaling pathway inhibitor treatment, the expression of the phosphorylation levels of downstream pathway proteins α-SMA and p38, JNK, ERK, Smad2 / 3, PI3K, AKT is reduced. 8.The method for regulating Fmod expression of the TGF-β1 signaling pathway inhibitor of claim 6, wherein the 554 genes downregulated by TGF-β1 but upregulated by the TGF-β1 signaling pathway inhibitor treatment are protective factors. 9.The method for regulating Fmod expression of the TGF-β1 signaling pathway inhibitor of claim 6, wherein the 218 genes upregulated by TGF-β1 but downregulated by the TGF-β1 signaling pathway inhibitor treatment represent fibrosis markers, and Fmod-TGF-β1 interaction can reverse fibrosis.

10. The use of the TGF-β1 signal pathway inhibitor according to claim 1, characterized in that, The TGF-β1 signaling pathway inhibitor is suitable for application in a ureteral stent to promote scarless tissue repair and remodeling of the ureter.

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