Ucerative colitis related biomarker and application thereof

Abstract

The invention provides an application of a substance for up-regulating the expression level of at least one biomarker in a signal channel of a protease activated receptor 2 (PAR2)-mediated lysosome function in preparation of a composition for one or more of the following: (1) regulating lysosome acidification in a cell; (2) regulating the autophagy function in the cells; (3) regulating autophagosome-lysosome fusion in the cells; (4) the accumulation of reactive oxygen species (ROS) in cells is reduced; (5) reducing the DNA damage level in the cells; (6) reducing the expression level of inflammatory factors in cells; (7) improving or treating acute injury of colitis in the subject; (8) improving or treating the inflammatory bowel disease of the subject; (9) preventing, ameliorating or treating inflammatory cancer transformation in a subject; and (10) preventing, ameliorating or treating colorectal cancer in the subject; wherein the biomarker is selected from PAR2 (Protein Activated Receptor 2), FOXA2 (Forkhead Frame Protein A2) and a lysosome V-type proton pump subunit ATP6V0E1.

Description

Technical Field

[0001] The present application relates to the fields of molecular biology and drug screening. Specifically, the present application provides the use of a substance that upregulates the expression level of at least one biomarker in the signal pathway mediated by protease-activated receptor 2 (PAR2) for lysosomal function in the preparation of a composition. Background Art

[0002] Ulcerative colitis (UC) is one of the common inflammatory bowel diseases (IBD). This disease is characterized by a long course, protracted illness, and progressive recurrence, seriously affecting the quality of life of patients. In 2023, the number of patients with ulcerative colitis globally was approximately 5 million cases and showed an upward trend. In the past three decades, the number of IBD patients in China has increased rapidly. Among them, the prevalence of UC has increased by an average of 25.3% per year. It is expected that the prevalence of UC will reach as high as 234 / 100,000 by 2030. The repeated attacks of UC not only increase the pain and disease burden of patients but also significantly increase the difficulty of clinical UC treatment.

[0003] Colitis-associated cancer (CAC) is a subtype of colorectal cancer (CRC) and develops from chronic inflammation such as IBD, accounting for approximately 1% of all CRCs. Epidemiological investigations have shown that the incidence of IBD patients progressing to CRC is 2-3 times that of sporadic colon cancer, and the average age of onset is advanced by 20 years.

[0004] Therefore, clarifying the mechanism of UC recurrence, effectively controlling UC recurrence and CAC progression therefrom, and screening drugs for preventing, improving, or treating UC and / or CAC have important biological and medical value. Summary of the Invention

[0005] The present application provides the use of a substance that upregulates the expression level of at least one biomarker in the signal pathway mediated by protease-activated receptor 2 (PAR2) for lysosomal function in the preparation of a composition for one or more of the following:

[0006] (1) Regulating lysosomal acidification in cells;

[0007] (2) Regulating autophagy function in cells;

[0008] (3) Regulating autophagosome-lysosome fusion in cells;

[0009] (4) Reducing the accumulation of reactive oxygen species (ROS) in cells;

[0010] (5) Reduce the level of DNA damage in cells;

[0011] (6) Reduce the expression level of inflammatory factors in cells;

[0012] (7) Improve or treat acute colitis injury in a subject;

[0013] (8) Improve or treat inflammatory bowel disease in a subject;

[0014] (9) Prevent, improve or treat inflammation-induced cancer transformation in a subject; and

[0015] (10) Prevent, improve or treat colorectal cancer in a subject;

[0016] Wherein, the biomarker is selected from: PAR2, Forkhead Box A2 (FOXA2), and lysosomal V-type proton pump subunit ATP6V0E1. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Shows a schematic diagram of the mechanism of action of the present application. Neutrophil-derived serine protease inactivates PAR2, inhibits FOXA2 expression, thereby reducing the expression of lysosomal V-ATPase subunit ATP6V0E1, resulting in lysosomal acidification disorder, and ultimately causing autophagy blockade. The blockade of autophagy causes excessive ROS accumulation, leading to increased DNA damage and increased tumorigenesis.

[0018] Figure 2The results of the neutrophil-derived serine protease inhibiting the FOXA2 / ATP6V0E1 signaling axis are shown. Among them, (A) The mRNA expression level of FOXA2 in control cells or FOXA2-overexpressing cells of NCM460 or HT29 was detected after treatment with 1 μg / mL elastase (MCE, catalog number: HY-P2974), or 2 μg / mL CTSG (Wuhan Cloud-Clone, catalog number: RPD993Hu01), or 1 μg / mL PRTN3 (MCE, catalog number: P70509FAQs), or a mixture of the above three (ECP) for 24 h. (B) Immunofluorescence images of FOXA2 in control cells or FOXA2-overexpressing cells of NCM460 or HT29 after treatment with 1 μg / mL elastase, or 2 μg / mL CTSG, or 1 μg / mL PRTN3, or a mixture of the above three for 24 h. Scale bar: 10 µm. (C) The expression levels of FOXA2, ATP6V0E1 and autophagy-related proteins in control cells or FOXA2-overexpressing cells of NCM460 or HT29 after treatment with 1 μg / mL elastase, or 2 μg / mL CTSG, or 1 μg / mL PRTN3, or a mixture of the above three for 24 h. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0019] Figure 3 The results of autophagy mediated by the neutrophil-derived serine protease inhibiting the FOXA2 / ATP6V0E1 signaling axis are shown. Among them, (A) Lysosomal pH was detected by flow cytometry in control cells or FOXA2-overexpressing cells of NCM460 or HT29 after treatment with 1 μg / mL elastase, or 2 μg / mL CTSG, or 1 μg / mL PRTN3, or a mixture of the above three (ECP) for 24 h. (B) The autophagosome-lysosome fusion was monitored using the LC3-EGFP-mCherry autophagy flux reporter system in control cells or FOXA2-overexpressing cells of NCM460 or HT29 after treatment with 1 μg / mL elastase, or 2 μg / mL CTSG, or 1 μg / mL PRTN3, or a mixture of the above three (ECP) for 24 h. Normal autophagosome-lysosome fusion appears as red spots, and abnormal autophagosome-lysosome fusion appears as yellow spots. Scale bar: 5 µm.

[0020] Figure 4It shows the results of neutrophil-derived serine proteases inhibiting autophagy impairment mediated by the FOXA2 / ATP6V0E1 signaling axis through PAR2. Among them, (A) Sequencing verification of the CRC cell line HT29 with PAR2 knocked out by CRISPR / Cas9. (B) mRNA expression levels of FOXA2 and ATP6V0E1 in control cells, PAR2-knockout cells, and PAR2-overexpressing cells after PAR2 knockout of HT29 treated with a mixture of 1 μg / mL elastase, 2 μg / mL CTSG, and 1 μg / mL PRTN3 (ECP) for 12 h. (C) Lysosomal pH detected by flow cytometry in control cells, PAR2-knockout cells, and PAR2-overexpressing cells after PAR2 knockout of HT29 treated with a mixture of 1 μg / mL elastase, 2 μg / mL CTSG, and 1 μg / mL PRTN3 (ECP) for 24 h. (D) Expression levels of FOXA2, ATP6V0E1, and autophagy-related proteins in control cells, PAR2-knockout cells, and PAR2-overexpressing cells after PAR2 knockout of HT29 treated with a mixture of 1 μg / mL elastase, 2 μg / mL CTSG, and 1 μg / mL PRTN3 (ECP) for 24 h. V0E1 represents ATP6V0E1, sgCtrl represents the plasmid with knockout control, and sgPAR2 represents the plasmid with PAR2 knockout. The PAM sequence is the key nucleotide sequence for Cas protein to recognize the target gene and integrate knockout. *p < 0.05, **p < 0.01, ***p < 0.001.

[0021] Figure 5 It shows the results of neutrophil-derived serine proteases inhibiting autophagy impairment mediated by the FOXA2 / ATP6V0E1 signaling axis through PAR2 in an organoid model. Among them, (A) Immunofluorescence images of autophagy-related proteins in colorectal organoids derived from wild-type mice or F2rl1 △IEC mice treated with 1 μg / mL elastase (NE) under glutamine deprivation (GD) or non-starvation conditions for 24 h. (B) mRNA expression levels of FOXA2 and ATP6V0E1 in colorectal organoids derived from wild-type mice or mice with epithelial-specific PAR2 knockout treated with 1 μg / mL elastase or a mixture of 1 μg / mL elastase, 2 μg / mL CTSG, and 1 μg / mL PRTN3 (ECP) under glutamine deprivation or non-starvation conditions for 12 h. V0E1 represents ATP6V0E1. ***p < 0.001, ****p < 0.0001.

