Application of GATA6 in the prevention and treatment of inflammatory bowel disease

Abstract

The present invention discloses the use of GATA6 as a target in screening drugs for preventing / treating inflammatory bowel disease, the use of GATA6 in preparing drugs for preventing / treating inflammatory bowel disease, and a drug for preventing / treating inflammatory bowel disease, the drug comprising GATA6. The present invention has found that GATA6 can regulate Th17 cell differentiation and inhibit fibroblast activation, and has a good preventive and therapeutic effect on IBD, providing a new strategy for preventing and treating IBD.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the application of GATA6 in preventing and treating inflammatory bowel disease. Background Art

[0002] Inflammatory bowel disease (IBD) is a chronic nonspecific intestinal inflammatory disease with unclear etiology and pathogenesis, including ulcerative colitis (UC) and Crohn's disease. The main clinical manifestations are diarrhea, abdominal pain, and bloody stools. It is one of the main causes of intestinal fibrosis and intestinal dysfunction. It has the characteristics of unknown pathogenesis, difficult diagnosis, difficult treatment, and poor prognosis. In recent years, the incidence of IBD in my country has continued to increase. The main patients are people aged 15 to 30 or over 60 years old, and are not affected by gender.

[0003] The onset of IBD is the result of the combined effects of multiple factors, including genetic susceptibility genes, immune response, intestinal microecology, diet, etc. Abnormal gene expression can play an important role in the occurrence and development of IBD by mediating cell differentiation disorders and excessive activation of immune cells.

[0004] The GATA transcription factor family consists of 6 members (GATA1 to GATA6), which play an important role in regulating the growth and differentiation of various cells, cell survival, and the maintenance of body functions. Transcription factor GATA6 belongs to the GATA family and is a zinc finger transcription factor. It plays an important regulatory role in cell differentiation and organogenesis during vertebrate development. At present, there is no research on the relationship between GATA6 and inflammation and body immunity in IBD. Summary of the invention

[0005] The purpose of the present invention is to provide an application of GATA6 in preventing and treating inflammatory bowel disease in view of the above problems.

[0006] In order to achieve its purpose, the present invention adopts the following technical solution:

[0007] A first aspect of the present invention provides the use of GATA6 as a target in screening drugs for preventing / treating inflammatory bowel disease.

[0008] Preferably, the drug promotes the expression of GATA6.

[0009] Preferably, the drug has at least one of the following functions:

[0010] (1) Reduce intestinal mucosal damage;

[0011] (2) Relieve colon inflammation;

[0012] (3) Inhibit fibroblast activation and alleviate intestinal mucosal fibrosis;

[0013] (4) Inhibit Th17 cell polarization.

[0014] The second aspect of the present invention provides the use of GATA6 in the preparation of a drug for preventing / treating inflammatory bowel disease.

[0015] The amino acid sequence of GATA6 is shown in SEQ ID NO.1:

[0016] .

[0017] A third aspect of the present invention provides a drug for preventing / treating inflammatory bowel disease, wherein the drug comprises GATA6.

[0018] The medicament further includes a pharmaceutically acceptable carrier.

[0019] The beneficial effects of the present invention are:

[0020] The present invention systematically studies and finds that GATA6 can regulate Th17 cell differentiation and inhibit fibroblast activation, thus having a good preventive and therapeutic effect on IBD.

[0021] In the onset and progression of IBD, many studies in recent years have found that excessive polarization of Th17 cells is directly related to the onset of IBD, and fibroblast activation is related to IBD fibrosis. The present invention found that GATA6 has the effect of preventing and treating IBD, and further proved that this effect is related to regulating Th17 cell polarization and fibroblast activation.

[0022] Current studies have confirmed that Th17 cells are an important group of cells that mediate autoimmune diseases and are associated with both IBD inflammation and fibrosis. Inhibiting Th17 cell proliferation and autoimmune reactions can reduce their cell activity, control the intensity of the immune response, and reduce tissue damage. Therefore, in-depth exploration of the role of Th17 cells in the IBD mechanism can provide new strategies for prevention and treatment. The present study found that GATA6 has the ability to regulate Th17 polarization, which may be the key immune mechanism for its therapeutic effect on IBD.

[0023] Fibrosis is a key factor in the progression and treatment of IBD, but there is currently no good drug for the prevention and treatment of fibrosis. Fibroblasts are direct effector cells related to fibrosis, inhibiting fibroblast proliferation, activation and collagen production, and alleviating the progression of tissue fibrosis. The present invention found that GATA6 has the ability to inhibit fibroblast activation, which may be an important mechanism for its alleviation of IBD fibrosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Comparison results of GATA6 expression in IBD patients.

