Application of carboxypyranocyanidin-3-O-glucoside

Abstract

This invention provides the application of carboxypyranocyanidin-3-O-glucoside, relating to the field of biomedical technology. Specifically, this invention provides the application of carboxypyranocyanidin-3-O-glucoside in the preparation of inhibitors of inflammatory cytokine expression. For inflammatory diseases, carboxypyranocyanidin-3-O-glucoside exhibits good therapeutic effects and high safety, making it worthy of widespread application.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and more specifically, to the application of carboxypyranocyanidin-3-O-glucoside. Background Technology

[0002] Leukocytosis is an independent risk factor for cardiovascular disease. Disorders of hematopoietic function, particularly increased myeloid cell differentiation, are directly associated with an increase in monocytes and neutrophils in cardiovascular disease. Blood-derived Ly6C... hi An increased supply of monocytes directly exacerbates the formation of atherosclerotic plaques. Blood-derived Ly6C hi Monocytes are also an important source of macrophages in the gut and participate in the process of enteritis. In the course of ulcerative colitis (UC), Ly6C... hi The terminal differentiation of monocytes into mature intestinal macrophages will be interrupted. Hematopoietic stem cells and progenitor cells (HSPCs) in the bone marrow can differentiate into mature myeloid cells and lymphocytes, maintaining the supply of immune cells to tissues and organs and homeostasis. However, the development of various inflammatory diseases, such as atherosclerosis and ulcerative colitis, can induce the proliferation of HSPCs and their conversion to Ly6C by altering the microenvironment in which they reside. hi Mononuclear macrophage differentiation leads to increased inflammation.

[0003] Therefore, finding effective drugs to prevent and treat inflammatory diseases caused by leukocytosis is of great significance. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide the application of carboxypyranocyanidin-3-O-glucoside, which can effectively improve inflammatory diseases.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] Application of carboxypyranocyanidin-3-O-glucoside in the preparation of inflammatory factor expression inhibitors.

[0007] The structure of the carboxyl-Pyranocyanidin-3-O-glucoside (Cpycy-3-glu) of the present invention is shown below:

[0008]

[0009] This invention does not limit the source of carboxypyranoanthocyanins-3-O-glucoside; it can be a commercially available product or prepared by the method described in the literature "Comparative study on the stability and antioxidant activity of six pyranoanthocyanins based on malvidin-3-glucoside".

[0010] In this invention, the inflammatory factors include one or more of TNF-α, MCP-1, IL-6, TNF-α, and MCP-1.

[0011] In this invention, the preferred dosage of pyranocyanidin-3-O-glucoside for mice is 25–200 mg / kg·bw; the preferred dosage for humans is 2–25 mg / kg·bw, preferably 2.75–21.98 mg / kg·bw.

[0012] The concentration of pyranocyanidin-3-O-glucoside in the inflammatory factor expression inhibitor of the present invention is 12.5-150 μM, preferably 100-150 μM.

[0013] The present invention also provides the use of carboxypyranocyanidin-3-O-glucoside in the preparation of products for the prevention and / or treatment of diseases caused by hematogenous mononuclear macrophage proliferation.

[0014] In this invention, the disease is an inflammatory disease; the inflammatory disease is caused by an elevated proportion of mononuclear macrophages in the blood; the mononuclear macrophages are preferably peripheral blood Ly6C. hi Mononuclear cells; the inflammatory diseases include atherosclerosis and / or ulcerative colitis.

[0015] In this invention, the product includes a drug.

[0016] Experimental examples of this invention have confirmed that carboxypyranocyanidin-3-O-glucoside can reduce the area of ​​atherosclerotic plaques and alleviate inflammation in ulcerative colitis.

[0017] This invention also provides the use of carboxypyranocyanidin-3-O-glucoside in the preparation of products for the prevention and / or treatment of inflammatory diseases.

[0018] In this invention, the inflammatory disease is caused by an elevated proportion of mononuclear macrophages in the blood; the mononuclear macrophages are preferably peripheral blood Ly6C. hi Mononuclear cells; the inflammatory diseases include atherosclerosis and / or ulcerative colitis.

[0019] In this invention, the product includes a drug.

[0020] This invention also provides the use of carboxypyranocyanidin-3-O-glucoside in the preparation of products that inhibit the differentiation of bone marrow-derived macrophages (BMDM).

[0021] In this invention, inhibiting the differentiation of bone marrow hematopoietic stem cells specifically means inhibiting the differentiation of BMDM into mononuclear macrophages.

[0022] In this invention, the inhibition of BMDM differentiation is achieved by downregulating immune response genes and upregulating cell cycle pathway genes; the immune response-related genes include one or more of Cd4, Lfi204, Myc, Vegfa, Zfp36l1, C1qc, Cebpa, Enpp1, and Gab3; the cell cycle protein-related genes include one or more of Dcstamp, Pir, and Tlr2.

[0023] The present invention also provides pharmaceutical formulations comprising pyranocyanidin-3-O-glucoside and pharmaceutically acceptable excipients or carriers.

[0024] The concentration of pyranocyanidin-3-O-glucoside of the present invention in the compound preparation is 12.5-150 μM, preferably 100-150 μM.

[0025] In this invention, the dosage form of the pharmaceutical preparation includes oral or inhaled dosage forms or other pharmaceutically acceptable dosage forms; the oral dosage forms include one or more of tablets, capsules, powders, syrups, dry syrups, liquids or suspensions; the inhaled dosage forms include one or more of powder inhalers, aerosols or sprays.

[0026] The carboxypyranocyanidin-3-O-glucoside described in this invention can significantly improve the abnormal differentiation of bone marrow hematopoietic stem cells into monocytes and macrophages, thereby improving the level of Ly6C in peripheral blood. hi The proportion of mononuclear macrophages can be reduced, thus alleviating the progression of atherosclerosis and ulcerative colitis. Experimental examples of this invention demonstrate that carboxypyranocyanidin-3-O-glucoside is effective and safe in preventing inflammatory diseases involving hematogenous mononuclear macrophage proliferation, and is worthy of widespread application. Attached Figure Description

[0027] Figure 1 The changes in cell morphology after treatment with different concentrations of carboxypyranocyanidin-3-O-glucoside (Cpycy-3-glu, Vitisin A) and cyanidin-3-O-glucoside (C3G) are shown.

[0028] Figure 2Cpycy-3-glu (Vitisin A) and C3G inhibited M-CSF-induced differentiation of bone marrow stem cells into BMDM: (A) Flow cytometry analysis of differentiation degree after treatment with different concentrations of Cpycy-3-glu; (B) Statistical analysis of differentiation degree;

[0029] Figure 3 This is a diagram identifying the differentiation arrest of M-CSF-induced bone marrow stem cells into BMDM by carboxypyranocyanidin-3-O-glucoside (Cpycy-3-glu, Vitisin A) and C3G.