[0022] Figure 6The results showing that trypsin promotes autophagy mediated by the FOXA2 / ATP6V0E1 signaling axis are presented. Among them, (A) The mRNA expression levels of ATP6V0E1 in NCM460 and IEC6 cells were treated with PAR2-AP (100 μM, 12 h; Shanghai Apeptide) or trypsin (50 μg / mL, 24 h; MCE, catalog number: HY-129047). (B) Immunofluorescence images of FOXA2 in NCM460 and IEC6 cells treated with PAR2-AP (100 μM, 12 h) or trypsin (50 μg / mL, 24 h). Scale bar is 10 µm. (C) Flow cytometry was used to detect the lysosomal pH in IEC6 cells treated with PAR2-AP (100 μM, 12 h), trypsin (50 μg / mL, 24 h), or bafilomycin A1 (BafA1; MedChemExpress, catalog number: HY-100558) (25 nM, 24 h). (D) Under glutamine deprivation (GD) conditions, the autophagosome-lysosome fusion was monitored using the LC3-EGFP-mCherry autophagy flux reporter system in IEC6 cells treated with PAR2-AP (100 μM, 12 h), trypsin (50 μg / mL, 24 h), or BafA1 (25 nM, 24 h). Normal autophagosome-lysosome fusion appears as red spots, and abnormal autophagosome-lysosome fusion appears as yellow spots. Scale bar is 5 µm. (E) Protein expression levels of autophagy-related proteins, as well as FOXA2 and ATP6V0E1, in IEC6 cells treated with PAR2-AP (100 μM, 12 h), trypsin (50 μg / mL, 24 h), or BafA1 (25 nM, 24 h). *p < 0.05, **p < 0.01, ****p < 0.0001.

[0023] Figure 7 The results showing the correlation between the expression of FOXA2 and neutrophil infiltration in UC patients are presented. Among them, (A) Infiltration indices of neutrophils in normal volunteers and UC patients in different GEO datasets. (B) Relative expression levels of FOXA2 in normal volunteers and UC patients in different GEO datasets. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0024] Figure 8The results of FOXA2 expression in CRC patients and CAC cancer patients are shown. Among them, the relative expression levels of FOXA2 in CRC patients and CAC patients in the GEO dataset. **p < 0.01.

[0025] Figure 9 The results of the relative expression levels of FOXA2 in CRC patients from the TCGA database divided into two groups of high and low expression according to the infiltration level of neutrophils are shown. Among them, CRC patients were divided into 4 groups according to the neutrophil index, and the top 1 / 4 and bottom 1 / 4 patients were selected to analyze their FOXA2 expression levels. *p < 0.05.

[0026] Figure 10 The results of the correlation between FOXA2 and ATP6V0E1 in UC patients are shown. Among them, the correlation between FOXA2 and ATP6V0E1 in UC patients was analyzed using the GEO database.

[0027] Figure 11 The results of the correlation between FOXA2 and PAR2 expression (left) and the correlation between FOXA2 and ATP6V0E1 (right) in CRC patients from the TCGA database are shown.

[0028] Figure 12 The results of multiplex immunofluorescence of FOXA2, intestinal epithelial cells, and neutrophils in UC patients are shown. Among them, (A) Representative images of multiplex immunofluorescence of epithelial cells (PANCK, magenta), neutrophils (MPO, yellow), and FOXA2 (green) in 8 quiescent and 8 active UC patients. Scale bar is 25 µm. (B) The number of FOXA2-positive epithelial cells per square millimeter in 8 quiescent and 8 active UC patients and the number of FOXA2-positive epithelial cells within a range of ≤25 µm of MPO-positive cells in 8 active UC patients. (C) The correlation between the number of MPO-positive cells and the number of FOXA2-positive epithelial cells in 8 active UC patients. (D) The mean fluorescence intensity of FOXA2 in 8 active UC patients and the mean fluorescence intensity of FOXA2 within a range of ≤25 µm of MPO-positive cells in 8 active UC patients. Inactive represents quiescent patients, and Active represents active patients. *p < 0.05, ***p < 0.001.

[0029] Figure 13Shows the results of multiplex immunofluorescence of FOXA2, intestinal epithelial cells, and neutrophils in CRC patients. Among them, (A) Representative images of multiplex immunofluorescence of epithelial cells (PANCK, magenta), neutrophils (MPO, yellow), and FOXA2 (green) in 3 CRC patients with neutrophil infiltration. Scale bar is 25 µm. (B) The mean fluorescence intensity of FOXA2 and the mean fluorescence intensity of FOXA2 within the range of MPO-positive cells less than or equal to 25 µm in 3 CRC patients with neutrophil infiltration. (C) The number of PANCK-positive and FOXA2-positive cells within the ranges of 0 - 25 µm, 25 - 50 µm, 25 - 75 µm, and 75 - 100 µm of MPO-positive cells in 3 CRC patients with neutrophil infiltration. **p < 0.01, ***p < 0.001.

[0030] Figure 14 Shows the results of high-throughput screening of drugs that inhibit neutrophil elastase-induced lysosomal dysfunction. Among them, (A) Flow chart of high-throughput drug screening. On the first day, cells were seeded into 384-well plates at 1000 cells per well. After 24 h, drug treatment was performed and continued for 48 h. Subsequently, a lysosomal pH indicator was added, and high-content imaging was used for photography and analysis. (B) Results of high-throughput drug screening. (C) Representative immunofluorescence pictures of lysosomal pH after treating HT29 cells with 1 μg / mL elastase (NE), CBZ (20 μM; MCE, Catalog No.: B0246), isotretinoin (13-cis-Retinoic acid, 20 μM; MCE, Catalog No.: HY-15127; abbreviated as 13-cis in the figure), metformin (10 μM; MCE, Catalog No.: HY-B0627; abbreviated as Met in the figure), resveratrol (10 μM; MCE, Catalog No.: HY-16561), 5-Aminoimidazole-4-carboxamide 1-β-D-ribofuranoside (AICAR, 10 μM; MCE, Catalog No.: HY-13417), 5-aminosalicylic acid (5ASA, 5 μM; MCE, Catalog No.: HY-15027), MLSA1 (10 μM; MCE, Catalog No.: HY-108462), or BafA1 (50 nM) for 24 h. Scale bar is 10 µm. (D) Statistical results of the fluorescence intensity of lysosomal pH by flow cytometry after treating HT29 cells with 1 μg / mL elastase, CBZ (20 μM), or BafA1 (50 nM) for 24 h. ****p < 0.0001.

[0031] Figure 15 It shows the results of carbamazepine reversing the downregulation of FOXA2 / ATP6V0E1 caused by neutrophil-derived elastase. Among them, (A) The mRNA of FOXA2 and ATP6V0E1 in HT29 cells treated with 1 μg / mL elastase (NE) and CBZ (20 μM) for 24 h. (B) The proteins of FOXA2 and ATP6V0E1 in HT29 cells treated with 1 μg / mL elastase and CBZ (20 μM) for 36 h. **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0032] Figure 16 It shows the results of carbamazepine reversing the increase in ROS caused by neutrophil elastase (NE). Among them, representative immunofluorescence pictures of ROS in HT29 cells treated with 1 μg / mL elastase and CBZ (20 μM) for 24 h. The scale bar is 10 µm.

[0033] Figure 17 It shows the results of carbamazepine reversing the increase in γH2AX caused by neutrophil-derived elastase (NE). Among them, representative immunofluorescence pictures of γH2AX in HT29 cells treated with 1 μg / mL elastase and CBZ (20 μM) for 24 h. The scale bar is 10 µm.

[0034] Figure 18 It shows the results of carbamazepine promoting the repair of injury after acute inflammation. Among them, (A) Intragastric administration of 50 mg / kg carbamazepine was performed on the day when DSS ended and on the 1st and 3rd days, a total of 3 times. Intestinal tissues were collected on the 0th, 2nd, 3rd, and 4th days after DSS ended. (B) Representative pictures of the intestine after DSS modeling. (C) Body weight, DAI score, and intestinal length after DSS modeling. Ctrl represents the intestine of normal mice. *p < 0.05, **p < 0.01.

[0035] Figure 19 It shows the results of carbamazepine inhibiting the upregulation of inflammatory factors induced by DSS. Among them, the mRNA expression levels of Il6 and Mcpt1 after DSS modeling. **p < 0.01, ***p < 0.001.

[0036] Figure 20 It shows the results of carbamazepine reversing the downregulation of Foxa2 and Atp6v0e1 induced by DSS. Among them, the mRNA expression levels of Foxa2 and Attp6v0e1 after DSS modeling. *p < 0.05, ****p < 0.0001.

