Application of potassium calcium activated channel subfamily N member 4 inhibitor in preparation of medicine for preventing and treating abdominal aortic aneurysm and / or delaying progress of abdominal aortic aneurysm

Abstract

The invention provides application of a potassium calcium activated channel subfamily N member 4 (KCNN4) inhibitor in preparation of a medicine for preventing and treating abdominal aortic aneurysm and / or delaying the progress of the abdominal aortic aneurysm, and belongs to the technical field of biological medicine. KCNN4 has an important regulation effect in the AAA generation process, and can be used as a new target for preventing or treating AAA. The macrophage expression of KCNN4 in AAA is increased, and a KCNN4 inhibitor (TRAM34 or Senicapoc) can effectively reduce the occurrence of abdominal aortic aneurysm, reduce the death rate of abdominal aortic aneurysm, reduce the diameter of aorta and protect aorta, so that the KCNN4 inhibitor can be used for preventing and treating abdominal aortic aneurysm or delaying the progress of abdominal aortic aneurysm. The AAA is treated by the medicine, so that the risk of an operation can be avoided, and the progress of the AAA and the medical cost can be controlled.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technologies, and particularly relates to the application of a potassium calcium-activated channel subfamily N member 4 (KCNN4) inhibitor in the preparation of a drug for preventing and treating abdominal aortic aneurysm and / or delaying the progression of abdominal aortic aneurysm. Background Art

[0002] Abdominal aortic aneurysm (AAA) refers to the aneurysmal dilation of the abdominal aorta, with a diameter exceeding 3 cm or greater than 50% of the normal diameter. AAA is a disease that seriously endangers the life and health of patients. Initially, AAA usually has no obvious symptoms, but as the aneurysm grows, the risk of rupture gradually increases. The mortality rate of patients with ruptured abdominal aortic aneurysm is as high as 90%. Therefore, preventing or treating abdominal aortic aneurysm has important medical significance. Currently, no drugs specifically targeting abdominal aortic aneurysm have been found in research. Therefore, in-depth understanding of the pathogenesis of AAA and finding new therapeutic targets are of great significance for the treatment of AAA. Summary of the Invention

[0003] The purpose of the present invention is to provide the application of a potassium calcium-activated channel subfamily N member 4 inhibitor in the preparation of a drug for preventing and treating abdominal aortic aneurysm and / or delaying the progression of abdominal aortic aneurysm, filling the blank of drugs for abdominal aortic aneurysm.

[0004] The present invention provides the application of a KCNN4 inhibitor in the preparation of a drug for preventing and treating abdominal aortic aneurysm and / or delaying the progression of abdominal aortic aneurysm.

[0005] Preferably, the KCNN4 inhibitor includes TRAM34 and / or Senicapoc; The chemical structural formula of TRAM34 is shown as Formula I; Formula I; The chemical structural formula of Senicapoc is shown as Formula II; Formula II.

[0006] Preferably, the KCNN4 inhibitor is the sole active ingredient of the drug.

[0007] The present invention provides the application of a reagent for knocking out or knocking down KCNN4 in macrophages in the preparation of a drug for preventing, treating abdominal aortic aneurysm or delaying the progression of abdominal aortic aneurysm.

[0008] Preferably, the dosage form of the drug includes an injection.

[0009] Preferably, the abdominal aortic aneurysm is induced by hyperlipidemia and hypertension.

[0010] Preferably, the prevention, treatment of abdominal aortic aneurysm or delay of the progression of abdominal aortic aneurysm are achieved by inhibiting the activation of macrophage inflammasome and / or reducing the release of macrophage IL1b.

[0011] The present invention provides the use of potassium calcium-activated channel subfamily N member 4 inhibitor in the preparation of a drug for preventing and treating abdominal aortic aneurysm and / or delaying the progression of abdominal aortic aneurysm. KCNN4 plays an important regulatory role in the occurrence of AAA. The key genes of the PIEZO-KCNN4-NLRP3 signaling axis, namely PIEZO1, KCNN4, NLRP3 and IL-1β, are significantly up-regulated in macrophages in abdominal aortic aneurysm; KCNN4 is involved in the activation of macrophage inflammasome and the release of IL1b, and participates in the occurrence of abdominal aortic aneurysm through the inflammatory effect of IL1b. Therefore, KCNN4 can be used as a new target for the prevention or treatment of AAA. The expression of KCNN4 in macrophages in AAA increases. KCNN4 inhibitors (TRAM34 or Senicapoc) can effectively reduce the occurrence of abdominal aortic aneurysm, reduce the mortality of abdominal aortic aneurysm, reduce the aortic diameter and protect the aorta. Therefore, KCNN4 inhibitors can be used to prevent, treat abdominal aortic aneurysm or delay the progression of abdominal aortic aneurysm. Treating AAA with drugs can avoid the risks of surgery and is beneficial to controlling the progression of AAA and medical costs. Description of the Drawings

