Application of MDSCs depleting agent in preparation of medicine for treating intestinal fibrosis diseases

Abstract

The invention discloses an application of an MDSCs depleting agent in preparation of a medicine for treating intestinal fibrosis diseases. The application proves that the MDSCs are obviously increased in the processes of mice with intestinal fibrosis and patients with clinical intestinal stenosis, and promote collagen secretion of the mice and the patients, so that the development of intestinal fibrosis is driven. The anti-Gr-1 antibody depleted MDSCs are used in a mouse intestinal fibrosis model for treatment, and the symptoms of mouse intestinal fibrosis are obviously improved. The depleted MDSCs has a good anti-fibrosis effect, is expected to be developed into a novel biological treatment medicine, and is used for clinical treatment of IBD-related intestinal fibrosis diseases.

Description

Technical Field

[0001] The invention relates to application of an MDSCs depleting agent in preparing a medicine for treating intestinal fibrosis diseases, and belongs to the technical field of biomedicine. Background Art

[0002] Inflammatory bowel disease (IBD) has become a global public health challenge. IBD, including Crohn's disease (CD) and ulcerative colitis (UC), is a recurrent gastrointestinal inflammatory disease and also an autoimmune disease. Intestinal fibrosis is formed by excessive deposition of extracellular matrix (ECM) derived from activated mesenchymal cells in the intestinal wall, mainly occurring in the submucosal layer and muscularis propria. The fibrogenic process is driven by a variety of cells, including local fibroblast proliferation and activation, epithelial-mesenchymal transition (EMT), recruitment and differentiation of fibroblasts, and endothelial cell transformation (Endo-MT), among which fibroblast activation is the most direct cause of intestinal fibrosis. Clinical studies have shown that despite the increasing anti-inflammatory ability of biological agents, the incidence of clinical intestinal strictures has not been substantially reduced. The effectiveness of anti-inflammatory drugs for IBD has not improved the severity of gastrointestinal fibrosis. Existing therapeutic drugs have limited efficacy against intestinal fibrosis, which is also a major challenge faced in clinical practice. Therefore, there is an urgent need to develop specific therapeutic drugs for intestinal fibrosis, which is also one of the issues that need urgent attention in the field of intestinal fibrosis research.

[0003] The interaction between immune cells and effector cells is an important factor in the process of intestinal fibrosis. The relationship between fibrosis and immune response involves complex immune cell networks and signaling pathways. In recent years, immune dysregulation has been considered a key factor in the fibrosis of many organs. The innate and adaptive immune systems can enhance the secretion of profibrotic factors, trigger healing and scar formation, and thus lead to fibrosis. Myeloid-derived suppressor cells (MDSCs) are a heterogeneous class of cells that are involved in the development of a variety of diseases such as tumors, infections, trauma, autoimmune diseases, and organ fibrosis. MDSCs play an important role in the fibrosis of many organs, such as renal fibrosis, pulmonary fibrosis, and liver fibrosis. However, the role and molecular mechanism of MDSCs in the process of intestinal fibrosis are still unclear and need to be further studied. Summary of the invention

[0004] The purpose of the present invention is to provide the use of MDSCs depleting agents in the preparation of drugs for treating intestinal fibrosis diseases, and to provide a new idea for the clinical preparation and treatment of IBD-related intestinal fibrosis drugs.

[0005] Preferably, the MDSCs depleting agent is an Anti-Gr-1 antibody.

[0006] The inventors found that MDSCs were highly accumulated in the colon tissues of patients with intestinal stenosis and mice with intestinal fibrosis. After depletion of MDSCs by Anti-Gr-1 antibody, the fibrosis symptoms in the colons of mice with intestinal fibrosis induced by TNBS were alleviated. We further infer that MDSCs in the colon tissues of mice with intestinal fibrosis may be closely related to the severity of intestinal fibrosis.

[0007] Preferably, the MDSCs depleting agent is the antibody BP0075 or BE0320. Both antibodies can be obtained from commercial sources. As a more preferred solution, the BP0075 and BE0320 are produced by BioXCell, namely InVivoPlus anti-mouse Ly6G / Ly6C (Gr-1) BioXCell BXC-BP0075 and InVivoPlus anti-mouse Ly6G / Ly6C (Gr-1) BioXCell BXC-BE0320.

