Use of CCR6 gene in preparation of drugs for resisting abdominal aortic aneurysm

Abstract

The application discloses application of CCR6 gene in preparation of an anti-abdominal aortic aneurysm medicine and belongs to the technical field of medicines. The application selects CCR6-siRNA interference technology, constructs an adeno-associated virus expression vector, inhibits expression of a mouse CCR6 gene, and constructs a mouse abdominal aortic aneurysm model by subcutaneous pumping of angiotensin II, finds that inhibition of the CCR6 gene can significantly reduce the incidence of abdominal aortic aneurysm and inhibit the occurrence and development of abdominal aortic aneurysm, and provides a theoretical and experimental basis for preparation and screening of a medicine or preparation for inhibiting expression of the CCR6 gene as a candidate medicine or preparation for resisting abdominal aortic aneurysm. The application prepares an adeno-associated virus for inhibiting the CCR6 gene, and finds that inhibition of the CCR6 gene can significantly inhibit the occurrence and development of abdominal aortic aneurysm by injecting the adeno-associated virus for inhibiting the CCR6 gene into a mouse tail vein.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and particularly relates to application of CCR6 gene in preparation of anti-abdominal aortic aneurysm drugs. BACKGROUND

[0002] The information disclosed in this Background section is only for the purpose of increasing the understanding of the background of the application and should not be taken as an acknowledgement or any form of suggestion that this information forms prior art that is already known to a person of ordinary skill in the art.

[0003] At present, there is a significant gap in the drug treatment strategy for abdominal aortic aneurysm (AAA), and clinical intervention completely depends on open surgery or endovascular aneurysm repair, but these means are only suitable for patients with specific indications and cannot meet the urgent need to inhibit the progression of early or small aneurysms. The pathophysiological process of this disease is complex and involves multiple mechanisms such as chronic inflammation, immune cell infiltration, and extracellular matrix degradation. In recent years, research has found that the formation of neutrophil extracellular traps (NETs) and the dual regulation of key molecules (such as TREM2) in the monocyte-macrophage system on cell infiltration and survival play an important role in the development of AAA, providing potential new targets for drug development.

[0004] With the deepening of the understanding of the pathogenesis of AAA, new treatment strategies targeting specific immune pathways have begun to emerge. For example, research has shown that precise intervention in neutrophil inflammation through nanomedicine delivery systems, or regulation of related signaling pathways such as TREM2, can effectively slow disease progression. These findings not only reveal the central role of the immune microenvironment in AAA, but also highlight the need to explore the role of key chemokine receptors in mediating the recruitment and activation of inflammatory cells, opening up new directions for the development of AAA therapies based on immune regulation. SUMMARY

[0005] In order to solve the deficiencies of the prior art, the purpose of the present application is to provide the application of CCR6 gene in preparation of anti-abdominal aortic aneurysm drugs, which first discloses the key role of CCR6 in the occurrence and development of abdominal aortic aneurysm. Inhibition of CCR6 can effectively inhibit abdominal aortic aneurysm. CCR6 is activated by CCL20 (C-C chemokine ligand 20) secreted by macrophages and other cells, and the immune cells expressing CCR6 infiltrate the abdominal aorta, promoting the occurrence and development of abdominal aortic aneurysm. Inhibition of its expression can block this pathway, thereby inhibiting the development of abdominal aortic aneurysm.

[0006] In order to achieve the above-mentioned purpose, the technical scheme of the present application is as follows:

[0007] In a first aspect, the present application provides use of a substance that inhibits CCR6 expression and / or activity in the manufacture of any one or more of the following:

[0008] (a1) a product that inhibits abdominal aorta dilation;

[0009] (a2) a product that reduces abdominal aorta elastin degradation;

[0010] (a3) a product that reduces T lymphocyte and B lymphocyte aggregation in abdominal aortic aneurysm;

[0011] (a4) a product that reduces the incidence of abdominal aortic aneurysm;

[0012] (a5) a product that inhibits the progression of abdominal aortic aneurysm;

[0013] (a6) a product that treats abdominal aortic aneurysm.

[0014] In some embodiments of the present application, the substance that inhibits CCR6 expression or reduces its activity includes, but is not limited to, an RNA interference molecule or an antisense oligonucleotide against CCR6, a small molecule inhibitor, siRNA, shRNA, a substance that performs lentiviral infection or gene knockout, etc., which are not specifically limited herein.

[0015] In some embodiments of the present application, the sequence of the siRNA is shown in SEQ ID NO. 1.

