Application of Gli1 + interstitial cell or SMOC2 protein thereof in diagnosis, treatment and prognosis evaluation of Crohn disease intestinal stenosis

Abstract

The invention discloses application of Gli1 + interstitial cells or SMOC2 protein thereof in diagnosis, treatment and prognosis evaluation of Crohn disease intestinal stenosis. The invention provides application of Gli1 + interstitial cells and SMOC2 protein as targets in preparation of a diagnostic kit or a therapeutic drug for Crohn disease fibrostenosis, and further provides the diagnostic kit for Crohn disease fibrostenosis and the therapeutic drug for Crohn disease fibrostenosis. Meanwhile, the invention further provides application of the reagent for detecting the expression quantity of the SMOC2 in preparation of the kit for evaluating postoperative recurrence of the Crohn disease fibrostenosis and the corresponding kit for evaluating postoperative recurrence of the Crohn disease fibrostenosis. The invention discusses the effect of the Gli1 + interstitial cells in the attack of intestinal fibrostenosis, evaluates the potential of the Gli1 + interstitial cells as a diagnosis and treatment target, and provides a new thought for the treatment of Crohn disease intestinal stenosis.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and biodiagnosis technology, and relates to the use of Gli1+ interstitial cells or their SMOC2 proteins as targets in the treatment or diagnosis of intestinal stenosis in Crohn's disease, and also relates to the use of SMOC2 protein as a predictive biomarker for postoperative recurrence of Crohn's disease. Background Art

[0002] Crohn's disease (CD) is a chronic, relapsing gastrointestinal disorder. Over 50% of patients will develop clinically significant fibrostenotic lesions during their lifetime, with the terminal ileum being the most commonly affected site. According to the latest expert consensus, fibrostenosis in Crohn's disease is defined as luminal narrowing, pre-stenotic dilatation, and intestinal wall thickening. Endoscopic fibrostenosis is generally considered to be diagnosed when an adult or pediatric colonoscope cannot be passed smoothly through the stricture under reasonable pressure. Fibrostenosis typically manifests as a mixed state of inflammation and fibrosis, containing inflammatory, fibrotic, and muscular components.

[0003] Clinically, most patients with fibrostenotic Crohn's disease have clinical symptoms, while approximately 20% are asymptomatic. Symptoms of fibrostenotic Crohn's disease include abdominal distension, intestinal cramping, dietary restrictions or changes, vomiting, and abdominal pain after eating and after meals.

[0004] Methods used to diagnose fibrostenotic Crohn's disease include non-intraluminal abdominal computed tomography (CT), CT enterography (CTE), intraluminal ultrasound (IUS), non-intraluminal abdominal magnetic resonance imaging (MRI), magnetic resonance enterography (MRE), endoscopy, intraoperative evaluation, and full-thickness histopathology. However, no cross-sectional imaging technique can accurately assess the extent of fibrostenotic Crohn's disease, nor are there clinically validated biomarkers for early diagnosis.

[0005] Treatments for primary and postoperative anastomotic fibrostenosis include medical therapy, endoscopic therapy, and surgery. However, there are currently no proven specific antifibrotic drugs; more than 50% of patients with intestinal fibrostenosis require surgical treatment, and more than half of these patients experience recurrence after surgery.

[0006] Gli1-positive stromal cells (Gli1-positive stromal cells) are a type of interstitial cell that express the transcription factor Gli1 and are found in various tissues. They play a crucial role in tissue homeostasis, injury repair, and fibrosis. Recently, a study first identified Gli1 as a marker for perivascular MSC-like cells in mouse incisors. Using the Gli1-CreERT2 gene for in vivo tracing in mice, Gli1-positive incisor cells were found to possess typical MSC surface markers and trilineage differentiation capacity. Subsequent studies further demonstrated that perivascular Gli1-positive cells from the bone marrow, muscle, heart, lung, liver, and kidney of mice express typical mesenchymal stem cell markers in vivo and exhibit characteristics of plasticity. Subsequently, Gli1-positive stromal cells have been shown to play a key role in the pro-fibrotic pathogenesis of various organs, including renal fibrosis, myelofibrosis, pulmonary fibrosis, hepatic fibrosis, and cardiac fibrosis in chronic kidney disease. Gli1+ interstitial cells are found in the intestine near intestinal crypts, regulating the niches of intestinal stem cells and epithelial cells, and playing a crucial role in maintaining intestinal cellular homeostasis. However, the role of Gli1+ interstitial cells in the pathogenesis of intestinal fibrosis remains unclear, and their potential therapeutic value awaits further investigation. Summary of the Invention

[0007] This invention addresses the aforementioned challenges and addresses the technical challenges of Crohn's disease-induced intestinal fibrostenosis, a key clinical challenge in early diagnosis and targeted precision treatment. Given the lack of early diagnostic biomarkers, a shortage of therapeutic drugs, and a high recurrence rate after surgery, which results in poor prognosis and quality of life for these patients, this invention aims to explore the role of Gli1+ interstitial cells in the pathogenesis of intestinal fibrostenosis and evaluate their potential as diagnostic and therapeutic targets.