[0037] Figure 2 and 3 In 6 and 14 - 17, "Vehicle" refers to the solvent control dimethyl sulfoxide (DMSO). Figure 18 In, "Vehicle" refers to the solvent control 85% PEG3000 + 10% DMSO + 5% Tween 80. Figure 2-4 In, "OE" (over - expression) represents over - expression. Figure 2-3 In, "Vec" represents the control plasmid vector (Vector). Autophagy - related proteins include LC3I / II and p62 proteins. Detailed implementation mode

[0038] Cell autophagy is a cellular process that depends on lysosomes to degrade intracellular macromolecules and organelles, maintaining cell and tissue homeostasis, intestinal epithelial barrier function, and controlling inflammation, and is involved in the processes of IBD and CAC. From IBD to CAC, it experiences stages such as "indeterminate dysplasia - low - grade dysplasia - high - grade dysplasia - invasive adenocarcinoma". During this process, inflammatory cells such as neutrophils infiltrate the lamina propria of the intestinal mucosa of IBD patients in large numbers, which not only serves as a pathological diagnosis criterion but also widely participates in the pathogenesis of IBD and the occurrence of CAC. Research shows that the degree of DNA double - strand breakage gradually increases with the progression of the inflammation - cancer transformation process. Among them, oxidative stress - induced DNA damage caused by chronic inflammation, which activates proto - oncogenes and inactivates tumor suppressor genes, is an important factor in the occurrence of CAC. The occurrence of CAC is positively correlated with the disease activity of IBD, including factors such as the activity of intestinal mucosal inflammation and neutrophil infiltration. Although excessive inflammation will exacerbate tissue damage, the mechanism by which inflammation promotes CAC has not been fully elucidated.

[0039] The inventors of this application first discovered that serine proteases derived from neutrophils (such as elastase, cathepsin G, or proteinase 3) can inactivate PAR2, thereby leading to autophagy disorders, which in turn exacerbates ROS generation and DNA damage in intestinal epithelial cells and tumor cells, ultimately resulting in the tumorigenesis induced by dextran sulfate sodium (DSS). Further research found that serine proteases derived from neutrophils inhibit the expression of FOXA2 by inactivating PAR2, thereby reducing the expression of the lysosomal V - type proton pump subunit ATP6V0E1, resulting in lysosomal acidification disorders, ultimately causing autophagy to be blocked. The blockage of autophagy causes excessive ROS accumulation, leading to increased DNA damage and exacerbating colitis and CAC. The anti - epileptic drug carbamazepine can reverse FOXA2 and promote the repair after acute enteritis injury. This application provides a new theoretical basis for the participation of neutrophils in the progression of colitis and CAC, and provides new markers and strategies for predicting, preventing the occurrence and progression of colitis and CAC, and also provides new treatment options.

[0040] Unless otherwise specified, the practice of this application will employ conventional techniques of molecular biology, microbiology, cell biology, biochemistry, and immunology in the art.

[0041] Unless otherwise specified, the terms used in this application have the meanings commonly understood by those skilled in the art.

[0042] Unless otherwise specified, experimental reagents are all commercially available products.

[0043] Definitions

[0044] As used herein, the term "autophagy blockade" refers to the inability of the contents of autophagosomes to be normally degraded.

[0045] As used herein, the term "lysosomal acidification disorder" refers to the pH of lysosomes being greater than its normal range of 4.5 - 5.5, resulting in a decrease in the activity of internal hydrolases (such as acid phosphatase, cathepsin D, etc.).

[0046] As used herein, the term "autophagosome-lysosome fusion disorder" refers to the inability of autophagosomes and lysosomes to fuse normally, resulting in the accumulation of autophagosomes.

[0047] In a first aspect, the present application provides the use of a substance that upregulates the expression level of at least one biomarker in the signaling pathway of protease-activated receptor 2 (PAR2)-mediated lysosomal function in the preparation of a composition for one or more of the following:

[0048] (1) Regulating lysosomal acidification in cells;

[0049] (2) Regulating autophagy function in cells;

[0050] (3) Regulating autophagosome-lysosome fusion in cells;

[0051] (4) Reducing the accumulation of reactive oxygen species (ROS) in cells;

[0052] (5) Reducing the level of DNA damage in cells;

[0053] (6) Reducing the expression level of inflammatory factors in cells;

[0054] (7) Improving or treating acute colitis injury in a subject;

[0055] (8) Improving or treating inflammatory bowel disease in a subject;

[0056] (9) Preventing, improving, or treating the inflammatory-cancer transformation in a subject; and

[0057] (10) Preventing, improving, or treating colorectal cancer in a subject;

[0058] Among them, the biomarker is selected from: PAR2, Forkhead box protein A2 (FOXA2), and lysosomal V-type proton pump subunit ATP6V0E1.

[0059] In one or more embodiments of the present application, the expression level of at least one biomarker in the signal pathway that upregulates PAR2-mediated lysosomal function is the expression level of upregulating at least one, at least two, or all of PAR2, FOXA2, and lysosomal V-type proton pump subunit ATP6V0E1.

[0060] In one or more embodiments of the present application, the expression level of PAR2 is upregulated.

[0061] In one or more embodiments of the present application, the expression level of FOXA2 is upregulated.

[0062] In one or more embodiments of the present application, the expression level of lysosomal V-type proton pump subunit ATP6V0E1 is upregulated.

[0063] In one or more embodiments of the present application, the composition can be a pharmaceutical composition or a kit.

[0064] In one or more embodiments of the present application, a decrease in the expression level of at least one of PAR2, FOXA2, and lysosomal V-type proton pump subunit ATP6V0E1 in the signal pathway of PAR2-mediated lysosomal function will result in one or more of the following:

[0065] (1) Lysosomal acidification disorder in cells;

[0066] (2) Autophagy arrest in cells;

[0067] (3) Autophagosome-lysosome fusion disorder in cells;

[0068] (4) Accumulation of reactive oxygen species (ROS) in cells;

[0069] (5) Increased DNA damage in cells;

[0070] (6) Increased expression level of inflammatory factors in cells;

[0071] (7) The subject has acute colitis injury;

[0072] (8) The subject has inflammatory bowel disease;

[0073] (9) The subject has a risk of inflammation-cancer transformation; and

[0074] (10) The subject has colorectal cancer.

[0075] In one or more embodiments of the present application, the regulation of lysosomal acidification in cells can be to improve lysosomal acidification disorders in cells; the regulation of autophagy function in cells can be to improve autophagy block in cells; and / or the regulation of autophagosome-lysosome fusion in cells can be to improve autophagosome-lysosome fusion disorders in cells.

[0076] In one or more embodiments of the present application, the subject is a mammal, such as a human, a mouse, or a rat.

[0077] In one or more embodiments of the present application, the cell is an intestinal epithelial cell, such as a colonic epithelial cell.

[0078] In one or more embodiments of the present application, the inflammatory factor is a colitis-related inflammatory factor, such as Il-6 and / or Mcpt1.

[0079] In one or more embodiments of the present application, the acute injury of colitis is one or more of the following: diarrhea, rectal prolapse, bloody stools, intestinal edema, mucosal erythema, intestinal stricture, and fistula.

[0080] In one or more embodiments of the present application, the inflammatory bowel disease is colitis, such as ulcerative colitis.

[0081] In one or more embodiments of the present application, the inflammation-cancer transformation is the transformation of inflammatory bowel disease to colorectal cancer (such as colitis-associated colon cancer (CAC)). In one or more embodiments of the present application, the inflammation-cancer transformation is the transformation of colitis to colorectal cancer (such as CAC). In one or more embodiments of the present application, the inflammation-cancer transformation is the transformation of ulcerative colitis to colorectal cancer (such as CAC). In one or more embodiments of the present application, the inflammation-cancer transformation is the transformation of ulcerative colitis to CAC.

[0082] In one or more embodiments of the present application, the colorectal cancer is colitis-associated colon cancer (CAC).

[0083] In one or more embodiments of the present application, the substance upregulates the reduction in the expression level of at least one biomarker in the signaling pathway of PAR2-mediated lysosomal function caused by serine protease. In one or more embodiments of the present application, the substance upregulates the reduction in the expression level of at least one of PAR2, FOXA2, and lysosomal V-type proton pump subunit ATP6V0E1 caused by serine protease. In one or more embodiments of the present application, the substance upregulates the reduction in the expression level of at least two of PAR2, FOXA2, and lysosomal V-type proton pump subunit ATP6V0E1 caused by serine protease. In one or more embodiments of the present application, the substance upregulates the reduction in the expression level of all of PAR2, FOXA2, and lysosomal V-type proton pump subunit ATP6V0E1 caused by serine protease. In one or more embodiments of the present application, the serine protease is a neutrophil-derived serine protease, such as neutrophil elastase (NE), cathepsin G (CTSG), or proteinase-3 (PRTN3).

[0084] In one or more embodiments of the present application, the serine protease inactivates PAR2, inhibits FOXA2 expression, and thereby reduces the expression of lysosomal V-type proton pump subunit ATP6V0E1.

[0085] In one or more embodiments of the present application, the substance is an antiepileptic drug, an activator of PAR2, or a serine protease inhibitor.

[0086] In one or more embodiments of the present application, the antiepileptic drug is carbamazepine.

[0087] In one or more embodiments of the present application, the activator of PAR2 is a synthetic peptide PAR2-AP or trypsin.

[0088] In one or more embodiments of the present application, the serine protease inhibitor is an inhibitor of neutrophil-derived serine protease, such as an inhibitor of neutrophil-derived elastase, CTSG, or PRTN3. In one or more embodiments of the present application, the serine protease inhibitor is ulinastatin, nafamostat mesylate, or aprotinin.

