Application of pro-inflammatory stromal 4 cell inhibitor and substance for detecting MMP1

Abstract

The invention provides application of a pro-inflammatory stromal 4 cell inhibitor and a substance for detecting MMP1, and belongs to the technical field of biological medicine. Wherein the pro-inflammatory stromal 4 cell inhibitor can be used for preparing medicines for treating intestinal epithelial aplastic disorder, congenital megacolon, enterocolitis or congenital megacolon-related enterocolitis. The substance for detecting the MMP1 can be used for preparing a product for predicting the congenital megacolon-related enterocolitis. The invention discovers that pro-inflammatory stromal 4 cells show specificity in dilated intestinal tissues of a congenital megacolon patient for the first time, but the cells cannot maintain the growth of healthy organs; however, by using a pro-inflammatory stromal 4 cell inhibitor, the growth of the epithelial organoids can be restored. In addition, the proinflammatory stromal 4 cells appear before the onset of the congenital megacolon-related enterocolitis, which provides a powerful basis for predicting and treating the disease. Therefore, the method has a wide application prospect in the technical field of biological medicines.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to the application of a pro-inflammatory matrix metalloproteinase 4 cell inhibitor and a substance for detecting MMP1. Background Art

[0002] Hirschsprung's disease (HD) is a congenital malformation of the enteric nervous system characterized by abnormal migration of enteric neural crest cells, resulting in a lack of parasympathetic intrinsic ganglion cells in the submucosal and myenteric plexuses of the distal colon. It is the most common cause of intestinal obstruction in neonates and infants. This functional intestinal obstruction hinders the passage of stool through the affected intestinal segment, leading to compensatory dilatation of the proximal colon and the development of Hirschsprung's disease-associated enterocolitis (HAEC), a life-threatening complication of HD. Although surgical resection of aganglionic intestinal segments can relieve intestinal obstruction and is currently a widely used standard surgical intervention, HAEC may also occur after corrective surgery in which the affected intestinal segment is resected. So far, the research focus in this field has been on finding the causes of neural crest cell migration disorders and their potential substitutes, while epithelial homeostasis in HD has not been fully studied, and therefore the pathophysiological mechanisms of HAEC remain poorly understood.

[0003] The intestinal epithelium has a remarkable ability to self-renew and regenerate to maintain homeostasis. Stem cells (ISCs) residing in the intestinal crypts are the source of all intestinal epithelial cells. Under physiological and stress conditions, they can generate new cells, which gradually migrate toward the tip along the crypt-villus axis, differentiate into a specific type of epithelial cell lineage and acquire corresponding functions in the process. During intestinal injury, this balance shifts to regenerate more new cells to replace or repair the intestinal epithelium. However, the role and related mechanisms of epithelial regeneration and changes in ISC dynamics in the initiation and development of HAECs remain unexplored. Wnt signaling is a key pathway to maintain the niche and regenerative capacity of epithelial stem cells. It directly regulates LGR5-positive ISCs, initiates signaling cascades, and promotes their self-renewal and proliferation. There are multiple sources of Wnt signaling molecules in the intestine, including Paneth cells, stromal cells, and submucosal immune cells. The balance between Wnt signaling molecules from different sources is essential for maintaining intestinal homeostasis, enhancing stem cell function, and promoting tissue repair, and has an important impact on disease progression; however, the loss of Paneth cells in the colon poses a huge challenge to understanding the source of Wnt signals associated with epithelial regeneration during HAEC injury.

[0004] Intestinal stromal cells are located beneath the epithelium and secrete a variety of growth factors, such as Wnt and R-responsive proteins, which are essential for maintaining the self-renewal ability of stem cells. In addition, stromal cells play a key role in creating a three-dimensional scaffold for intestinal structure by producing extracellular matrix components such as collagen. Recent studies have shown that colonic stromal cell remodeling plays an important role in damage formation and epithelial regeneration in inflammatory bowel disease; however, the functional changes of stromal-epithelial regeneration crosstalk in the pathogenesis of HAEC are still unclear. In recent years, researchers have begun to explore strategies to regulate stromal cells and stem cell niches to offset the effects of intestinal damage and achieve therapeutic goals, including promoting tissue repair, improving the efficacy of stem cell-based treatments, and potential intervention methods for diseases involving the intestinal epithelium.