[0016] In some embodiments of the present application, the product is a drug or an experimental reagent for non-medical use. The experimental reagent can be used for basic research. For example, the product can be used to externally regulate the malignant biological behavior of abdominal aorta cells, thereby preparing an abdominal aortic aneurysm-related biological model and laying a foundation for mechanism research of abdominal aortic aneurysm and other diseases.

[0017] In some embodiments of the present application, the drug includes, but is not limited to, any one or more of a nucleic acid molecule, a carbohydrate, a lipid, a small molecule chemical drug, an antibody drug, a polypeptide, a protein, and a virus.

[0018] The nucleic acid molecule includes, but is not limited to, an antisense oligonucleotide, double-stranded RNA, or short hairpin RNA.

[0019] The virus is an adeno-associated virus, and the effective component contains siRNA and is packaged by the virus to prevent the occurrence of abdominal aortic aneurysm by inhibiting the CCR6 gene.

[0020] In a second aspect, the present application provides use of CCR6 as a target in screening drugs for treating abdominal aortic aneurysm.

[0021] In some embodiments of the present application, the method for screening drugs for treating abdominal aortic aneurysm comprises:

[0022] (b1) treating a system overexpressing or containing CCR6 with a candidate substance; setting a parallel control without treating with the candidate substance;

[0023] (b2) after step (b1), detecting the expression level of CCR6 in the system; if the expression level of CCR6 in the system treated with the candidate substance is significantly reduced compared with the parallel control, the candidate substance can be used as a candidate drug for treating abdominal aortic aneurysm.

[0024] In some embodiments of the present application, the system is a cell system, a solution system, a tissue system or an animal system.

[0025] In some embodiments of the present application, the cells in the cell system are macrophages, and the tissues in the tissue system are abdominal aortic tissues.

[0026] In some embodiments of the present application, the animals in the animal system are mammals, including any one or several of mice, rats, guinea pigs, rabbits, monkeys, orangutans and humans.

[0027] The present application has the following advantages:

[0028] The present application provides the use of CCR6 in the preparation of drugs for resisting abdominal aortic aneurysm. The present application uses CCR6-siRNA interference technology to construct an adenovirus expression vector, inhibits the expression of mouse CCR6 gene, and constructs a mouse abdominal aortic aneurysm model by subcutaneous pumping of angiotensin II, finds that the inhibition of CCR6 gene can significantly reduce the incidence of abdominal aortic aneurysm and inhibit the occurrence and development of abdominal aortic aneurysm, and provides a theoretical and experimental basis for preparing and screening drugs or preparations for inhibiting the expression of CCR6 gene as candidate drugs or preparations for resisting abdominal aortic aneurysm. The present application prepares an adenovirus for inhibiting CCR6 gene, and finds that the inhibition of CCR6 gene can significantly inhibit the occurrence and development of mouse abdominal aortic aneurysm by injecting the adenovirus for inhibiting CCR6 gene into the tail vein of a mouse.

[0029] The present application also provides the use of CCR6 as a target in the screening of drugs for treating abdominal aortic aneurysm. The present application uses CCR6 gene as a target to screen drugs for resisting abdominal aortic aneurysm. Drug screening is mainly directed to unknown drugs, and CCR6 is used on the target gene, and the drugs for resisting abdominal aortic aneurysm are screened according to whether the drugs can inhibit the expression of the target gene; drug preparation is mainly based on the target gene, and the drugs for resisting abdominal aortic aneurysm are prepared or constructed to inhibit the expression of the target gene; the screened or prepared drugs have important significance in the treatment of abdominal aortic aneurysm. BRIEF DESCRIPTION OF DRAWINGS

[0030] The accompanying drawings, which form a part of this specification, are included to provide a further understanding of the application and are incorporated in and constitute a part of this specification. The embodiments of the application, together with its

[0031] Figure 1 Single cell RNA-seq analysis of CCR6 expression in 11 cells.

[0032] Figure 2 Immunofluorescence staining of CCR6 and T lymphocytes in patient abdominal aortic aneurysm tissue.

[0033] Figure 3 Immunofluorescence staining of CCR6 and B lymphocytes in patient abdominal aortic aneurysm tissue.

[0034] Figure 4 Single cell RNA-seq analysis of CCR6 expression in 11 cells in patient abdominal aortic aneurysm tissue and normal abdominal aorta tissue.

[0035] Figure 5 Analysis of CCR6 expression in human normal abdominal aorta tissue and patient abdominal aortic aneurysm tissue using regular transcriptome data (GSE269845 and GSE183464).

