Use of an adra2a antagonist in the manufacture of a medicament for the treatment of inflammatory bowel disease

Abstract

The present application provides the use of antagonists of tyramine-ADRA2A signaling axis in the preparation of a medicament for preventing and / or treating inflammatory bowel disease. By using antagonists of tyramine-ADRA2A signaling axis, including but not limited to yohimbine, midaglizole and 2-methoxyimidazolidine, the activation of ADRA2A receptors on intestinal stem cells by tyramine can be inhibited, and the proliferation of intestinal stem cells, the repair of damaged intestines and the relief of intestinal inflammation can be restored. The present application lays a new material foundation for the treatment of inflammatory bowel disease, especially Crohn's disease.

Description

Technical Field

[0001] This invention relates to the field of medicine. Specifically, this invention relates to the use of ADRA2A antagonists in the preparation of medicaments for the prevention and / or treatment of inflammatory bowel disease, particularly Crohn's disease. Background Technology

[0002] Inflammatory bowel disease (IBD) is a chronic inflammatory disease of the gastrointestinal tract with an unknown etiology, characterized by a lifelong tendency to relapse, and this chronic intestinal inflammation also increases the risk of colon cancer. Studies have shown that countries with higher levels of urbanization and industrialization have observed higher prevalence and incidence rates. Currently, there are over 10 million IBD patients worldwide. Meanwhile, with the continuous advancement of urbanization and industrialization, the living environment, dietary lifestyles, and habits of Chinese people have undergone tremendous changes. IBD has now become the most common type of digestive system disease in my country, and it is estimated that by 2025, the number of IBD patients in China may reach at least 1.5 million.

[0003] Inflammatory bowel disease (IBD) mainly includes two types: Crohn's disease (CD) and ulcerative colitis (UC). IBD has unique physiological and pathological clinical characteristics, significantly reducing patients' quality of life, increasing the burden of treatment, and is also a high-risk factor for inducing colorectal cancer. Currently, common clinical treatments for IBD include: 1. Partial resection of the gastrointestinal tract; 2. Non-targeted immunosuppressive therapy, such as immunomodulators like 5-aminosalicylic acid and corticosteroids; 3. Targeted biotherapy, using neutralizing antibodies to neutralize pro-inflammatory cytokines (such as neutralizing antibodies against TNF-α), neutralizing cytokines of specific pathogenic immune subsets (such as anti-IL-12 and anti-IL-23 antibodies), blocking downstream signal transduction in related signaling pathways (such as JAK inhibitors), or regulating lymphocyte transport (such as anti-α4β7 integrin antibodies). Biotherapy is effective for many patients, but up to 30-40% of patients do not respond to initial treatment, and 50% lose response. Current treatments cannot cure IBD.

[0004] Therefore, there is an urgent need in this field for new targets to treat IBD, so as to develop novel treatment methods. Summary of the Invention

[0005] The purpose of this invention is to provide an ADRA2A antagonist that has excellent preventive and / or therapeutic effects on inflammatory bowel disease, especially Crohn's disease.

[0006] In a first aspect, the present invention provides the use of tyramine-ADRA2A signaling axis antagonists in the preparation of medicaments for the prevention and / or treatment of inflammatory bowel disease and for the restoration of stem cell / organoid growth.

[0007] In a preferred embodiment, the stem cells are intestinal stem cells; the organoids are intestinal organoids.

[0008] In a specific implementation, the antagonist of the tyramine-ADRA2A signaling axis is a tyramine inhibitor.

[0009] In a preferred embodiment, the tyramine inhibitor is a substance that destroys tyramine or kills or inhibits tyramine-producing microorganisms.

[0010] In a preferred embodiment, the tyramine-producing microorganisms include, but are not limited to, Enterococcus.

[0011] In a preferred embodiment, the tyramine inhibitor is an antibiotic targeting enterococci, such as penicillins and vancomycin.

[0012] In a preferred embodiment, the tyramine inhibitor is a therapy targeting enterococcal bacteriophage virus.

[0013] In a specific implementation, the tyramine inhibitor is a substance that inhibits the production of tyramine by relevant microorganisms, such as 3-fluoro-alpha-fluoromethyltyrosine (CAS: 73804-76-9).

[0014] In a specific implementation, the tyramine-ADRA2A signal axis antagonist is an ADRA2A antagonist.

[0015] In specific embodiments, the ADRA2A antagonist includes, but is not limited to: small molecule inhibitors of ADRA2A, monoclonal antibodies against ADRA2A, and siRNA encoding the ADRA2A gene.

[0016] In specific embodiments, the small molecule antagonists of ADRA2A include, but are not limited to: yohimbine, imidazolidin, and 2-methoxyimidazolidine; yohimbine is preferred.

[0017] In a preferred embodiment, the CAS numbers and structural formulas of yohimbine, imidazolidin, and 2-methoxyimidazolidine are shown below:

[0018]

[0019] In specific embodiments, the inflammatory bowel disease includes, but is not limited to, Crohn's disease and ulcerative colitis; Crohn's disease is preferred.

[0020] In a second aspect, the present invention provides a pharmaceutical composition comprising an antagonist of the tyramine-ADRA2A signaling axis and other therapeutic agents for inflammatory bowel disease, as well as pharmaceutically acceptable excipients.

[0021] In a preferred embodiment, the tyramine-ADRA2A signal axis antagonist is a tyramine antagonist.

[0022] In a preferred embodiment, the tyramine inhibitor is a substance that destroys tyramine or kills or inhibits tyramine-producing microorganisms.

[0023] In a preferred embodiment, the tyramine-producing microorganisms include, but are not limited to, Enterococcus.

[0024] In a preferred embodiment, the tyramine inhibitor is an antibiotic targeting enterococci, such as penicillins and vancomycin.

[0025] In a preferred embodiment, the tyramine inhibitor is a therapy targeting enterococcal bacteriophage virus.

[0026] In a preferred embodiment, the tyramine inhibitor is a substance that inhibits the production of tyramine by relevant microorganisms, such as 3-fluoro-alpha-fluoromethyltyrosine (CAS: 73804-76-9).

[0027] In a preferred embodiment, the antagonist of the tyramine-ADRA2A signal axis is an ADRA2A antagonist.

[0028] In a preferred embodiment, the ADRA2A antagonist includes, but is not limited to: small molecule inhibitors of ADRA2A, monoclonal antibodies against ADRA2A, and siRNA of the gene encoding ADRA2A.

[0029] In a preferred embodiment, the small molecule antagonist of ADRA2A includes, but is not limited to: yohimbine, imidazolidin, and 2-methoxyimidazolidine; yohimbine is preferred.

[0030] In a preferred embodiment, the CAS numbers and structural formulas of yohimbine, imidazolidin, and 2-methoxyimidazolidine are shown below:

[0031]

[0032] In a third aspect, the present invention provides an antagonist of the tyramine-ADRA2A signaling axis or a pharmaceutical composition comprising an antagonist of the tyramine-ADRA2A signaling axis for the prevention and / or treatment of inflammatory bowel disease.

[0033] In a preferred embodiment, the antagonist of the tyramine-ADRA2A signaling axis is a tyramine inhibitor.

[0034] In a preferred embodiment, the tyramine inhibitor is a substance that destroys tyramine or kills or inhibits tyramine-producing microorganisms.

[0035] In a preferred embodiment, the tyramine-producing microorganisms include, but are not limited to, Enterococcus.

[0036] In a preferred embodiment, the tyramine inhibitor is an antibiotic targeting enterococci, such as penicillins and vancomycin.

[0037] In a preferred embodiment, the tyramine inhibitor is a therapy targeting enterococcal bacteriophage virus.

[0038] In a preferred embodiment, the tyramine inhibitor is a substance that inhibits the production of tyramine by relevant microorganisms, such as 3-fluoro-alpha-fluoromethyltyrosine (CAS: 73804-76-9).

[0039] In a preferred embodiment, the antagonist of the tyramine-ADRA2A signal axis is an ADRA2A antagonist.

[0040] In a preferred embodiment, the ADRA2A antagonist includes, but is not limited to: small molecule inhibitors of ADRA2A, monoclonal antibodies against ADRA2A, and siRNA of the gene encoding ADRA2A.

[0041] In a preferred embodiment, the small molecule antagonist of ADRA2A includes, but is not limited to: yohimbine, imidazolidin, and 2-methoxyimidazolidine; yohimbine is preferred.

[0042] In a preferred embodiment, the CAS numbers and structural formulas of yohimbine, imidazolidin, and 2-methoxyimidazolidine are shown below:

[0043]

[0044] In a preferred embodiment, the inflammatory bowel disease includes, but is not limited to, Crohn's disease and ulcerative colitis; Crohn's disease is preferred.

[0045] In a preferred embodiment, the pharmaceutical composition comprises an antagonist of the tyramine-ADRA2A signaling axis and other therapeutic agents for inflammatory bowel disease, as well as pharmaceutically acceptable excipients.

[0046] In a fourth aspect, the present invention provides the use of tyramine in inhibiting intestinal stem cells or in the preparation of intestinal stem cell inhibitors.

[0047] In a preferred embodiment, the intestinal stem cell inhibitor is a drug for the prevention or treatment of intestinal cancer.

[0048] In a fifth aspect, the present invention provides a method for preventing and / or treating inflammatory bowel disease, the method comprising administering to a subject requiring prevention and / or treatment of inflammatory bowel disease an effective amount of a tyramine-ADRA2A signaling axis antagonist or a pharmaceutical composition comprising a tyramine-ADRA2A signaling axis antagonist.

[0049] In a preferred embodiment, the antagonist of the tyramine-ADRA2A signaling axis is a tyramine inhibitor.

[0050] In a preferred embodiment, the tyramine inhibitor is a substance that destroys tyramine or kills or inhibits tyramine-producing microorganisms.

[0051] In a preferred embodiment, the tyramine-producing microorganisms include, but are not limited to, Enterococcus.

[0052] In a preferred embodiment, the tyramine inhibitor is an antibiotic targeting enterococci, such as penicillins and vancomycin.

[0053] In a preferred embodiment, the tyramine inhibitor is a therapy targeting enterococcal bacteriophage virus.

[0054] In a preferred embodiment, the tyramine inhibitor is a substance that inhibits the production of tyramine by relevant microorganisms, such as 3-fluoro-alpha-fluoromethyltyrosine (CAS: 73804-76-9).

[0055] In a preferred embodiment, the antagonist of the tyramine-ADRA2A signal axis is an ADRA2A antagonist.

[0056] In a preferred embodiment, the ADRA2A antagonist includes, but is not limited to: small molecule inhibitors of ADRA2A, monoclonal antibodies against ADRA2A, and siRNA of the gene encoding ADRA2A.