[0005] Intestinal organoids derived from purified crypt cells have become an important tool for studying disease progression and screening potential therapeutic agents. Based on human intestinal single-cell RNA sequencing (scRNA-seq), researchers have gained the latest insights into the development of the enteric nervous system and its defects, revealing the expression regulation patterns of a series of dynamic genes distributed along the spatial axis of the digestive tract. At the same time, they have also mastered valuable information about the diversity of intestinal cell types and their complex interactions, especially the lineage of enteric neural cells and their relationship with the occurrence of HD diseases. However, despite the above progress, there are still many unknown areas in the understanding of the cellular composition, biological characteristics, gene transcription regulation patterns and intercellular crosstalk within the intestinal system under disease states. It is of great significance to explore the relevant mechanisms of HAEC and develop drugs and methods to predict and treat HAEC. Summary of the invention

[0006] In view of the shortcomings of the prior art, the present invention aims to provide applications of a pro-inflammatory matrix metalloproteinase 4 cell inhibitor and a substance for detecting MMP1.

[0007] To achieve this object, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides a use of a pro-inflammatory stromal 4 cell inhibitor in the preparation of a drug for treating intestinal epithelial regeneration disorders.

[0009] It should be noted that, in the present invention, pro-inflammatory Stromal 4 cell, Stromal 4 cell, MMP1+Stromal 4 cell, and MMP1 positive Stromal 4 cell all refer to the same cell.

[0010] In a second aspect, the present invention provides a use of a pro-inflammatory stromal 4 cell inhibitor in the preparation of a drug for treating Hirschsprung's disease.

[0011] In a third aspect, the present invention provides a use of a pro-inflammatory stromal 4 cell inhibitor in the preparation of a drug for treating enterocolitis.

[0012] In some embodiments of the invention, the enterocolitis is Hirschsprung's disease-associated enterocolitis.

[0013] In some embodiments of the present invention, the pro-inflammatory stromal 4 cell inhibitor is ibuprofen.

[0014] In a fourth aspect, the present invention provides a use of a substance for detecting MMP1 (matrix metallopeptidase 1) in the preparation of a product for predicting Hirschsprung's disease-related enterocolitis.

[0015] In some embodiments of the present invention, the substance for detecting MMP1 includes an antibody against MMP1.

[0016] In some embodiments of the present invention, the substance for detecting MMP1 further includes a secondary antibody capable of binding to the antibody against MMP1.

[0017] In some embodiments of the present invention, the secondary antibody is a fluorescently labeled secondary antibody.

[0018] In a fifth aspect, the present invention provides a kit for predicting Hirschsprung's disease-related enterocolitis, the kit comprising a substance for detecting MMP1, wherein the substance for detecting MMP1 is the same as the substance for detecting MMP1 described in the fourth aspect.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The present invention discovered for the first time that proinflammatory stromal 4 cells are specific in the dilated intestinal tissue of patients with congenital megacolon, and these cells cannot maintain the growth of healthy organoids; however, by using proinflammatory stromal 4 cell inhibitors, the growth of epithelial organoids can be restored. In addition, the appearance of proinflammatory stromal 4 cells precedes the onset of congenital megacolon-related enterocolitis, which provides a strong basis for predicting and treating the disease. Based on the above findings, the present invention proposes for the first time the use of proinflammatory stromal 4 cell inhibitors in the preparation of drugs for treating intestinal epithelial regeneration disorders, congenital megacolon, enterocolitis or congenital megacolon-related enterocolitis, as well as the use of substances for detecting MMP1 in the preparation of products for predicting congenital megacolon-related enterocolitis, which has broad application prospects in the field of biomedical technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1ARepresentative tissue micrographs of the proximal, distal, and aganglionic rectum of HAEC patients stained with the neuronal marker TUJ1;

[0022] Figure 1B Representative H&E-stained tissue micrographs of the proximal, distal, and rectum of HAEC patients;