[0036] Figure 6 Immunofluorescence detection of CCR6 in human normal abdominal aorta tissue and patient abdominal aortic aneurysm tissue.

[0037] Figure 7 Western-blot detection of CCR6 expression in human normal abdominal aorta tissue and patient abdominal aortic aneurysm tissue.

[0038] Figure 8 Figure 7 Statistical graph of CCR6 expression in human normal abdominal aorta tissue and patient abdominal aortic aneurysm tissue.

[0039] Figure 9 Results of intercellular communication analysis using single cell sequencing data.

[0040] Figure 10 Immunofluorescence detection of T lymphocytes, B lymphocytes and macrophages in patient abdominal aortic aneurysm tissue.

[0041] Figure 11 Flow cytometry detection of B lymphocytes.

[0042] Figure 12 Flow cytometry detection of T lymphocytes.

[0043] ​Figure 13 Grouping of animal experiments and corresponding interventions and time.

[0044] Figure 14 Pictures of representative aortic specimens of the four groups of experimental animals.

[0045] Figure 15 Maximum aortic diameter (A) and tumor formation rate (B) of the four groups of experimental animals.

[0046] Figure 16 Comparison of HE, EVG and Masson staining pictures (A), elastic fiber degradation (B) and collagen deposition (C) of aortic specimens of the four groups of experimental animals.

[0047] Figure 17 RNA expression level of CCR6 in aortas of the four groups of experimental animals.

[0048] Figure 18 Immunofluorescence detection results of CCR6, CD3 and CD19 in aortas of the four groups of experimental animals. DETAILED DESCRIPTION

[0049] In order to enable those skilled in the art to more clearly understand the technical solutions of the present application, the technical solutions of the present application will be described in detail below in combination with specific examples.

[0050] The drugs and reagents involved in the examples are all ordinary commercially available products unless otherwise specified; the experimental operations involved in the examples are all carried out according to the conventional operations in the art unless otherwise specified.

[0051] Example 1: Localization and detection of CCR6 in AAA.

[0052] First, the expression level of CCR6 in 11 cells in AAA tissue was analyzed by bioinformatics analysis (single-cell data sets GSE166676 and GSE226492). The detection results are shown in Table 1. Figure 1 As shown in Table 1, CCR6 was mainly and highly expressed in T lymphocytes and B lymphocytes among the 11 cells.

[0053] In order to further verify the expression of CCR6 in the abdominal aortic aneurysm tissue of AAA patients, immunofluorescence co-localization was applied in the AAA patient specimens to further confirm that CCR6 mainly exists in T lymphocytes and B lymphocytes (Fig. 2). Figure 2 Figure 3

[0054] The expression of CCR6 in 11 cells in AAA group and normal tissue was analyzed by bioinformatics analysis (single-cell data sets GSE166676 and GSE226492). As shown in Table 2, CCR6 was mainly and highly expressed in T lymphocytes and B lymphocytes among the 11 cells. Figure 4 ​​As shown, the CCR6 expression level of AAA group T lymphocytes and B lymphocytes was significantly higher than that of the normal group.

[0055] In the analysis of conventional transcriptome data (GSE183464 and GSE269845), it was confirmed that the CCR6 expression level in the AAA group was significantly higher than that in the normal group Figure 5 ).

[0056] In the AAA patients and control group, it was also confirmed by immunofluorescence and Western-blot that CCR6 was enriched in AAA tissue than in normal aortic tissue Figure 6 、 Figure 7 、 Figure 8 ).

[0057] Example 2: Explore the potential mechanism of CCR6 in the formation of AAA.

[0058] Using single-cell sequencing data for intercellular communication analysis, it was found that macrophages in AAA had extensive interactions with other cell types. Macrophages in AAA and normal aortic tissue communicated with various cell types through the CCL3L3-CCR1 axis. However, macrophages only communicated with B lymphocytes, T lymphocytes and natural killer cells through the CCL20-CCR6 axis in AAA tissue, and there was no such communication in normal aortic tissue Figure 9 . In order to further verify the immune cells in the aneurysm tissue of AAA patients, immunofluorescence was applied in the AAA patients and control group to detect that T lymphocytes, B lymphocytes and macrophages were obviously aggregated in AAA tissue, indicating that macrophages may have a potential recruitment effect on T lymphocytes and B lymphocytes Figure 10 ).