[0057] In a preferred embodiment, the small molecule antagonist of ADRA2A includes, but is not limited to: yohimbine, imidazolidin, and 2-methoxyimidazolidine; yohimbine is preferred.

[0058] In a preferred embodiment, the CAS numbers and structural formulas of yohimbine, imidazolidin, and 2-methoxyimidazolidine are shown below:

[0059]

[0060] In a preferred embodiment, the inflammatory bowel disease includes, but is not limited to, Crohn's disease and ulcerative colitis; Crohn's disease is preferred.

[0061] In a preferred embodiment, the pharmaceutical composition comprises an antagonist of the tyramine-ADRA2A signaling axis and other therapeutic agents for inflammatory bowel disease, as well as pharmaceutically acceptable excipients.

[0062] In a preferred embodiment, the subject is a mammal; preferably, the subject is a human.

[0063] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0064] Figure 1 This study revealed the presence of a tyramine (bacterial metabolite)-ADRA2A signaling axis on intestinal stem cells, which is positively correlated with intestinal inflammation. Specifically, Figure 1 A shows Lgr5-GFP hi Intestinal stem cells and Lgr5-GFP lo Expression patterns of nine adrenergic receptors (GSE67324) in progenitor cells. Figure 1 B shows a bubble diagram illustrating the expression profiles of nine adrenergic receptors in single cells of mouse intestinal epithelium. Figure 1 C shows the ADRA2A immunostaining of the intestinal epithelium of Lgr5-GFP reporter mice. Figure 1 D shows a schematic diagram of the PRESTO-Tango GPCR detection of ADRA2A. Figure 1 E shows that 87 microbial metabolites were screened using the ADRA2A PRESTO-Tango GPCR detection system to identify potential ADRA2A agonists. The fold change represents the ratio of metabolite-activated luminescence to control luminescence; the results indicate that tyramine specifically activates ADRA2A signaling. Figure 1 F shows the structural formulas of tyramine and norepinephrine. Figure 1 G shows the dose-dynamic response of tyramine and norepinephrine in the ADRA2A PRESTO-Tango trial, n=9. Figure 1 H shows a schematic diagram of the BRET2 experiment detecting ADRA2A activation. The ratio of GFP2 to RLuc8 luminescence values ​​is calculated as the BRET2 ratio. Figure 1 I shows the dose-dynamic response of tyramine and norepinephrine in the ADRA2ABRET2 trial. N=6. Figure 1 J shows the LC-MS quantification of tyramine (ug / g wet weight stool) in the stool of healthy controls (n=36) and CD patients (n=51) in the IBD cohort (USTC). Each point represents an independent individual. Figure 1K represents the ADRA2A mRNA expression levels in biopsy samples from healthy individuals (n=50) and those with Crohn's disease (CD) (n=124). Data were analyzed from the public dataset of the Human Microbiome Project 2 (HMP2). All data are expressed as Mean ± SEM. Statistical analysis was performed on the two groups using an unpaired Student's t-test. ****P<0.0001; ***P<0.001; **P<0.01; *P<0.05; Data represent results from two to three independent experiments.

[0065] Figure 2 This study demonstrates that tyramine activation of the ADRA2A receptor inhibits intestinal stem cell proliferation. Among other things, Figure 2 A shows a schematic diagram of tyramine activation of ADRA2A and ADRA2A receptor knockout blocking strategies. Adra2a KO: Adra2a gene-deleted mice. Figure 2 B shows representative images of organoid cultures at different tyramine concentrations. Scale bar: 500 μm. Figure 2 C shows the quantitative data for organoid formation efficiency on day 4. "Organoid formation efficiency" is calculated as the ratio of the number of living organoids to the number of crypts initially seeded per hole, n=3. Figure 2 The image shown on D represents a representative image of intestinal organoids cultured from the crypts of Lgr5-GFP reporter mice. Scale bar: 100 μm. Figure 2 E shows the flow cytometry analysis of intestinal organoid DAPI. - Epcam + CD24 low Lgr5-GFP in epithelial cells + The proportion of intestinal stem cells; Figure 2 Lgr5-GFP was shown on F. + Quantitative data on the proportion of intestinal stem cells. N=3 animals per group. All data are expressed as mean±sem. ****P<0.0001; ***p<0.001; **p<0.01; *p<0.05; Data are representative of two or three independent experiments. Figure 2 G shows the GO analysis of genes that were significantly downregulated in tyramine-treated samples, N=3 independent samples. Figure 2 H shows a heatmap of gene expression related to intestinal stem cell (ISC) proliferation and cell cycle regulation in the transcriptomes of the tyramine-treated group and the control group; Figure 2 Figure I shows the qRT-PCR analysis of cell cycle genes in organoids from WT and Adra2a KO mice under 100 μM tyramine. All data are expressed as mean ± sem. ****P < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05; Data are representative of two or three independent experiments.

[0066] Figure 3 The study showed that bioinformatics analysis clearly identified tyramine-producing Enterococci as being enriched in patients with enteritis. Figure 3 A shows the phylogenetic distribution of tyrDC in the human microbiome. Homology of the Enterococcus faecalis V583 TyrDC protein in the Human Microbiome Project reference genome was queried using BLASTP, and the results are displayed on a phylogenetic tree based on 16S rRNA alignment, where tyrDC is mainly distributed in the Enterococcus genus. Figure 3 Figure B shows the differential distribution of gut microbiota in Crohn's disease (CD) cohorts from geographically different regions, including the United States and France. Figure 3 The image below shows the differential distribution of gut microbiota in patients with different disease states in different geographically dispersed CD cohorts, including those in the progression and remission phases. Figure 3 D shows the 16S sequencing analysis results of the gut microbiome of Crohn's disease patients. Principal coordinate analysis (PCOA) based on unweighted and weighted unifrac distances between fecal microbiota of healthy donors (n=36) and Crohn's disease (CD) patients (n=51) revealed significant alterations in the microbiome of Crohn's disease patients. Figure 3 E shows the differential distribution of gut microbiota in patients from the USTC CD cohort, with Enterococcus enriched in Crohn's disease patients. Figure 3 F shows the phylogenetic tree of bacterial taxa that were found to be significantly associated with CD patients by LefSe analysis;

[0067] Figure 4 This study showed that the enterococcus-tyramine-ADRA2A signaling axis exacerbates the progression of intestinal inflammation. Among other things, Figure 4 A shows a schematic diagram of the workflow for inducing enteritis under tyramine administration conditions. ADRA2A ΔIEC Mice and their littermates ADRA2A WT Mice were given tyramine via drinking water (including a water control group), and a mouse colitis model was induced 4 weeks later using 2.5% DSS. Figure 4 BG showed ADRA2A exposed to water or tyramine. WT and ADRA2A ΔIEC Mice were induced to develop a colitis phenotype using 2.5% DSS. Figure 4 B shows the changes in mouse body weight, with N=9 mice in each group; Figure 4 C and Figure 4 D shows the length of the mouse colon; Figure 4 E and Figure 4 F represents the colon pathology score. Figure 4 G shows a schematic diagram of the workflow for Enterococcus monoculture inducing enteritis. ADRA2A WTMice were pretreated with antibiotics and then inoculated by gavage with WT and tyrDC-deficient (ΔtyrDC) Enterococcus faecalis strains every 2 days for 2 weeks. Fecal microbiota transplantation (FMT) was then performed, and a 2.5% DSS colitis model was established one day later. Figure 4 H shows the weight changes of mice in each group, with N=6 mice in each group; Figure 4 I and Figure 4 J shows the changes in colon length in each group of mice; Figure 4 K and Figure 4 L shows the changes in colon length in mice across groups. Scale bar, 50 μm. Data are expressed as mean ± sem. ****P<0.0001; ***p<0.001; **p<0.01; *p<0.05; Data are representative of two or three independent experiments;

[0068] Figure 5 This demonstrates the potential of ADRA2A antagonists in treating intestinal inflammation. Figure 5 A shows a schematic diagram illustrating the blocking effect of three ADRA2A antagonists, yohimbine, imidazolidin, and 2-methoxyimidazolidine, on the tyramine-ADRA2A signaling axis. Figure 5 Image B shows a representative image of organoid culture. Figure 5 C shows the quantitative statistics of organoid culture. Three ADRA2A antagonists, yohimbine, imidazolidin, and 2-methoxyimidazolidine, were used to treat in vitro organoid cultures. The results showed that all three antagonists could reverse the inhibition of intestinal stem cells by tyramine. Figure 5 D shows a schematic diagram illustrating the intestinal inflammation induced by single-cell colonization of clinically isolated Enterococcus in mice, with ADRA2A knockout and yohimbine treatment groups designed respectively. WT and ADRA2A △IEC Mice were pretreated with antibiotics and then inoculated by gavage with clinically isolated Enterococcus faecalis strains every two days for two weeks. Fecal microbiota transplantation (FMT) was then performed, and a 2.5% DSS colitis model was established one day later. In the yohimbine treatment group, yohimbine (2 mg / kg) was administered intraperitoneally starting one week after Enterococcus colonization, once every other day for a total of eight injections. Figure 5 E shows the weight changes of mice in each group, with N=6 mice in each group; Figure 5 F and Figure 5 G shows the changes in colon length in each group of mice; Figure 5 H and Figure 5 Figure 1 shows the changes in colon length in mice across different groups. Scale bar, 50 μm. Data are expressed as mean ± sem. ****P < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05; Data are representative of two or three independent experiments. Detailed Implementation

[0069] Through extensive and in-depth research, the inventors unexpectedly discovered a novel regulatory mechanism for intestinal stem cells. Specifically, they found that tyramine produced by Enterococcus (a widely reported pathogenic enteritis bacterium) activates the α-2a adrenergic receptor (ADRA2A), inhibiting ISC proliferation and exacerbating DSS-induced colitis pathological damage. Furthermore, the inventors designed targeted drugs against the discovered pathogen-host target signaling axis, developing a treatment regimen that uses ADRA2A receptor-specific antagonists to target and intervene in the pathogen-host interaction receptor, thereby alleviating colitis. This also demonstrates the important role of intestinal stem cells in promoting mucosal repair and treating intestinal inflammation. Based on this, the present invention was completed.

[0070] the term

[0071] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0072] Tyramine

[0073] Tyramine is a common natural compound found in plants and animals, with the chemical formula C8H. 11 NO, and has the following structure:

[0074]

[0075] Inflammatory bowel disease

[0076] Inflammatory bowel disease (IBD) is a chronic inflammatory disease of the gastrointestinal tract with an unknown etiology. Numerous studies have shown that intestinal inflammation is primarily a result of damage to the intestinal epithelial barrier, leading to an imbalance in the interaction between the immune system and symbiotic microorganisms within the intestinal lumen. In specific embodiments, the inflammatory bowel disease of this invention includes, but is not limited to, Crohn's disease and ulcerative colitis; Crohn's disease is preferred.