[0008] The research process of this invention is as follows: First, a mouse model of intestinal fibrosis caused by chronic DSS and chronic TNBS was established. Analysis showed that Gli1+ interstitial cells are highly expressed in patients with Crohn's disease fibrostenosis and have a profibrotic effect. Cell ablation can alleviate intestinal fibrosis, making them a potential diagnostic and therapeutic target. Further analysis revealed that the specific secretion of SMOC2 protein by Gli1+ interstitial cells is a key factor in this process. Next, a cohort of Crohn's disease patients undergoing intestinal stricture surgery was established. Analysis showed that SMOC2 expression levels in fibrotic areas were significantly increased in patients with Crohn's disease who relapsed after surgery. Patients with high SMOC2 expression levels had a postoperative recurrence rate significantly exceeding 60%, suggesting that SMOC2 may serve as a predictive biomarker for postoperative recurrence of Crohn's disease and a potential therapeutic target.

[0009] Based on the above research, the technical solutions specifically adopted in the present invention are as follows:

[0010] Based on the research results of animal model experiments, the first aspect of the present invention provides the use of Gli1+ interstitial cells as a target in the preparation of a diagnostic kit or therapeutic drug for Crohn's disease fibrostenosis.

[0011] Preferably, the diagnostic kit for Crohn's disease fibrostenosis comprises a reagent for detecting Gli1+ interstitial cells or their secretions; and the therapeutic drug for Crohn's disease fibrostenosis is a reagent for ablating Gli1+ interstitial cells or inhibiting the expression of secretions.

[0012] Furthermore, the reagent for detecting Gli1+ interstitial cells is selected from immunohistochemistry reagents, Masson's reagent, or immunofluorescence staining reagents for intestinal tissue testing. The secretion is SMOC2 protein, and the reagent for detecting the secretion is selected from a reagent that specifically detects SMOC2 gene or protein expression. The reagent for inhibiting secretion expression is selected from SMOC2 shRNA or recombinant expression vector, siRNA or recombinant expression vector, small molecule inhibitor, or antibody.

[0013] The reagent for detecting the expression level of the SMOC2 gene is selected from a PCR system, a probe or a high-throughput sequencing system for detecting the SMOC2 gene, wherein the primer sequences for detecting SMOC2 in the PCR system are shown in SEQ ID NOs. 1 and 2 below:

[0014] Forward primer: 5′-AAAGATCCCCAGCTAGAGATTGC -3′ (SEQ ID NO. 1);

[0015] Reverse primer: 5′-TAGGTGCCGTCGTCATTGC-3′ (SEQ ID NO. 2).

[0016] The reagent for detecting the expression of SMOC2 protein is selected from immunohistochemical staining reagents.

[0017] Experimental results in animal models show that the number of Gli1+ cells increases significantly in fibrotic intestinal models; ablation of these cells in vivo significantly reduces intestinal fibrosis, suggesting that increasing Gli1+ cells is a potential diagnostic and therapeutic target. SMOC2 protein expression increases significantly in the intestines of mice induced by chronic DSS, but decreases after Gli1+ cell ablation. Furthermore, SMOC2 protein expression is significantly elevated in fibrotic areas of Crohn's disease patients. This demonstrates for the first time that both Gli1+ cells and SMOC2 can serve as diagnostic and therapeutic targets.

[0018] In a second aspect, the present invention provides a diagnostic kit for Rohn's disease fibrostenosis, comprising a reagent for detecting Gli1+ interstitial cells or a reagent for detecting SMOC2 protein secreted by Gli1+ interstitial cells;

[0019] The reagent for detecting Gli1+ interstitial cells is selected from immunohistochemistry reagents, Masson reagents or immunofluorescence staining reagents for detecting intestinal tissue; the reagent for detecting secretory SMOC2 protein is selected from reagents that specifically detect the expression level of SMOC2 gene or protein.