[0030] Figure 4 Transcriptome analysis and validation of the inhibition of M-CSF-induced differentiation of bone marrow stem cells into BMDM by carboxypyranocyanidin-3-O-glucoside (Cpycy-3-glu, Vitisin A): Transcriptome sequencing heatmap analysis;

[0031] Figure 5 Transcriptome analysis and validation of the inhibition of M-CSF-induced differentiation of bone marrow stem cells into BMDM by carboxypyranocyanidin-3-O-glucoside (Cpycy-3-glu, Vitisin A): Transcriptome sequencing volcano plot analysis;

[0032] Figure 6 Transcriptome analysis and validation of the inhibition of M-CSF-induced differentiation of bone marrow stem cells into BMDM by carboxypyranocyanidin-3-O-glucoside (Cpycy-3-glu, Vitisin A): enrichment of differentially expressed genes in the GO pathway;

[0033] Figure 7 Transcriptome analysis and validation of the inhibition of M-CSF-induced differentiation of bone marrow stem cells into BMDM by carboxypyranocyanidin-3-O-glucoside (Cpycy-3-glu, Vitisin A): qPCR validation results of genes related to monocyte-macrophage differentiation;

[0034] Figure 8 Transcriptome analysis and validation of the inhibition of M-CSF-induced differentiation of bone marrow stem cells into BMDM by carboxypyranocyanidin-3-O-glucoside (Cpycy-3-glu, Vitisin A): (A) Tlr2 gene qPCR validation results; (B) Tlr4 gene qPCR validation results; (C) Tlr7 gene qPCR validation results; (D) Tlr9 gene qPCR validation results;

[0035] Figure 9The effect of carboxypyranocyanidin-3-O-glucoside (Cpycy-3-glu, Vitisin A) on the cell cycle during the differentiation of bone marrow stem cells into monocytes and macrophages: (A) Flow cytometry results of cell cycle; (B) Cell cycle statistics.

[0036] Figure 10 The effect of carboxypyranocyanidin-3-O-glucoside (Cpycy-3-glu, Vitisin A) on the cell cycle of bone marrow stem cells in the differentiation process into monocytes and macrophages: (A) Detection graphs of Cyclin B protein and CDK1 protein; (B) Statistical results of Cyclin B protein validation; (C) Statistical results of CDK1 protein validation;

[0037] Figure 11 It is carboxypyranocyanidin-3-O-glucoside (Cpycy-3-glu, Vitisin A) that inhibits ApoE. - / - Effects of atherosclerotic plaques on mice and their improvement on arterial inflammation: (A) Coronary artery HE staining, Oil Red staining, and Masson staining; (B) Statistical analysis results of arterial plaque area; (C) Immunofluorescence map of F4 / 80 expression in coronary arteries; (D) Immunostatistical analysis results of F4 / 80 expression in coronary arteries; (E) Immunofluorescence map of CD45 expression in coronary arteries; (F) Immunostatistical analysis results of CD45 expression in coronary arteries;

[0038] Figure 12 The effects of carboxypyranocyanidin-3-O-glucoside (Cpycy-3-glu, Vitisin A) and cyanidin-3-O-glucoside (C3G) on the levels of plasma inflammatory factors in ApoE- / - mice: (A) TNF-α; (B) MCP-1; (C) GM-CSF; (D) INF-γ; (E) GRO-α; (F) IL-22;

[0039] Figure 13 The effects of carboxypyranocyanidin-3-O-glucoside (Cpycy-3-glu, Vitisin A) and cyanidin-3-O-glucoside (C3G) on the levels of plasma inflammatory factors in ApoE- / - mice were investigated: (A) IL-33; (B) IL-18; (C) IL-6; (D) MIP-1α; (E) MIP-1β.

[0040] Figure 14 It is carboxypyranocyanidin-3-O-glucoside (Cpycy-3-glu, Vitisin A) that inhibits ApoE. - / - Effects of peripheral blood immune cells on mice: peripheral blood flow cytometry mononuclear cell typing;

[0041] Figure 15 It is carboxypyranocyanidin-3-O-glucoside (Cpycy-3-glu, Vitisin A) that inhibits ApoE. - / - Effects of peripheral blood immune cells in mice: (A)CD45 + Cell ratio; (B)CD11b + Cell proportion; (C) Neutrophil proportion; (DE) Ly6C hi Cell ratio;

[0042] Figure 16 The ameliorative effect of carboxypyranocyanidin-3-O-glucoside (Cpycy-3-glu, Vitisin A) on ulcerative colitis: colon HE staining;

[0043] Figure 17 The ameliorative effect of carboxypyranocyanidin-3-O-glucoside (Cpycy-3-glu, Vitisin A) on ulcerative colitis: colonic alicin blue staining;

[0044] Figure 18 The ameliorative effects of carboxypyranocyanidin-3-O-glucoside (Cpycy-3-glu, Vitisin A) on ulcerative colitis were assessed using the following metrics: (A) inflammation score; (B) mucosal layer damage score; (C) crypt loss score; (D) pathological damage extent score; and (E) total histological evaluation score.

[0045] Figure 19 The effect of carboxypyranocyanidin-3-O-glucoside (Cpycy-3-glu, Vitisin A) on peripheral blood immune cells in mice with ulcerative colitis: (A) CD45 + Cell ratio; (B)CD11b + Cell proportion; (C) Neutrophil proportion; (DE) Ly6C hi Cell ratio;

[0046] Figure 20 Effects of carboxypyranocyanidin-3-O-glucoside (Cpycy-3-glu, Vitisin A) on colonic immune cells in mice with ulcerative colitis: Immunofluorescence staining analysis of colonic F4 / 80 cells;

[0047] Figure 21 The effect of carboxypyranocyanidin-3-O-glucoside (Cpycy-3-glu, Vitisin A) on colonic immune cells in mice with ulcerative colitis: (A) CD45 in the colonic epithelium + Statistical analysis of cells; (B) CD11b in colonic epithelium +Statistical analysis of cells; (C) Statistical analysis of macrophages in the colonic epithelium; (D) Statistical analysis of inflammatory macrophages in the colonic epithelium; (E) Statistical analysis of neutrophils in the colonic epithelium; (F) Statistical analysis of dendritic cells in the colonic epithelium;