[0077] The intestinal epithelium is a rapidly renewing monolayer of tightly packed cells. The small intestinal epithelium consists of different types of cells forming a series of luminal projections (villi) and goblet-shaped invaginations (crypts). Small intestinal cells comprise six distinct cell lines, which can be broadly classified into two categories: cells that absorb the lumen's contents and secretory cells. Our intestinal epithelial cells have a rapid regeneration capacity, undergoing a complete renewal approximately every 3–5 days. Old epithelial cells shed from the villus tips into the lumen, while the production of entirely new intestinal epithelial cells is achieved through the continuous proliferation and differentiation of intestinal stem cells. Lgr5 +Intestinal stem cells (ISCs) are long-lived, pluripotent adult stem cells of the intestine, residing in the stem cell niche at the base of the crypts. Within the lineage "stripes" extending from the crypts to the villus tips, they rapidly generate all intestinal epithelial cell types of the small and colonic intestines, thus being responsible for most of the ongoing homeostatic epithelial cell transitions. Competition for limited niche space within the stem cell niche enables precise regulation of the number and fate of intestinal stem cells. Simultaneously, in cases of epithelial damage, the ISC niche undergoes hyperstability adjustments to resist pathogenic stimuli and transform into epithelial regeneration. MAP3K2-regulated intestinal basal cells (MRISCs) are a novel regulatory component of the intestinal stem cell niche. During intestinal inflammatory damage, they can rely on MAP3K2 to enhance WNT signaling in intestinal stem cells, thereby promoting epithelial repair and alleviating enteritis. Stable intestinal stem cells (Lgr5) + ISC (Intracytoplasmic Sclerosing Cell) pools play a crucial role in the repair of the epithelial system, particularly in the repair of epithelial damage during intestinal inflammation. Therefore, it is necessary to understand the stem cell plasticity of reserve ISCs and the role of Lgr5. + Biological characteristics of ISCs in their different adaptations to their niche during epithelial injury and subsequent repair.

[0078] Meanwhile, numerous studies have shown that functional deficiencies in inflammatory bowel disease (IBD) are also reflected in gut microbiota dysbiosis. Patients with IBD exhibit significant gut microbiota imbalance, primarily manifested as reduced microbial diversity, enrichment of harmful bacteria, and reduction of beneficial bacteria. The gut microbiota promotes abnormal immune responses in genetically susceptible IBD patients through various mechanisms, and the dysbiosis, in turn, exacerbates colitis symptoms. However, whether inflammation-associated bacteria can directly affect intestinal stem cells, inhibit epithelial cell repair, and thus worsen colitis progression remains unclear.

[0079] Tyramine-ADRA2A signaling axis and ADRA2A receptor

[0080] The term “tyramine-ADRA2A signaling axis” used in this article refers to the complete pathway in which symbiotic microorganisms in the mammalian gut produce a small molecule metabolite, tyramine, which specifically activates the ADRA2A receptor and affects the proliferation of intestinal stem cells.

[0081] The term "ADRA2A receptor" used in this article refers to a receptor expressed on intestinal stem cells. ADRA2A receptor, α2-adrenergic receptor, is a member of the G protein-coupled receptor superfamily and is involved in regulating the release of neurotransmitter molecules from adrenergic neurons in the sympathetic and central nervous systems. The sympathetic nervous system regulates cardiovascular function by activating adrenergic receptors in the heart, blood vessels, and kidneys. Studies in mice have shown that ADRA2A receptor is essential for the release of presynaptic neurotransmitters from the cardiac sympathetic nervous system and central noradrenergic neurons. ADRA2A receptor also participates in catecholamine signaling by regulating the extracellular protein kinase 1 and 2 (ERK1 / 2) pathway. Currently, no definitive association has been established between ADRA2A receptor and disease.

[0082] The inventors have discovered that intestinal stem cells possess a tyramine-ADRA2A signaling axis. Tyramine-producing Enterococci downregulate the expression of cell cycle-related genes by tyramine activating ADRA2A receptors, thereby inhibiting intestinal stem cell proliferation and ultimately preventing the repair function of intestinal stem cells on damaged intestines.

[0083] Inhibitors of the tyramine-ADRA2A signaling axis

[0084] The term "inhibitor" as used in this article is mainly based on substances that block and regulate specific signaling pathways to control cellular function and biological processes; the term "antagonist" is mainly based on substances that, after binding to receptor proteins, do not cause biological effects themselves, but block the effects mediated by receptor agonists.

[0085] The inventors have discovered that enterococci accumulate in the intestines of Crohn's disease patients. Tyramine produced by enterococci can activate ADRA2A receptors on intestinal stem cells (ISCs), inhibiting ISC proliferation, hindering intestinal epithelial repair, and thus exacerbating intestinal inflammation. Therefore, tyramine-ADRA2A signaling axis antagonists can inhibit the activation of ADRA2A receptors on intestinal stem cells by tyramine, thereby increasing intestinal stem cell proliferation, repairing damaged intestines, and improving intestinal inflammation. Based on this, the present invention provides the use of tyramine-ADRA2A signaling axis antagonists in the preparation of medicaments for the prevention and / or treatment of inflammatory bowel disease. Simultaneously, tyramine-ADRA2A signaling axis antagonists can also be used to restore stem cell / organoid growth. Therefore, the present invention also provides the use of tyramine-ADRA2A signaling axis antagonists in the preparation of medicaments for the prevention and / or treatment of restoring stem cell / organoid growth. Preferably, the stem cells are intestinal stem cells.

[0086] Based on the teachings of this invention, those skilled in the art will understand that the inhibitor of the tyramine-ADRA2A signaling axis can be a tyramine inhibitor. For example, the tyramine inhibitor can be a substance that destroys tyramine, or a substance that can kill or inhibit tyramine-producing microorganisms (including but not limited to enterococci), or a substance that can inhibit the production of tyramine by related microorganisms. For example, in specific embodiments, the tyramine inhibitor can be penicillin and vancomycin, or an enterococcal bacteriophage virus biological agent, in order to clear enterococcal colonization in the intestine; or, the tyramine inhibitor can be 3-fluoro-alpha-fluoromethyltyrosine (CAS: 73804-76-9), in order to inhibit the production of tyramine by related microorganisms.

[0087] Based on the teachings of this invention, those skilled in the art will also understand that inhibition of the tyramine-ADRA2A signaling axis can also be an antagonistic effect on the ADRA2A receptor. Therefore, inhibitors of the tyramine-ADRA2A signaling axis can also be ADRA2A antagonists. Those skilled in the art are aware of various specific forms of ADRA2A antagonists, including but not limited to: small molecule inhibitors of ADRA2A, monoclonal antibodies against ADRA2A, siRNA of the ADRA2A encoding gene, etc. In specific embodiments, ADRA2A-specific antagonists include yohimbine, imidazolidinone, and 2-methoxyimidazolidine. The CAS numbers and structural formulas of yohimbine, imidazolidinone, and 2-methoxyimidazolidine are shown below:

[0088]

[0089] These ADRA2A antagonists enhance the repair of damaged intestinal epithelium by blocking ADRA2A activation on intestinal stem cells during the pathogenesis of intestinal inflammation, thereby playing an important role in the treatment of Crohn's disease with enterococcal or tyramine accumulation.

[0090] Furthermore, in in vitro experiments, the inventors discovered that ADRA2A-specific antagonists, particularly yohimbine, can significantly inhibit tyramine's activation of ADRA2A, thus reversing tyramine's inhibitory effect on intestinal stem cell proliferation. Therefore, ADRA2A-specific antagonists, including yohimbine, imidazolidin, and 2-methoxyimidazolidine, significantly restore the proliferative function of intestinal stem cells during intestinal inflammation and enhance the damage repair function of intestinal epithelial cells, providing a new approach for the treatment of inflammatory bowel disease, especially Crohn's disease—namely, treating Crohn's disease by regulating intestinal stem cells.

[0091] Furthermore, given that the inventors have discovered that tyramine inhibits intestinal stem cell proliferation by activating the ADRA2A receptor on intestinal stem cells (ISCs), tyramine can be used to inhibit intestinal stem cells or to prepare intestinal stem cell inhibitors. Those skilled in the art know that intestinal stem cells are located at the base of the intestinal mucosal crypts, i.e., the basal crypts are a cell reservoir for intestinal stem cells. Abnormal proliferation of intestinal stem cells is considered a significant risk factor for intestinal cancer. Therefore, tyramine inhibition of intestinal stem cells can effectively suppress abnormal proliferation, reduce the probability of intestinal cancer, and thus become a potential new treatment option for intestinal cancer. In other words, under certain pathological conditions, inhibiting intestinal stem cells is also of paramount importance.

[0092] Therefore, the present invention also provides the use of tyramine in inhibiting intestinal stem cells or in the preparation of intestinal stem cell inhibitors. The intestinal stem cell inhibitors can be used as drugs for the prevention or treatment of intestinal cancer.

[0093] Pharmaceutical Composition

[0094] Based on the tyramine-ADRA2A signaling axis antagonist of the present invention, the present invention provides a pharmaceutical composition comprising the tyramine-ADRA2A signaling axis antagonist of the present invention. However, based on the teachings of the present invention and as is known in the art, the pharmaceutical composition of the present invention may also comprise other therapeutic agents for inflammatory bowel disease. Therefore, the present invention also covers pharmaceutical compositions comprising the tyramine-ADRA2A signaling axis antagonist of the present invention and other therapeutic agents for inflammatory bowel disease.

[0095] Based on the teachings of this invention and common knowledge in the art, those skilled in the art will know that the pharmaceutical compositions of this invention can be formulated into dosage forms suitable for various routes of administration, such as oral dosage forms, like capsules.

[0096] The pharmaceutical compositions of the present invention may include a preventative or therapeutically effective amount of the tyramine-ADRA2A signaling axis antagonist of the present invention or other therapeutic agents for inflammatory bowel disease. The effective amount is sufficient to improve or, in some way, alleviate symptoms associated with enteritis. Such an amount may be administered as a single dose or as part of an effective treatment regimen. The dosage may cure enteritis, but administration is often intended to improve enteritis symptoms, such as significantly alleviating weight loss, shortening of colon length, and release of inflammatory factors in the enteritis patient. The dosage may be determined by the clinician based on the patient's age, health and weight, the type of concurrent treatment, the frequency of treatment, and the desired therapeutic benefit.