[0020] In a third aspect, the present invention provides a drug for treating Crohn's disease fibrostenosis, comprising an active ingredient and a pharmaceutically acceptable excipient. The active ingredient is an agent that ablates Gli1+ interstitial cells or inhibits SMOC2 expression. Preferably, the agent that inhibits SMOC2 expression is selected from SMOC2 shRNA or recombinant expression vectors, siRNA or recombinant expression vectors, small molecule inhibitors, or antibodies.

[0021] Based on the research results of the surgical cohort, the fourth aspect of the present invention provides the use of a reagent for detecting the expression level of SMOC2 in the preparation of a kit for evaluating the postoperative recurrence of Crohn's disease fibrostenosis.

[0022] Preferably, the reagent for detecting the expression level of SMOC2 is selected from a PCR system, a probe or a high-throughput sequencing system for detecting the SMOC2 gene. Further preferably, the primer sequences for detecting SMOC2 in the PCR system are as shown in SEQ ID NOs. 1 and 2 above.

[0023] In a fifth aspect, the present invention provides a kit for assessing recurrence of Crohn's disease fibrostenosis after surgery, which is selected from any one of the following forms:

[0024] (1) comprising a reverse transcription system, a primer system and an amplification system for detecting the SMOC2 gene, wherein the primer system comprises primers for detecting SMOC2, the sequences of which are shown in SEQ ID NOs. 1 and 2;

[0025] (2) Includes immunohistochemical staining reagents for detecting SMOC2 protein.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] This study systematically analyzes the key role of Gli1+ interstitial cells in intestinal fibrosis for the first time. It also demonstrates that specific ablation of Gli1+ interstitial cells can effectively alleviate intestinal fibrosis, providing a new therapeutic target for Crohn's disease-associated intestinal fibrosis. It also proposes that SMOC2 can serve as a biomarker for predicting postoperative recurrence of Crohn's disease and a potential therapeutic target, enabling clinical testing and the development of anti-fibrotic small molecule drugs or antibody therapies. This study provides new insights into the treatment of intestinal strictures in Crohn's disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Showing that Gli1+ interstitial cells proliferate and exhibit a pro-fibrotic phenotype in intestinal fibrosis: Immunofluorescence staining of the intestine of mice induced by chronic DSS and chronic TNBS models, showing the distribution and colocalization of tdTomato and Collagen I (A), and statistical quantitative analysis (B);

[0029] Figure 2 Showing that Gli1+ interstitial cell ablation can alleviate intestinal fibrosis: Immunofluorescence staining of the intestine of mice subjected to chronic DSS model and chronic TNBS model after ablation of Gli1+ interstitial cells, showing the intestinal distribution of Collagen I (A), and statistical quantitative analysis (B);

[0030] Figure 3 The Spearman correlation analysis between Smoc2 and fibrosis-related genes was shown, suggesting that Smoc2 is a potential key gene for Gli1+ interstitial cells to promote fibrosis;

[0031] Figure 4 The results show that Smoc2 is significantly upregulated in fibrotic sites in humans and mice. A: Immunofluorescence staining of the intestine of mice with or without Gli1+ interstitial cell ablation after chronic DSS enteritis modeling shows the distribution and colocalization of tdTomato, SMOC2, and Collagen I. B: qPCR analysis of intestinal tissue of mice with or without Gli1+ interstitial cell ablation after chronic DSS enteritis modeling shows the statistical changes in Smoc2 gene mRNA expression levels. C: Immunofluorescence staining of fibrotic and non-fibrotic sites of surgical tissue from Crohn's disease patients shows the distribution and colocalization of αSMA, SMOC2, and Collagen I.

[0032] Figure 5Figure 3 shows the relationship between SMOC2 expression levels and postoperative recurrence in Crohn's disease patients: A, Schematic diagram of the surgical cohort for patients with Crohn's disease fibrostenosis; B, qPCR analysis of intestinal tissue from patients with Crohn's disease fibrostenosis after surgery, showing statistical differences in SMOC2 mRNA expression levels in fibrotic and nonfibrotic areas of the intestine between patients with recurrence and those without recurrence; C, Kaplan-Meier survival curves based on SMOC2 and CO L1A1 mRNA expression levels in surgical tissue from patients with Crohn's disease; D, SMOC2 immunohistochemical staining. *, P < 0.05; **, P < 0.01; ***, P < 0.001; **, P < 0.0001. DETAILED DESCRIPTION

