Use of mesenteric adipose tissue exosomes in crohn's disease in evaluating the level of intestinal fibrosis in crohn's disease and constructing an animal model of intestinal fibrosis in crohn's disease

Abstract

The present application belongs to the technical field of biology, and particularly relates to the use of Crohn's disease mesenteric fat exosomes in evaluating the level of intestinal fibrosis of Crohn's disease and constructing an animal model of intestinal fibrosis of Crohn's disease. miR-184 can be used as a risk assessment marker for intestinal fibrosis associated with Crohn's disease, and can also be used as a tool for intervening in an animal model of Crohn's disease to intervene in levels such as intestinal fibrosis. NCOR2 protein can be used as a risk assessment marker for intestinal fibrosis associated with Crohn's disease, and can also be used as a tool for intervening in an animal model of Crohn's disease to intervene in levels such as intestinal fibrosis. The present disclosure verifies the feasibility of constructing an experimental model scheme for Crohn's disease with intestinal fibrosis based on the above two target points, and provides support for deepening the research on Crohn's disease, especially deepening the understanding of the pathological process of intestinal fibrosis secondary to Crohn's disease.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and in particular relates to the use of Crohn's disease mesenteric fat exosomes in assessing the level of intestinal fibrosis in Crohn's disease and in constructing an animal model of intestinal fibrosis in Crohn's disease. Background Technology

[0002] Crohn's disease is a chronic, relapsing inflammatory bowel disease. As the disease progresses, patients often develop complications such as intestinal stricture and intestinal fibrosis, which severely impact their quality of life and significantly increase the risk of surgical resection. Currently, there are still no effective clinical interventions for intestinal fibrosis, and its pathogenesis is not fully understood. Therefore, exploring the molecular mechanisms of intestinal fibrosis in Crohn's disease is of great significance.

[0003] Recent studies have revealed that creeping fat in the mesentery is a key pathological feature of Crohn's disease. It manifests as abnormal proliferation of mesenteric adipose tissue surrounding the diseased intestinal segment, and the degree of creeping fat formation is closely related to disease activity, intestinal stenosis, and fibrosis. Increasing evidence suggests that creeping fat is not merely a symptom of inflammation, but rather an important regulatory tissue actively involved in the pathological processes of the intestine.

[0004] Exosomes are nanoscale membrane vesicles actively secreted by cells, carrying various bioactive molecules such as miRNAs, proteins, and lipids. They participate in pathological processes such as inflammation, immunity, and tissue remodeling through intercellular communication. Previous studies have suggested that adipose-derived exosomes may participate in liver, lung, and tumor-associated fibrosis. However, systematic research on the role and molecular mechanisms of crawling adipose-derived exosomes in intestinal fibrosis in Crohn's disease remains lacking.

[0005] Existing research has mainly focused on the regulatory role of crawling fat on fibroblast activation and extracellular matrix deposition. However, increasing evidence suggests that intestinal fibrosis is not simply a matter of abnormal fibroblast activation, but a dynamic pathological process involving multiple cell types and complex signaling networks. In particular, the role of epigenetic regulatory mechanisms in organ fibrosis has received increasing attention in recent years, with abnormal histone acetylation participating in disease progression by regulating the transcription of fibrosis-related genes.

[0006] Therefore, further exploration of the role of crawling fat-derived exosomes in intestinal fibrosis in Crohn's disease, and whether they participate in the progression of intestinal fibrosis by regulating histone acetylation, is of great significance for revealing novel pathogenesis of intestinal fibrosis in Crohn's disease and developing new anti-fibrotic treatment strategies. Summary of the Invention