[0097] The pharmaceutical formulations of this invention can be administered to any mammal, provided they can obtain the therapeutic effects of the compounds of this invention. Humans are the most important of these mammals.

[0098] The pharmaceutical compositions of the present invention can be manufactured using known methods. The pharmaceutical compositions of the present invention contain pharmaceutically acceptable excipients, such as sugars like lactose or sucrose, mannitol or sorbitol; cellulose preparations or calcium phosphates, such as tricalcium phosphate or calcium hydrogen phosphate; and binders, such as starch pastes, including corn starch, wheat starch, rice starch, potato starch, gelatin, astragalus gum, methylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, or polyvinylpyrrolidone. If desired, disintegrants, such as the starches mentioned above, as well as carboxymethyl starch, croscarmellose, agar, or alginate or its salts, such as sodium alginate, may be added. Excipients, particularly flow regulators and lubricants, such as silica, talc, stearates, such as calcium magnesium stearate, stearic acid, or polyethylene glycol, may be added. If desired, a suitable coating that resists gastric juices can be provided to the tablet core. For this purpose, a concentrated sugar solution can be applied. This solution may contain gum arabic, talc, polyvinylpyrrolidone, polyethylene glycol and / or titanium dioxide, lacquer solution, and suitable organic solvents or solvent mixtures. To prepare a gastric juice-resistant coating, a suitable cellulose solution, such as cellulose acetate phthalate or hydroxypropyl methylcellulose phthalate, can be used. Dyes or pigments may be added to the coating of the tablet or tablet core, for example, for identification or to characterize the dosage of the active ingredient.

[0099] Treatment of inflammatory bowel disease

[0100] Based on the tyramine-ADRA2A signaling axis antagonist or pharmaceutical composition of the present invention, the inventors also provide a method for preventing and / or treating inflammatory bowel disease. The method comprises administering a preventative and / or therapeutically effective amount of the tyramine-ADRA2A signaling axis antagonist or a pharmaceutical composition containing the tyramine-ADRA2A signaling axis antagonist to a subject requiring prevention and / or treatment of inflammatory bowel disease. The subject may be a mammal; preferably a human. The dosage, timing of administration, and route of administration during treatment can be determined autonomously by the clinician based on the patient's age, health and weight, types of concurrent treatments, frequency of treatment, and desired therapeutic benefits.

[0101] Advantages of this invention:

[0102] 1. In this invention, the presence of a tyramine-ADRA2A signaling axis in intestinal stem cells was discovered for the first time;

[0103] 2. In this invention, it is discovered for the first time that tyramine-producing Enterococci inhibit the proliferation of intestinal stem cells by tyramine activating ADRA2A receptors, thereby preventing the repair function of intestinal stem cells on damaged intestines and aggravating intestinal inflammation;

[0104] 3. In this invention, tyramine-ADRA2A signaling axis antagonists are used to inhibit the activation of ADRA2A receptors on intestinal stem cells by tyramine, thereby restoring the proliferation of intestinal stem cells, repairing damaged intestines, and alleviating intestinal inflammation;

[0105] 4. In this invention, a variety of tyramine-ADRA2A signaling axis antagonists are provided and verified, among which yohimbine, imidazolidin and 2-methoxyimidazolidine significantly restore the proliferative function of intestinal stem cells during intestinal inflammation and enhance the damage repair function of intestinal epithelial cells;

[0106] 5. This invention lays a completely new material and theoretical foundation for the treatment of inflammatory bowel disease, especially Crohn's disease.

[0107] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0108] Example

[0109] Materials and Methods

[0110] In the following examples, GraphPad 8.0.2 statistical software was used to process the data. The experimental results are expressed as mean ± standard deviation. The data were analyzed using ANOVA, the pairwise comparisons were analyzed using the T test, and the multiple comparisons were analyzed using the Tukey test. P < 0.05 (*) indicates a significant difference, P < 0.01 (**) indicates a highly significant difference, and P < 0.001 (***) indicates a highly significant difference.

[0111] All experimental mice in the following examples are C57BL / 6 mice, mainly including the following genotypes:

[0112] Table 1. Information on experimental mice

[0113]

[0114] The Lgr5 lineage tracking mice (Lgr5-EGFP-IRES-CreERT2; Rosa26) used in the following examples LSL -tdTomato ) and Villin1-cre:ADRA2A fl / flTransgenic mice were bred through crossbreeding of the above-mentioned mice. All animal experiments ultimately used mice aged 6–10 weeks. All mice were housed in the SPF-grade animal facility of the Experimental Animal Center, School of Life Sciences, University of Science and Technology of China (USTC), and the care of each mouse strictly adhered to the regulations for the husbandry and management of experimental animals of USTC. All mouse experiments were conducted in strict accordance with the management regulations of the Experimental Animal Ethics Committee of USTC (Ethics No.: USTCACUC192401055).

[0115] Example 1. Intestinal stem cells possess a tyramine-ADRA2A signaling axis

[0116] 1. Experimental Procedure:

[0117] Bioinformatics methods were used to analyze the transcriptome of intestinal stem cells and the single-cell transcriptome of intestinal epithelial cells to obtain the expression pattern of ADRA2A. Immunofluorescence staining was used to clarify the expression pattern of ADRA2A in intestinal stem cells, using the following antibody information: rabbit anti-ADRA2A (1:50, Lifespan LS-C804463) and goat anti-rabbit secondary antibody (Invitrogen).

[0118] 2. Screening for potential ADRA2A agonists from gut bacterial-derived small molecule metabolites

[0119] The inventors constructed a microbial metabolite complex containing 87 small molecule metabolites derived from gut microbes. These 87 metabolites were screened using the PRESTO-Tango GPCR method to identify potential ADRA2A receptor agonists. HTLA cells were transfected with 20 ng of human ADRA2A Tango expression plasmid per well. The stock solutions of the microbial metabolites were diluted and added to each well at a final concentration of 100 μM. After 18 h, 20 μL / well of BrightGlo reagent (Promega) was used to replace the culture medium in the culture dishes. After incubation at room temperature for 10 min, the samples were read on Envision (Perkin Elmer). Each microbial metabolite was divided into four replicates, with drug buffer used as a negative control. The results showed a dual variation (i.e., the reaction of each metabolite / the reaction of the negative control).

[0120] 3. Bioluminescent Resonance Energy Transfer (BRET) Measurement Experiment

[0121] This study used bioluminescent resonance energy transfer (BRET) to monitor the dissociation of the heterotrimeric G protein induced by ADRA2A activation. In the GPCR-inactivated state, due to the aggregation of the heterotrimeric G protein, the fluorescent groups carried by the luminescent group (RLuc8-Gα) came into very close contact, and the luminescent group effectively excited the fluorescent receptor GFP2-Gβγ, resulting in the detection of the GFP signal. After GPCR activation, the dissociation of the heterotrimeric G protein complex prevented the luminescent group RLuc8-Gα from effectively exciting the fluorescent receptor GFP2-Gβγ, leading to a reduction in the GFP2 signal. The experimental procedure is as follows:

[0122] 1) Four plasmids were co-transfected into HEK293T cells (ATCC CRL-11268; mycoplasma free) in a mass ratio of 1:1:1:1.

[0123] 2) After transfection, the cells were seeded in 96-well clear cell culture plates and cultured in DMEM medium containing 10% fetal bovine serum (100 μL / well, 30,000 cells). After incubation overnight, the culture medium was replaced with 40 μL / well of 7.5 μM coelenterazine 400a (Promega).

[0124] 3) After 2 minutes of diffusion, add 20 μL / well of the agonist (3-fold) to the drug buffer. Using a Mithras LB940 multimode microplate reader with emission filters at 395 nm (RLuc8) and 510 nm (GFP2), read each well for 15 seconds. Calculate the GFP2 / RLuc8 ratio per well using GraphPad Prism 8.0 and plot the drug concentration as a function.

[0125] 4. Quantitative determination of tyramine content by liquid chromatography-mass spectrometry (LC-MS)

[0126] The expression level of ADRA2A in Crohn's disease patients was analyzed in the Human Microbiome Project (Hmp2). Simultaneously, a Crohn's disease cohort was established (36 healthy individuals and 51 Crohn's disease patients). Tyramine content was detected in stool samples from patients in the cohort using liquid chromatography-mass spectrometry (LC-MS).

[0127] 1) To separate tyramine from fecal samples, resuspend 80 mg of human or 50 mg of mouse fecal sample in 1 mL of cold extraction solvent methanol / acetonitrile / H2O (2:2:1, v / v / v), rotate thoroughly, and incubate on ice for 20 minutes. Centrifuge at 14000 g for 20 minutes at 4°C. Collect the supernatant and dry it in a vacuum centrifuge at 4°C.

[0128] 2) To separate tyramine from the bacterial supernatant, the bacterial culture was centrifuged at 2000g for 30 minutes at 4°C. 100 μL of the supernatant was resuspended in 400 μL of extraction solvent methanol / acetonitrile (2:2, v / v), thoroughly vortexed, incubated on ice for 20 minutes, and then centrifuged at 14000g for 20 minutes at 4°C. The supernatant was collected and dried in a vacuum centrifuge at 4°C.

[0129] 3) The dried sample was redissolved in 100 μL of acetonitrile / water (1:1, v / v) solvent and injected at a dose of 1 μL. Tyramine was quantified using an AB ExionLC™ liquid chromatography system (AB SCIEX) and an AB 5600+ TripleTOF system (ABSCIEX). Liquid chromatography separation: XBridge BEH C18 column (100 mm × 2.1 mm; 2.5 μm; Waters Corp., USA) at a flow rate of 0.4 mL / min. The mobile phase solvent was a mixture of 0.1% (v / v) formic acid-10 mM ammonium acetate aqueous solution (a) and 0.1% formic acid-80% (v / v) methanol-20% (v / v) acetonitrile (B). The gradient flow rate was set to 35% (v / v) B for 0.5 min, linearly increasing to 60% B over the next 2.5 min, linearly increasing to 80% B over the next 7 min, linearly increasing to 90% B over the next 6 min, linearly decreasing to 35% B over the next 4.5 min, and finally holding at this composition for 2.5 min. All data were collected in positive ion mode.

[0130] 4) Based on the m / z values ​​and sample retention times, the raw data were processed using Peak View 2.2 software. Metabolites were identified by matching the observed m / z signal (+ / -10 ppm) and chromatographic retention time with the retention times observed in commercial metabolite standards (Selleckchem). Tyramine was detected using parallel reaction monitoring at m / z = 138.0913 and chromatographic retention time = 1.5 min, and the signal was confirmed by MS / MS fragmentation plots.