[0025] Figure 2 This is the comparison result of GATA6 expression in each group of mice.

[0026] Figure 3 This is the comparison result of DAI scores of mice in each group.

[0027] Figure 4 The colon morphology of mice in each group was observed.

[0028] Figure 5 Comparison of colon length among mice in each group.

[0029] Figure 6 Figure 3 is a comparison of pathological changes in colon tissue HE staining of mice in each group.

[0030] Figure 7 The figure is the comparison of Masson staining of colon tissues in each group of mice.

[0031] Figure 8The results of flow cytometry analysis of the activated proportion of Th17 cells in the colon of IBD mice after treatment.

[0032] Fig. 9 Yes CD4 + Flow cytometry results of detecting the activation ratio of T cells (normal and overexpressing GATA6) after inducing Th17 in vitro.

[0033] Fig.10 It is the result of WB detection of the expression of fibroblast activation marker a-SMA. DETAILED DESCRIPTION

[0034] The present invention will be further described below in conjunction with embodiments, but the present invention is not limited thereto.

[0035] The experimental methods in the following examples are conventional methods unless otherwise specified.

[0036] Example 1

[0037] 1 Experimental Materials and Reagents

[0038] 1.1 Experimental animals: SPF grade 8-week-old C57 male mice (20±5 g).

[0039] GATA6 knockout mice were purchased from Shanghai Model Organisms Technology Co., Ltd.

[0040] 1.2 Experimental Reagents

[0041] Water-soluble dextran sulfate sodium (DSS);

[0042] GATA6 overexpression virus was purchased from Heyuan Biotechnology (Shanghai) Co., Ltd. (China);

[0043] Flow cytometry antibodies AF488-CD4, BV421-RORrt, APC-IL-17A, and antibody CD3 / CD28 were purchased from BD (Becton, Dickinson and Company) (USA);

[0044] The histochemical antibody a-SMA was purchased from Sigma (USA);

[0045] Cytokines IL-23, IL-6, and TGF-β were purchased from MCE (MedChemExpress) (USA);

[0046] The Fixation / Permeabilization Solution Kit and the Transcription Factor Buffer Set for intracellular analysis were purchased from Becton, Dickinson and Company (USA).

[0047] BD Antibody 550583 Leukocyte Activation Cocktail, Protein Transport Inhibitor BD Golgi Plug TM Purchased from Becton, Dickinson and Company (USA).

[0048] 2 Experimental methods

[0049] 2.1 Model preparation:

[0050] The inflammatory bowel disease model (IBD model) was prepared by feeding mice with 3% DSS in water for 7 weeks.

[0051] 2.2 Experimental Grouping:

[0052] The experimental animals were divided into: IBD model group, GATA6 deficiency model group, GATA6 drug intervention group and control group, with 6 mice in each group.

[0053] The GATA6 drug intervention group was treated with GATA6 overexpression virus: 14 days before modeling, the mice were given a single dose of 5E10vg / 100ul / mouse via the tail vein, and then the model was established according to the IBD model preparation method, and the mice were fed normally for 3 days after modeling;

[0054] GATA6 deficiency modeling kit is to transform GATA6 flox / flox CreERT2 conditional-deficient mice were subjected to Tamoxifen (drug name: Tamoxifen, CAS registration number 10540-29-1)-induced knockout of GATA6 and then established according to the IBD model preparation method. After establishment of the model, the mice were fed normally for 3 days.

[0055] The IBD model group consisted of wild-type mice that were given 100uL PBS via tail vein 14 days before modeling and then were modeled according to the IBD model preparation method. They were fed normally for 3 days after modeling.

[0056] The wild-type mice in the control group were given 100uL PBS via the tail vein and then fed normally for 10 days.

[0057] 2.3 Drug efficacy testing:

[0058] First, the general indicators of mice before and after modeling, such as food / water intake, activity, body weight, and blood in stool, were observed; then, the mice were killed by cervical dislocation on the 11th day and the colon was isolated, and the colon morphology was observed by photographing and the colon length was measured; the distal colon was flushed and fixed with 4% paraformaldehyde or frozen at -80°C for subsequent testing.

[0059] 3 Results 3.1

[0061] Figure 1 Intestinal tissues of clinically healthy people and IBD patients were obtained, fixed with 4% paraformaldehyde, and sections were immunohistochemically stained for GATA6. The results showed that the expression of GATA6 in healthy tissues was higher than that in IBD patient tissues.