[0007] To solve the above problems, the present invention adopts the following technical solution: The first aspect of this invention provides novel uses for miR-184-3p or miR-184. miR-184-3p or miR-184 can serve as a risk assessment biomarker for Crohn's disease accompanied by intestinal fibrosis, and can also be used as a tool to intervene in animal models of Crohn's disease to address intestinal fibrosis and other related conditions. It should be noted that the sequences of miR-184 and miR-184-3 are both UGGACGGAGAACUGAUAAGGGU. The non-coding RNA derived from humans is conventionally named miR-184, while the non-coding RNA derived from mice is conventionally named miR-184-3p. Further details are provided below: One application of reagents for detecting miR-184 expression levels in the preparation of products for analyzing the risk of Crohn's disease complicated by intestinal fibrosis. Specifically, miR-184 expression levels are positively correlated with the risk of Crohn's disease complicated by intestinal fibrosis (or positively correlated with the risk of developing Crohn's disease complicated by intestinal fibrosis). Elevated miR-184 expression levels indicate an increased risk of Crohn's disease complicated by intestinal fibrosis or the presence of Crohn's disease complicated by intestinal fibrosis, while decreased miR-184 expression levels indicate a decreased risk. In some cases, the miR-184 is derived from mesenteric crawling fat, and exosomes derived from mesenteric crawling fat can significantly promote the progression of intestinal fibrosis and induce extensive pro-fibrotic molecular remodeling. Furthermore, miR-184 can be derived from mesenteric crawling fat exosomes, allowing for analysis of miR-184 expression levels at specific sites to better characterize the risk of intestinal fibrosis. Elevated or decreased miR-184 expression levels can be referenced against normal levels and can be adjusted according to clinical changes. For individuals, the risk can also be assessed by observing changes in their own miR-184 expression levels over a period of time. Regarding the location of intestinal fibrosis, it can be intestinal fibrosis of the colon.

[0008] Secondly, building upon the aforementioned, reagents for detecting miR-184 expression levels can also be used to screen individuals at risk of Crohn's disease with intestinal stenosis. Analyzing miR-184 expression levels helps assess the risk of Crohn's disease with intestinal stenosis. Similarly, elevated miR-184 expression levels indicate an increased risk of Crohn's disease with intestinal stenosis, classifying individuals as high-risk; conversely, decreased miR-184 expression levels indicate an increased risk, classifying individuals as low-risk. Both increases and decreases in miR-184 expression levels can be referenced to normal levels and can be adjusted according to clinical changes. For individuals, risk can also be assessed by observing changes in their own miR-184 expression levels over a period of time. Regarding the location of intestinal fibrosis, it can be intestinal fibrosis of the colon.

[0009] Thirdly, reagents that upregulate miR-184-3p expression levels are used in products designed to aggravate the severity of intestinal damage or fibrosis in Crohn's disease animal models. This is primarily used to worsen the severity of intestinal damage or fibrosis associated with Crohn's disease. Alternatively, reagents that upregulate miR-184-3p expression levels are used in products designed to aggravate the severity of intestinal damage or fibrosis associated with Crohn's disease in animal models. Upregulating miR-184-3p expression levels can increase the severity of intestinal fibrosis or fibrosis associated with Crohn's disease in these models. When using these reagents, miR-184-3p enhancement can be administered simultaneously during the construction of the Crohn's disease animal model. For example, enhancing miR-184-3p during DNBS treatment of mice can induce Crohn's disease symptoms and exacerbate intestinal fibrosis. The location of intestinal fibrosis can be in the colon. Regarding methods to intervene in miR-184-3p expression and reduce its expression, the reagent that upregulates miR-184-3p expression is miR-184-3p agomir (miR-184-3p agomir is a chemically modified double-stranded small RNA preparation), which can be achieved by exogenously supplementing miR-184-3p. Specifically, miR-184-3p agomir can be used to intervene in animal models via injection. miR-184-3p agomir can be directly constructed by commercial biotechnology companies, as long as it has the ability to enhance miR-184-3p expression.