[0089] In one or more embodiments of the present application, the antiepileptic drug (such as carbamazepine) can be used for use in the preparation of a composition for one or more of the following:

[0090] (1) Regulating lysosomal acidification in cells;

[0091] (2) Regulate autophagy function in cells;

[0092] (3) Regulate autophagosome-lysosome fusion in cells;

[0093] (4) Reduce the accumulation of reactive oxygen species (ROS) in cells;

[0094] (5) Reduce the level of DNA damage in cells;

[0095] (6) Reduce the expression level of inflammatory factors in cells;

[0096] (7) Improve or treat acute colitis injury in a subject;

[0097] (8) Improve or treat inflammatory bowel disease in a subject;

[0098] (9) Prevent, improve or treat inflammation-cancer transformation in a subject; and

[0099] (10) Prevent, improve or treat colorectal cancer in a subject;

[0100] Wherein, the anti-epileptic drug (such as carbamazepine) up-regulates the expression level of at least one biomarker among PAR2, forkhead box protein A2 (FOXA2) and lysosomal V-type proton pump subunit ATP6V0E1 in the signaling pathway of protease-activated receptor 2 (PAR2)-mediated lysosomal function.

[0101] It should be understood that the above detailed description is only to make those skilled in the art understand the content of the present application more clearly, and is not intended to limit in any way. Those skilled in the art can make various changes and modifications to the described embodiments. Examples

[0102] Example 1. Experimental Materials and Methods

[0103] I. Experimental Materials

[0104] 1. Biological Materials

[0105] 1.1 Tissue Samples

[0106] Colonic tissue specimens of 16 patients with ulcerative colitis who received treatment at Peking Union Medical College Hospital, Chinese Academy of Medical Sciences were collected, and tumor specimens of 3 cancer patients who underwent colorectal cancer surgery at Cancer Hospital, Chinese Academy of Medical Sciences were collected. The clinicopathological features are shown in Table 1 and Table 2 in detail. All patients have signed written informed consent, and the procedures for human sample collection and human tissue use have been approved by the Ethics Committees of Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Cancer Hospital, Chinese Academy of Medical Sciences.

[0107] Table . Basic clinical information of 16 patients with colitis

[0108]

[0109] Table . Basic clinical information of 3 patients with colon cancer

[0110]

[0111] 1.2 Cell lines and culture methods

[0112] Human colorectal cancer cell lines HT29, RKO, CaCO2; human normal colon epithelial cell NCM460 and rat small intestine crypt epithelial cell IEC6 used in this study were all purchased from the ATCC cell bank. Among them, HT29 and RKO cells were cultured in DMEM / F12 medium, and NCM460 and IEC6 cells were cultured in high-glucose DMEM medium. The above cell culture components were all 90% medium + 10% fetal bovine serum. The culture condition of CaCO2 cells was 80% MEM medium + 20% fetal bovine serum + 1% amino acid solution. All cells were placed in a constant temperature incubator at 37°C and 5% CO2 for culture. The culture of stable transfected cell lines was screened under a certain concentration of puromycin, G418 or hygromycin.

[0113] 1.3 Experimental animals

[0114] All experimental animal studies complied with the requirements of the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and were conducted under the guidelines of the Institutional Review Board of the Cancer Institute and Hospital, Chinese Academy of Medical Sciences. SPF-grade male mice at 6 - 8 weeks old with C57BL / 6J background used for colitis were purchased from Beijing Huafukang Biotech Co., Ltd. The feeding conditions were: temperature 25 - 27°C, humidity 45 - 55%, 12-hour light and 12-hour dark cycle.

[0115] 2. Reagents

[0116] 2.1 Antibodies

[0117] The antibodies used in this application are shown in Table 3 as follows:

[0118] Table 3. Names and sources of the antibodies used

[0119]

[0120] 2.2 Kits

[0121] The kits used in this application are shown in Table 4 as follows:

[0122] Table 4. Names and sources of the kits used

[0123]

[0124] 2.3 Strains and plasmids

[0125] Table 5. Strains and plasmids used in this application

[0126]

[0127] 2.4 Primer sequence design and synthesis

[0128] All primers were searched in PrimerBank (https: / / pga.mgh.harvard.edu / primerbank / ) and then verified in NCBI's Primer-blast (https: / / www.ncbi.nlm.nih.gov / tools / primer-blast / ). The specific steps are as follows: First, select the target sequence in PrimerBank according to the species and Gene Symbol, and then detect the specificity of the primers for the candidate sequences in Primer-blast. qPCR primers should span two exons to avoid false positives caused by residual genomic DNA. The primers used in this application were synthesized by Beijing Novogene Bioinformatics Technology Co., Ltd., and the specific sequences used are shown in Table 6.

[0129] Table 6. qPCR primer sequences used in this application

[0130]

[0131] 2.5 Sequence information of PAR2, FOXA2 and lysosomal V-type proton pump subunit ATP6V0E1

[0132] The NCBI ID of the PAR2 gene: 2150, the protein UniProtKB / Swiss-Prot: P55085; the NCBI ID of the FOXA2 gene: 3170, the protein UniProtKB / Swiss-Prot: Q9Y261; and the NCBI ID of the lysosomal V-type proton pump subunit ATP6V0E1 gene: 8992, the protein UniProtKB / Swiss-Prot: O15342.

[0133] II. Experimental methods

[0134] 1. Construction, transformation, extraction of plasmids and freezing of bacterial solutions

[0135] 1.1 Construction of plasmids

[0136] ① Design of sgRNA sequences. The corresponding sgRNA sequences were designed and synthesized by Beijing Genomics Institute Co., Ltd. The sequences of sgRNA are shown in Table 7.

[0137] Table 7. sgRNA sequences

[0138]

[0139] ② Digest the backbone vector and perform electrophoresis. Prepare the digestion reaction system as shown in Table 8. After mixing well by oscillation, digest at 22 °C for 30 min and then perform agarose gel electrophoresis. After the DNA marker is completely separated, take a photo in the ultraviolet imager, and cut the target band into a 1.5 mL centrifuge tube.

[0140] Table 8. Digestion reaction system

[0141]

[0142] ③ Gel recovery. Use the agarose gel DNA recovery kit for gel recovery, and measure the concentration of the gel recovery product with a NanoDrop instrument.

[0143] ④ Oligo annealing. Anneal the sgRNA to form a double strand. The reaction system and procedure are shown in Table 9 and Table 10.

[0144] Table 9. Annealing reaction volume

[0145]

[0146] Table 10. Annealing reaction procedure

[0147]

[0148] ⑤ Ligation. Ligate the annealed solution and the gel-recovered vector backbone overnight at 4 °C. The ligation system is shown in Table 11.

[0149] Table 11. Ligation reaction volume

[0150]

[0151] 1.2 Transformation of plasmids

[0152] Take out stbl3 competent cells from the -80 °C refrigerator, place them on ice to thaw, then pipette 50 μL into a clean 1.5 mL centrifuge tube, and add the above-mentioned ligated mixed product into the competent cells, gently mix by flicking. Place on ice for 25 min, then heat shock in a 42 °C water bath for 90 s and then let it stand on ice for 3 min. Add 1 mL of LB liquid medium in a bacterial laminar flow hood and incubate on a shaker at 37 °C and 180 rpm for 1 h. Take the supernatant and evenly spread it on the surface of a solid culture plate using a spreader (pre-coat the plate with the corresponding antibiotic according to the resistance of the plasmid). Invert the solid culture plate and incubate it in an incubator at 37 °C for 16 h. Then, pick single colonies into LB medium containing antibiotics in the laminar flow hood, incubate on a shaker at 37 °C and 200 rpm overnight, and then perform colony PCR verification.

[0153] 1.3 Plasmid extraction

[0154] The plasmid was extracted using the Endotoxin-Free Midiprep Kit from Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0155] 2. Lentivirus packaging

[0156] The packaging system used in this application is a four-plasmid system, consisting of four plasmids: vector plasmid LentiV2, auxiliary plasmids pMDL, pRSV-Rev, and VSVG. The pMDL plasmid contains the gag gene and pol gene of the HIV virus, mainly encoding the structural proteins and specific enzymes of the virus; the pRSV-Rev plasmid contains the rev gene that can regulate the expression of the gag and pol genes; the VSVG plasmid can provide the envelope protein required for virus packaging.