[0048] Figure 22 The effect of carboxypyranocyanidin-3-O-glucoside (Cpycy-3-glu, Vitisin A) on colonic immune cells in mice with ulcerative colitis: (A) CD45 in the colonic lamina propria. + Statistical analysis of cells; (B) CD11b of the lamina propria of the colon + Statistical analysis of cells; (C) Statistical analysis of macrophages in the lamina propria of the colon; (D) Statistical analysis of inflammatory macrophages in the lamina propria of the colon; (E) Statistical analysis of neutrophils in the lamina propria of the colon; (F) Statistical analysis of dendritic cells in the lamina propria of the colon;

[0049] Figure 23 The effects of carboxypyranocyanidin-3-O-glucoside (Cpycy-3-glu, Vitisin A) on the mRNA levels of colonic inflammatory factors in mice with ulcerative colitis: (A) Tnf-α; (B) IL-1α; (C) IL-1β; (D) Ccl-2;

[0050] Figure 24 The effects of carboxypyranocyanidin-3-O-glucoside (Cpycy-3-glu, Vitisin A) on the mRNA levels of colonic inflammatory factors in mice with ulcerative colitis: (A) IL-6; (B) IL-10; (C) IL-13; (D) IL-18;

[0051] Figure 25 The effects of carboxypyranocyanidin-3-O-glucoside (Cpycy-3-glu, Vitisin A) on the mRNA levels of colonic inflammatory factors in mice with ulcerative colitis: (A) Tgf-β; (B) NF-κb;

[0052] Figure 26 The effect of carboxypyranocyanidin-3-O-glucoside (Cpycy-3-glu, Vitisin A) on the differentiation of bone marrow hematopoietic stem cells into mononuclear macrophages in mice with ulcerative colitis: cell flow cytometry diagram;

[0053] Figure 27 The effect of carboxypyranocyanidin-3-O-glucoside (Cpycy-3-glu, Vitisin A) on the differentiation of bone marrow hematopoietic stem cells into mononuclear macrophages in mice with ulcerative colitis: (A) Myeloid progenitor (CMP Flt3)+ CD135 lo (B) Common myeloid progenitor (CMPFlt) 3- (C) Granulocyte / macrophage progenitor (GMP); (D) Monocyte / dendritic cell progenitor (MDP); (E) Monocyte progenitor (MP+cMoP); (F) Granulocyte progenitor (GP). Detailed Implementation

[0054] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0055] To further illustrate the present invention, detailed descriptions are provided below through the following embodiments. All raw materials used in the following embodiments of the present invention are commercially available products. The mice used in the present invention are ApoE mice. - / - Forty male mice, 4 weeks old, were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. The experimental animals were housed in the SPF animal facility of Jinan University, with alternating 12-hour light and dark cycles, maintained at a temperature of 22-26℃ and a relative humidity of 60%-80%, and provided with free access to food and water. Experimental procedures were conducted in accordance with the relevant requirements of the Jinan University Experimental Animal Ethics Committee (Ethics No. 20200907-09).

[0056] The method for carboxypyranoanthocyanins-3-O-glucoside in the specific embodiments of this invention, as described in the reference "Comparative study on the stability and antioxidant activity of sixpyranoanthocyanins based on malvidin-3-glucoside", is optimized for the crude anthocyanin extract from black soybean husks as follows:

[0057] a) Accurately weigh 10g of the aforementioned crude anthocyanin extract and dissolve it in 700mL of deionized water. Add 280mL of pyruvate solution, adjust the pH to 3.0 using saturated NaHCO3, and bring the volume to 1L with deionized water. Transfer the solution to an Erlenmeyer flask.

[0058] b) Seal the bottle mouth with sealing film, place the system in a constant temperature shaker at 90 rpm and 30°C for 15 days in a micro-oxygen environment, away from light.

[0059] c) Using the aforementioned macroporous resin purification method, pyruvate in the reaction system was removed, the alcohol eluent was collected, concentrated under reduced pressure at 42°C until alcohol-free, and freeze-dried for 48 hours to obtain a powder. Separation and purification were performed using the aforementioned medium-pressure preparative chromatography conditions. Based on the elution of the liquid phase peaks, the detection signal A was collected by cutting the sample into separate tubes. 504nm >A 520nm Components. The purity of carboxypyranocyanidin-3-O-glucoside (Vitisin A) was identified and detected by the aforementioned UPLC-MS method, and Vitisin A monomer was prepared; cyanidin-3-O-glucoside (C3G) was prepared according to the method in the literature "Medium-pressure liquid chromatography preparation and structural identification of anthocyanins from black soybean peel".

[0060] The steps of each detection method in this invention are as follows:

[0061] The steps for HE staining are as follows:

[0062] (1) The heart was fixed in 4% paraformaldehyde for more than 24 hours. After being removed, it was placed in 15% sucrose solution at 4°C to dehydrate and settle. Then it was transferred to 30% sucrose solution and dehydrated and settled at 4°C.

[0063] (2) After absorbing the dehydrated tissue with filter paper, smooth the plane of the aortic root, place it on the sample holder, drop OCT embedding agent on it, and quickly freeze and embed it on the freezing stage of the cryostat. After the OCT turns white and hard, cut it into sections with a thickness of 8-10 μm and place them on a glass slide.

[0064] (3) Take frozen sections of the aortic root of the heart, rewarm and dry them, fix them in 4% paraformaldehyde for 15 min, wash them with water and air dry them.

[0065] (4) Stain the sections with hematoxylin solution for 3-5 minutes, wash with tap water, differentiate with hematoxylin differentiation solution, wash with water, blue with blue solution, and rinse with running water.

[0066] (5) The sections were dehydrated in 85% and 95% graded alcohol for 5 min each, and then stained in eosin staining solution for 5 min.

[0067] (6) Gently immerse the slide in pure water twice, for 5s and 10s each time, and examine the staining effect under a microscope.

[0068] (7) The slide was mounted with glycerin gelatin mounting medium and the images were acquired and analyzed under a microscope.

[0069] The steps for flow cytometry typing of peripheral blood mononuclear cells are as follows:

[0070] (1) Take 150 μL of anticoagulated ocular blood, add 900 μL of erythrocyte lysis buffer, lyse at room temperature for 15 min, and then centrifuge at 5000 rpm for 5 min.

[0071] (2) Discard the upper layer of liquid, add 900 μL of red blood cell lysis buffer, lyse again at room temperature for 15 min, and then centrifuge at 5000 rpm for 5 min.

[0072] (3) Discard the upper layer of liquid, carefully remove the red blood clots with the pipette tip, add 50 μL of CD16 / 32 (1:500) antibody diluted with PBS, and block at 4℃ for 15 min.

[0073] (4) Centrifuge at 5000 rpm for 5 min and discard the upper layer of liquid.

[0074] (5) Dilute the flow cytometry antibodies (PE / Cyanine7-F4 / 80, FITC-Ly6C, PE-Ly6G, PerCP / Cyanine7-CD11b, APC / Cyanine7-CD45) with PBS at a ratio of 1:200. Add 100 μL of the prepared antibody to each tube and stain at 4°C in the dark for 15 min.