[0033] To make the purpose, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the described embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0034] Example 1 Experimental samples and animal model construction

[0035] 1. Experimental samples

[0036] This study enrolled surgical specimens from patients with Crohn's disease (CD) intestinal strictures (including both non-fibrotic and fibrotic sites). Gli1-CreERT2;R26-tdTomato, Gli1-CreERT2;iDTR, and Gli1-CreERT2;Smoc2-Flox transgenic mice were generated and subjected to chronic DSS and chronic TNBS intestinal fibrosis models for in vivo lineage tracing and cell ablation. Gli1+ interstitial cells were isolated by flow cytometry combined with Smart-seq sequencing to reveal their pathogenic characteristics. A surgical cohort of patients with Crohn's disease intestinal strictures was established, and intestinal tissue was collected for further analysis.

[0037] 2. Animal model construction

[0038] Crossing Gli1CreERT2 mice with Rosa26tdTomato mice yielded Gli1-CreERT2, R26-tdTomato bigenic mice, enabling lineage tracing of Gli1+ cells in vivo. Crossing Gli1CreERT2 mice with iDTR mice yielded Gli1-CreERT2, iDTR bigenic mice, enabling in vivo ablation of Gli1+ cells. Crossing Gli1CreERT2 mice with Smoc2-Flox mice yielded Gli1-CreERT2, Smoc2-Flox bigenic mice, enabling in vivo knockout of the Smoc2 gene in Gli1+ cells. Transgenic mice were subjected to a chronic intestinal inflammation model, including three cycles of DSS treatment or six cycles of TNBS treatment. Control mice were simultaneously treated with water.

[0039] Example 2 Gli1+ interstitial cells are key factors in intestinal stricture in Crohn's disease

[0040] Immunofluorescence staining of the intestines of mice treated with chronic DSS and TNBS models revealed the distribution and colocalization of tdTomato and Collagen I, and statistical quantitative analysis was performed. Immunofluorescence staining of the intestines of mice treated with chronic DSS and TNBS models, following in vivo ablation of Gli1+ interstitial cells, revealed the intestinal distribution of Collagen I, and statistical quantitative analysis was performed.

[0041] The results showed that the number of Gli1+ cells in the fibrotic model intestine increased significantly ( Figure 1 After ablation of Gli1+ cells in vivo, the degree of intestinal fibrosis was significantly reduced ( Figure 2 ).

[0042] Example 3 SMOC2 protein is a key factor in Gli1+ interstitial cells promoting intestinal fibrosis

[0043] Gli1-CreERT2; R26-tdTomato transgenic mice were subjected to chronic DSS and TNBS treatments, and Gli1+ and Gli1- cells in the mouse intestine were subsequently sorted by flow cytometry for SMART-seq sequencing. Immunofluorescence staining was performed on samples from patients undergoing intestinal stricture surgery for Crohn's disease.

[0044] SMART-seq sequencing results suggest that Gli1+ cells have profibrotic functions, and Smoc2 is a key molecule ( Figure 3). SMOC2 protein expression increased significantly in the intestine of mice after chronic DSS modeling, and decreased after deleting Gli1+ cells. SMOC2 protein expression was also significantly increased in fibrotic areas of Crohn's disease patients ( Figure 4 ). This suggests that Gli1+ interstitial cells specifically secrete SMOC2 protein, which promotes fibrosis through its endocrine and paracrine functions.

[0045] Example 4 SMOC2 can be used as a predictive biomarker for postoperative recurrence of Crohn's disease and is a potential therapeutic target

[0046] A surgical cohort of patients with Crohn's disease (CD) undergoing intestinal stricture surgery was established. Patients with Crohn's disease who underwent intestinal resection for fibrotic strictures were enrolled. The resection included the stricture area and the adjacent non-fibrotic terminal ileum. SMOC2 and the traditional fibrosis marker COL1A1 were detected in intestinal tissue by qPCR, and SMOC2 tissue expression was assessed by immunohistochemistry.

[0047] Among them, the primer sequences for detecting SMOC2 are as follows:

[0048] Human SMOC2:

[0049] Forward: 5'-AAAGATCCCCAGCTAGAGATTGC-3' (SEQ ID NO.1);

[0050] Reverse: 5'-TAGGTGCCGTCGTCATTGC-3' (SEQ ID NO. 2);

[0051] Mouse Smoc2:

[0052] Forward: 5'-AGTGGAGACATTGGCAAGAAG-3' (SEQ ID NO.3);

[0053] Reverse: 5'-ACACACTTTTTGGGCTTGGATT-3' (SEQ ID NO. 4).