[0157] The specific steps for lentivirus packaging are as follows: Digest HEK-293T cells into a 10 cm dish. After 24 h, aspirate the culture medium, rinse twice with PBS, add 12 mL of serum-free high-glucose DMEM medium, and place it in an incubator at 37°C for culture. Subsequently, add LentiV2: pMDL: VSVG: pRSV-Rev into a 5 mL centrifuge tube containing 1.5 mL of serum-free medium (total plasmid amount is 24 μg) at a ratio of 10:5:3:2. At the same time, add 1.5 mL of serum-free medium into another 5 mL centrifuge tube, then add 60 μL of liposome, let it stand at room temperature for 5 min, mix and invert gently up and down. After standing at room temperature for 20 min, gently rotate and add the nucleic acid-Lipo2000 mixture into the 10 cm dish, gently shake the 10 cm dish, and then place it in the incubator for 6 - 8 h. After that, aspirate the culture medium and add fresh complete medium. At 72 h after transfection, collect the supernatant containing virus particles in a biosafety cabinet, and then centrifuge at 1200 rpm for 8 min. Filter the centrifuged supernatant with a 0.22 μm filter membrane. One part is used to infect target cells, and the other part can be stored in an -80°C refrigerator for subsequent infection of target cells.

[0158] 3. Construction of stable cell lines

[0159] Digest the target cells to be infected into 6-well plates in a biosafety cabinet. Add 1 mL of complete medium, 1000 μL of virus solution, and 2 μL of lentivirus infection enhancer Polybrene to each well. After 72 h of infection, aspirate the culture medium and add the medium containing antibiotics, and continuously screen for 14 days according to the lowest killing concentration. After 14 days, take out a part of the cells to extract RNA and proteins to identify the expression efficiency of the mixed cell population. At the same time, use the limiting dilution method to transfer some cells to 96-well plates to ensure 1 cell per well. After culturing and expanding to a certain time, identify each monoclonal cell line, and freeze the positive cell lines. After screening positive cell lines, the antibiotic concentration can be adjusted to half of the lowest killing concentration for maintenance culture.

[0160] 4. Extraction and quantification of total proteins from cells and tissues

[0161] The extraction steps for total proteins from animal tissues and cells are basically the same, and are carried out by conventional methods in this field. The protein concentration is measured by the BCA method.

[0162] 5. Western Blot

[0163] Use the one-step rapid gel preparation kit of Nanjing Novozymes Biotech Co., Ltd. and perform according to the conventional methods in this field.

[0164] 6. RNA Extraction of Cells and Tissues

[0165] The extraction procedures for RNA from animal tissues and cells are basically the same and are carried out using conventional methods in the art. For the measured extracted RNA, generally, A260 / 280 should be 1.8 - 2.2. If A260 / 280 is less than 1.8, it may be contaminated by proteins or organic solvents; if A260 / 280 is greater than 2.2, it may indicate RNA degradation.

[0166] 7. Reverse Transcription of RNA

[0167] The reverse transcription of RNA uses the Fast DNA Removal Reverse Transcription Kit from Nanjing Novoprotein Scientific Inc. Prepare the reaction system in a centrifuge tube according to Table 12, mix well by shaking and then centrifuge. Place the centrifuge tube in a PCR instrument and set the reaction program according to Table 13. After the reaction is completed, store the product at -20°C.

[0168] Table 12. Reverse Transcription System

[0169]

[0170] Table 13. Reverse Transcription Program

[0171]

[0172] 8. Real-Time Fluorescent Quantitative PCR

[0173] Real-time fluorescent quantitative PCR is carried out using the SYBR Green PCR Master Mix Kit from Takara. Take out the reverse transcription product and reagents from -20°C, mix well and then centrifuge, and place the reagents on ice. Prepare the reaction system in a centrifuge tube according to Table 14, mix well by inverting up and down and then centrifuge.

[0174] Table 14. Real-Time Fluorescent Quantitative PCR System

[0175]

[0176] The reaction program is shown in Table 15. After the reaction is completed, use the 2 -△△Ct method to calculate the relative expression level.

[0177] Table 15. Real-Time Fluorescent Quantitative PCR Program

[0178]

[0179] 9. LC3-EGFP-mcherry Autophagy Detection System

[0180] The LC3-EGFP-mcherry autophagy detection system is one of the classical methods for detecting autophagy and is carried out by methods well-known in the art. Its main principle is as follows: When autophagy occurs, the LC3-EGFP-mcherry fusion protein will transfer to the surface of the autophagosome membrane. When the autophagosome can normally fuse with the lysosome, at this time, due to the sensitivity of EGFP to the acidic environment of the lysosome, the EGFP fluorescence quenches, while the mcherry fluorescence is not affected. At this time, red spots are observed under the fluorescence microscope. When the autophagosome cannot normally fuse with the lysosome, at this time, EGFP is in a neutral pH environment such as the cytoplasm, so the fluorescence of both EGFP and mCherry is not affected. At this time, yellow spots are observed under the fluorescence microscope.

[0181] 10. Immunofluorescence of cells

[0182] It is carried out by methods well-known in the art and photographed using an UltraVIEW VoX live cell high-speed laser confocal real-time imaging system. After photographing, quantitative analysis is performed using ImageJ software.

[0183] 11. Detection of lysosome pH and ROS by flow cytometry

[0184] For the detection of lysosome pH by flow cytometry, it is carried out by methods well-known in the art, measured using a BD LSRII flow cytometer, and analyzed using Flow J software.

[0185] The content of ROS in cells is detected using a DCFH-DA probe. The main principle is that the DCFH-DA probe can freely penetrate the cell membrane and is then hydrolyzed by intracellular esterases to generate DCFH. At this time, the ROS in the cell can oxidize the non-fluorescent DCFH into DCF with green fluorescence. For the detection of ROS by flow cytometry, it is measured using a BD LSRII flow cytometer and analyzed using Flow J software.

[0186] 12. Multiplex immunofluorescence

[0187] (1) Deparaffinization. The paraffin component on the section will affect the staining effect. To ensure the staining effect, the paraffin on the section needs to be removed before staining. First, place the paraffin section in an oven at 65 °C for 2 h for deparaffinization. Subsequently, immediately immerse the completely deparaffinized section in xylene for 20 min. To avoid the re-solidification of paraffin, the section needs to be quickly placed in the xylene solution. Then place the section in a new xylene solution and soak for 20 min.

[0188] (2) Hydration. To enable the reagent to fully bind to the antigens in the tissue on the section, hydration is required. Take the section out of xylene, soak it in absolute ethanol for 10 min, and then place the section successively in 95% ethanol solution, 90% ethanol solution, 85% ethanol solution, 80% ethanol solution, 75% ethanol solution, and 70% ethanol solution for 10 min each. Subsequently, wash the section with PBST (PBS containing 0.05% Tween 20) solution on a horizontal shaker for 5 min, three times in total.

[0189] (3) Microwave antigen retrieval. Prepare EDTA buffer (pH = 9.0), then soak the section in the antigen retrieval solution, heat it to boiling in a microwave oven at high power, and immediately turn the temperature to low heat for 30 min. After heating, cool it to room temperature, and then wash the section with PBST (PBS containing 0.05% Tween 20) solution on a horizontal shaker for 5 min, three times in total.

[0190] (4) Elimination of endogenous peroxidase. Since endogenous peroxidase and biotin will interfere with the immunohistochemical reaction, inactivation must be carried out. Circle the tissue to be stained with an immunohistochemical pen, and then add 3% hydrogen peroxide solution and react at room temperature for 20 min. Subsequently, wash the section with PBST (PBS containing 0.05% Tween 20) solution on a horizontal shaker for 5 min, three times in total.

[0191] (5) Blocking. Add a certain amount of normal goat serum solution for blocking to the tissue of the section. Then place the section in an incubator at 37°C for blocking for 1 h. Subsequently, wash the section with PBST (PBS containing 0.05% Tween 20) solution on a horizontal shaker for 5 min, three times in total.

[0192] (6) Incubation with primary antibody. Prepare a solution containing anti-FOXA2 primary antibody with an antibody diluent, and add the diluted solution containing anti-FOXA2 primary antibody to the tissue of the section. Incubate at 4°C for 16 - 18 h. After incubation, wash the section with PBST (PBS containing 0.05% Tween 20) solution on a horizontal shaker for 5 min, three times in total.

[0193] (7) Incubation with poly-HRP secondary antibody. Prepare the corresponding poly-HRP secondary antibody solution with an antibody diluent and add it to the tissue of the section, and incubate in a humid box at room temperature in the dark for 1 h. Subsequently, wash the section with PBST (PBS containing 0.05% Tween 20) solution on a horizontal shaker for 5 min, three times in total.

[0194] (8) TSA Fluorescent Dye Conjugation. Add 50 μL of ready-to-use TYR-488 fluorescent dye dropwise onto the tissue sections, and incubate in a moist chamber at room temperature in the dark for 15 min.

[0195] (9) Antibody Elution. Add a certain amount of mIHC-specific antibody elution solution onto the tissue sections, and incubate in a moist chamber at 37°C in the dark for 20 min. Discard the elution solution, add a certain amount of mIHC-specific antibody elution solution onto the tissue sections again, and incubate in a moist chamber at 37°C in the dark for 20 min. Subsequently, wash the sections on a horizontal shaker with PBST (PBS containing 0.05% Tween 20) solution for 5 min, three times in total.