[0059] The only ligand of CCR6, CCL20, is mainly expressed in macrophages. In order to further verify whether macrophages recruit lymphocytes to infiltrate AAA tissue through the CCL20-CCR6 axis, the experiment in this example was performed in vitro using a Transwell experiment. Macrophages and B lymphocytes or T lymphocytes were co-cultured, and CCL20 neutralizing antibody was added after stimulation with lipopolysaccharide (LPS). Flow cytometry detection results confirmed that blocking the CCL20-CCR6 axis with the antibody significantly reduced the migration of B lymphocytes and T lymphocytes Figure 11 、 Figure 12 ). The above results show that macrophages recruit B lymphocytes and T lymphocytes to infiltrate AAA tissue through the CCL20-CCR6 axis, which may promote the progression of AAA.

[0060] Example 3: Verify the role of CCR6 in the formation of AAA through in vivo experiments in mice.

[0061] 1) AAA mouse model establishment method: select 8-week-old male ApoE- / - mice, after intraperitoneal injection of 0.08% sodium pentobarbital (40 mg / kg) to anesthetize the mice, sterilely cut the back skin along the lower edge of the scapula, bury the implantable capsule osmotic pump subcutaneously, give high-fat diet, and continuously pump in angiotensin II (Angiotensin II, AngII) (1000 ng / kg / min) for 28 days.

[0062] 2) Experimental grouping and treatment measures before pumping Ang Ⅱ or saline in each group:

[0063] Group A (normal saline group): no CCR6-related treatment is needed.

[0064] Group B (model group): as described in 1) above.

[0065] Group C (CCR6 gene interference group): first synthesize CCR6-siRNA (sequence: 5'-GATCCATGACTGACGTCTACCT-3', SEQ ID NO. 1), package into an adeno-associated virus vector, and inject into the mouse body through the tail vein, repeat once after 2 weeks. Randomly select 2 mice for detection 7 days after injection.

[0066] Group D (empty vector group): inject empty adeno-associated virus vector through the tail vein, and inject once every 2 weeks.

[0067] 3) Animal experiment process: select 60 8-week-old male ApoE- / - mice, randomly divide them into 4 groups (as described above A-D groups), and give them high-fat feed, according to the grouping, the treatment as described in 2) above, on the 2nd day after the first injection, all groups start to pump in constant speed, pump in normal saline (A and D groups) or Ang Ⅱ (B and C groups) for 28 days, and finally sacrifice the mice. Figure 13 ).

[0068] 4) Use a mouse ultrasound instrument to observe AAA formation and measure the diameter.

[0069] 5) Sacrifice the mice to take out the abdominal aorta, and perform the following examination and detection:

[0070] ① Measure the abdominal aorta diameter and calculate the tumor rate;

[0071] ② Prepare AAA tissue frozen sections and perform HE, EVG and Masson staining to detect elastic fiber degradation and collagen deposition;

[0072] ③ Immunofluorescence was used to detect the expression of CCR6 and lymphocyte infiltration in various aortic tissues. RT-PCR was used to detect the expression of CCR6 in various aortic tissues.

[0073] The results showed that interfering with CCR6 significantly inhibited abdominal aortic dilation and reduced the tumor rate (P < 0.05) Figure 14 and Figure 15 HE, EVG and Masson staining showed that interfering with CCR6 could reduce the degradation of elastic fibers and collagen deposition (P < 0.05) Figure 16 PCR results showed that the expression of CCR6 in the model group was significantly higher than that in the control group (P < 0.05) Figure 17 Immunofluorescence showed that the expression of CCR6 increased and T lymphocytes and B lymphocytes infiltrated in the model group, while the aggregation of T lymphocytes and B lymphocytes was reduced after inhibiting CCR6 (P < 0.05) Figure 18 .

[0074] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. The use of a substance that inhibits CCR6 expression and / or reduces its activity in the preparation of a drug for treating abdominal aortic aneurysm; wherein the substance that inhibits CCR6 expression or reduces its activity is a CCR6-targeting siRNA; the sequence of the siRNA is shown in SEQ ID NO.

1.

2. The application as described in claim 1, characterized in that, The substance that inhibits CCR6 expression or reduces its activity can inhibit abdominal aortic dilation; Or, reduce the degradation of abdominal active elastic fibers; Alternatively, it can reduce the aggregation of T lymphocytes and B lymphocytes within the abdominal aortic aneurysm. Or, reduce the incidence of abdominal aortic aneurysm; Alternatively, it can inhibit the progression of abdominal aortic aneurysm.

3. The application as described in claim 1, characterized in that, The drug includes any one or more of nucleic acid molecules, small molecule chemical drugs, polypeptides, or proteins.

Images