[0008] Preferably, the MDSCs depleting agent is a compound that is a labeled or chemically modified version of the antibody BP0075 and also has the function of depleting MDSCs.

[0009] Preferably, the MDSCs depleting agent is a salt, solvate, tautomer, or isomer of the antibody BP0075, and the salt, solvate, tautomer, or isomer also has the effect of depleting MDSCs.

[0010] Intestinal fibrosis refers to IBD-related intestinal fibrosis. Inflammatory bowel disease (IBD) includes Crohn's disease and ulcerative colitis. As time and the course of the disease prolong, it develops into intestinal fibrosis and even intestinal stenosis. Intestinal stenosis is the most serious stage.

[0011] Currently, there is no specific therapeutic drug for the symptoms of patients with intestinal fibrosis. The present invention discovered for the first time that MDSCs over-accumulate in intestinal fibrotic colonic tissue, and promote the activation of colonic fibroblasts and collagen production, thereby promoting the progression of intestinal fibrosis. In the mouse intestinal fibrosis model, intestinal fibrosis was effectively alleviated by depleting MDSCs using antibodies. Currently, there is no effective treatment plan or medicine for complications related to inflammatory bowel disease. Most anti-inflammatory drugs are used clinically, but they have failed to truly reduce the incidence of intestinal stenosis. The technical solution of the present invention specifically treats intestinal fibrosis by targeting specific target MDSCs, which has a groundbreaking significance for improving the health of patients with intestinal fibrosis and even intestinal stenosis. The present invention achieves the purpose of treating intestinal fibrosis by depleting intestinal MDSCs, providing a new approach to the treatment of intestinal fibrosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 , MDSCs detection results in mouse colon tissue, where A is the flow cytometry detection result and B is the statistical analysis of the flow cytometry data.

[0013] Figure 2 , MDSCs detection results in healthy colon tissue, inflammatory non-stricture tissue and stricture tissue of patients with clinical intestinal stricture, where A is the immunofluorescence data and B is the statistical data of immunofluorescence.

[0014] Figure 3 , in vitro study of the functional effects of MDSCs on intestinal fibroblasts.

[0015] Figure 4 , in vivo study of the therapeutic effect of Anti-Gr-1 antibody on intestinal fibrosis diseases, where A is the flow cytometry results and statistical analysis, and B is Masson staining and statistical analysis. Specific implementation methods

[0017] The present invention is further described below in conjunction with embodiments, but the description of the embodiments does not impose any limitation on the protection scope of the present invention.

[0018] Unless otherwise defined, all technical terms and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the present invention, and the terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention. In addition, although demonstrations of parameters including specific values ​​may be provided herein, it should be understood that the parameters do not need to be exactly equal to the corresponding values, but may be approximated to the corresponding values ​​within an acceptable error tolerance or design constraint.

[0019] Unless otherwise specified, the materials and instruments used in the following examples can be obtained from conventional commercial channels. Example 1

[0020] S1. TNBS was used to repeatedly induce the establishment of a mouse intestinal fibrosis model. After the model was established, the mouse colon tissues were collected and CD11b was detected by flow cytometry. + Gr-1 + Changes in MDSCs, such as Figure 1 As shown, the Control group refers to the same treatment but without the addition of TNBS solution. Figure 1 This indicates that MDSCs increase in the colon tissue of mice with TNBS-induced intestinal fibrosis.

[0021] The construction method of TNBS intestinal fibrosis model is as follows:

[0022] Eight-week-old Balb / c female mice were selected as experimental subjects, and the model group mice were pre-sensitized on the first day of model construction. The specific method is: acetone and olive oil are mixed in a volume ratio of 4:1, and then mixed with 5% (wt / vol) TNBS in a volume ratio of 4:1 to prepare a 1% TNBS solution. Use an electric shaver to clean an area of ​​about 1.5cm×1.5cm on the back and neck of the mouse to expose the skin. Then evenly apply 150μL of 1% TNBS solution on the exposed area. Starting from the second week, the mice were given an enema once a week. The mice were fasted for 24 hours before enema and anesthetized by intraperitoneal injection of 80mg / kg sodium pentobarbital. Then, a 3.5F catheter was lubricated with glycerol, connected to a 1mL syringe, and slowly inserted into the mouse anus for about 4cm. Then, 80μL of TNBS solution was slowly injected into the colon, and the mouse was kept in an upward position with its tail for about 30 seconds to ensure absorption of the drug solution. The concentration of TNBS enema was set as: 0.75% in the first week, 1.5% in the second week, and 2.5% in the third to seventh weeks, with a total of six increasing concentration treatments. Samples were collected in the eighth week.