[0075] (6) Centrifuge at 5000 rpm for 5 min, discard the supernatant, add 200 μL PBS and perform flow cytometry analysis.

[0076] The steps of immunohistochemistry are as follows:

[0077] (1) Frozen sections were air-dried at room temperature, baked in an oven at 37°C for 10-20 minutes, fixed in 4% paraformaldehyde for 20 minutes, and washed three times in PBS (pH 7.4) on a decolorizing shaker for 5 minutes each time.

[0078] (2) Antigen retrieval: Tissue slides were placed in a retrieval box filled with EDTA (pH 9.0) antigen retrieval solution and microwaved for antigen retrieval. Microwave on medium heat for 5 minutes, then turn off the power for 5 minutes and microwave on low for another 5 minutes. During this process, excessive evaporation of the buffer solution should be prevented, and the slides should not be allowed to dry. After natural cooling, the slides were placed in PBS and washed three times on a decolorizing shaker for 5 minutes each time.

[0079] (3) Blocking endogenous peroxidase: Place the slide in 3% hydrogen peroxide solution and incubate at room temperature in the dark for 25 min. Then place the slide in PBS and wash it three times on a decolorizing shaker for 5 min each time.

[0080] (4) Serum blocking: After the section is slightly dried, draw a circle around the tissue with a histochemical pen, and drop 10% rabbit serum into the circle to evenly cover the tissue. Block at room temperature for 30 minutes.

[0081] (5) Add primary antibody: Gently shake off the blocking solution, add the primary antibody prepared in PBS at a certain ratio to the slide, and incubate the slide in a humidified chamber at 4°C overnight.

[0082] (6) Add secondary antibody: Place the slide in PBS and wash it three times on a decolorizing shaker for 5 minutes each time. After drying the slide, add the secondary antibody (HRP-labeled) of the same species as the primary antibody from the histochemistry kit to the circle to cover the tissue, and incubate at room temperature for 1 hour.

[0083] (7) DAB staining: Place the slide in PBS and wash it three times on a decolorizing shaker for 5 minutes each time. After the slide is dried, add freshly prepared DAB staining solution to the circle. Control the staining time under a microscope. The positive color is brownish-yellow. Rinse the slide with tap water to stop the staining.

[0084] (8) Counterstaining cell nuclei: Counterstain with hematoxylin for about 3 minutes, wash with tap water, differentiate with hematoxylin differentiation solution for a few seconds, rinse with tap water, re-blue with hematoxylin blue solution, and rinse with running water.

[0085] (9) Dehydration and mounting: Place the sections in 75% alcohol for 5 min, 85% alcohol for 5 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, n-butanol for 5 min, and xylene I for 5 min to dehydrate and make them transparent. Remove the sections from the xylene and let them dry slightly before mounting them with neutral resin.

[0086] (10) Microscopic examination and image acquisition and analysis.

[0087] The steps for flow cytometry typing of peripheral blood mononuclear cells are as follows:

[0088] (1) Take 150 μL of anticoagulated ocular blood, add 900 μL of erythrocyte lysis buffer, lyse at room temperature for 15 min, and then centrifuge at 5000 rpm for 5 min.

[0089] (2) Discard the upper layer of liquid, add 900 μL of red blood cell lysis buffer, lyse again at room temperature for 15 min, and then centrifuge at 5000 rpm for 5 min.

[0090] (3) Discard the upper layer of liquid, carefully remove the red blood clots with the pipette tip, add 50 μL of CD16 / 32 (1:500) antibody diluted with PBS, and block at 4℃ for 15 min.

[0091] (4) Centrifuge at 5000 rpm for 5 min and discard the upper layer of liquid.

[0092] (5) Dilute the flow cytometry antibodies (PE / Cyanine7-F4 / 80, FITC-Ly6C, PE-Ly6G, PerCP / Cyanine7-CD11b, APC / Cyanine7-CD45) with PBS at a ratio of 1:200. Add 100 μL of the prepared antibody to each tube and stain at 4°C in the dark for 15 min.

[0093] (6) Centrifuge at 5000 rpm for 5 min, discard the supernatant, add 200 μL PBS and perform flow cytometry analysis.

[0094] High-throughput liquid protein chip assay for inflammatory factors:

[0095] The determination of plasma inflammatory factors (GM-CSF, GRO-α, IFN-γ, IL-1α, IL-1β, IL-10, IL-18, IL-22, IL-33, IL-6, MCP-1, MIP-1α, MIP-1β, TNF-α) was performed by Leiz Biotechnology Co., Ltd. using a high-throughput liquid phase protein chip. The main steps are as follows:

[0096] (1) The pre-prepared microspheres were vortexed for 30s to completely disperse them, and 50μL of premixed microspheres were added to each well of a 96-well plate.

[0097] (2) Place the 96-well plate in the magnetic separation plate and let it stand for 2 minutes to allow the microspheres to sink to the bottom. Then quickly invert the magnetic plate and discard the liquid.

[0098] (3) Add 150 μL Wash Buffer to each well, let stand for 30 seconds, quickly invert the magnetic plate to discard the liquid, and use a paper towel to absorb the residual liquid on the surface of the well plate.

[0099] (4) Add 25 μL of Universal Assay Buffer to each well, then add 25 μL of sample or serially diluted standard, shake at 500 rpm at room temperature for 30 min, then let stand overnight at 4 °C, and then take it out the next day and shake at 500 rpm at room temperature for 30 min.

[0100] (5) Place the 96-well plate in the magnetic separation plate, let it stand for 2 minutes, and then quickly invert the magnetic plate to discard the liquid.

[0101] (6) Add 150 μL Wash Buffer to each well, let stand for 30 seconds, quickly invert the magnetic plate to discard the liquid, repeat three times, and use a paper towel to absorb the residual liquid on the surface of the well plate for the last time.

[0102] (7) Add 25 μL of detection antibody mixture to each well, remove the 96-well plate from the magnetic separation plate, and incubate at room temperature with shaking at 500 rpm for 30 min.

[0103] (8) Repeat steps (5) to (6) to wash the plate.

[0104] (9) Add 50 μL of SA-PE to each well, remove the 96-well plate from the magnetic separation plate, and incubate at room temperature with shaking at 500 rpm for 30 min.

[0105] (10) Repeat steps (5) to (6) to wash the plate.

[0106] (11) Add 120 μL of Reading Buffer to each well, remove the 96-well plate from the magnetic separation plate, and incubate at room temperature with shaking at 500 rpm for 5 min.