[0010] A second aspect of this invention provides novel uses for the NCOR2 protein. The NCOR2 protein can serve as a risk assessment biomarker for Crohn's disease complicated by intestinal fibrosis, and also as a tool for intervention in animal models of Crohn's disease to address intestinal fibrosis and other related conditions. This is further described in detail below: One application of reagents for detecting NCOR2 protein expression levels is in the preparation of products for analyzing the risk of Crohn's disease complicated by intestinal fibrosis. Decreased NCOR2 protein expression levels are associated with an increased risk of Crohn's disease complicated by intestinal fibrosis. In some cases, NCOR2 protein expression levels are negatively correlated with the degree of risk of Crohn's disease complicated by intestinal fibrosis; elevated NCOR2 expression levels indicate a decreased risk, while decreased NCOR2 expression levels indicate an increased risk or the presence of intestinal fibrosis. In some cases, the NCOR2 originates from intestinal tissue; more specifically, NCOR2 may originate from fibroblasts in intestinal tissue (such as the numerous COL1A2-positive fibroblasts in the intestine). Analysis can target NCOR2 expression levels at specific sites to better characterize the risk of intestinal fibrosis. Both increases and decreases in NCOR2 expression levels can be referenced to normal levels and can be adjusted according to clinical changes. For individuals, risk can also be assessed by observing changes in their own NCOR2 expression levels over a period of time. In terms of the location of intestinal fibrosis, it can be intestinal fibrosis of the colon.

[0011] Secondly, agents that inhibit miR-184-3p expression or miR-184 expression can also be used in the preparation of products that reduce the risk of Crohn's disease with intestinal fibrosis or increase NCOR2 protein expression levels in fibroblasts in vitro. Reducing the risk of Crohn's disease with intestinal fibrosis should also include reducing the severity of Crohn's disease with intestinal fibrosis, and can also reduce the likelihood of developing Crohn's disease with intestinal fibrosis by inhibiting miR-184-3p expression or miR-184 expression. In vitro, inhibiting miR-184-3p expression or miR-184 expression can interfere with NCOR2 protein expression levels in fibroblasts; specifically, this can be manifested as an increase in NCOR2 protein expression levels after inhibiting miR-184-3p expression or miR-184 expression. Therefore, inhibiting miR-184-3p expression or miR-184 expression can be used as a tool to regulate NCOR2 protein expression in in vitro fibroblast models. For example, when it is necessary to increase NCOR2 protein expression in fibroblast models, this can be achieved by inhibiting miR-184-3p or miR-184 expression. Of course, this application is primarily for non-diagnostic and therapeutic purposes. However, where legally permissible, inhibiting miR-184-3p or miR-184 expression can also be used to adjust NCOR2 protein expression in in vivo fibroblast models, and it is even expected that adjusting NCOR2 protein expression can achieve some disease remission. The corresponding reagents for inhibiting miR-184 and miR-184-3p expression are designed for use in humans and other animals (such as mice) to achieve the above goals. Inhibition of miR-184-3p or miR-184 expression can be achieved using reagents such as miR-184 inhibitors.

[0012] Thirdly, the application of reagents that inhibit NCOR2 expression in the preparation of animal models of Crohn's disease accompanied by intestinal fibrosis. Lowering NCOR2 expression levels can increase the incidence or severity of intestinal fibrosis in Crohn's disease animal models. When using this approach, NCOR2 inhibition can be administered simultaneously during the construction of the Crohn's disease animal model. For example, administering NCOR2 inhibition to mice treated with DNBS can induce Crohn's disease symptoms accompanied by intestinal fibrosis (specifically, the Crohn's disease animal model with intestinal fibrosis can be obtained by knocking down NCOR2 expression in the animal model followed by DNBS stimulation. Knockdown primarily means reduction, but in animal models, it often means elimination). The location of intestinal fibrosis can be in the colon. Regarding the methods of intervening in NCOR2 expression to reduce its expression, the reagent that inhibits NCOR2 expression level is SiNcor2 (sense,5'-GCUUCACAACACAGGCAUGAA-3'; antisense,5'-UUCAUGCCUGUGUUGUGAAGC-3').

[0013] Fourthly, the application of NCOR2 protein or recombinant NCOR2 protein in the preparation of products that reduce the severity of intestinal fibrosis associated with Crohn's disease. Intervention with NCOR2 protein or recombinant NCOR2 protein can reduce the severity of intestinal fibrosis associated with Crohn's disease, specifically manifested as a significant increase in colon length and a decrease in intestinal fibrosis scores. Commercially available recombinant NCOR2 protein can be directly purchased, as long as it performs the aforementioned functions, regardless of its specific model or type. Furthermore, NCOR2 protein or recombinant NCOR2 protein can be used via enema intervention, such as direct enema administration to animal models.