[0023] Figure 1C The histological injury grading chart of the proximal, distal and rectum of HAEC patients;

[0024] Figure 1D Representative micrographs of Ki67 immunohistochemical staining of the proximal, distal, and rectum of HAEC patients;

[0025] Figure 1E Quantification of Ki67-positive cells per crypt in all colonic regions of HAEC patients;

[0026] Figure 1F Representative immunohistochemical staining micrographs of Lgr5 in the proximal, distal, and rectum of HAEC patients;

[0027] Figure 1G Quantification of Lgr5-positive cells per crypt in three colon regions of HAEC patients;

[0028] Figure 2A UMAP images showing stromal cell subtype clusters;

[0029] Figure 2B Heatmap visualization of gene expression for stromal cell subtype-specific markers;

[0030] Figure 2C Violin plots of the activities of signaling pathways inferred by UCell in different stromal cell subtypes;

[0031] Figure 2D It is a bar graph showing the percentage of stromal 4 cells to the total number of stromal cells in different groups;

[0032] Figure 2E Representative immunofluorescence staining micrographs of MMP1 in distal ganglionated colon cells of non-HD patients and HD patients;

[0033] Figure 2F Quantification of MMP1-positive cells per crypt in the distal colon and rectum of non-HD patients and HD patients;

[0034] Figure 2G Representative micrographs of stromal cells from the distal colon of non-HD patients and HD patients on days 1 and 5 of culture;

[0035] Figure 2HThis is a growth curve of stromal cells from the distal colon of non-HD patients and HD patients;

[0036] Figure 3A Representative micrographs of distal colon epithelial organoids from non-HD and HD patients cultured in medium enriched with recombinant “WENR” growth factors Wnt3a, epidermal growth factor (EGF), Noggin, and Rspo1;

[0037] Figure 3B Representative micrographs of distal colon epithelial organoids from non-HD patients and HD patients co-cultured with distal colon stromal cells from non-HD patients and HD patients in the absence of “WENR” growth factor;

[0038] Figure 3C Immunofluorescence micrographs of MMP1+stromal 4 cells from the distal colon of HD patients treated with or without ibuprofen;

[0039] Figure 3D The graph shows the quantitative percentage of MMP1-positive cells relative to the total number of stromal cells in the distal colon of HD treated with or without ibuprofen;

[0040] Figure 3E Representative micrographs of epithelial organoids from non-HD patients co-cultured with HD distal colon stromal cells pretreated with or without ibuprofen in the absence of recombinant growth factors;

[0041] Figure 3F Figure 2 quantification of organoid numbers under each culture condition. DETAILED DESCRIPTION

[0042] The technical solution of the present invention is further described below with reference to the accompanying drawings and through specific implementations. It should be understood by those skilled in the art that the specific implementations are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0043] 1. Experimental Methods

[0044] Human colon tissue

[0045] For single-cell RNA sequencing analysis, patients who underwent surgery for short-segment HD in the Department of General Surgery, Children's Hospital, Capital Institute of Pediatrics were recruited (n = 5). The clinical diagnosis of each patient was confirmed by intraoperative frozen pathology and postoperative immunohistochemical staining. In addition, patients with Hirschsprung's disease (HAD) in this center were included in the non-HD control group (n = 2). Although these children still had some ganglion cells in the intestine, their symptoms of intestinal peristalsis dysfunction did not respond to traditional alternative treatments, so they still needed to undergo surgical resection of the intestine and became suitable controls for this study. In order to minimize the impact of potential biologically unrelated variations and luminal content exposure on the study results, full-thickness intestinal biopsies of similar locations were obtained from different patients. The aganglionic rectal segment (HD_R) and distal ganglionic colon (HD_D) of HD patients and the rectum (Control_R) and distal colon (Control_D) of non-HD patients were analyzed.