[0196] (10) Microwave Antigen Retrieval. Prepare citrate buffer (pH = 6.0), then immerse the sections already labeled with FOXA2 in the antigen retrieval solution, heat to boiling in a microwave oven on high power, immediately turn the temperature to low heat and repair for 30 min. After heating, cool to room temperature, and then wash the sections on a horizontal shaker with PBST (PBS containing 0.05% Tween 20) solution for 5 min, three times in total.

[0197] (11) Elimination of Endogenous Peroxidase. Outline the tissue with an immunohistochemistry pen, then add 3% hydrogen peroxide solution and react at room temperature for 20 min. Subsequently, wash the sections on a horizontal shaker with PBST (PBS containing 0.05% Tween 20) solution for 5 min, three times in total.

[0198] (12) Blocking. Add a certain amount of normal goat serum solution for blocking onto the tissue sections. Then place the sections in an incubator at 37°C for blocking for 1 h. Subsequently, wash the sections on a horizontal shaker with PBST (PBS containing 0.05% Tween 20) solution for 5 min, three times in total.

[0199] (13) Primary Antibody Incubation. Prepare a solution containing MPO primary antibody with an antibody diluent, and add the diluted solution containing MPO primary antibody onto the tissue sections. Incubate at 4°C for 16 - 18 h. After incubation, wash the sections on a horizontal shaker with PBST (PBS containing 0.05% Tween 20) solution for 5 min, three times in total.

[0200] (14) Incubation with Polymer HRP Secondary Antibody. Prepare a corresponding polymer HRP secondary antibody solution with an antibody diluent and add it onto the tissue sections, and incubate in a moist chamber at room temperature in the dark for 1 h. Subsequently, wash the sections on a horizontal shaker with PBST (PBS containing 0.05% Tween 20) solution for 5 min, three times in total.

[0201] (15) TSA Fluorescent Dye Conjugation. Add 50 μL of ready-to-use TYR-594 fluorescent dye dropwise onto the tissue sections, and incubate in the dark at room temperature for 15 min in a humidified chamber.

[0202] (16) Antibody Elution. Add a certain amount of mIHC-specific antibody elution solution onto the tissue sections, and incubate in the dark at 37 °C for 20 min in a humidified chamber. Discard the elution solution, add a certain amount of mIHC-specific antibody elution solution onto the tissue sections again, and incubate in the dark at 37 °C for 20 min in a humidified chamber. Subsequently, wash the sections on a horizontal shaker with PBST (PBS containing 0.05% Tween 20) solution for 5 min, three times in total.

[0203] (17) Microwave Antigen Retrieval. Prepare citrate buffer (pH = 6.0), then immerse the sections labeled with FOXA2 and MPO in the antigen retrieval solution, heat to boiling in a microwave oven at high power, immediately turn the temperature to low heat and retrieve for 30 min. After heating, cool to room temperature, and then wash the sections on a horizontal shaker with PBST (PBS containing 0.05% Tween 20) solution for 5 min, three times in total.

[0204] (18) Elimination of Endogenous Peroxidase. Outline the tissue with an immunohistochemistry pen, then add 3% hydrogen peroxide solution and react at room temperature for 20 min. Subsequently, wash the sections on a horizontal shaker with PBST (PBS containing 0.05% Tween 20) solution for 5 min, three times in total.

[0205] (19) Blocking. Add a certain amount of normal goat serum solution for blocking onto the tissue sections. Then place the sections in an incubator at 37 °C for blocking for 1 h. Subsequently, wash the sections on a horizontal shaker with PBST (PBS containing 0.05% Tween 20) solution for 5 min, three times in total.

[0206] (20) Primary Antibody Incubation. Prepare a solution containing PANCK primary antibody with an antibody diluent, and add the diluted solution containing PANCK primary antibody onto the tissue sections. Incubate at 4 °C for 16 - 18 h. After incubation, wash the sections on a horizontal shaker with PBST (PBS containing 0.05% Tween 20) solution for 5 min, three times in total.

[0207] (21) Incubation with Polymer HRP Secondary Antibody. Prepare the corresponding polymer HRP secondary antibody solution with an antibody diluent and add it onto the tissue sections, and incubate in the dark at room temperature for 1 h in a humidified chamber. Subsequently, wash the sections on a horizontal shaker with PBST (PBS containing 0.05% Tween 20) solution for 5 min, three times in total.

[0208] (22) TSA Fluorescent Dye Conjugation. Add 50 μL of ready-to-use TYR-647 fluorescent dye dropwise onto the tissue sections, and incubate in the dark at room temperature for 15 min in a moist chamber.

[0209] (23) Antibody Elution. Add a certain amount of mIHC-specific antibody elution solution onto the tissue sections, and incubate in the dark at 37 °C for 20 min in a moist chamber. Discard the elution solution, add a certain amount of mIHC-specific antibody elution solution onto the tissue sections again, and incubate in the dark at 37 °C for 20 min in a moist chamber. Subsequently, wash the sections with PBST (PBS containing 0.05% Tween 20) solution on a horizontal shaker for 5 min, three times in total.

[0210] (24) Nuclear Staining. Add DAPI working solution dropwise onto the tissue sections, and incubate in the dark at room temperature for 10 min in a moist chamber. Subsequently, wash the sections with PBST (PBS containing 0.05% Tween 20) solution on a horizontal shaker for 5 min, three times in total. Rinse the sections in the dark under distilled water for 30 min.

[0211] (25) Add anti-quenching mounting medium dropwise onto the sections.

[0212] (26) Observe and image using a multispectral tissue imaging system.

[0213] Multiplex Immunofluorescence Analysis

[0214] (1) HALO Software Analysis. Perform machine training for region recognition on the scanned samples, including tumor, stroma, and blank regions.

[0215] (2) Identify and determine the number of cell nuclei.

[0216] (3) Count the number of cells positive for FOXA2, MPO, and PANCK.

[0217] (4) Proximity Analysis. Using MPO + cells as the center, detect the proximity of PANCK + / FOXA2 + to MPO + cells.

[0218] (5) Fluorescence Intensity Analysis. According to the results of proximity analysis, compare the fluorescence intensity of FOXA2 in PANCK + cells, and calculate the average fluorescence intensity of FOXA2.

[0219] 13. Mouse Organoid Extraction

[0220] (1) Anesthetize the mice with good growth status, then open the abdominal cavity of the mice, and use sterilized scissors and forceps to intercept a 3-cm section of the colon. Use forceps to remove the mesentery and fat outside the intestine and then open the intestine. Remove the feces. Place the intestinal segment in PBS solution pre-cooled to 4°C.

[0221] (2) Cut the intestinal segment into fragments the size of rice grains, transfer them to a 50-mL centrifuge tube, add 8 mL of DPBS (containing 1% penicillin and streptomycin), mix well by inverting up and down, then let the centrifuge tube stand still to allow the fragments to naturally settle to the bottom of the centrifuge tube, and aspirate the supernatant. Repeat this step 3 - 4 times.

[0222] (3) Add 15 mL of 5 mM EDTA solution and digest at room temperature on a horizontal shaker for 25 min.

[0223] (4) Let the centrifuge tube stand still to allow the fragments to naturally settle to the bottom of the centrifuge tube, and aspirate the supernatant. Add 8 mL of DPBS (containing 1% penicillin and streptomycin), mix well by inverting up and down, then let the centrifuge tube stand still to allow the fragments to naturally settle to the bottom of the centrifuge tube, and aspirate the supernatant.

[0224] (5) Add 8 mL of DPBS (containing 1% penicillin and streptomycin), vigorously pipette to resuspend the crypts in the DPBS solution. Let the centrifuge tube stand still to allow the fragments to naturally settle to the bottom of the centrifuge tube, then collect the supernatant, filter it through a 70-μm cell strainer, and collect the filtrate.

[0225] (6) Centrifuge at 280×g for 10 min, aspirate the supernatant, add Matrigel to resuspend the precipitate, pipette 50 μL of the Matrigel-organoid mixed suspension to the center of a 24-well plate, and then place it in an incubator at 37°C for gelation for 30 min.

[0226] (7) Add 600 μL of mouse large intestine organoid medium to each well for culture, and change the medium every 3 - 4 days. When the organoids grow to a certain density, perform subculture.

[0227] 14. High-throughput drug screening targeting lysosomal function

[0228] This experiment was assisted by the Pharmaceutical Technology Center of Tsinghua University, and the drug library used was a collection of 3098 drugs approved by the FDA of Selleck. The steps are as follows: On the first day, seed the cells into a 384-well plate, 1000 cells per well. After 24 h, perform drug treatment for 48 h continuously. Then add a lysosomal pH indicator and use a high-content imager to take pictures and analyze.

[0229] 15. Bioinformatics analysis

[0230] For immune cell infiltration analysis, the GEO dataset was first downloaded, and the "CIBERSORT" package in R language was used to evaluate immune cell infiltration through the gene expression matrix, with 22 immune cell gene sets (LM22) as the reference gene set.