[0107] (12) Place the sample in a Luminex 200 instrument and take a reading. Use a five-parameter nonlinear regression method to fit the standard curve and calculate the concentration value.

[0108] The steps for Allicin Blue-Periodic Acid-Schiff staining are as follows:

[0109] (1) Bake paraffin sections at 70℃ for 2 hours, then dewax to water: xylene 10 min × 3 times; anhydrous ethanol 5 min × 2 times; 90% ethanol 5 min; 80% ethanol 5 min; 70% ethanol 5 min; deionized water 5 min. (2) Stain with alexandrite blue solution for 10 min, then wash with water. (3) Oxidize with periodic acid solution for 10 min, then wash with water; stain with Schiff base solution for 10 min, then wash with water. (4) Stain with hematoxylin for 1 min, then wash with water. (5) Dehydrate and clear: 70% ethanol 2 min; 80% ethanol 2 min; 90% ethanol 2 min; anhydrous ethanol 3 min; xylene 5 min × 2 times. (6) Mount with neutral resin, then examine and photograph under a microscope.

[0110] The steps for qPCR analysis are as follows:

[0111] (1) RNA extraction from the tissue to be tested

[0112] a) Add 1.0 mL of Trizol to the tissue sample, add two zirconia magnetic beads, and homogenize in a tissue homogenizer at 60 Hz for 180 s at low temperature until no visible tissue fragments remain. b) Add 200 μL of chloroform, gently invert and mix, let stand for 5 min, centrifuge at 4 °C and 12000 g for 10 min, and transfer 300 μL of the upper aqueous phase to a new enzyme-free EP tube. c) Add 300 μL of isopropanol, gently invert and mix for 15 s, let stand at 4 °C for 10 min, centrifuge at 4 °C and 12000 g for 15 min, discard the supernatant, and retain the precipitate. d) Add 1.0 mL of 75% ethanol prepared with DEPC water, gently shake to wash the precipitate, centrifuge at 4 °C and 12000 g for 15 min, gently discard the supernatant, and invert the EP tube onto filter paper to air dry, obtaining the precipitate. e) Add 50-100 μL of DEPC water according to the amount of precipitate, vortex to dissolve the precipitate, and obtain RNA stock solution. f) Take 2.0 μL of RNA stock solution and use Nanodrop to determine the RNA purity and A260 / 280 purity.

[0113] (2) RNA reverse transcription

[0114] a) DNA removal reaction

[0115] Prepare the reaction system according to Table 1, and react at 42℃ for 2 min in a PCR instrument.

[0116] Table 1 DNA removal reaction

[0117]

[0118] b) Reverse transcription reaction

[0119] Prepare the reaction system according to Table 2. In the PCR instrument, perform reverse transcription reaction at 37℃ for 15 min; 85℃ for 5 s; 4℃ for 5 min. After the reaction, store the apparatus at -20℃ for later use.

[0120] Table 2 RNA reverse transcription reaction

[0121]

[0122] (3) Real-time quantitative PCR

[0123] Prepare the reaction system according to Table 3 and perform amplification under the following conditions: Pre-denaturation (Repeat: 1): 95℃, 30s; PCR reaction (Repeat: 40): 95℃, 5s; 60℃, 30s; Dissociation.

[0124] Table 3 Real-time PCR reaction system

[0125]

[0126] (4) Processing of qPCR results

[0127] Based on the Ct value obtained from the reaction, we can calculate Ct(target) - Ct(internal reference) = ΔCt, and then calculate ΔCt and 2 through intergroup calculations. -ΔΔCt The differences between data groups in this invention were analyzed using SPSS 22.0 and GraphPad Prism 7.0, and statistical graphs were created using GraphPad Prism 7.0. Data results are expressed as mean ± standard error (Mean ± SEM), and one-way ANOVA was used for comparisons between groups. *p<0.05 indicates statistical significance, and **p<0.01 indicates extremely significant difference.

[0128] Example 1: Carboxypyranocyanidin-3-O-glucoside (Cpycy-3-glu, Vitisin A) inhibits M-CSF-induced differentiation of bone marrow stem cells into BMDM.

[0129] I. Experimental Methods

[0130] Bone marrow cells were extracted from the femur and tibia of normal mice and seeded into 12-well plates with 2 mL of culture medium per well, at a rate of 2 × 10⁶ cells / well. 6 Bone marrow cells were induced to differentiate into BMDM using DMEM medium containing 20 ng / mL M-CSF. Different concentrations of carboxypyranocyanidin-3-O-glucoside (Cpycy-3-glucoside A) and cyanidin-3-O-glucoside (C3G) were added to the medium at concentrations of 0, 12.5, 25, 50, 100, and 150 μM. After six days of induction, cell morphology was observed, and cells were collected for flow cytometry analysis of surface receptors MHC-II and CD80 expression to analyze differentiation levels. Furthermore, cell subpopulations at different differentiation stages were identified using CD117, Ly6A / E, CD34, CD135, Ly6C, and CD115 to analyze differentiation arrest stages.

[0131] II. Experimental Results

[0132] We treated BMDM cells with M-CSF and added different concentrations of Vitisin A or C3G for 6 days to investigate the effect of Vitisin A on the differentiation and maturation process of macrophages. Figure 1As shown, cells without anthocyanin intervention matured 6 days after differentiation under the influence of M-CSF and appeared round and morphologically intact. After treatment with different concentrations of C3G (100-200 μM), cells exhibited a certain degree of aggregation; while Vitisin A treatment at a concentration of 12.5 μM showed a certain degree of aggregation, and at higher concentrations (50-150 μM), cell aggregation was more pronounced, resulting in spindle-shaped, flattened cells. Some cells were significantly smaller and opaque, revealing that Vitisin A treatment had a more significant impact on cell morphology during BMDM differentiation.

[0133] To further characterize the degree of cell differentiation, flow cytometry was used to analyze the two marker surface molecules of BMDM differentiation maturity, MHC-II and CD80. The flow cytometry results are as follows: Figure 2 As shown, Figure A and Figure 2 B indicates that after 6 days of culture, the percentage of mature cells (MHC-II and CD80 double positive) in the untreated group was 52.77%. Different concentrations of C3G treatment and low concentrations of Vitisin A (12.5-50 μM) had no significant effect on the differentiation rate of BMDM, while the differentiation rate of BMDM decreased to 23.47% and 26.00% after treatment with 100 μM and 150 μM, respectively.