[0014] It should be noted that the reagents mentioned above that exert inhibitory or expression-promoting effects are not limited to traditional reagent forms. Any product that can exert the corresponding effect should be the same as or equivalent to the scope of this invention.

[0015] This publication clarifies that miR-184-3p, miR-184, and NCOR2 can all serve as effective diagnostic biomarkers for Crohn's disease with intestinal fibrosis. Furthermore, the study reveals a potential pathway to intervene in the progression of Crohn's disease with intestinal fibrosis by targeting and regulating miR-184-3p, miR-184, and NCOR2. In addition, the feasibility of constructing an experimental model of Crohn's disease with intestinal fibrosis based on these two key targets was successfully verified. These findings not only provide new entry points for further in-depth research into the mechanisms of Crohn's disease, but also lay an important theoretical and experimental foundation for a deeper understanding of the complex pathological process of Crohn's disease-related intestinal fibrosis. Attached Figure Description

[0016] Figure 1 DNBS successfully induced intestinal fibrosis in mice, accompanied by significant mesenteric creeping fat proliferation; Figure 2 Figure showing the effect of crawling fat-derived exosomes on intestinal fibrosis; Figure 3 The dual-luciferase reporter gene that miR-184-3p targets and inhibits NCOR2 expression, and the Western blot validation diagram; Figure 4 The receiver operating characteristic (ROC) curve analysis of NCOR2's diagnostic efficacy for intestinal fibrosis is shown in the figure. Figure 5 Receiver operating characteristic (ROC) curve analysis of the diagnostic efficacy of mesenteric fat-derived exosomes miR-184; Figure 6 The changes in NCOR2 expression and histone acetylation modification levels in Crohn's disease and DNBS-induced colitis models; Figure 7 NCOR2 deficiency can significantly exacerbate the progression of intestinal fibrosis under chronic inflammatory conditions; Figure 8 The result of the NCOR2 deficiency activating the profibrotic process in intestinal fibroblasts by enhancing histone acetylation is shown in the figure. Figure 9 Scatter plot of the linear regression fit line and confidence interval between NCOR2 gene and intestinal fibrosis; Figure 10 The effect of NCOR2 gene overexpression or intervention on the progression of intestinal fibrosis in DNBS mice; Figure 11 This is a phenotypic analysis of intestinal structural damage and fibrosis induced by Agomir. Detailed Implementation

[0017] The invention will be further illustrated below with specific research examples.

[0018] I. DNBS successfully induced intestinal fibrosis in mice, accompanied by significant mesenteric creeping fat hyperplasia. Masson and Sirius Red staining showed that, compared with the control group, DNBS treatment significantly increased collagen deposition in the colonic tissue of mice, resulting in marked disordered intestinal wall structure and significant fibrotic changes. Figure 1 (A, B). Quantitative analysis further confirmed that the colon length of mice in the DNBS group was significantly shortened, while the fibrosis score and Sirius Red positive area were significantly increased ( ). Figure 1 (C~E).

[0019] Western blot results showed that DNBS treatment significantly increased the expression of fibrosis-related proteins FN, α-SMA, and COL1A1. Figure 1 The results (F~I) further indicate that intestinal fibrosis has been successfully established.

[0020] In addition, visual observation revealed significant mesenteric fat proliferation in the DNBS group mice. Figure 1 Quantitative analysis showed that the mesenteric fat weight / body weight ratio and mesenteric fat volume were significantly increased in the DNBS group mice. Figure 1 The presence of K,L indicates intestinal fibrosis associated with mesenteric fat hyperplasia in Crohn's disease.

[0021] II. Exosomes derived from crawling fat can significantly promote the progression of intestinal fibrosis and induce extensive pro-fibrotic molecular remodeling. After confirming the significant proliferation of crawling fat, the effects of crawling fat-derived exosomes on intestinal fibrosis were further investigated. When mice were administered control-derived exosomes (Con-Exos) or fibrotic mouse-derived exosomes (CF-Exos) via tail vein injection during DNBS-induced intestinal fibrosis, CF-Exos significantly exacerbated intestinal fibrosis.