[0131] 5. Experimental Results:

[0132] The inventors used flow cytometry to sort Lgr5-GFP from Lgr5-eGFP-IRES-CreERT2 knock-in mice (referred to as Lgr5-GFP reporter mice). hi ISCs and Lgr5-GFP lo Based on publicly available mRNA-Seq datasets from progenitor cells, the inventors discovered that the α2a-adrenergic receptor (Adra2a) is Lgr5. + Adra2a is one of the most abundant GPCRs expressed in intestinal stem cells (ISCs), and it is also a receptor in the adrenaline receptor family specifically expressed in intestinal stem cells. Figure 1 A). The inventors reanalyzed the single-cell transcriptome dataset of the mouse small intestine and again noted the Adra2a specificity in Lgr5. + High expression in intestinal epithelial cells (i.e., ISCs), but low expression levels in other cell differentiation lineages of the intestinal epithelium, while other receptors of the adrenergic receptor family are expressed at low levels in all intestinal epithelial cells. Figure 1 B). By immunostaining ADRA2A in the intestinal epithelial tissue of Lgr5-GFP reporter mice, consistent with observations in the transcriptome dataset, the inventors found that ADRA2A protein expression highly overlapped with Lgr5-GFP, indicating that ADRA2A is specifically expressed in intestinal stem cells (B). Figure 1 C). ADRA2A antibody staining results also showed that ADRA2A expression in stem cells exhibited a characteristic distribution, with receptors concentrated on the apical side of the cell, biased towards the intestinal lumen. The intestinal lumen is rich in in vitro components, including food, microorganisms, and mucus. Given that gut microbial metabolites have become a rich source of potential GPCR ligands and can exert various physiological and pathological effects locally in the intestine, the inventors further investigated whether bacterial metabolites would activate ADRA2A receptors expressed on intestinal stem cells (ISCs).

[0133] To further identify potential ADRA2A ligands from microbial-derived metabolites, the inventors screened ADRA2A using the PRESTO-Tango GPCR method. ADRA2A was genetically modified so that its C-terminus was linked to the transcriptional regulator tetracycline transcription activator (tTA) via the tobacco etched viral nucleoinclusion body A endopeptidase cleavage site sequence. Simultaneously, the β-Arrestin protease was genetically modified so that its C-terminus was coupled to the tobacco etched viral nucleoinclusion body-TEV element. In cells expressing the tetracycline transcription activator (tTA)-luciferase reporter plasmid, the ADRA2A receptor binds to the ligand, activating the receptor and simultaneously promoting the recruitment of β-Arrestin protease, triggering the release of the tethered transcription factor tetracycline transcription activator (tTA), and inducing luciferase reporter gene expression. Figure 1 D). The inventors established a customized metabolite library containing 87 microbial metabolites enriched in the colonic lumen, and screened all metabolites in the library using the PRESTO-Tango ADRA2A screening system. The inventors discovered that only tyramine can activate ADRA2A-Tango activity at a high level. Figure 1 E). Tyramine is a trace monoamine with a structure highly similar to norepinephrine (the known ADRA2A ligand). It possesses sympathomimetic properties and also functions as a neurotransmitter in lower organisms, guiding sensory and behavioral decisions in the host of *C. elegans*. Figure 1 F).

[0134] Subsequently, the inventors used different concentrations of tyramine and norepinephrine (NorEpi) to perform ADRA2A-tango system detection. The maximum effect (Emax) of tyramine activating ADRA2A was 40% of that of the norepinephrine positive control; the mean effective concentration (EC) detected by norepinephrine was... 50 The mean effective concentration (EC) of tyramine was 180 nM. 50 ) is 302μM ( Figure 1 G). The inventors also used bioluminescent resonance energy transfer 2 (BRET2) detection to monitor the degree of dissociation of the heterotrimeric G protein induced by ADRA2A activation. In the GPCR inactivated state, due to the aggregation of the heterotrimeric G protein, the fluorescent groups carried by the luminescent group are in very close contact, and the luminescent group RLuc8-Gα can effectively excite the fluorescent receptor GFP2-Gβγ, and the system detects the GFP signal; after GPCR activation, the dissociation of the heterotrimeric G protein complex leads to the inability of the luminescent group RLuc8-Gα to effectively excite the fluorescent receptor GFP2-Gβγ, and the GFP2 signal is reduced ( Figure 1 H). In the ADRA2A bioluminescent resonance energy transfer 2 (BRET2) detection system, tyramine activated ADRA2A to approximately 30% of the norepinephrine-activated Emax. The EC50 value of norepinephrine was 13 nM, and the EC50 value of tyramine was 42 μM. Figure 1 I). These data indicate that tyramine is a ligand for ADRA2A and can effectively activate the Gi-cAMP signaling pathway.

[0135] Next, the inventors investigated the presence of the tyramine-ADRA2A signaling axis in patients with enteritis and its potential impact. The inventors explored the physiological and pathological concentrations of tyramine in the feces of healthy individuals and those with IBD. IBD includes two main disease types: Crohn's disease (CD) and ulcerative colitis (UC). Here, the inventors focused primarily on patients with Crohn's disease (CD). Through the isolation and LC-MS quantification of tyramine in the feces of patients with Crohn's disease (CD), the inventors found that the level of tyramine in the feces of patients with Crohn's disease (CD) was significantly higher than that in healthy controls. Figure 1 J). Furthermore, the inventors reanalyzed some public datasets and observed a significant increase in ADRA2A expression in both biopsy samples from Crohn's disease (CD) patients and colonic organoids expanded from intestinal stem cell ISCs from Crohn's disease (CD) patients. Figure 1 These results indicate that the tyramine-ADRA2A signaling axis is enriched in some patients with Crohn's disease (CD) and influences the progression of colitis.

[0136] Results and Discussion: The experimental results of Example 1 show that intestinal stem cells have a tyramine-ADRA2A signaling axis that mediates the interaction between intestinal bacteria and the host, and are enriched in intestinal inflammation, especially Crohn's disease, indicating potential enteritis-related factors.

[0137] Example 2: Activation of the tyramine-ADRA2A signaling axis in intestinal stem cells inhibits cell proliferation.

[0138] 1. Experimental Procedure:

[0139] like Figure 2 As shown in Figure A, the activation pattern of the tyramine-ADRA2A signaling axis is illustrated. The inventors used an intestinal organoid platform to explore the effect of tyramine activation of ADRA2A on intestinal stem cells. At the same time, ADRA2A knockout mice were introduced to verify the ADRA2A dependence of the pathway at the gene level.

[0140] 2. Experimental Model

[0141] Organoids represent one of the biggest revolutions in the biomedical field over the past decade. An organoid is a 3D cell culture system that closely resembles its source tissue or organ in vivo; these 3D systems are typically derived from the replication and differentiation of pluripotent stem cells from specific tissue sites. These in vitro cultured 3D miniature organ structures are remarkably similar to the structures of tissues and organs. Using 3D matrix gel and various cytokines, leveraging the regenerative capacity of stem cells, a 3D organoid structure called an intestinal organoid has been created that can simulate the characteristics of real intestinal organs, including morphology, function, and personalized responses to specific stimuli. Furthermore, intestinal organoids are also an excellent model for studying the self-renewal and proliferative differentiation functions of intestinal stem cells.

[0142] To investigate the effects of tyramine on the function of intestinal stem cells, the inventors established a method for culturing intestinal organoids, the specific method of which is as follows:

[0143] 1) Prepare 8-12 week old mice. Euthanize the mice by cervical dislocation. Take 6 cm of small intestine and immerse it in a culture dish containing cold PBS. Rinse the intestine with a 1 ml pipette, remove the fat, and place it in clean PBS. (The pipette should be rinsed with 0.1% BSA beforehand.) Cut the intestine longitudinally and cut it into 2-4 mm segments. Place the segments into a 50 ml centrifuge tube containing 10 ml of cold PBS. Add 10 ml of PBS, pipette three times, let stand for 30 seconds to 1 minute, remove as much supernatant as possible with a pipette, add 15 ml of PBS, pipette three times, and discard the supernatant.

[0144] 2) Remove as much supernatant as possible, add 20ml chelation buffer, and incubate gently at 4°C for 30 minutes.

[0145] 3) Take 15ml centrifuge tubes (6 small intestines), add a small amount of 0.1% BSA, shake gently on a shaker for 10 minutes, and discard the 0.1% BSA;

[0146] 4) Remove the centrifuge tube, let it stand, discard the supernatant, add 10ml chelation buffer, pipette and aspirate 3 times, let it stand for 30s, then use a 1ml pipette (pre-treated with 0.1% BSA) to transfer the supernatant through a nylon mesh to the above 15ml centrifuge tube, label it 1, repeat the process 5 times in the small intestine to obtain suspensions containing crypt numbered 1-5.

[0147] 5) Centrifuge each tube at 250g at 4℃ for 5 minutes. You will see crypt settle at the bottom. Discard the supernatant, add 10ml of 0.1% BSA for washing, and pipette three times. Centrifuge at 250g for 5 minutes. Add 0.1% BSA again (10ml for the first 3 tubes, 5ml for the last 2 tubes).

[0148] 6) Take 250 μl of each tube suspension and add it to a 24-well plate. Examine the suspension under a microscope and screen for those containing crypt cells but with mixed cell clusters or few single cells. Count the cells roughly under the microscope.

[0149] 7) Separate a suspension containing approximately 20-50 crypts, centrifuge at 250g for 5 minutes, roughly estimate the location of the crypts, carefully remove the supernatant, and use a pipette tip pre-cooled to -20℃ to add 24μl of organoid culture medium (IntestiCult) pre-cooled to 4℃. TM Add organoid growth medium (StemCell Technologies, Cat. 06005) and 24 μl of matrigel (Corning, #356231 growth factor reduced). Mix carefully, being careful of air bubbles. Take 47 μl of the mixed solution and carefully seed it into the center of each well of a 24-well plate, ensuring there are no air bubbles, forming a hemispherical shape. Incubate at 37°C for 15 min. Remove the plate and add 500 μl of organoid growth medium. Add 500 μl of PBS to the surrounding empty wells. Observe the crypt state under a microscope. Incubate in a 5% CO2, 37°C incubator. Change the organoid growth medium every 2 days.

[0150] 8) Add tyramine (Selleckchem, S3625) to the organoid culture medium at the concentration shown in the illustration. The yohimbine-treated group required 2 hours of pretreatment, followed by the addition of an appropriate concentration of tyramine or bacterial culture supernatant. After 6 hours of incubation, the medium was replaced with fresh medium. After 4 days of culture, the organoids were imaged using a Leica THUNDER Imager 3D tissue microscope, and the organoid formation efficiency was calculated and evaluated.