[0231] To analyze neutrophil infiltration and FOXA2 levels in the TCGA database, the transcriptome expression profile data of COAD and READ patients were first downloaded, and the "CIBERSORT" package in R language was used to evaluate immune cell infiltration through the gene expression matrix, with 22 immune cell gene sets (LM22) as the reference gene set. Samples were grouped according to the neutrophil infiltration level, and the Wilcoxon test was used to compare the FOXA2 expression between the high-expression and low-expression quartiles.

[0232] 16. Animal model

[0233] Dextran Sulfate Sodium (DSS; MP Biomedicals, catalog number: MFCD00081551) is an artificially synthesized sulfate polysaccharide that can induce colitis characterized by bloody stools, intestinal mucosal ulceration, and massive granulocyte infiltration in mice. For the establishment of the DSS model, 2.5% DSS was prepared in drinking water and administered continuously for 7 days. On the day when DSS treatment ended and on the 1st and 3rd days, 50 mg / kg carbamazepine or the solvent control (85% PEG3000 + 10% DMSO + 5% Tween 80) was administered by gavage, and intestinal tissues were collected on the 0th, 2nd, 3rd, and 4th days after DSS treatment ended.

[0234] III. Statistical methods

[0235] In this application, GraphPad Prism 8 software was used to generate charts and perform statistical analysis. Unpaired Student’s t-tests were used to detect statistical significance between two groups. One- or two-way ANOVAs were used for three groups and above. The results were expressed as mean ± standard deviation. Significance was indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0236] Example 2. Neutrophil-derived serine proteases cleave and inhibit the PAR2 / FOXA2 signaling axis

[0237] 1. Inactivation of PAR2 by neutrophil-derived serine proteases reduces the expression of FOXA2

[0238] Normal colon epithelial cells NCM460 and colon cancer cells HT29 were treated with neutrophil elastase (NE; denoted as Elastase in the figure), cathepsin G (CTSG), proteinase-3 (PRTN3), and a mixture of the three, respectively. The results showed that the expression of FOXA2 and ATP6V0E1 was significantly reduced whether elastase, CTSG, PRTN3, or the mixture of the three was used alone ( Figure 2 A-C). Interestingly, overexpression of FOXA2 could reverse the downregulation of ATP6V0E1 caused by neutrophil-derived serine proteases ( Figure 2 A-C). This suggests that neutrophil-derived serine proteases can regulate the expression of ATP6V0E1 through FOXA2.

[0239] Treatment with neutrophil-derived serine proteases in either normal colon epithelial cells NCM460 or colon cancer cells HT29 could lead to a decrease in lysosomal acidification function ( Figure 3 A), autophagosome-lysosome fusion disorder ( Figure 3 B), and autophagy inhibition ( Figure 2 C). More importantly, overexpression of FOXA2 could reverse the decrease in lysosomal acidification function ( Figure 3 A), autophagosome-lysosome fusion disorder ( Figure 3 B), and autophagy inhibition ( Figure 2 A-C) caused by neutrophil-derived serine proteases. In summary, these results suggest that neutrophil-derived serine proteases can regulate the expression of ATP6V0E1 through FOXA2, thereby inhibiting lysosomal acidification, autophagosome-lysosome fusion, and autophagy.

[0240] The PAR2-knockout HT29 cell line was constructed using CRISPR / Cas9 ( Figure 4 A). Although neutrophil-derived serine proteases could inhibit the expression of FOXA2 and ATP6V0E1, lysosomal acidification, and autophagy in control cells ( Figure 4 B-D); however, knocking out PAR2 eliminated all of the above effects of neutrophil-derived serine proteases ( Figure 4 B-D). To exclude off-target effects, the PAR2 gene was re-supplemented in the PAR2-knockout cells, and these effects were restored again ( Figure 4 B-D). In summary, this suggests that neutrophil-derived serine proteases indeed inhibit lysosomal acidification mediated by FOXA2 / ATP6V0E1 through PAR2, causing autophagy disorders.

[0241] Isolate F2rl1△IEC Organoids were established from the intestinal epithelium of mice and treated with neutrophil-derived elastase to detect the expression of FOXA2, ATP6V0E1 and autophagy-related proteins. The results showed that neutrophil-derived elastase significantly inhibited the expression of F2rl1 f / f Expression of FOXA2 and ATP6V0E1 in mouse colorectal organoids, whereas in F2rl1 △IEC The mouse colorectal organoids derived from WT mice did not result in downregulation of FOXA2 and ATP6V0E1 ( Figure 5 B). This further suggests that neutrophil-derived elastase inhibits the FOXA2 / ATP6V0E1 signaling axis through PAR2. Since the basal autophagic flux of normal cells is weak, a glutamine starvation-induced autophagy model was established. Specifically, the cultured colorectal organoids were treated with glutamine-free medium for 24 h. The results showed that: in F2rl1 f / f In mouse-derived colorectal organoids, glutamine deficiency significantly induced the upregulation of the autophagy-related protein LC3II, while treatment with neutrophil-derived elastase significantly led to the accumulation of p62 ( Figure 5 A) This suggests that neutrophil-derived elastase significantly blocks autophagy. △IEC In mouse colorectal organoids, glutamine starvation-induced autophagy was significantly blocked, and treatment with neutrophil-derived elastase did not further impair autophagy ( Figure 5 A).

[0242] Next, we used Trypsin and PAR2 activator PAR2-AP to detect the effects of PAR2 activation on lysosomal acidification and autophagy mediated by the FOXA2 / ATP6V0E1 signaling axis in normal intestinal epithelial cells. The results showed that, like PAR2-AP, Trypsin can promote the expression of FOXA2 and ATP6V0E1 in normal intestinal epithelial cells NCM460 and IEC6 ( Figure 6 AB and Figure 6 E). In addition, Trypsin can promote lysosomal acidification and autophagosome-lysosome fusion in normal intestinal epithelial cells NCM460 and IEC6 ( Figure 6 CD). More importantly, Trypsin can promote autophagy in normal intestinal epithelial cells NCM460 and IEC6 ( Figure 6 E). In summary, neutrophil-derived serine proteases downregulate the FOXA2 / ATP6V0E1 signaling axis by cleaving PAR2, while Trypsin can activate PAR2 to upregulate the FOXA2 / ATP6V0E1 signaling axis.

[0243] 2. Validation of the expression correlation between neutrophils and FOXA2 in clinical samples

[0244] It has been clarified that the inactivation of PAR2 by neutrophils inhibits FOXA2, which is the core event of the signal axis regulating autophagy mediated by lysosomal function. Next, correlation analysis was performed in clinical samples. First, the gene set of neutrophils defined in the CIBERSORT package was used to analyze the transcriptome data of normal volunteers and UC patients. Consistent with expectations, the neutrophil index was significantly higher in UC patients than in normal volunteers ( Figure 7 A), and more importantly, the expression of FOXA2 in UC patients was significantly lower than that in normal volunteers ( Figure 7 B). This suggests that there is a negative correlation between neutrophil infiltration and FOXA2 expression in UC patients.

[0245] Next, the expression of FOXA2 in CRC and CAC was analyzed. By using the transcriptome data of 6 CRC patients and 5 CAC patients in the GEO database, it was found that: compared with CRC, CAC patients developed from IBD with an inflammatory environment, and the expression of FOXA2 was also lower ( Figure 8 ).

[0246] Next, the correlation between neutrophil infiltration and FOXA2 in CRC patients in the TCGA database was analyzed. First, CRC patients were divided into 4 groups according to the neutrophil index, and the patients in the top 1 / 4 and bottom 1 / 4 were selected. Subsequently, the expression levels of their FOXA2 were analyzed. The results showed that: the expression of FOXA2 in CRC patients with a high neutrophil index was significantly lower than that in CRC patients with a low neutrophil index ( Figure 9 ). These results suggest that: whether in UC patients, CAC patients or CRC patients, there is a negative correlation between the infiltration level of neutrophils and the expression of FOXA2.

[0247] Subsequently, the correlation between the expression of FOXA2 and ATP6V0E1 in UC patients was analyzed. The results showed that: there was a significant positive correlation between the expression of FOXA2 and ATP6V0E1 ( Figure 10 ). In addition, there was also a significant correlation between FOXA2 and ATP6V0E1 in the TCGA database, as well as between PAR2 and FOXA2 ( Figure 11 ).

[0248] Analysis through the database showed a significant negative correlation between the infiltration level of neutrophils and FOXA2. Subsequently, multiplex immunofluorescence was performed on the collected clinical samples for verification. First, 8 patients with active UC and 8 patients with remission UC were collected, and stained for epithelial cells (PANCK, magenta), neutrophils (MPO, yellow), and FOXA2 (green). The results showed that the marker of neutrophils was significantly higher in patients with active disease than in those in remission. In addition, FOXA2 was mainly expressed in epithelial cells ( Figure 12 A). More importantly, it was found that the expression level of FOXA2 in patients with active disease was significantly lower than that in patients in remission ( Figure 12 A - B), and these results were consistent with those of the database analysis.