[0023] S2. Collect clinical intestinal stricture colon tissue samples, and take three sections of intestinal tissue in different states from the same patient, including healthy tissue, inflammatory non-stricture tissue, and stricture tissue. Detect healthy tissue by immunofluorescence

[0024] Healthy intestine, Inflamed non-stenosis, and Stenosis

[0025] The changes of CD11b+CD33+MDSCs in Stenosis are shown in the following table. Figure 2 shown. Figure 2 This indicates that in patients with clinical intestinal stricture, MDSCs are significantly increased in both inflammatory non-stricture tissues and stricture tissues compared with healthy tissues.

[0026] S3. Colon MDSCs from normal mice and TNBS intestinal fibrosis mice were sorted, and co-cultured with colon fibroblasts at a ratio of 10:1. Transwell chambers were used to separate the two types of cells during co-culture, and Fibrobalsts cells were placed in the lower layer. In the Fibrobalsts group, there were no cells in the upper layer. In the Fibrobalsts+Ctr-MDSCs group, colon MDSCs from normal mice were sorted and placed in the upper layer for co-culture with Fibrobalsts; in the Fibrobalsts+TNBS-MDSCs group, colon MDSCs from TNBS intestinal fibrosis mice were sorted and placed in the upper layer for co-culture with Fibrobalsts. Fibroblasts were taken 24 hours later for immunofluorescence experiments to detect the expression level of α-SMA. The results are as follows: Figure 3 shown. Figure 3 It shows that the co-culture of fibroblasts and MDSCs in vitro shows that colon MDSCs in TNBS intestinal fibrosis mice can promote the activation of fibroblasts.

[0027] S4. In vivo depletion of MDSCs using Anti-Gr-1 antibody (InVivoPlus anti-mouse Ly6G / Ly6C (Gr-1) BioXCell BXC-BP0075) was performed by intraperitoneal injection of 150ug / mouse on the day before TNBS treatment, the third day after TNBS treatment, and the fifth day after TNBS treatment, respectively, for 6 cycles. For MDSCs reinfusion, 5x10 6 After the MDSCs model was constructed, the mouse colon tissue was collected and pathologically stained to observe its therapeutic effect on intestinal fibrosis in mice. Figure 4 shown. Figure 4 The Control group and TNBS group Figure 1 The treatment was the same as that of the control group. The TNBS+MDSCs group used the tail vein to return MDSCs cells to increase the number of MDSCs in the intestine; the TNBS+Anti-Gr-1 group used Anti-Gr-1 antibodies to deplete MDSCs to reduce the amount of MDSCs in the intestine. This shows the effect of changes in MDSCs content on the symptoms of intestinal fibrosis in mice. Figure 4 The results show that the increase of colon MDSCs can promote intestinal fibrosis through in vivo MDSCs knockout and MDSCs reinfusion experiments, and anti-Gr-1 antibody knockout of MDSCs can alleviate intestinal fibrosis.

[0028] The above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable people familiar with this technology to understand the contents of the present invention and implement them accordingly. They cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be included in the protection scope of the present invention.

Claims

1. Application of MDSCs depleting agents in the preparation of drugs for the treatment of intestinal fibrosis.

2. The use according to claim 1, characterized in that: The MDSCs depleting agent is Anti-Gr-1 antibody.

3. The use according to claim 2, characterized in that: The MDSCs depleting agent is antibody BP0075 or BE0320.

4. The use according to claim 3, characterized in that: The MDSCs depleting agent is a compound obtained by labeling or chemically modifying the antibody BP0075 and also having the function of depleting MDSCs.

5. The use according to claim 3, characterized in that: The MDSCs depleting agent is a salt, solvate, tautomer, or isomer of the antibody BP0075, and the salt, solvate, tautomer, or isomer also has the effect of depleting MDSCs.

6. The use according to any one of claims 1 to 5, characterized in that: Intestinal fibrosis refers to intestinal fibrosis associated with IBD.

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