[0022] Masson and Sirius Red staining results showed that, compared with the DNBS and Con-Exos groups, the CF-Exos treatment group mice had further increased collagen deposition in the colon tissue, more obvious intestinal wall structural damage, and more severe fibrosis. Figure 2 (A, B). Quantitative analysis further confirmed that the colon length of mice in the CF-Exos group was significantly shortened, while the fibrosis score and Sirius Red positive area were significantly increased, whereas there was no significant difference between the Con-Exos group and the DNBS group. Figure 2 (C~E).

[0023] Immunofluorescence staining results showed that the expression of α-SMA and Vimentin in intestinal tissue was significantly enhanced after CF-Exos treatment. Figure 2 Quantitative analysis also confirmed a significant increase in the fluorescence intensity of α-SMA and Vimentin (F). Figure 2 The presence of G and H in the sample suggests that CF-Exos can promote the formation of a profibrotic phenotype in the gut.

[0024] To further clarify the molecular changes induced by CF-Exos, proteomic sequencing analysis was performed on colon tissue. Differential protein clustering analysis showed that the CF-Exos group and the Con-Exos group had significantly different protein expression profiles. Figure 2GO enrichment analysis indicated that the differentially expressed proteins were mainly enriched in biological processes closely related to fibrosis, such as extracellular structure organization, connective tissue development, muscle cell differentiation, collagen metabolic processes, and cytokine-mediated signaling pathways. Figure 2 KEGG enrichment analysis further revealed that multiple classic pro-fibrosis signaling pathways, including the TGF-β signaling pathway, PI3K-AKT pathway, integrin signaling, focal adhesion, and Hippo signaling pathway, were significantly activated. Figure 2 (Middle K).

[0025] The above results indicate that crawling fat-derived exosomes can significantly promote the progression of intestinal fibrosis and induce extensive profibrotic molecular remodeling.

[0026] 3. NCOR2 is a key downstream target gene of miR-184 and may be involved in the process of intestinal fibrosis mediated by reptilian adipose-derived exosomes. To identify the key miRNAs playing a pro-fibrotic role in exosomes derived from crawling adipose tissue, small RNA sequencing analysis was first performed on exosomes derived from Crohn's disease patients and DNBS-induced fibrosis mice. Volcano plot results showed that miR-184 was significantly upregulated in exosomes derived from Crohn's disease patients (…). Figure 3 (A), while mmu-miR-184-3p was also significantly elevated in exosomes derived from fibrotic mice (A). Figure 3 (B). qPCR results further confirmed that the expression of miR-184 in exosomes from Crohn's disease patients and mmu-miR-184-3p in exosomes from fibrotic mice were significantly increased. Figure 3 (C, D) suggests that miR-184 may be involved in the regulation of intestinal fibrosis.

[0027] To further screen key downstream target genes of miR-184, TargetScan was first used to predict potential target genes of miR-184 and miR-184-3p. Simultaneously, miR-184 inhibitor (miR...) was transfected into fibroblasts... KD ) or negative control (miR- NCKDFollowing this, RNA sequencing and proteomics analysis were performed. Intersection analysis of integrated human exosome, mouse exosome, RNA sequencing, and proteomics data revealed that NCOR2 was the only commonly enriched candidate target gene. Figure 3 (E).

[0028] Further bioinformatics analysis revealed a conserved miR-184 binding site in the 3′UTR region of NCOR2. Figure 3 (F). Dual-luciferase reporter assay results showed that inhibition of miR-184 significantly increased the fluorescence activity of the wild-type 3′UTR reporter gene of NCOR2, while this effect disappeared after mutation of the binding site. Figure 3 The results showed that miR-184 could directly target NCOR2. Western blot results further confirmed that inhibition of miR-184 significantly increased NCOR2 protein expression in fibroblasts. Figure 3 (H, I). The above results indicate that NCOR2 is a key downstream target gene of miR-184 and may be involved in the process of intestinal fibrosis mediated by crawling adipose-derived exosomes.