[0046] Pathological sections of human HD colon were obtained from the Department of Pathology at the above-mentioned center. The diagnosis of HD was confirmed by intraoperative frozen sections, histological hematoxylin and eosin (H&E) examination, and postoperative immunohistochemical staining. Based on colonic morphology, the specimens were divided into 3 segments: non-ganglionic rectum, ganglionic distal colon, and ganglionic proximal colon. The severity of enterocolitis was scored using an established histopathological scoring system. Based on the histopathological assessment of the severity of epithelial damage, the specimens were divided into two groups: HD patients without HAECs and HD patients with HAECs. In this study, distal colon and rectal tissues resected for reasons other than HD were used as non-HD patient controls.

[0047] Histological staining and evaluation

[0048] Paraffin-embedded human colon tissue cross-sections (5 μm thickness each) were stained with hematoxylin and eosin. Tissue sections were blindly evaluated by three researchers according to an established histopathological scoring system: grade 0 = no damage; grade 1 = epithelial cell detachment or separation; grade 2 = epithelial cell detachment to the level of the middle of the crypt; grade 3 = necrosis of the entire crypt; and grade 4 = transmural necrosis.

[0049] Immunostaining

[0050] Colon tissue sections were incubated with primary antibodies (Table S1) diluted 1 / 500 at 4°C. For immunofluorescence staining, sections were incubated with secondary antibodies (Table S1) diluted 1 / 1000 and DAPI (Vector Laboratories, Burlington, ON) for visualization of nuclei at room temperature for 1 hour. For immunohistochemical staining, sections were incubated with HRP-coupled secondary antibodies diluted 1 / 1000, followed by counterstaining with streptavidin-biotin complex peroxidase kit (LASB+Kit, Dako, Denmark) and hematoxylin. Slides were imaged and scanned using a Nikon TE-2000 digital microscope equipped with a Hamamatsu C4742-80-12AG camera. Three researchers blindly counted the number of positively labeled cells in at least five images and at least ten intestinal crypts from each subject.

[0051] Gene quantification

[0052] RNA was isolated from colon tissue using TRIzol (Invitrogen). Total RNA (1 μg) was reverse transcribed using qScript cDNA SuperMix (Quanta Biosciences, Gaithersburg). SYBR green-based quantitative polymerase chain reaction (qPCR) was performed using a CF384 C1000 ThermalCycler (Bio-Rad) and Advanced qPCR MasterMix (Wisent, Quebec) according to the manufacturer's protocol and the primers listed in Table S1. Data were analyzed using CFX Manager 3.1 (Bio-Rad). Results are from three independent experiments repeated at least three times. Gene expression levels were calculated by the ΔΔCt method and normalized to the reference housekeeping gene 3-glyceraldehyde phosphate dehydrogenase (Gapdh).

[0053] Table S1 List of key reagents

[0054]

[0055]

[0056] Flow cytometry

[0057] Flow cytometry was performed to confirm the expression of MMP1 in stromal cells. Briefly, stromal cells were mildly pretreated and incubated with MMP1 antibody at 4 °C overnight, followed by incubation with FITC secondary antibody for 2 h at room temperature, washed and analyzed by flow cytometry on a Beckman Coulter Gallios flow cytometer. Data were analyzed using FlowJo software. The mean fluorescence intensity was calculated and expressed as the amount of MMP1 expression within stromal 4 cells.

[0058] Human colon organoids and stromal cell co-culture

[0059] Human colon organoids were cultured using Human IntestiCult Organoid Growth Medium (Stem Cell Technologies, Cambridge, MA) and 10 μM Y-27632 (Stem Cell Technologies, Cambridge, MA). Stromal cells were cultured in DMEM medium containing 10% heat-inactivated fetal bovine serum (FBS) (vol / vol), penicillin-streptomycin (100 U / mL), and 1× insulin-transferrin-selenium A. Ibuprofen (100 ug / mL, Sigma #14883) was added to the culture medium of stromal cells. Approximately 25-40 organoids and 5×10 4 Stromal cells were resuspended in Matrigel (Corning, New York), transferred to 24-well plates (40 μL / well), cultured in organoid culture medium, and maintained in an incubator at 37° C. for 5 days. The co-culture system was photographed every day.