[0134] like Figure 3 As shown, a new group of cells treated with Cpycy-3-glu was observed on day 4 of differentiation. Flow cytometry analysis identified this group of cells as Sca-1. - c-kit + CD34 + FcγR lo CD115 lo Flt3 + Cells, preliminarily identified as CMPFlt3 + CD115 lo Cellular activity, as observed in DSS-induced colitis animal experiments, showed that Cpycy-3-glu did not alter the elevated CMP Flt3 levels after modeling. + CD115 lo The ratio was adjusted, but the downstream CMP Flt3 was reduced. - The cell proportion results were consistent. Therefore, the arrest of BMDM by Cpycy-3-glu may occur at the CMP Flt3 stage of HSC differentiation into monocytes. + CD115 lo stage.

[0135] Example 2: Effects of carboxypyranocyanidin-3-O-glucoside (Cpycy-3-glu, Vitisin A) on genes involved in the differentiation of bone marrow stem cells into monocytes and macrophages.

[0136] I. Experimental Methods

[0137] Bone marrow cells were extracted from the femur and tibia of normal mice and seeded into 12-well plates with 2 mL of culture medium per well, at a rate of 2 × 10⁶ cells / well. 6 Bone marrow cells were induced to differentiate into BMDM using DMEM medium containing 20 ng / mL M-CSF. Cpycy-3-glu was added to the medium at a concentration of 100 μM. After six days of induction, the supernatant was discarded, and 1.0 mL of Trizol was added to each well for RNA extraction. Next-generation transcriptome sequencing was performed by Shanghai Ouyi Biomedical Technology Co., Ltd. using a Novaseq 6000 sequencing instrument, and the results were analyzed and validated by qPCR.

[0138] II. Experimental Results

[0139] The results are as follows Figures 4 to 8 As shown. Figure 4 and Figure 5 The results showed that Cpycy-3-glu treatment significantly altered gene expression at the cellular transcriptome level. After Cpycy-3-glu treatment, 584 genes were significantly upregulated and 484 genes were significantly downregulated. Enrichment analysis of the GO pathway was performed to target the upregulated and downregulated genes affected by Cpycy-3-glu, and the results are as follows: Figure 6 As shown, Cpycy-3-glu downregulated genes mainly affect immune response-related pathways, while Cpycy-3-glu upregulated genes mainly affect cell cycle-related pathways. Further analysis of transcriptome data identified differentially expressed pathways, including monocyte differentiation, Toll-like receptor signaling, and cell cycle regulation. Key genes at different stages of HSC differentiation into monocytes (GO:0060218 Hematopoietic stem cell differentiation, GO:0002244 Hematopoietic progenitor cell differentiation, GO:0002318 Myeloid progentior cell differentiation, GO:0030224 Monocyte differentiation, GO:0030225 Macrophage differentiation) were screened and validated by qPCR. The results are shown below. Figure 7As shown in the figure, qPCR validation results showed that Cpycy-3-glu treatment mainly affected genes related to the Monocyte Differentiation and Macrophage Differentiation pathways. Specifically, the expression of Cd4, Lfi204, Myc, Vegfa, Zfp36l1, C1qc, Cebpa, Enpp1, and Gab3 was significantly downregulated after Cpycy-3-glu treatment, while the expression of Dcstamp, Pir, and Tlr2 was significantly upregulated. Toll-like receptor signaling participates in and regulates the differentiation process of monocytes, playing a crucial role, especially in the CMPs and GMPs stages. Figure 8 As shown, Cpycy-3-glu treatment significantly increased TLR2 expression, while the levels of TLR4, TLR7, and TLR9 all decreased significantly. Notably, the TLR2-mediated signaling pathway is involved in early progenitor cell differentiation, and the production of monocytes and macrophages has been confirmed to be TLR2-dependent. In this study, Cpycy-3-glu upregulated TLR2 and subsequently caused differentiation arrest, suggesting that abnormalities in the TLR2-mediated signaling pathway may be key to the differentiation arrest effect of Cpycy-3-glu.

[0140] Example 3: Effects of carboxypyranocyanidin-3-O-glucoside (Cpycy-3-glu, Vitisin A) on the cell cycle of bone marrow stem cells in the differentiation process into monocytes and macrophages.

[0141] I. Experimental Methods

[0142] Bone marrow cells were extracted from the femur and tibia of normal mice and seeded into 12-well plates with 2 mL of culture medium per well, at a rate of 2 × 10⁶ cells / well. 6 Bone marrow cells were induced to differentiate into BMDM using DMEM medium containing 20 ng / mL M-CSF. Different concentrations of Cpycy-3-glu were added to the medium, with a concentration of 100 μM. After six days of induction, the supernatant was discarded, and cells were collected and stained with PI staining solution. Cell cycle analysis was performed using flow cytometry. Cells were seeded in 6-well plates, induced and treated with the same concentration, and cellular proteins were collected for Western blot validation of cell cycle-related proteins.

[0143] II. Experimental Results

[0144] Cell cycle changes after treatment with Vitisin A and C3G were analyzed by PI staining and flow cytometry. Results are as follows: Figure 9As shown, cells exhibited significant G2 / M phase arrest after treatment with both C3G and Vitisin A, with Vitisin A showing a more pronounced arrest. Further Western blotting was used to detect the expression levels of key G2 / M phase proteins Cyclin B and CDK1, with the following results: Figure 10 The results indicate that Vitisin A treatment significantly decreased the expression of Cyclin B and CDK1, while C3G treatment also resulted in a certain degree of decrease in the levels of both proteins. These results suggest that the differentiation arrest induced by Vitisin A treatment ultimately led to G2 / M phase arrest in undifferentiated cells, while C3G, although it did not produce a significant differentiation-inhibiting effect, did not.

[0145] Example 4: The effect of carboxypyranocyanidin-3-O-glucoside (Cpycy-3-glu, Vitisin A) on improving atherosclerosis.

[0146] I. Experimental Methods

[0147] Using 4-week-old ApoE - / - Male mice were divided into three groups: Control + Vehicle, Model + Vehicle, and Model + Cpycy-3-glu, with 10 mice in each group. The Control + Vehicle group was fed a control diet and administered saline via gavage daily; the Model + Vehicle group was fed a Western diet and administered saline via gavage daily; the Model + Cpycy-3-glu group was fed a Western diet and administered 100 mg / kg bw Cpycy-3-glu via gavage daily. All mice had free access to food and water, and the treatment lasted for 12 weeks. After the treatment, blood was collected for analysis of cholesterol levels using reagent kits, analysis of plasma inflammatory factors using high-throughput liquid chromatography-protein microarrays, and analysis of peripheral blood mononuclear cell composition using flow cytometry. Hearts were collected for coronary artery tissue structure and immunohistochemical analysis.