[0151] 9) Intestinal organoids cultured for 4 days were used in TrypLE TM Single cells were isolated from the cells using an Express (ThermoFisher Scientific, #12604013) apparatus and cultured at 37°C for 10 minutes. Further analysis was performed using flow cytometry. The obtained single cells could be re-seeded into a matrix gel for second organoid culture, or treated with tyramine (100 μM) for 16 hours, after which mRNA could be extracted from the samples.

[0152] 3. Flow cytometry analysis to detect Lgr5 + proportion of intestinal stem cells

[0153] After obtaining single-cell suspensions according to different tissue and organ processing methods, subsequent flow cytometry-based fluorescent antibody labeling and flow cytometry detection can be performed. Generally, only experiments involving cell membrane surface antigen labeling detection are needed, using a concentration of 1×10⁻⁶ cells. 6Cells are sufficient for the experimental requirements. Before antibody labeling, transfer the cells to a U-bottom 96-well plate at an appropriate cell number, and then perform the following operations:

[0154] 1) Prepare antibody mixtures of various fluorescent antibodies in advance, including FCR blocking solution anti-mouse CD16 / 32 before antibody labeling;

[0155] 2) Centrifuge at 500g for 5 minutes at 4℃, collect the cell pellet, add 50μL of FCR blocking solution to the cell pellet, gently pipette the cells with a 300μL pipette, and then incubate at 4℃ for 15-20 minutes.

[0156] 3) Add 50 μL of the prepared antibody mixture, gently pipette the cells using a 300 μL pipette, and then incubate at 4°C in the dark for 20–30 minutes.

[0157] 4) Fill a 96-well plate with 1×PBS containing 1% FBS, centrifuge at 500g for 5 minutes at 4°C to collect the cell pellet;

[0158] 5) For cells that are only labeled with cell membrane surface antigens, resuspend the cells in 200 μL of 1×PBS containing 1% FBS, filter them through a 200-mesh nylon net into a new centrifuge tube, and then perform flow cytometry analysis.

[0159] 6) All data were collected on a BECKMAN COULTER CytoFLEX S flow cytometer and analyzed using Flowjo software (TreeStar). The following antibodies were used: APC-CY7 anti-mCD45.2 (BioLegend, Clone 104), APC anti-CD326 (EpCAM) (BioLegend, Clone G8.8), PE-CY7 anti-mCD24 (BioLegend, Clone M1 / 69), purified anti-CD16 / 32 (BioLegend, Clone 93), and DAPI (Bosterbio, AR1177).

[0160] 4. RNA extraction, reverse transcription, and RT-PCR

[0161] RNA extraction:

[0162] 1) Add 1 mL of TRNzol Universal to the obtained cell sample, then add 200 μL of chloroform (1 / 5 the volume of TRNzol Universal), tighten the centrifuge tube cap, and vortex vigorously to mix thoroughly until the solution emulsifies and turns milky white. (The process for extracting RNA from tissue samples is similar, but tissue samples need to be lysed.)

[0163] 2) Let stand at room temperature for 5 minutes. Then centrifuge at 12000g for 15 minutes at 4℃. Carefully remove the centrifuge tube from the centrifuge. At this point, the solution has separated into three layers.

[0164] 3) Carefully aspirate the top layer of liquid with a 200 μL pipette and transfer it to a new RNase-free centrifuge tube. Be careful not to aspirate the middle white protein layer to prevent RNA contamination.

[0165] 4) Add 0.5 to 1 volume of isopropanol to the supernatant, mix thoroughly by inverting, and let stand on ice for 10 minutes. If the amount of RNA is very small, this step can be performed by precipitation at -80°C overnight.

[0166] 5) Centrifuge at 12000g for 10 minutes at 4℃. RNA precipitate will usually appear at the bottom of the centrifuge tube after centrifugation.

[0167] 6) Washing the RNA precipitate: Carefully discard the supernatant, being careful not to touch the RNA precipitate. Add 500 μL of 75% ethanol solution prepared with DEPC water, gently invert the centrifuge tube to wash the RNA precipitate and the tube walls, centrifuge at 7500g for 5 minutes at 4°C, then carefully discard the supernatant, being careful not to touch the precipitate. This washing process can be repeated once.

[0168] 7) Dissolving RNA precipitate: Carefully open the centrifuge tube cap and allow the RNA precipitate to dry at room temperature for 10-15 minutes (to allow the ethanol to evaporate). Then, add an appropriate amount of DEPC water and incubate at 4°C to dissolve the RNA precipitate. Finally, determine the RNA concentration before proceeding to the next experiment.

[0169] Reverse transcription:

[0170] Prepare the reaction working solution on ice according to the following reaction system. To ensure the accuracy and sufficiency of the working solution, you can prepare the working solution by the reaction number + 1, and then aliquot it into each RNase-free PCR tube. Finally, add water and RNA sample in sequence.

[0171] a) Removal of genomic DNA

[0172] Prepare the reaction solution according to the following reaction system in a 200 μL RNase-free PCR tube, and gently mix with a pipette. Incubate at 42°C for 2 minutes in a PCR instrument.

[0173]

[0174] b) RNA reverse transcription reaction

[0175] Add 5×HiScript III qRT SuperMix directly to the reaction mixture from the previous step. Gently mix with a pipette and proceed with the reaction in a PCR instrument according to the following procedure: incubate at 37°C for 15 minutes, incubate at 85°C for 5 seconds, and incubate at 4°C for 5 minutes.

[0176]

[0177] 1) Real-time quantitative PCR:

[0178] a) Prepare the working solution according to the following reaction system.

[0179]

[0180] b) Perform RT-PCR reaction under the following conditions.

[0181]

[0182]

[0183] 2) The primer pairs used for the target gene are as follows:

[0184] mHprt-F:5'-TGTAATGATCAGTCAACGGGGG-3' (SEQ ID NO: 1);

[0185] mHprt-R:5'-AGAGGGTCCTTTTCACCAGCAA-3' (SEQ ID NO:2);

[0186] mRpl13a-F:5'-CGAGGTTGGCTGGAAGTACC-3' (SEQ ID NO:3);

[0187] mRpl13a-R:5'-CTTCTCGGCCTGTTTCCGTAG-3' (SEQ ID NO:4);

[0188] mCdc25a-F:5'-CCTACTGATGGCAAGCGTGTCA-3' (SEQ ID NO:5);

[0189] mCdc25a-R:5'-CTCATTGCCGAGCCTATCTCTC-3' (SEQ ID NO: 6);

[0190] 5. Preparation and sequencing analysis of RNA sequencing samples

[0191] 1) Two pairs of male ADRA2AWT and ADRA2AKO mice from the same littermate at 8 weeks of age were selected for in vitro organoid culture. For specific experimental procedures, refer to "Intestinal Organoid Culture Experiment". After four days of culture, the cells were digested into single cells and treated with 100 uM tyramine and control, respectively. After 18 hours of treatment, the total RNA of intestinal stem cells (ISCs) was directly lysed and expanded in vitro using TRIzol reagent (Invitrogen).

[0192] 2) Total RNA was extracted from ISC cells using the phenol-chloroform method.

[0193] 3) The total RNA was sent to Berry Genomics (Beijing) for processing and mRNA library construction: it was then sequenced using Illumina on the Novaseq 6000 system.

[0194] 4) Use STAR (v2.5.3a) to align the raw RNA sequencing reads with the mouse genome (mm10, GRCm38).

[0195] 5) Gene expression levels and differential expression analysis were performed using edgeR (v3.29.2). Gene expression differences were considered significant when the FoldChange was ≥ 1.5-fold and the FDR was < 0.05.

[0196] 6) Gene enrichment analysis was performed using online bioinformatics tools (metascape) and GSEA (v4.0.3), and volcano plots and pathway plots were plotted using the R package "ggplot2".

[0197] 6. Experimental Results

[0198] like Figure 2 As shown in Figure B, by adding different concentrations of tyramine (0, 10, 50, 100 μM) during organoid culture, organoid formation was observed. The experimental results showed that tyramine inhibited the formation of intestinal organoids in a dose-dependent manner, which directly reflects the activity of intestinal stem cells. Figure 2 B, Figure 2 C). Meanwhile, the organoid-forming ability of ADRA2A-deficient cells was not affected by tyramine treatment. This reflects the role of the tyramine-ADRA2A signaling axis in inhibiting the organoid-forming ability of intestinal stem cells.

[0199] like Figure 2 As shown in DF, by culturing intestinal organoids from Lgr5-GFP reporter mice, the inventors found that high concentrations of tyramine treatment inhibited Lgr5. + The proportion of intestinal stem cells indicates that, at effective concentrations, tyramine inhibits the proportion of intestinal stem cells in single intestinal organoid cells.

[0200] When analyzing the transcriptomic changes in the tyramine-treated group, the inventors found that high concentrations of tyramine treatment inhibited Lgr5. + The proportion of intestinal stem cells indicates that, at effective concentrations, tyramine downregulates cell cycle-related genes in intestinal stem cells, thereby inhibiting their proliferation. Figure 2 G, Figure 2 H). In response to the changes in the CDC25a gene, the inventors performed qPCR analysis of the intestinal stem cell transcriptome, which verified that tyramine treatment downregulated CDC25a gene expression, while ADRA2A knockout could restore the downregulated changes in CDC25a.

[0201] Discussion of Results:

[0202] In Example 2, the inventors demonstrated that in in vitro experiments on intestinal stem cells, tyramine activation of ADRA2A downregulated the expression of cell cycle-related genes, thereby inhibiting the proliferation of intestinal stem cells.

[0203] Example 3. Bioinformatics analysis confirmed that tyramine-producing enterococci are enriched in patients with enteritis.

[0204] 1. Experimental Procedure:

[0205] Using the Human Microbiome Project, we analyzed the distribution of tyrDCs in the human microbiome. We analyzed the differences in the microbiome among enteritis patient cohorts from different regions to explore the distribution of Enterococcus in different regions and disease states. Simultaneously, we established our own enteritis cohort to validate the distribution of Enterococcus in the Chinese population, facilitating subsequent functional validation and targeted therapy.

[0206] 2. Experimental Results:

[0207] The inventors performed a BLASTP (Protein Basic Local Alignment Search Tool) search on the human gut bacteria TyrDC homolog against the Human Microbiome Project (HMP) reference genome, and found that most matching sequence strains were located in the neighboring Enterococcus genus (84-100% homology). Figure 3 A). The analysis results indicate that Enterococcus is a major producer of tyramine in the human gut microbiome, consistent with recently published gut microbiome metabolomics (data not provided).