[0029] Furthermore, to further evaluate the potential diagnostic value of NCOR2 in intestinal fibrosis, we constructed ROC curve analyses of NCOR2 protein expression based on samples from Crohn's disease patients and DNBS-induced fibrotic mice, respectively. The results showed that in Crohn's disease patients, NCOR2 protein expression had good discriminative ability against intestinal fibrosis, with an area under the ROC curve (AUC) of 0.8024 (P < 0.0001). Figure 4 (A). In a DNBS-induced mouse intestinal fibrosis model, NCOR2 also demonstrated high diagnostic efficacy, with ROC curve analysis showing an AUC of 0.8854 (P < 0.0001). Figure 4 (Middle B). The above results further suggest that a decrease in NCOR2 not only participates in the process of intestinal fibrosis, but also has potential value as a biomarker of fibrosis.

[0030] IV. Mesenteric fat-derived exosomes miR-184 have potential biomarker value in the pathological process of intestinal stricture and fibrosis associated with Crohn's disease. To evaluate the ability of mesenteric adipose-derived exosomal miR-184 to distinguish Crohn's disease-related intestinal stenosis, mesenteric adipose tissue was collected from Crohn's disease patients who underwent surgery for intestinal stenosis and from control groups. Mesenteric adipose-derived exosomal tissue was then isolated, and the relative expression level of miR-184 in the exosomal tissue was detected by qPCR. Crohn's disease patients were used as the positive group, and the control group as the negative group. ROC curve analysis was further performed to calculate AUC and statistical significance.

[0031] ROC curve analysis shows that ( Figure 5 Mesenteric fat-derived exosomes miR-184 effectively distinguished Crohn's disease patients with intestinal stenosis from controls (AUC = 0.8056, P < 0.0001). These results suggest that mesenteric fat-derived exosomes miR-184 have potential biomarker value in the pathological processes related to intestinal stenosis and fibrosis in Crohn's disease, further supporting its pathological role in crawling fat-mediated intestinal fibrosis.

[0032] V. NCOR2 deficiency is involved in the formation of abnormal acetylation states during intestinal fibrosis. To further clarify the changes in histone acetylation levels during intestinal fibrosis, the expression levels of H3K9ac, H3K27ac, and NCOR2 in intestinal tissues of Crohn's disease patients and DNBS-induced intestinal fibrosis tissues of mice were detected.

[0033] Western blot results showed that, compared with the control group, the levels of H3K9ac and H3K27ac in the intestinal tissue of Crohn's disease patients were significantly increased, while the expression of NCOR2 was significantly decreased. Figure 6 (A, B). Quantitative analysis further confirmed that the relative expression levels of Acetyl H3K9 and Acetyl H3K27 were significantly increased, while the expression of NCOR2 protein was significantly decreased.

[0034] Similarly, in a DNBS-induced mouse intestinal fibrosis model, significantly elevated levels of H3K9ac and H3K27ac were observed, while NCOR2 expression was significantly decreased. Figure 6 (C, D). The above results suggest that there is significant enhancement of histone acetylation in both human Crohn's disease intestinal tissue and mouse intestinal fibrosis model, accompanied by downregulation of NCOR2, suggesting that NCOR2 deficiency may be involved in the formation of abnormal acetylation state during intestinal fibrosis.

[0035] To further clarify the function of fibroblast-derived Ncor2 in intestinal fibrosis, fibroblast-specific Ncor2 knockout mice were constructed, and a DNBS-induced chronic intestinal fibrosis model was established. Results are as follows: Figure 7 As shown, without DNBS stimulation, Ncor2-deficient mice did not exhibit significant fibrotic changes in their intestinal tissue, and their intestinal fibrosis scores were not significantly different from the control group. However, after DNBS induction, the degree of intestinal fibrosis in fibroblast-specific Ncor2-deficient mice was significantly aggravated, with fibrosis scores significantly higher than the Con + DNBS group. These results indicate that fibroblast-derived Ncor2 plays an important protective role in maintaining intestinal fibrosis homeostasis, and its deficiency can significantly exacerbate the progression of intestinal fibrosis under chronic inflammatory conditions.