[0062] Figure 2 3% DSS was used to induce IBD in mice (7 days of DSS, 3 days of normal water). After the induction, colon tissue was taken, fixed with 4% paraformaldehyde, and sections were immunohistochemically stained for GATA6. The results showed that GATA6 expression in the control group tissue was higher than that in the IBD model group tissue.

[0063] Figure 3 3% DSS was used to induce IBD in mice (7 days of DSS, 3 days of normal water). The induction started as day 0. The weight and fecal characteristics of the experimental mice were recorded every other day, and the status of the mice was scored according to the DAI score table. The DAI score during the modeling process was counted. Control group: 100ul PBS was given through the tail vein 14 days before modeling, corn oil was given intraperitoneally every other day for 5 consecutive times, and water was given normally during the modeling process; model group: 100ul PBS was given through the tail vein 14 days before modeling, corn oil was given intraperitoneally every other day for 5 consecutive times, and DSS was given normally during the modeling process; GATA6 knockout group (i.e. defective modeling group): Tamoxifen was given intraperitoneally every other day for 5 consecutive times before modeling 14 days, and DSS was given normally during the modeling process; GATA6 drug intervention group: 100ul GATA6 AAV virus was given through the tail vein 14 days before modeling, corn oil was given intraperitoneally every other day for 5 consecutive times, and DSS was given normally during the modeling process; PBS and corn oil were the media, respectively.

[0064] Figure 4 yes Figure 3 At the end of the modeling, colon tissues were taken and photographed. DSS modeling caused colon shortening, and the GATA6 knockout group was shorter than the DSS model group. GATA6 intervention restored the colon length.

[0065] Figure 5 Yes Figure 3 The colon tissue length was divided into groups and statistically analyzed. DSS modeling led to shortened colon, and the GATA6 knockout group was shortened more than the DSS model group. GATA6 intervention restored the colon length. There were significant differences in all of them.

[0066] Control group: The mice were responsive, had smooth and clean fur, ate more, had significantly increased body weight, had hard, dark brown, ellipsoidal feces; macroscopic observation of the mouse colon showed no obvious blood in the stool, edema, bleeding, or ulcer formation.

[0067] IBD model group: The mice had significantly reduced food / water intake, were emaciated, had rough and dull fur, had dirty perianal area, had few spontaneous activities, were lethargic and lazy, and had slow reactions; their feces were partially occult bloody or soft, their colons were shortened, and some of the colons had visible congestion, edema, bleeding and ulcers.

[0068] GATA6-deficient modeling group: mice ate / drank less, were less active, lost more weight, had rough and dull fur, had dirty and bloody perianal area, had less spontaneous activity, were lethargic and had slow reactions; their feces were mucus, pus and blood or watery bloody; their colon was significantly shortened and less elastic; and the colon showed visible congestion, edema, bleeding and ulcers.

[0069] In the GATA6 drug intervention group, the mice's food and water intake gradually returned to normal, their activities increased, their fur became more shiny, and their weight loss eased. Their stools became formed, with less loose and soft stools. The degree of shortening of the colon eased, and symptoms such as congestion, edema, bleeding, and ulcers visible to the naked eye in the colon were significantly alleviated.

[0070] 3.2 Intestinal mucosal histopathological scoring:

[0071] The colon tissue was fixed in 4% paraformaldehyde solution, embedded in paraffin, sliced ​​and stained with HE (eg Figure 6 As shown), and optical microscopy was used for observation and detection. HE staining of the colon tissue of the control group mice showed intact cell structure under the microscope, without lymphocyte and neutrophil infiltration. IBD model group: HE staining showed disordered cell structure, disappearance of goblet cells, and infiltration of lymphocytes and neutrophils under the microscope. GATA6-deficient modeling group: HE staining showed disordered cell structure, disappearance of goblet cells, and more significant infiltration of lymphocytes and neutrophils. GATA6 drug intervention group: HE staining showed that the cell arrangement structure tended to be neat, and a small amount of lymphocytes and neutrophils infiltrated. This shows that GATA6 relieves colon inflammation.

[0072] 3.3 Intestinal mucosal fibrosis score:

[0073] The colon tissue was fixed in 4% paraformaldehyde solution, embedded in paraffin, sliced, and stained with HE and Masson (eg Figure 7As shown), and optical microscopy was used for observation and detection. In the colon tissue of mice in the control group, the Masson result showed that the distribution of cellulose was normal under the microscope. IBD model group: The Masson result showed that the collagen fibers increased and were abnormally distributed under the microscope. GATA6-deficient modeling group: The Masson result showed that the collagen fibers increased and were significantly abnormally distributed under the microscope. GATA6 drug intervention group: The Masson result showed that the distribution of cellulose tended to be normal under the microscope. This shows that GATA6 alleviates fibrosis.