[0208] To further clarify the distribution of Enterococci in the gut of IBD patients, the inventors analyzed multiple genomics datasets. They found that Enterococci are widely enriched in the gut of Crohn's disease (CD) patients and exhibit regional conservation in multiple cohort omics analyses. By analyzing the gut microbiota composition of IBD cohorts from three different geographical locations in France, Israel, and the United States (n=537), with a control group consisting of unrelated healthy individuals, consistent with previous reports, shotgun metagenomic analysis of fecal samples from the four cohorts showed low microbial richness in Crohn's disease (CD) patients (p<0.0001). Analysis of differentially expressed strains revealed that Enterococci are generally enriched in the gut of Crohn's disease (CD) patients in the French, Israeli, and American cohorts. Figure 3 B). Furthermore, this study analyzed the differentially expressed bacterial strains in Crohn's disease (CD) patients at different disease stages in the appeals cohort, including the stable phase (mild inflammation) and the progressive phase (severe inflammation). The results showed that Enterococci were enriched in patients with progressive Crohn's disease (CD). Figure 3 C). This result suggests that the distribution of Enterococci may exacerbate the progression of intestinal inflammation. Additionally, this study independently collected and analyzed the gut microbiota composition (n=51) of patients in the IBD cohort (USTC), finding that Enterococci were enriched in Crohn's disease (CD) patients. Figure 3 C), which is consistent with the results of the appeal analysis.

[0209] The inventors established an IBD cohort center (University of Science and Technology of China, USTC), with a control group consisting of unrelated healthy individuals. After ethical review and approval of clinical sample collection and analysis, fecal samples were collected from Crohn's disease (CD, n=51) patients, and intestinal tyramine levels were determined by liquid chromatography-mass spectrometry. Figure 1 J) and gut microbiota composition ( Figure 3 DF). The inventors conducted an experiment on 16S rDNA sequencing of the gut microbiota of Crohn's disease (CD) cohort patients. First, they independently collected and analyzed feces from healthy donors (n=36) and CD patients (n=51), storing them at -80°C to establish a USTC CD cohort. Then, fecal DNA extraction, library construction, and 16S rDNA sequencing analysis were performed. The inventors simultaneously used unweighted and weighted UniFrac distance analysis (PCOA), and the p-value was determined using permutation multivariate ANOVA (PERMANOVA) test, employing 100,000 permutations (…). Figure 3D), the results showed significant differences in fecal bacterial composition between the healthy donor and CD patients groups. Further differential component analysis using LefSe revealed that the inflammatory commensal bacteria Escherichia-Shigella and Proteus, closely related to IBD, were enriched in the fecal samples of CD patients; meanwhile, the probiotics Prevotella and Faecalibacterium were enriched in the fecal samples of healthy individuals. Figure 3 CE). Meanwhile, the inventors discovered that Enterococcus is also enriched in CD patients ( Figure 3 This is consistent with the analysis results of CD cohorts from multiple different regions (CE), which is consistent with the results of the analysis.

[0210] Discussion of Results:

[0211] In summary, Enterococci are enriched in the intestines of patients from different geographic cohorts of Crohn's disease (CD) and are positively correlated with disease progression, potentially exacerbating intestinal inflammation. This finding is consistent with the role of enterococcal-derived tyramine in promoting colitis in experimental colitis.

[0212] Example 4. The role of the Enterococcus-tyramine-ADRA2A signaling axis in a mouse model of enteritis.

[0213] 1. DSS-induced mouse colitis model

[0214] Dextran sulfate sodium (DSS)-induced colitis is one of the most commonly used rodent models for studying IBD. The symptoms of this colitis model are similar to those of UC patients, mainly manifesting as diarrhea, weight loss, and fecal occult blood. To simulate the pathogenesis of human inflammatory bowel disease, the inventors used a DSS (MP Biomedicals) chemically induced colitis model, the specific method of which is as follows:

[0215] 1) Prepare 4-6 pairs of littermate, same-sex control mice aged 8 weeks. On day 0, mark the mice and weigh them. Then, prepare an aqueous solution containing 2.5% DSS and feed the mice with it. Note: Each mouse needs about 5-8 mL of DSS solution per day. Ensure that the DSS solution is not completely consumed.

[0216] 2) Weigh the mice regularly every day and observe the state of their feces: the feces will gradually become loose, and in severe cases, bloody stools may occur. Colonoscopy can also be used to monitor and assess colitis.

[0217] 3) The DSS solution needs to be changed once on the 3rd or 4th day. For acute DSS models, two solutions are usually sufficient.

[0218] 4) During the experiment, replace the DSS solution with regular drinking water according to the changes in mouse weight and bloody stool. Note that DSS should generally be discontinued when the mouse weight drops below 95%. The mouse weight should continue to drop after discontinuation to indicate successful modeling. Mice must be sacrificed when their weight drops below 70%.

[0219] 5) After DSS modeling is completed, mice are euthanized. Colon length needs to be measured and colon tissue samples collected for histological and pathological analysis. According to IACUC guidelines, mice must be euthanized once they lose more than 30% of their initial body weight following colitis induction. For histological examination, immediately after euthanasia, mouse colon tissue is fixed with 4% formalin buffer and stored overnight at 4°C. After fixation, the samples are transferred to 70% ethanol and then coated with paraffin. Sagittal sections are cut to a thickness of 5 mm and stained with hematoxylin and eosin.

[0220] 6) The severity of colitis was assessed based on the pathological grading of H&E-stained sections (Wirtz et al., 2017). After H&E staining, the sections were photographed and scanned for double-blind pathological scoring. In short, these sections were scored by two researchers using a double-blind method, based on two criteria: the number of inflammatory cells (0-none; 1-isolated focal epithelial damage; 2-mucosal erosion and ulceration; 3-extensive damage penetrating the intestinal wall) and tissue damage (0-uncommon; 1-increased, some neutrophils; 2-inflammatory cell clusters in the submucosa; 3-transmural cell infiltration). The combined score, the sum of the two sub-scores, ranged from 0 (no change) to 6 (extensive cell infiltration and extensive tissue damage). The histopathological scoring criteria included:

[0221]

[0222]

[0223] 2. SPF mouse colonization, DSS model induction, and yohimbine treatment

[0224] 1) Prepare littermates of the same sex, aged 8-12 weeks. Administer by gavage ABX concentrate (dissolved in 1×PBS) containing ampicillin sodium (10 g / L), neomycin sulfate (10 g / L), metronidazole (10 g / L), and vancomycin hydrochloride (5 g / L) once daily for three days. Fecal samples should be collected from the mice before and after the start of antibiotic administration to assess the intestinal flora clearance effect.

[0225] 2) After the intestinal bacteria of the experimental mice were cleared, Enterococcus colonization was carried out (10^9 CFU of Enterococcus per mouse, administered by gavage every 2 days for 2 consecutive weeks).

[0226] 3) After single-bacterial colonization of the mouse gut, fecal microbiota transplantation (FMT) was used to reconstruct the mouse gut microbiota, facilitating the subsequent construction of the DSS-induced gut validation model. FMT was performed according to the reference protocol. In short, fecal microspheres from normal healthy wild-type mice were harvested under sterile conditions, suspended in sterile 10% glycerol at a concentration of 50 mg / mL, and orally administered at a dose of 10 mg per mouse. One day later, 2.5% DSS was added to the drinking water to induce a mouse colitis model. Additionally, yohimbine was administered to treat the colitis model. Specifically, starting one week after enterococcal colonization, yohimbine (2 mg / kg) was injected intraperitoneally every other day for a total of 8 injections.

[0227] 3. Experimental Results:

[0228] In IBD, inhibition of ISC proliferation disrupts epithelial barrier reconstruction and exacerbates colitis. Therefore, the inventors further investigated whether tyramine's inhibition of intestinal stem cells (ISCs) aggravates experimental intestinal inflammation, such as a dextran sulfate sodium (DSS)-induced colitis model. The inventors administered ADRA2A... △IEC Mice and their littermates ADRA2A WT Control group mice were administered tyramine via water (including the water control group) for 4 weeks, followed by induction of a DSS colitis model. Figure 4 A). Weight loss through measurement ( Figure 4 B) Colon length ( Figure 4 C, Figure 4 D) Histopathological changes in intestinal inflammation and tissue damage (including colon thickness, structural distortion, and inflammatory cell infiltration) Figure 4 E, Figure 4 F) to assess the severity of colitis, the inventors observed that in ADRA2A WT In mice, tyramine exposure significantly increased the severity of colitis compared to water, while in ADRA2A... △IEC In mice, the severity of tyramine-mediated inflammation was significantly reduced. These results suggest that tyramine exacerbates DSS-induced colitis via ADRA2A.

[0229] To further demonstrate the pathogenic role of enterococcal-derived tyramine in promoting colitis, the inventors administered single-cell bacterial inoculation of WT and tyrDC-deficient Enterococcus faecalis strains via gavage to antibiotic-pretreated wild-type mice, thereby inducing a DSS colitis model. Figure 4A). After single-strain colonization, the inventors used liquid chromatography-mass spectrometry to detect the tyramine content in mouse feces. They found that both strains successfully colonized mice, but the tyramine content in the mouse feces was significantly different (data not provided). After 2.5% DSS induction, compared with mice colonized with the WT strain, mice colonized with the tyrDC-deficient Enterococcus faecalis showed a reduced colitis phenotype, including weight loss, colon length reduction, and histopathological changes. WT strain ( Figure 4 GL). The above results indicate that Enterococci produce tyramine through tyrDC, thereby exacerbating DSS-induced colitis.

[0230] Meanwhile, the inventors also wanted to know whether the aggravation of enterococcal enteritis depends on the ADRA2A receptor on intestinal stem cells (ISCs). The inventors administered single-cell oral inoculations of WT and tyrDC-deficient Enterococcus faecalis strains to antibiotic-pretreated ADRA2A receptors. △IEC In mice, a DSS colitis model was then induced ( Figure 4 M). Following 2.5% DSS induction, mice colonized with the tyrDC-deficient Enterococcus faecalis strain exhibited the same colitis phenotype as mice colonized with the WT strain, including weight loss, colon length, mortality, and histopathological changes. Figure 4 (NR). The above results indicate that Enterococci produce tyramine via tyrDCs, which, in conjunction with ADRA2A expressed on intestinal stem cells (ISCs), exacerbates DSS-induced colitis.

[0231] Discussion of Results:

[0232] This in vivo experiment showed that the Enterococcus-tyramine-ADRA2A signaling axis exacerbates the development of intestinal inflammation in mice, while blocking this signaling axis can restore intestinal epithelial repair and alleviate the progression of enteritis.