[0148] II. Experimental Results

[0149] The results are as follows Figure 11As shown in Figure A, HE staining of coronary artery sections revealed that the control group had an intact coronary artery root structure with slight intimal thickening. In the model group, due to a high-fat, high-cholesterol diet, HE staining showed significant intimal thickening, a marked increase in atherosclerotic plaques, and disruption of the arterial root structure. Oil Red staining indicated significant lipid components in the model group, and Masson staining showed the formation of fibrous caps. These results indicate that a high-fat, high-cholesterol diet successfully induced atherosclerosis in mice. The Cpycy-3-glu intervention groups all showed reduced plaque area, slight intimal thickening, and no significant fibrous cap formation. Based on HE staining results, plaque area was statistically analyzed. The control group had approximately 5.92±1.35% plaque area, while the model group significantly increased to 20.18±1.31%, and after Cpycy-3-glu intervention, the area decreased to 10.25±1.63%. These results demonstrate that Cpycy-3-glu can reduce plaque area and improve the formation of atherosclerotic plaques. Immunofluorescence was used to detect the expression of F4 / 80 and CD45 in the root of the coronary arteries to examine the infiltration of immune cells into the arteries. Results are as follows: Figure 11 B and Figure 11 As shown in Figure C, the model group developed atherosclerosis after a high-fat diet, with significant aggregation of F4 / 80 and CD45 positive cells at the site of intimal thickening. Their expression was significantly increased compared to the control group, while in the Cpycy-3-glu intervention group, their expression was significantly decreased. These results indicate that Cpycy-3-glu intervention effectively reduced macrophage infiltration into blood vessels.

[0150] The relevant inflammatory factors in the plasma were measured, and the results were as follows: Figure 12 As shown, plasma TNF-α and the chemokine MCP-1 were significantly elevated in the model group, but returned to normal levels after Cpycy-3-glu intervention, indicating that plasma inflammation was suppressed to some extent. GRO-α has been shown to be involved in the activation, residence, and foaming of macrophages recruited to plaques. In this model, plasma GRO-α levels in the model group were significantly higher than those in the control group, but significantly lower after Cpycy-3-glu intervention, which corroborates the decrease in inflammation levels after Cpycy-3-glu treatment. In addition, IL-33 and MIP-1β showed significant changes in atherosclerosis compared to the control group, but neither showed improvement after intervention. Other indicators such as GM-CSF, IFN-γ, IL-22, IL-18, IL-6, and MIP-1α did not show significant differences among the groups.

[0151] Plasma TNF-α and the chemokine MCP-1 were significantly elevated in the model group, but returned to normal levels after Vitisin A intervention, indicating that plasma inflammation was suppressed to some extent. GRO-α has been shown to be involved in the activation, residence, and foaming of macrophages recruited to plaques. In this model, plasma GRO-α levels in the model group were significantly higher than those in the control group, but significantly lower after Vitisin A intervention, which corroborates the decrease in inflammation levels after Vitisin A treatment. Notably, none of the above three indicators showed significant improvement in the C3G intervention group. In addition, IL-33 and MIP-1β showed significant changes in atherosclerosis compared to the control group, but neither intervention group showed any improvement. Other indicators such as GM-CSF, IFN-γ, IL-22, IL-18, IL-6, and MIP-1α showed no significant differences among the groups.

[0152] Immune cells in peripheral blood migrate to the vascular endothelium to induce an inflammatory response. Ly6Chi cells, which highly express the MCP-1 receptor CCR2, are considered to be a type of cell more likely to infiltrate the vascular endothelium, differentiate into M1 macrophages, and contribute to inflammation. Therefore, flow cytometry was used to classify immune cells in peripheral blood. The results are as follows: Figure 13-15 As shown, after a high-fat, high-cholesterol diet, ApoE... - / - CD11b in mouse peripheral blood + The number of cells increased significantly, and the proportion of neutrophils also showed a certain upward trend, but the difference was not statistically significant. Ly6C is the main cell subtype inducing intimal infiltration in atherosclerosis. hi The proportion of neutrophils was significantly higher than that of the control group, which is consistent with the progression and changing trends of atherosclerosis. After intervention with Cpycy-3-glu, the proportion of neutrophils in peripheral blood decreased significantly, and Ly6C was also detected. hi The proportion of cells decreased to some extent compared to the model group. These results indicate that Cpycy-3-glu intervention can alter the proportion of immune cells in the blood, maintain a low level of inflammation, and reduce Ly6C, which is involved in monocyte migration to vascular endothelial differentiation during the progression of atherosclerosis. hi The proportion of key cell subpopulations.

[0153] The above studies indicate that Cpycy-3-glu can significantly improve atherosclerosis, and its mechanism may be through influencing the differentiation process of monocytes.

[0154] Example 5: The ameliorative effect of carboxypyranocyanidin-3-O-glucoside (Cpycy-3-glu, Vitisin A) on ulcerative colitis.

[0155] I. Experimental Methods

[0156] Male C57 mice were used and divided into four groups: Control+Vehicle, DSS+Vehicle, DSS+Cpycy-3-glu, and Control+Cpycy-3-glu, with 12 mice in each group. Pre-protection phase: The experiment began with 7 consecutive days of Cpycy-3-glu pre-protection, with all groups receiving normal drinking water. The Control+Vehicle and DSS+Vehicle groups were administered saline by gavage, while the DSS+Cpycy-3-glu and Control+Cpycy-3-glu groups were administered 100 mg / kg bw Cpycy-3-glu by gavage. Modeling Phase: After pre-protection, the Control+Vehicle group was given regular drinking water and saline by gavage; the DSS+Vehicle group was given 3.0% DSS in drinking water and saline by gavage; the DSS+Cpycy-3-glu group was given 3.0% DSS in drinking water and 100 mg / kg bw Cpycy-3-glu by gavage; and the Control+Cpycy-3-glu group was given regular drinking water and 100 mg / kg bw Cpycy-3-glu by gavage. This phase lasted for 8 days. On day 5, the DSS was removed from the drinking water for all DSS groups and replaced with regular drinking water. After the DSS was removed, the corresponding gavage treatment was continued in each group until the experiment was terminated on day 8. After the experimental animals were treated, the composition of peripheral blood mononuclear cells was analyzed by flow cytometry, and bone marrow was used for lineage analysis. - Cell sorting analysis; cecum and colon for immune cell analysis of intestinal epithelial and lamina propria; colon for tissue structure analysis and RNA extraction analysis.