[0074] 3.4 Analysis of streaming results:

[0075] Flow cytometry was used to detect the expression of IL-17A and Foxp3, markers of Th17 and Treg cells, in the mesenteric lymph nodes of mice in the control group, IBD model group, and GATA6 administration intervention group. The Treg marker Foxp3 in the mesenteric lymph nodes of mice in the IBD model group and GATA6-deficient mice was significantly decreased compared with the control group, while the Th17 marker IL-17A was significantly increased. Moreover, under the condition of GATA6 intervention, the Treg marker Foxp3 in the mesenteric lymph nodes was significantly increased, while the Th17 marker IL-17A was significantly inhibited. Flow cytometry was used to detect the expression of IL-17A and Foxp3, markers of Th17, in the mesenteric lymph nodes of mice in the control group and GATA6-overexpressing group. + Induction of Th17 T cells in vitro (results as Figure 8 As shown in the figure, DSS had more Th17 cell infiltration and lower Treg infiltration than the control group. The GATA6 knockout group had more Th17 cell infiltration and lower Treg infiltration compared with the DSS group. GATA6 intervention improved the infiltration trend of Th17 cells and Treg cells, making it closer to the control group. This shows that GATA6 intervention can inhibit the recruitment of Th17 cells in the colon.

[0076] Fig. 9 The spleen of healthy mice was taken and naive CD4 + T cells, and half of the cells were transfected with GATA6 overexpression plasmid; at the same time, Th17 cells were induced in vitro; Th17 cell-specific markers RORrt and IL-17A were detected 3-4 days after induction. The results showed that GATA6 overexpression inhibited the polarization of Th17 cells.

[0077] 3.5WB result analysis:

[0078] Fibroblasts were extracted from colon tissues of healthy mice. Fibroblast activation was induced in the model group, and activation was induced in the GATA6 intervention group after transfection of GATA6 overexpression plasmid. Fibroblast activation marker a-SMA was detected by WB.

[0079] The results are as follows Fig.10As shown, the cell marker a-SMA in the GATA6 overexpression group was significantly inhibited compared with the control group. The results show that GATA6 inhibits fibroblast activation and thus inhibits inflammatory bowel disease-mediated fibrosis, and can be prepared as a therapeutic drug for inflammatory bowel disease fibrosis.

[0080] 4 Analysis

[0081] The present invention simulated human inflammatory bowel disease by feeding mice with DSS and found that IBD was susceptible under the condition of GATA6 deficiency, and GATA6 had obvious preventive and therapeutic effects on inflammatory bowel disease, which was specifically manifested as follows: 1) IBD model group: mice had reduced food / drinking, reduced activity, and significant weight loss. Some of the feces were occult blood or soft stools, the colon was shortened, and part of the colon showed visible congestion, edema, bleeding and ulcers. Under the microscope, cell structure disorder, goblet cell loss, inflammatory cell infiltration, etc. were observed. 2) GATA6 defective modeling group: mice had significantly reduced food / drinking, reduced activity, and more significant weight loss. The feces were mucus, pus and blood or thin bloody stools. The colon was significantly shortened and its elasticity was weakened. The colon showed visible congestion, edema, bleeding and ulcers. Under the microscope, cell structure disorder, goblet cell disappearance, and inflammatory cell infiltration were observed. 3) GATA6 drug intervention group: Compared with the IBD model group, the mice's food and water intake returned to normal, their activities increased significantly, their weight loss was significantly alleviated, their bowel movements became normal, with less loose and soft stools, the shortening of the colon was alleviated, and the symptoms of congestion, edema, bleeding, and ulcers visible to the naked eye in the colon were significantly alleviated. The cell structure disorder, disappearance of goblet cells, and inflammatory cell infiltration visible under the microscope were significantly alleviated.

[0082] The experimental results of the present invention show that GATA6 deficiency leads to susceptibility to IBD, and GATA6 administration can effectively counteract the damage of colitis to the mucosa, relieve colon inflammation and fibrosis, and protect mice. Therefore, GATA6 can be used as a therapeutic target for IBD and develop preventive and therapeutic drugs.

Claims

1. Use of GATA6 in the preparation of a drug for preventing / treating intestinal mucosal fibrosis mediated by inflammatory bowel disease, characterized in that: The GATA6 directly inhibits fibroblast activation by inhibiting Th17 cell polarization and IL-17A expression, thereby alleviating intestinal mucosal tissue fibrosis. The amino acid sequence of the GATA6 is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that: The drug is a GATA6 overexpression virus.

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