[0060] Human tissue isolation, single-cell suspension preparation, and sequencing

[0061] Colon tissue is stored in GEXSCOPE tissue preservation solution according to the standard protocol of New Grid Yuan Laboratory.After digestion using 2mL GEXSCOPE tissue dissociation solution (Singleron), filter the cell suspension and centrifuge through 40 micron sterile filters (Falcon, catalog number 352340). The obtained particles are resuspended in 1mL PBS. By adding 2mL RBC lysis buffer (Roche, catalog number 11814389001), then another round of centrifugation and resuspension are carried out to remove red blood cells. Under NikonECLIPSE Ts2 microscope, trypan blue staining (Bio-Rad, catalog number 1450013) is used to verify cell viability to ensure a concentration of 100000 cells / mL and a viability of more than 80%. scRNA-seq libraries were prepared using the GEXSCOPE single-cell RNA library kit (Singleron Biotechnologies), which includes cell lysis, mRNA capture, cell labeling with barcodes, and mRNA labeling with UMIs, followed by reverse transcription of mRNA into cDNA, amplification, and fragment capture. Sequencing was performed on an Illumina HiSeq X, generating 150 bp paired-end reads.

[0062] Single-cell RNA-seq data preprocessing

[0063] After quality control, single-cell sequencing data were aligned to the GRCh38 human reference genome and quantified using cellRanger (version 6.1.2, 10x Genomics Inc). The preliminary filtered data generated by Cell Ranger were further filtered: cells with UMI counts below 30,000, gene counts between 200 and 5,000, and mitochondrial content greater than 50% were removed. To remove potential dimorphic cells, each sequencing library was processed using Scrublet, and the expected dimorphic rate was set to 0.12. For dimensionality reduction, clustering, and data normalization, Seurat v4 software was used. Specifically, data normalization and scaling were achieved using the “NormalizeData()” and “ScaleData())” commands. Principal component analysis (PCA) was performed using “FindVariableFeatures()” to identify the top 2,000 variable genes. When integrating multiple samples, a CCA-based workflow was implemented in Seurat. Louvain community detection facilitated clustering through principal components applied to batch corrections by Seurat. The top 20 principal components were used as input for Louvain clustering using the "FindClusters()" command at a resolution of 1.0. Finally, visualization of single-cell transcriptional profiles and clusters was performed by unified manifold approximation and projection (UMAP) using default parameters (n_neighbors = 30L, min_dist = 0.5) in the "RunUMAP()" command of Seurat.

[0064] Cell type annotation

[0065] Cell type classification uses a semi-automatic annotation method, including two rounds of clustering to distinguish major and minor cell types. Reference was made to the previously published annotations of major and minor cell types (Elmentaite, R., et al., Cells of the human intestinal tract mapped across space and time. Nature, 2021. 597 (7875): p. 250-255.). The K-nearest neighbor (KNN) algorithm implemented by the "RunKNNPredict()" command in the R SCP package facilitates cell type prediction. The secondary clustering protocol used is the same as the first round of clustering, with a resolution of 0.8 to 1.5. Minor cell types were manually annotated based on specific markers in each subcluster and predictions of minor cell types.

[0066] Definition of Wnt pathway activity and inflammatory response module scores

[0067] To assess the characteristics and functions of each subtype of stromal cells, the "AddModuleScore()" function in the Seurat R package was used to determine the extent to which individual cells expressed a specific predefined gene set expression program. The gene set was obtained from the Molecular Signature Database (MSigDB) (https: / / www.gsea-msigdb.org / gsea / msigdb). Gene signature scores were generated by using the "ScoreSignatures_UCell()" command in the UCell package.

[0068] Cluster Organization

[0069] To quantify the organizational preference of each cluster at different locations, the ratio of the observed cell number to the expected cell number for each cluster, R, was calculated. o / e The chi-square test was used to obtain the expected number of cells for each combination of cell cluster and tissue. o / e >1, a cluster was identified as enriched in a specific tissue. o / e <1, a cluster was identified as depleted in a specific tissue.

[0070] Statistical methods

[0071] All analyses were performed using GraphPad Prism 6. All results were performed at least in triplicate and are presented as mean ± SEM. P values ​​were determined using nonparametric unpaired Student t-test (Mann–Whitney U test) or one-way ANOVA with Turkey test for post hoc testing (as appropriate). P < 0.05 was considered statistically significant.