[0036] VI. NCOR2 deficiency can activate the transcription of fibrosis-related genes by promoting histone acetylation. To further elucidate the epigenetic mechanism by which NCOR2 regulates intestinal fibrosis, NCOR2 (SiNcor2, sense, 5'-GCUUCACAACACAGGCAUGAA-3'; antisense, 5'-UUCAUGCCUGUGUUGUGAAGC-3') was knocked down in fibroblasts, and changes in histone acetylation levels were analyzed using the CUT&Tag sequencing system.

[0037] Immunofluorescence results showed that intracellular acetyl CoA levels were significantly increased after NCOR2 knockdown, and further increased under TGFβ stimulation. Figure 8 (A, B) suggests that NCOR2 deficiency can promote enhanced intracellular acetylation activity.

[0038] RNA-seq differential gene analysis showed that, compared with the control group (SiSCR), NCOR2 knockdown resulted in extensive transcriptional remodeling. Figure 8 (C). Further cut & tag heatmap analysis revealed that the H3K9ac and H3K27ac signals in the SiNcor2 group were significantly enhanced. Figure 8 (D) suggests that NCOR2 deficiency can promote the accumulation of histone acetylation.

[0039] GO enrichment analysis showed that H3K9ac enriched genes are mainly involved in fibrosis-related biological processes such as extracellular matrix organization, tissue remodeling, SMAD protein signal transduction, myoblast differentiation, and collagen fibril organization. Figure 8 The H3K27ac enriched genes are mainly enriched in processes such as cytoskeleton organization, extracellular matrix organization, muscle tissue development, and cell migration. Figure 8 (Middle F).

[0040] KEGG pathway analysis further revealed that H3K9ac and H3K27ac enriched genes were significantly enriched in classic profibrotic pathways such as TGFβ signaling pathway, PI3K-AKT signaling pathway, ECM receptor interaction, focal adhesion, MAPK signaling pathway, and Hippo signaling pathway. Figure 8 The presence of G and H in the data suggests that NCOR2 deficiency can activate multiple fibrosis-related signaling networks by enhancing histone acetylation.

[0041] Motif analysis showed that the H3K9ac and H3K27ac enriched regions were significantly enriched with transcription factor binding sites such as SMAD3, TEAD, c-Jun, PU.1, and GATA3. Figure 8 (I) suggests that these transcription factors may be involved in the epigenetic remodeling process following NCOR2 deletion.

[0042] Further visualization analysis of key fibrosis-related gene loci revealed that, under SiNcor2 conditions, the H3K9ac and H3K27ac signals in gene regions such as TGFB1, TWIST1, TEAD3, and FAP were significantly enhanced, accompanied by increased RNA expression levels. Figure 8 (J), indicating that NCOR2 deficiency can activate the transcription of fibrosis-related genes by promoting histone acetylation.

[0043] VII. NCOR2 protein expression level was significantly negatively correlated with intestinal fibrosis score. To further verify the relationship between NCOR2 expression levels and the severity of intestinal fibrosis, we detected NCOR2 protein expression in colonic tissue in a DNBS-induced mouse intestinal fibrosis model and simultaneously assessed intestinal fibrosis scores. After the experiment, mouse colonic tissue was collected; one portion was used for fibrosis scoring, and the other portion was used for Western blot detection of NCOR2 protein expression. Correlation analysis was then performed between the relative NCOR2 protein expression level and the corresponding intestinal fibrosis score for each mouse. The correlation analysis showed a significant negative correlation between NCOR2 protein expression levels and intestinal fibrosis scores (r = -0.941, P < 0.001). Figure 9As the fibrosis score increased, NCOR2 expression in the colonic tissue gradually decreased, suggesting that the decrease in NCOR2 is closely related to the aggravation of intestinal fibrosis. To further clarify the protective effect of exogenous NCOR2 supplementation on intestinal fibrosis, recombinant NCOR2 protein was administered to mice via enema during the DNBS-induced fibrosis process. Recombinant NCOR2 protein was purchased from Hanhuamei Biotechnology Co., Ltd., catalog number CSB-YP893883MO. After the experiment, the colon length of the mice was measured, and the intestinal fibrosis score was evaluated based on histological staining results. Further in vivo intervention experiments showed that compared with the DNBS group, the colon length of mice in the DNBS + NCOR2 group was significantly increased, while the intestinal fibrosis score was significantly reduced. Figure 10 ).