[0249] Due to the presence of a large number of protease inhibitors in the body, the range of action of serine proteases released by neutrophils is small, usually around a single cell. Then, patients with active disease were stratified, and the expression level of FOXA2 in epithelial cells around neutrophils (≤25 μm) was analyzed. The results showed that compared with the overall expression of FOXA2, the expression of FOXA2 in intestinal epithelial cells around neutrophils was significantly lower than the overall FOXA2 expression ( Figure 12 B, D). Subsequently, the correlation between the number of neutrophils and the number of FOXA2 - positive epithelial cells was analyzed. Consistent with the above conclusion, there was a significant negative correlation between the number of neutrophils and the number of FOXA2 - positive epithelial cells ( Figure 12 C).

[0250] In addition to patients with UC, samples from 3 CRC patients with neutrophil infiltration were collected and multi - color immunofluorescence staining was performed for verification. The results showed that the expression of FOXA2 in intestinal epithelial cells around neutrophils was significantly lower than the overall and average FOXA2 expression ( Figure 13 A - B). Subsequently, stratification analysis was performed according to the distance from neutrophils, divided into 0 - 25 µm, 25 - 50 µm, 25 - 75 µm, and 75 - 100 µm from neutrophils. The results showed that the number of FOXA2 - positive cells in epithelial cells at 25 - 50 µm, 25 - 75 µm, and 75 - 100 µm around neutrophils was significantly higher than that in epithelial cells at 0 - 25 µm around neutrophils ( Figure 13 C). These results suggest that serine proteases released by neutrophils can also inhibit the expression of FOXA2 in vivo.

[0251] This example used multiple models such as normal intestinal epithelium, colon cancer cells, and organoids to confirm that neutrophil-derived serine proteases can inactivate PAR2, reduce the expression of FOXA2, thereby reducing the expression of ATP6V0E1, leading to lysosomal acidification disorders, and ultimately causing autophagy blockade. In addition, multiple database analyses and multiplex immunofluorescence results of UC and CRC patients suggested that the PAR2 / FOXA2 / ATP6V0E1 signaling axis exists in IBD, CAC, and CRC samples. These results strongly suggest that neutrophil-induced downregulation of FOXA2 may be a key step in the inflammation-cancer transformation.

[0252] Example 3. Drug screening and mechanism study for targeting PAR2 / FOXA2 to reverse autophagy disorders

[0253] 1. In vitro screening of drugs that can reverse PAR2 / FOXA2-related lysosomal dysfunction

[0254] First, a drug library containing 3,098 FDA-approved drugs from Selleck was selected. Based on the detection of changes in lysosomal pH by a high-content imager, drugs that can reverse the lysosomal acidification disorder related to PAR2 knockdown were screened. The present application first found that the anti-epileptic drug carbamazepine (CBZ) can effectively restore lysosomal pH ( Figure 14 A-B). Then, the inventors of the present application conducted verification. Consistent with the previous results, neutrophil-derived elastase can significantly inhibit lysosomal pH, and carbamazepine can reverse the increase in lysosomal pH caused by neutrophil-derived elastase ( Figure 14 C-D).

[0255] To verify whether carbamazepine can promote lysosomal acidification in a FOXA2-ATP6V0E1 signaling axis-dependent manner. The inventors of the present application first detected the mRNA and protein expression levels of FOXA2 and ATP6V0E1 under the treatment of neutrophil-derived elastase. Consistent with the results in Example 2, the treatment with neutrophil-derived elastase significantly reduced the mRNA and protein expression levels of FOXA2 and ATP6V0E1, while carbamazepine can reverse the downregulation of the mRNA and protein expression levels of FOXA2 and ATP6V0E1 caused by neutrophil-derived elastase ( Figure 15 A-B). More importantly, carbamazepine can reverse the autophagy blockade caused by neutrophil-derived elastase ( Figure 15 B).

[0256] The inventors of the present application also confirmed that carbamazepine can reverse the excessive accumulation of ROS caused by neutrophil-derived elastase ( Figure 16)。Excessive ROS accumulation can lead to DNA damage, and carbamazepine can also reverse the accumulation of γH2AX, a DNA damage induced by neutrophil-derived elastase( Figure 17 )。

[0257] 2. Carbamazepine promotes the repair after acute enteritis injury

[0258] The repair of acute post-injury in the intestine is related to the occurrence of CAC. Carbamazepine is an anti-epileptic drug used clinically. By using the method of short-term administration, it was detected whether carbamazepine could promote the post-injury repair process of acute enteritis. First, mice were induced with acute colitis by DSS for 7 consecutive days( Figure 18 A), and then carbamazepine was intragastrically administered on the day when DSS ended and on the 1st and 3rd days, for a total of 3 times. Intestinal samples were taken on the 0th, 2nd, 3rd, and 4th days after DSS ended. Interestingly, carbamazepine could reverse the increased DAI score and shortened intestinal length induced by DSS( Figure 18 B-C), the up-regulation of inflammatory factors( Figure 19 ), and the down-regulation of FOXA2 and ATP6V0E1( Figure 20 )。

[0259] Conclusion: The inventors of this application first discovered and verified through screening that the anti-epileptic drug carbamazepine can reverse the acidification disorder of lysosomes mediated by FOXA2 / ATP6V0E1 induced by neutrophil-derived serine protease, as well as excessive ROS accumulation and DNA damage. The in vivo experimental results showed that carbamazepine can promote the post-injury repair of acute enteritis, partly by up-regulating the expression of FOXA2 and ATP6V0E1. These results indicate that carbamazepine has the potential to be a candidate drug for the treatment of colitis.

[0260] It can be understood that although the inventions involved in this application are described in the above specific forms, these inventions are not limited to the specific content described in these forms. It is obvious to those skilled in the art that, without departing from the spirit of the inventions described in this application, various equivalent changes can also be made to the technical features included in the inventions involved, and these changes should all fall within the scope of the said inventions.

Claims

1. Use of a substance that upregulates the expression level of at least one biomarker in the signal pathway of protease-activated receptor 2 (PAR2)-mediated lysosomal function in the preparation of a composition for one or more of the following: (1) Regulating lysosomal acidification in cells; (2) Regulating autophagy function in cells; (3) Regulating autophagosome-lysosome fusion in cells; (4) Reducing the accumulation of reactive oxygen species (ROS) in cells; (5) Reducing the level of DNA damage in cells; (6) Reducing the expression level of inflammatory factors in cells; (7) Improving or treating acute colitis injury in a subject; (8) Improving or treating inflammatory bowel disease in a subject; (9) Preventing, improving or treating inflammatory cancer transformation in a subject; and (10) Preventing, improving or treating colorectal cancer in a subject; Among them, The biomarker is selected from: PAR2, forkhead box protein A2 (FOXA2), and lysosomal V-type proton pump subunit ATP6V0E1; Preferably, the upregulation of the expression level of at least one biomarker in the signal pathway of PAR2-mediated lysosomal function is the upregulation of the expression level of at least one, at least two, or all of PAR2, FOXA2, and lysosomal V-type proton pump subunit ATP6V0E1.

2. The use according to claim 1, wherein the subject is a mammal, preferably a human.

3. The use according to claim 1 or 2, wherein the cell is an intestinal epithelial cell, preferably a colon epithelial cell.

4. The use according to any one of claims 1-3, wherein the inflammatory factor is a colitis-related inflammatory factor, preferably Il-6 and / or Mcpt1.

5. The use according to any one of claims 1-4, wherein the acute colitis injury is one or more of the following: diarrhea, rectal prolapse, bloody stools, intestinal edema, mucosal erythema, intestinal stricture, and fistula.

6. The use according to any one of claims 1-5, wherein the inflammatory bowel disease is colitis, preferably ulcerative colitis.

7. The use according to any one of claims 1-6, wherein the inflammatory cancer transformation is the transformation from inflammatory bowel disease to colorectal cancer; preferably the transformation from colitis to colorectal cancer; more preferably the transformation from ulcerative colitis to colorectal cancer; most preferably the transformation from ulcerative colitis to colitis-associated colon cancer (CAC).

8. The use according to any one of claims 1-7, wherein the colorectal cancer is colitis-associated colon cancer (CAC).

9. The use according to any one of claims 1-8, wherein the substance upregulates the reduced expression level of at least one biomarker in the signal pathway of PAR2-mediated lysosomal function caused by serine protease; preferably, the serine protease is a neutrophil-derived serine protease; preferably, the serine protease is neutrophil-derived elastase, cathepsin G, or protease 3.

10. The use according to any one of claims 1-9, wherein the substance is an antiepileptic drug, an activator of PAR2, or a serine protease inhibitor; Preferably, the anti-epileptic drug is carbamazepine; and / or the activator of PAR2 is a synthetic peptide PAR2-AP or trypsin; and / or the serine protease inhibitor is an inhibitor of neutrophil-derived serine protease; more preferably, the serine protease inhibitor is an inhibitor of neutrophil-derived elastase, cathepsin G or protease 3; most preferably, the serine protease inhibitor is ulinastatin, nafamostat mesylate or aprotinin.

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