[0054] The experimental results showed that in patients with Crohn's disease who relapsed after surgery, the expression level of SMOC2 in the fibrosis area increased most significantly, significantly higher than the expression level of COL1A1; patients with high SMOC2 expression levels had a postoperative recurrence rate of significantly more than 60% ( Figure 5 ).

[0055] The technical advantages of the present invention are as follows:

[0056] (1) For the first time, we systematically analyzed the key role of Gli1+ interstitial cells in intestinal fibrosis.

[0057] (2) For the first time, it was confirmed that specific ablation of Gli1+ interstitial cells can effectively alleviate intestinal fibrosis, providing a new therapeutic target for Crohn's disease-related intestinal fibrosis.

[0058] (3) It was proposed for the first time that SMOC2 can be used as a biomarker for predicting postoperative recurrence of Crohn's disease and a potential therapeutic target, which can be used for clinical testing and the development of anti-fibrotic small molecule drugs or antibody therapies.

[0059] The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the described embodiments. Those skilled in the art may make various equivalent modifications or substitutions without departing from the spirit of the present invention. These equivalent modifications or substitutions are all included in the scope defined by the claims of this application.

Claims

1. Application of Gli1+ interstitial cells as targets in the preparation of diagnostic kits or therapeutic drugs for Crohn's disease fibrostenosis.

2. The use according to claim 1, characterized in that The Crohn's disease fibrostenosis diagnostic kit comprises a reagent for detecting Gli1+ interstitial cells or their secretions in intestinal tissue.

3. The use according to claim 2, characterized in that The reagent for detecting Gli1+ interstitial cells is selected from immunohistochemical reagents, Masson reagents or immunofluorescence staining reagents for detecting intestinal tissue; The secretion is SMOC2 protein, and the reagent for detecting the secretion is selected from a reagent for specifically detecting the expression level of SMOC2.

4. The use according to claim 3, characterized in that The reagent for detecting the expression level of SMOC2 is selected from any one of the following forms: (1) A PCR system, probe or high-throughput sequencing system for detecting the SMOC2 gene, wherein the primer sequences for detecting SMOC2 in the PCR system are as shown in SEQ ID NOs. 1 and 2; (2) Immunohistochemical staining reagent for detecting SMOC2 protein.

5. A diagnostic kit for Rohn's disease fibrostenosis, characterized in that: Including reagents for detecting Gli1+ interstitial cells or reagents for detecting SMOC2 protein secreted by Gli1+ interstitial cells; The reagent for detecting Gli1+ interstitial cells is selected from immunohistochemistry reagents, Masson reagents or immunofluorescence staining reagents for detecting intestinal tissue; and the reagent for detecting secretory SMOC2 protein is selected from reagents that specifically detect SMOC2 expression.

6. The use according to claim 1, characterized in that The drug for treating Crohn's disease fibrostenosis is an agent that ablates Gli1+ interstitial cells or inhibits SMOC2 expression.

7. A pharmaceutical composition for treating Crohn's disease fibrostenosis, characterized in that: The invention comprises an active component and a pharmaceutically acceptable excipient, wherein the active component is an agent for ablating Gli1+ interstitial cells or inhibiting SMOC2 expression. The agent for inhibiting SMOC2 expression is selected from shRNA or recombinant expression vector, siRNA or recombinant expression vector of SMOC2. delivery vehicles, small molecule inhibitors or antibodies.

8. Application of reagents for detecting SMOC2 expression in the preparation of a kit for evaluating the recurrence of Crohn's disease fibrostenosis after surgery.

9. The use according to claim 8, characterized in that The reagent for detecting the expression level of SMOC2 is selected from a PCR system, a probe or a high-throughput sequencing system for detecting the SMOC2 gene, or an immunohistochemical staining reagent for detecting the SMOC2 protein. The primer sequences for detecting SMOC2 in the PCR system are shown in SEQ ID NOs. 1 and 2.

10. A kit for evaluating recurrence of Crohn's disease fibrostenosis after surgery, characterized in that: The kit is selected from any one of the following forms: (1) It includes a reverse transcription system, a primer system and an amplification system for detecting the SMOC2 gene, wherein the primer system includes The primers for detecting SMOC2 have the sequences shown in SEQ ID NOs. 1 and 2; (2) Includes immunohistochemical staining reagents for detecting SMOC2 protein.

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