[0157] II. Experimental Results

[0158] H&E staining and alcinocyanine blue staining were used to assess colonic damage in mice, and the results were as follows: Figure 16-18As shown, the colon tissue of mice in the blank control group was intact and clearly defined, with tightly arranged cell structures in each layer, an intact mucosal epithelium, and no inflammatory cell infiltration. The DSS model group, however, showed mucosal ulceration, destruction of the mucosal epithelium with disordered cell arrangement, elongated and structurally damaged crypts, and significant inflammatory cell infiltration in the submucosa and lamina propria, indicating that DSS caused severe damage to the mouse colon, consistent with the damage characteristics of a typical UC model. Although the Cpycy-3-glu group showed mucosal destruction, the inflammatory infiltration was improved to some extent compared to the DSS model group. Furthermore, the colon structure of the Cpycy-3-glu control group was intact, similar to the control group, indicating that Cpycy-3-glu treatment alone did not cause colonic damage. Blindly assessing the inflammatory infiltration, mucosal damage, crypt loss, and lesion extent of the colon in each mouse was performed, and a colonic injury score was obtained by summing the scores; a higher score indicated more severe damage. The scoring results are shown below. Figure 18 As shown, DSS lesions significantly increased the scores of all indicators, while the Cpycy-3-glu group showed a decrease in the inflammatory infiltration score.

[0159] Intestinal immune cells are mainly replenished and infiltrated through peripheral blood. Flow cytometry was used to perform typing analysis on immune cells in peripheral blood. Results are as follows: Figure 19 As shown, Ly6C in peripheral blood hi Monocytes decreased to some extent after DSS injury, presumably due to the extensive infiltration of peripheral blood immune cells into the intestine. Ly6C in the C3G treatment group... hi There was no difference between the monocyte and DSS model groups, but the level of Ly6C in peripheral blood after Vitisin A treatment was higher. hi The proportion of monocytes returned to normal. In addition, the number of neutrophils in peripheral blood increased, which was not affected by either anthocyanin treatment.

[0160] The progression of IBD is closely related to intestinal macrophage infiltration. Immunofluorescence was used to detect F4 / 80 to label the expression of mature macrophages in the colon. The results are as follows: Figure 20As shown, the blank control group mice showed virtually no macrophage expression in the lamina propria and intestinal epithelium of the colon, while the DSS model group staining revealed a large number of macrophage infiltrations in the lamina propria, and a certain degree of macrophage aggregation was also found in the intestinal epithelium. Mice treated with Cpycy-3-glu showed relatively reduced F4 / 80 expression in both the lamina propria and epithelium of the colon, indicating a reduction in macrophage infiltration. Considering the discontinuous nature of IBD lesions in the colon, immune cells from the intestinal epithelium and lamina propria of the entire colon were extracted for flow cytometry analysis to determine the overall immune cell infiltration status of the intestines in each mouse. The progression of enteritis increased the overall proportion of various immune cells in the intestinal epithelium, with a significant increase in the proportions of macrophages, infiltrating macrophages, neutrophils, and dendritic cells (DCs). In mice treated with Cpycy-3-glu (Vitisin A), the proportions of macrophages, infiltrating macrophages, and neutrophils in the intestinal epithelium were significantly downregulated, while there was no significant improvement or downregulation of DCs. The various immune cell types in the C3G-treated group remained unchanged compared to the model group. Figure 21 The number of immune cells in the lamina propria of the colon is higher than, for example... Figure 22 As shown, similar to the intestinal epithelium, the expression of macrophages, infiltrative macrophages, and neutrophils all increased significantly after DSS injury; the proportions of these three cell types decreased significantly after Cpycy-3-glu (Vitisin A) intervention, and C3G also did not significantly improve the expression of various immune cells. In addition, the content of DC cells in the intestinal lamina propria remained unchanged.

[0161] qPCR was used to analyze the expression of relevant inflammatory factors in intestinal tissue after inflammatory infiltration to determine the level of inflammation. Results are as follows: Figure 23-25 As shown, the results indicated that the mRNA levels of pro-inflammatory factors and chemokines TNF-α, IL-1α, IL-1β, IL-6, MCP-1, and IL-18 were significantly increased, indicating inflammatory infiltration in intestinal tissue. Cpycy-3-glu (Vitisin A) downregulated the expression of TNF-α, IL-1α, MCP-1, and IL-6, while C3G only downregulated IL-1α, indicating that the inflammation level was alleviated under Cpycy-3-glu intervention. This result is consistent with the analysis of intestinal immune cell counts, where C3G only downregulated IL-1α expression. Furthermore, there were no significant differences in the expression of IL-10, IL-13, TGF-β, and NF-κB among the groups.

[0162] After weaning, the macrophages in the intestines of mice mainly rely on bone marrow-derived HSCs for continuous development and differentiation, forming macrophages that then migrate to the intestines for renewal. This is because peripheral blood contains Ly6C... hiThe cell proportion changed significantly during DSS modeling and returned to normal levels after Cpycy-3-glu intervention. Linage was performed on bone marrow cells to target the development of HSCs into macrophages. - Mature hematopoietic cells were removed by negative selection, and then flow cytometry was used to identify cells in the developmental and differentiation process. The experimental results are as follows: Figure 26 As shown, CMP Flt was successfully identified from bone marrow by flow cytometry. 3+ CD115lo, CMPlt 3- The six cell subtypes—GMP, MDP, MP+cMOP, and GP—differentiate into monocytes / macrophages in the bone marrow as follows: Figure 27 As indicated by the red arrows in the diagram. The results showed a significant increase in all six cell types in the bone marrow of mice in the DSS-induced colitis model. In terms of outcomes, the increased proportion of Monocyte progenitor cells (MP+cMoP) revealed an increased number of macrophages, consistent with the increase in macrophages in the intestine. Granulocyte progenitor cells (GP) are precursors to neutrophil differentiation, and the results also showed a significant increase in the proportion of GP cells in the bone marrow of the colitis group, again consistent with the increase in neutrophils in the intestine. GMP can differentiate into MP, MDP can differentiate into cMoP, and both MP and cMoP can differentiate into Ly6C. hi The potential of monocytes. Experimental results showed that neither C3G nor Vitisin A, acting alone, had a significant effect on HSC differentiation in the bone marrow of normal mice. However, studies of Vitisin A in mice with DSS modeling showed that it significantly downregulated CMP Flt3 in the bone marrow. - The study also showed that Vitisin A significantly reduced the proportion of GP cells and GMP cells, ultimately leading to a decrease in the proportion of MP+cMoP cells, which are capable of differentiating into macrophages. Furthermore, the results indicated that Vitisin A also significantly reduced the proportion of GP cells. C3G treatment in DSS mice did not show any effect on the proportions of these six cell subsets. These results suggest that the anthocyanin Vitisin A alone does not affect the differentiation process of HSCs into macrophages in normal mice, while Vitisin A has a significant regulatory effect on the increased differentiation induced by DSS. Vitisin A may regulate the proportion of intestinal immune cells by influencing the differentiation process of macrophages in vivo.

[0163] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Application of carboxypyranocyanidin-3-O-glucoside in the preparation of products for the prevention and / or treatment of ulcerative colitis.

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