[0072] 2. Results

[0073] Impaired intestinal regeneration in the distal ganglionic colon of HD patients

[0074] Colon samples were obtained from six patients diagnosed with HAEC based on histological evaluation by a pathologist. The neuronal marker TUJ1 was present in the proximal ganglionated colon, whereas its abundance was reduced in the distal colon and absent in the aganglionic rectal tissue ( Figure 1A ). Staining of intestinal tissue sections revealed that the distal ganglionated colon epithelium of HAEC patients showed significant damage compared with the proximal ganglionated colon and the non-ganglionated rectum, as evidenced by a significantly higher histological injury score ( Figure 1B , Figure 1C In the same distal ganglionated colonic segment, the proliferation marker Ki67 ( Figure 1D , Figure 1E ) and intestinal stem cell marker LGR5 ( Figure 1F , Figure 1G These findings confirm that children with HAEC have significant impairment of intestinal regeneration in the distal ganglionated colon.

[0075] The composition of stromal cells in the distal ganglionic colon of HD is significantly altered

[0076] The viability and regeneration capacity of the intestinal epithelium depend on the coordinated interaction between different cellular components within the intestinal tissue. Given that the results of single-cell sequencing analysis suggest that intestinal stromal cells have the strongest interaction with epithelial cells, we first revealed the existence of five different subtypes of stromal cells by unsupervised clustering ( Figure 2A , Figure 2B ). The gene set scoring results suggest that stromal 4 cells exhibit a significant pro-inflammatory state ( Figure 2C ), whereas in this stage of HD, no significant epithelial inflammation was present in the same segment. In addition, in contrast to the fact that stromal 3 cells were only expressed in the aganglionic rectum in HD patients, this study revealed that the stromal 4 cell population was only present in the distal ganglionic colon of HD patients, a result supported by experiments with the matrix metallopeptidase 1 (MMP1) marker ( Figure 2D , Figure 2E , Figure 2F ). Subsequently, stromal cells from the distal colon of HD patients and non-HD patients were isolated and cultured. Notably, stromal cells from HD patients grew significantly slower than those from non-HD patients ( Figure 2G , Figure 2H ).

[0077] Proinflammatory stromal cells in the distal ganglionic colon of HD fail to support organoid growth

[0078] To verify the contribution of stromal 4 cells to the defective regeneration of the colonic epithelium, an in vitro cell-organoid co-culture model was constructed to study the interaction between stromal cells and colonic organoids. Compared with non-HD patients, the growth of organoids generated from the distal ganglionated colonic epithelium of HD patients was impaired and the number was significantly reduced ( Figure 3A HD patient-derived stromal cells cannot provide adequate support for the growth of non-HD patient-derived organoids ( Figure 3B These findings suggest that pro-inflammatory stromal 4 expression in the distal colon of HD patients is insufficient to promote epithelial cell growth and impedes the regenerative capacity of the colonic epithelium, which may contribute to the development of epithelial damage in HAECs.

[0079] In order to explore potential strategies to prevent epithelial regeneration damage caused by stromal 4 cells, we tried to use the nonsteroidal anti-inflammatory drug ibuprofen to intervene in the stromal 4 cell population. The results confirmed that adding ibuprofen to the culture medium of cells cultured in vitro could effectively reduce the number of MMP1-positive stromal 4 cells ( Figure 3C , Figure 3D ). Pretreatment of HD distal colon stromal cells with ibuprofen was able to effectively support the growth of colon epithelial organoids derived from individuals other than HD patients, indicating that ibuprofen has a positive effect on the ability of HD stromal cells to support organoid growth. Notably, direct treatment of organoids with ibuprofen showed no effect in the absence of stromal cells ( Figure 3E , Figure 3F ). This means that the positive effects of ibuprofen are related to its interaction with stromal cells rather than a direct effect on organoids. The above findings suggest that ibuprofen is a potential candidate therapeutic drug to alleviate the disruption of HD epithelial regeneration by inhibiting proinflammatory stromal cell populations, thereby preventing disease progression to HAEC.