[0233] Example 5. ADRA2A antagonists can block the aggravating effect of patient-derived enterococcal strains on enteritis.

[0234] 1. Intestinal organoid culture

[0235] The experimental subjects were mouse intestinal organoids cultured in vitro, to verify the blocking effect of ADRA2A antagonists on ADRA2A activation in tyramine-mediated intestinal stem cells. The specific methods are as follows:

[0236] 1) Prepare 8-12 week old mice. Euthanize the mice by cervical dislocation. Take 6 cm of small intestine and immerse it in a culture dish containing cold PBS. Rinse the intestine with a 1 ml pipette, remove the fat, and place it in clean PBS. (The pipette should be rinsed with 0.1% BSA beforehand.) Cut the intestine longitudinally and cut it into 2-4 mm segments. Place the segments into a 50 ml centrifuge tube containing 10 ml of cold PBS. Add 10 ml of PBS, pipette three times, let stand for 30 seconds to 1 minute, remove as much supernatant as possible with a pipette, add 15 ml of PBS, pipette three times, and discard the supernatant.

[0237] 2) Remove the supernatant as much as possible, add 20ml chelation buffer, and incubate gently at 4°C for 30 minutes.

[0238] 3) Take 15ml centrifuge tubes (6 small intestines), add a small amount of 0.1% BSA, shake gently on a shaker for 10 minutes, and discard the 0.1% BSA.

[0239] 4) Remove the centrifuge tube, let it stand, discard the supernatant, add 10ml chelation buffer, pipette and aspirate 3 times, let it stand for 30s, then use a 1ml pipette (pre-treated with 0.1% BSA) to transfer the supernatant through a nylon mesh to the above 15ml centrifuge tube, label it 1. Repeat this process 5 times in the small intestine to obtain suspensions containing crypt numbered 1-5.

[0240] 5) Centrifuge each tube at 250g at 4℃ for 5 minutes. You will see crypt settle at the bottom. Discard the supernatant, add 10ml of 0.1% BSA for washing, and pipette three times. Centrifuge at 250g for 5 minutes. Add 0.1% BSA again (10ml for the first 3 tubes and 5ml for the last 2 tubes).

[0241] 6) Take 250 μl of each tube suspension and add it to a 24-well plate. Examine the suspension under a microscope to screen for cells containing crypt but with few mixed cell clusters or single cells. Count the cells roughly under the microscope.

[0242] 7) Separate a suspension containing approximately 20-50 crypts, centrifuge at 250g for 5 minutes, roughly estimate the location of the crypts, carefully remove the supernatant, and use a pipette tip pre-cooled to -20℃ to add 24μl of organoid culture medium (IntestiCult) pre-cooled to 4℃. TMAdd 24 μl of organoid growth medium (StemCell Technologies, Cat. 06005) and 24 μl of matrigel (Corning, #356231 growth factor reduced). Mix carefully, being careful of air bubbles. Take 47 μl of the mixture and inoculate it into the center of each well of a 24-well plate, ensuring there are no air bubbles, forming a hemispherical shape. Incubate at 37°C for 15 min. Remove the plate and add 500 μl of organoid growth medium. Add 500 μl of PBS to the surrounding empty wells. Observe the crypt state under a microscope. Incubate in a 5% CO2, 37°C incubator. Change the organoid growth medium every 2 days.

[0243] 8) Tyramine (Selleckchem, S3625), yohimbine (Selleckchem, S2373), imidazolidinedion (MCE, HY-14561A), and 2-methoxyimidazolidine (MCE, HY-103197) were added to the organoid culture at concentrations of 100 μM, 10 μM, 10 μM, and 10 μM, respectively. ADRA2A antagonists, including yohimbine, imidazolidinedion, and 2-methoxyimidazolidine, required a 2-hour pretreatment, followed by the addition of appropriate concentrations of tyramine or bacterial culture supernatant. The culture medium was replaced with fresh medium every two days. After 4 days of culture, the organoids were imaged using a Leica THUNDER Imager 3D tissue microscope, and the organoid formation efficiency was calculated and evaluated.

[0244] 2. Isolation and culture of Enterococcus faecalis strains in IBD clinical settings

[0245] 1) Prepare fresh stool samples from IBD patients and store them in 1×PBS (25% glycerol, autoclaved).

[0246] 2) Using a tissue homogenizer, the samples were broken down and separated. The fecal samples were diluted and spread onto solid agar plates of GAM bacterial anaerobic medium. This medium consisted of 1.0% (w / v) animal tissue digestive enzyme digestion solution, 0.3% soybean meal digestion solution, 1.0% protease peptone, 1.35% digested serum, 0.5% yeast extract, 0.22% beef extract, 0.12% liver extract, 0.3% glucose, 0.25% potassium dihydrogen phosphate, 0.3% sodium chloride, 0.5% soluble starch, 0.03% L-cysteine ​​salt, 0.25% potassium dihydrogen phosphate, 0.03% L-cysteine, and 0.03% sodium thioacetate, with a final pH of 7.3±0.1.

[0247] 3) After culturing in a strictly anaerobic chamber under strict anaerobic conditions (80% N2, 10% H2, 10% CO2) for 2-4 days, single colonies are picked, cultured, and identified. The primers used for identification are universal primers (16S-27f: 5'agagtttgatcmtggctcag3', 16S-1492r: 5'-tacggytaccttgttacgtt-3'), used for 16S rRNA gene sequencing and classification identification.

[0248] 4) The isolated strain was identified as a single strain of Enterococcus by 16S PCR and whole genome sequencing. It was named separately and aliquoted and frozen at -80°C in GAM liquid medium containing 25% glycerol for subsequent use.

[0249] 3. DSS-induced mouse colitis model

[0250] Dextran sulfate sodium (DSS)-induced colitis is one of the most commonly used rodent models for studying IBD. The symptoms of this colitis model are similar to those of UC patients, mainly manifesting as diarrhea, weight loss, and fecal occult blood. To simulate the pathogenesis of human inflammatory bowel disease, the inventors used a DSS (MP Biomedicals) chemically induced colitis model, the specific method of which is as follows:

[0251] 1) Prepare 4-6 pairs of littermate, same-sex control mice aged 8 weeks. On day 0, mark the mice and weigh them. Then, prepare an aqueous solution containing 2.5% DSS and feed the mice with it. Note: Each mouse needs about 5-8 mL of DSS solution per day. Ensure that the DSS solution is not completely consumed.

[0252] 2) Weigh the mice regularly every day and observe the state of their feces: the feces will gradually become loose, and in severe cases, bloody stools may occur. Colonoscopy can also be used to monitor and assess colitis.

[0253] 3) The DSS solution needs to be changed once on the 3rd or 4th day. For acute DSS models, two solutions are usually sufficient.

[0254] 4) During the experiment, replace the DSS solution with regular drinking water according to the changes in mouse weight and bloody stool. Note that DSS should generally be discontinued when the mouse weight drops below 95%. The mouse weight should continue to drop after discontinuation to indicate successful modeling. Mice must be sacrificed when their weight drops below 70%.

[0255] 5) After DSS modeling is completed, mice are euthanized. Colon length is measured, and colon tissue samples are collected for histological and pathological analysis. According to IACUC guidelines, mice must be euthanized once they lose more than 30% of their initial body weight following colitis induction. For histological examination, immediately after euthanasia, mouse colon tissue is fixed with 4% formalin buffer and stored overnight at 4°C. After fixation, the samples are transferred to 70% ethanol and then coated with paraffin. Sagittal sections are cut to a thickness of 5 mm and stained with hematoxylin and eosin.

[0256] 6) The severity of colitis was assessed based on the pathological grading of H&E-stained sections (Wirtz et al., 2017). Pathological sections were H&E-stained, photographed, and then scored using a double-blind method. In short, these sections were scored by two researchers using a double-blind method, based on two criteria: the number of inflammatory cells (0-none; 1-isolated focal epithelial damage; 2-mucosal erosion and ulceration; 3-extensive damage penetrating the intestinal wall) and tissue damage (0-uncommon; 1-increased, some neutrophils; 2-inflammatory cell clusters in the submucosa; 3-transmural cell infiltration). The combined score, the sum of the two sub-scores, ranged from 0 (no change) to 6 (extensive cellular infiltration and extensive tissue damage). The histopathological scoring criteria included:

[0257]

[0258] 4. Experimental Results:

[0259] Treatment with ADRA2A receptor antagonists significantly inhibited the inhibitory effect of tyramine-activated ADRA2A on intestinal stem cells. Figure 5 AC (*p < 0.05, ****p < 0.0001). The results indicate that ADRA2A antagonists can interfere with the progression of intestinal inflammation by Enterococcus through the tyramine-ADRA2A signaling axis.

[0260] The inventors further obtained patient-derived strains through culture omics to verify disease relevance. The inventors obtained a patient-derived Enterococcus strain, named *Enterococcus faecium* CD30 (E. faecium CD30). A DSS (Diverterless Superconducting Syndrome) enteritis model was induced by single-strain colonization of *Enterococcus*. Figure 5 D), the inventors demonstrated that E. faecium CD30 can inhibit intestinal stem cell proliferation, hinder intestinal epithelial repair, and exacerbate intestinal inflammation by activating the tyramine-ADRA2A signaling axis on intestinal stem cells. Figure 5 (EI). The experimental results indicate that clinically relevant enterococci can exacerbate the progression of enteritis in patients through the tyramine-ADRA2A signaling axis on intestinal stem cells.

[0261] The inventors further verified the potential of ADRA2A antagonists in treating and intervening in patients with enterococcal-enriched intestinal inflammation. In the aforementioned DSS-induced mouse enteritis model, the inventors implemented yohimbine intervention (…). Figure 5 D). The results showed that reconstituted clinically sourced enterococci had a significant aggravating effect on enteritis. Figure 5 (EI). Experimental results show that ADRA2A antagonists play an important and significant role in the prevention and treatment of enterococcal-enriched intestinal inflammation.

[0262] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. Use of tyramine-ADRA2A signaling axis antagonists in the preparation of medicaments for the prevention and / or treatment of inflammatory bowel disease; The tyramine-ADRA2A signaling axis antagonist is an ADRA2A antagonist, and the small molecule ADRA2A antagonist is yohimbine.

2. The use as described in claim 1, characterized in that, The CAS number and structure of the yohimbine are shown below:

3. The use as described in claim 1 or 2, characterized in that, The inflammatory bowel disease includes, but is not limited to, Crohn's disease and ulcerative colitis.

4. The use as described in claim 3, characterized in that, The inflammatory bowel disease mentioned is Crohn's disease.

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