[0044] 8. miR-184-3p has a pro-fibrotic effect in vivo and supports the involvement of crawling adipose-derived exosomes miR-184-3p in the development and progression of intestinal fibrosis in Crohn's disease. To further verify the pathogenic role of miR-184-3p in intestinal fibrosis, a mouse model of chronic intestinal fibrosis was established using DNBS. During the modeling process, miR-184-3p agomir (Shanghai Jikai Gene) or the negative control agomir (agomir NC) was injected via the tail vein. After the experiment, the colon length and the degree of tissue fibrosis in the mice were assessed.

[0045] The results are as follows Figure 11 As shown, compared with the agomir NC group, the colon length of mice treated with miR-184-3p agomir was further shortened, indicating a significant increase in intestinal damage. Simultaneously, the fibrosis score was significantly elevated, suggesting that exogenous supplementation with miR-184-3p (Shanghai Jikai Gene) can further promote the progression of intestinal fibrosis.

[0046] The above results further demonstrate that miR-184-3p has a pro-fibrotic effect in vivo and support the involvement of crawling adipose-derived exosomes miR-184-3p in the development and progression of intestinal fibrosis in Crohn's disease.

[0047] Those skilled in the art will appreciate that various modifications can be made to the above embodiments without departing from the overall spirit and concept of the present invention. For any aspects not detailed herein, reference can be made to the prior art. All such modifications fall within the protection scope of the present invention. The protection scheme of the present invention is defined by the appended claims.

Claims

1. Use of a reagent for detecting the expression level of miR-184 in the preparation of a product for analyzing the risk of intestinal fibrosis associated with Crohn's disease; wherein, miR-184 expression levels are positively correlated with the risk of intestinal fibrosis associated with Crohn's disease.

2. The application according to claim 1; wherein, The miR-184 is derived from mesenteric crawling fat; preferably, the miR-184 is derived from mesenteric crawling fat exosomes.

3. Application of reagents for detecting miR-184 expression levels in the preparation of screening materials for high-risk individuals with Crohn's disease and intestinal stricture.

4. Application of reagents that upregulate miR-184-3p expression levels in the preparation of products that aggravate the severity of intestinal damage or intestinal fibrosis in animal models of Crohn's disease; among which, Preferably, the reagent for upregulating miR-184-3p expression level is miR-184-3p agomir, which is injected into the animal model.

5. Application of reagents for detecting NCOR2 protein expression levels in the preparation of products for analyzing the risk of Crohn's disease complicated by intestinal fibrosis; among which, Decreased NCOR2 protein expression levels increase the risk of intestinal fibrosis associated with Crohn's disease.

6. The application according to claim 5; wherein, NCOR2 protein expression levels are negatively correlated with the risk of intestinal fibrosis associated with Crohn's disease.

7. Application of reagents that inhibit miR-184 expression or miR-184-3p expression in the preparation of products that reduce the risk of Crohn's disease with intestinal fibrosis or increase the expression level of NCOR2 protein in fibroblasts in vitro.

8. Application of reagents that inhibit NCOR2 expression in the preparation of animal models of Crohn's disease with intestinal fibrosis.

9. The application according to claim 8; wherein, The Crohn's disease-associated intestinal fibrosis animal model was obtained by knocking down NCOR2 expression in the animal model and then stimulating it with DNBS; preferably, the reagent for knocking down NCOR2 expression is SiNcor2.

10. Application of NCOR2 protein or recombinant NCOR2 protein in the preparation of products that reduce the severity of intestinal fibrosis associated with Crohn's disease.

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