[0080] 3. Discussion

[0081] HD is a severe, life-threatening disease that is often associated with HAECs, yet is poorly understood. This study revealed three key insights: (1) In HD patients, regardless of whether they have HAECs or not, the distal ganglionated colon consistently shows impaired epithelial regeneration; (2) Stromal cell types and composition are significantly altered in HD patients, with proinflammatory stromal 4 cells specifically present in the distal ganglionated colon; and (3) Stromal cells derived from the distal ganglionated colon of HD patients are unable to sustain healthy organoid growth. Notably, application of ibuprofen inhibited stromal 4 cells and successfully restored epithelial organoid growth.

[0082] In this study, we observed a significant reduction in both active stem cell populations and epithelial proliferation in the distal ganglionated colon of HD patients. Early diagnosis and prevention of HAEC pose a major challenge due to its complexity. It is of great interest that seemingly healthy ganglionated colon segments, although without obvious epithelial damage, may show significantly reduced levels of Wnt / β-catenin signaling and impaired regenerative capacity. This reduction in regenerative capacity may limit the effective response of the colonic epithelium to injury, thereby increasing the risk of HAEC development.

[0083] The inflammation-associated stromal 4 cell population is not present in the aganglionic rectal tissue, but is specifically present in the adjacent distal ganglionated colon. In vitro experiments have confirmed that stromal cells from this segment of HD patients hinder the growth of epithelial organoids from non-HD patients, indicating that stromal 4 cells in the distal colon may cause the epithelial damage observed in HAEC. Intervention experiments with the nonsteroidal anti-inflammatory drug ibuprofen suggest that this class of drugs has the function of specifically inhibiting stromal 4 cells, providing a potential therapeutic approach to reduce human intestinal damage using stromal 4 cell inhibitors. Further exploration of the emergence of stromal 4 cell subpopulations in the distal ganglionated colon of HD and their role in coordinating the microenvironment of this segment is essential for a deeper understanding of the pathogenesis of HAEC. In addition, staining the edges of the resected intestinal segments of HD patients with the stromal 4 cell marker MMP1 may be an innovative method to provide valuable guidance for the determination of surgical margins, help formulate more precise and tailored surgical plans, and also serve as a predictive biomarker to assess the possibility of HAEC occurrence and development. It is worth noting that this study not only successfully identified the matrix 4 cell population, but also demonstrated the therapeutic potential of drugs that specifically target and inhibit this cell population. This discovery brings great hope for the treatment of HAEC and provides an important theoretical basis for further exploring its potential clinical application.

[0084] This study found that changes in epithelial regenerative capacity preceded the onset of enterocolitis-like intestinal damage, and regenerative dysfunction may be a contributing factor to the development of HAEC, or even its precursor. Changes in the composition of stromal cells, especially the appearance of proinflammatory stromal 4 cells, suggest that stromal cell remodeling may be a warning change in the distal ganglionated colon of HD, which has a significant role in promoting the subsequent occurrence and development of HAEC. Understanding this pathogenic progression from congenital megacolon to congenital megacolon-related enterocolitis and its related mechanisms are of great clinical significance for advancing early diagnosis and treatment strategies.

[0085] 4. Legend

[0086] Figure 1A to Figure 1G: Intestinal regeneration disorder in the colon of HAEC patients. (A) Representative neuronal marker TUJ1-stained tissue micrographs of the proximal, distal, and aganglionic rectum of HAEC patients; (B) Representative H&E-stained tissue micrographs of the proximal, distal, and rectum of HAEC patients; (C) Histological injury grading scores of the proximal, distal, and rectum of HAEC patients; (D) Representative micrographs of Ki67 immunohistochemical staining of the proximal, distal, and rectum of HAEC patients; (E) Quantification of Ki67-positive cells in each crypt of all colon regions of HAEC patients; (F) Representative immunohistochemical staining micrographs of Lgr5 in the proximal, distal, and rectum of HAEC patients, with arrows indicating positively stained cells; (G) Quantification of Lgr5-positive cells in each crypt of three colon regions of HAEC patients.

[0087] Figure 2A to Figure 2H :Single-cell sequencing found that there were significant changes in stromal cell components in the distal dilated intestinal tissue of HD patients. (A) UMAP diagram showing stromal cell subtype clusters, including Stromal1, Stromal 2, Stromal 3. Stromal4, Stromal_cycling, and Stromal_Transitional are pro-inflammatory stromal 1 cells, pro-inflammatory stromal 2 cells, pro-inflammatory stromal 3 cells, pro-inflammatory stromal 4 cells, cycling stromal cells, and transient stromal cells, respectively; (B) Visualization heat map of gene expression of stromal cell subtype-specific markers; (C) Violin plot of the activity of signaling pathways inferred by UCell in different stromal cell subtypes; (D) Bar chart of the percentage of stromal 4 cells to the total number of stromal cells in colon tissues of different individuals; (E) Representative immunofluorescence staining micrographs of MMP1 in distal ganglion cell-containing intestinal segments of non-HD patients and HD patients, with arrows indicating positively stained cells; (F) Quantification of MMP1-positive cells in each crypt in the distal colon and rectum of non-HD patients and HD patients; (G) Representative micrographs of stromal cells from the distal colon of non-HD patients and HD patients on days 1 and 5 of in vitro culture; (H) Growth curves of stromal cells from the distal colon of non-HD patients and HD patients, where **p<0.01,

[0088] ***p<0.001.

[0089] Figure 3A to Figure 3F:In vitro experiments confirmed that MMP1+ stromal cells cannot support organoid growth, and ibuprofen can exert significant anti-inflammatory function. (A) Representative micrographs of distal colon epithelial organoids from non-HD patients and HD patients cultured in culture medium enriched with recombinant "WENR" growth factors Wnt3a, epidermal growth factor (EGF), Noggin, and Rspo1; (B) Representative micrographs of distal colon epithelial organoids from non-HD patients and HD patients co-cultured with distal colon stromal cells from non-HD patients and HD patients in the absence of "WENR" growth factors; (C) MMP1+ stromal cells from the distal colon of HD patients treated with or without ibuprofen. + Immunofluorescence staining micrographs of stromal 4 cells; (D) Quantitative data graph of the percentage of MMP1-positive cells relative to the total number of HD distal colon stromal cells treated with or without ibuprofen, ***p<0.001; (E) Representative micrographs of non-HD patient epithelial organoids co-cultured with HD patient distal colon stromal cells pretreated with or without ibuprofen in the absence of recombinant growth factors (Wnt3a, EGF, Noggin and Rspo1, WENR); (F) Quantitative data graph of the number of organoids in each culture condition.

[0090] The above description is only a specific embodiment of the present disclosure, so that those skilled in the art can understand or implement the present disclosure. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present disclosure. Therefore, the present disclosure will not be limited to the embodiments described herein, but will conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Application of pro-inflammatory stromal 4 cell inhibitor in the preparation of drugs for the treatment of intestinal epithelial regeneration disorders.

2. Application of pro-inflammatory stromal 4 cell inhibitor in the preparation of drugs for the treatment of congenital megacolon.

3. Application of pro-inflammatory stromal 4 cell inhibitor in the preparation of drugs for the treatment of enterocolitis.

4. The use according to claim 3, characterized in that: The enterocolitis is Hirschsprung's disease-associated enterocolitis.

5. The use according to any one of claims 1 to 4, characterized in that: The pro-inflammatory stromal 4 cell inhibitor is ibuprofen.

6. Use of a substance for detecting MMP1 in the preparation of a product for predicting Hirschsprung's disease-related enterocolitis.

7. The use according to claim 6, characterized in that: The substance for detecting MMP1 includes an antibody against MMP1.

8. The use according to claim 7, characterized in that: The substance for detecting MMP1 also includes a secondary antibody capable of binding to the antibody against MMP1.

9. The use according to claim 8, characterized in that: The secondary antibody is a fluorescently labeled secondary antibody.

10. A kit for predicting Hirschsprung's disease-related enterocolitis, characterized in that: The kit contains a substance for detecting MMP1.

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