[0012] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0013] Figure 1 It is a cell clustering diagram of single-cell sequencing results of aortic tissue of control group mice and mice with aneurysm model constructed; Figure 2 It is the expression results of each gene in different cell populations in the mouse aorta; Figure 3 It is the detection results of IL-1β content after different treatments of wild-type and KCNN4-knockout THP1 cells; Figure 4 It is the detection results of IL-1β content after treating THP1 cells with KCNN4 inhibitor TRAM34; Figure 5 It is the construction flow chart of the mouse AAA model; Figure 6 It is the survival curve and survival rate results of mice in different groups; Figure 7 The incidence rate of abdominal aortic aneurysm in different groups of mice; Figure 8 The results of the maximum diameter of the abdominal aorta in different groups of mice; Figure 9 The results of the serum alanine aminotransferase activity in different groups of mice; Figure 10 The results of the serum aspartate aminotransferase activity in different groups of mice; Figure 11 The body weights of different groups of mice. Specific implementation mode

[0014] The present invention provides the use of a potassium calcium-activated channel subfamily N member 4 (KCNN4) inhibitor in the preparation of a drug for preventing and treating abdominal aortic aneurysm and / or delaying the progression of abdominal aortic aneurysm.

[0015] The amino acid sequence of KCNN4 described in the present invention is shown in SEQ ID NO.1, specifically as follows: MGGDLVLGLGALRRRKRLLEQEKSLAGWALVLAGTGIGLMVLHAEMLWFGGCSWALYLFLVKCTISISTFLLLCLIVAFHAKEVQLFMTDNGLRDWRVALTGRQAAQIVLELVVCGLHPAPVRGPPCVQDLGAPLTSPQPWPGFLGQGEALLSLAMLLRLYLVPRAVLLRSGVLLNASYRSIGALNQVRFRHWFVAKLYMNTHPGRLLLGLTLGLWLTTAWVLSVAERQAVNATGHLSDTLWLIPITFLTIGYGDVVPGTMWGKIVCLCTGVMGVCCTALLVAVVARKLEFNKAEKHVHNFMMDIQYTKEMKESAARVLQEAWMFYKHTRRKESHAARRHQRKLLAAINAFRQVRLKHRKLREQVNSMVDISKMHMILYDLQQNLSSSHRALEKQIDTLAGKLDALTELLSTALGPRQLPEPSQQSK.

[0016] In the specific implementation process of the present invention, the prevention, treatment of abdominal aortic aneurysm or delaying the progression of abdominal aortic aneurysm includes reducing the occurrence of abdominal aortic aneurysm, reducing the mortality rate of animals or patients with abdominal aortic aneurysm, reducing the aortic diameter and protecting the aorta in one or more of them; protecting the aorta includes avoiding the damage of hyperlipidemia and / or hypertension to the aortic structure.

[0017] In the present invention, the expression of KCNN4 in macrophages in AAA is increased, and KCNN4 inhibitors (TRAM34 or Senicapoc) can effectively reduce the mortality rate and aortic diameter in the AAA mouse model.

[0018] In the specific implementation process of the present invention, the KCNN4 inhibitor includes TRAM34 and / or Senicapoc, and the chemical structural formula of TRAM34 is shown in Formula I; Formula I; The chemical structural formula of Senicapoc is shown in Formula II; Formula II.

[0019] In the present invention, TRAM34 is purchased from MedChemExpress, catalog number HY-13519, specification 100 mg, and Senicapoc is purchased from MedChemExpress, catalog number HY-50694, specification 100 mg.

[0020] In the present invention, TRAM34 or Senicapoc has no obvious hepatotoxicity and has safety.

[0021] In the specific implementation process of the present invention, the KCNN4 inhibitor is the only active ingredient of the drug.

[0022] The present invention also provides the use of a reagent for knocking out or knocking down KCNN4 in macrophages in the preparation of a drug for preventing, treating abdominal aortic aneurysm or delaying the progression of abdominal aortic aneurysm.

[0023] In the present invention, the reagent for knocking out or knocking down KCNN4 in macrophages includes CRISPR-Cas9 reagent; knocking out KCNN4 in macrophages can significantly reduce the activation of inflammasomes and the release of downstream IL1b.

[0024] In the specific implementation process of the present invention, the dosage form of the drug includes an injection; the injection is prepared into a dosage form convenient for intraperitoneal injection; calculated by mice, the administration frequency of the drug is once a day, the administration dose of TRAM34 is 15 mg / kg / d, and the administration dose of Senicapoc is 15 mg / kg / d; the drug also includes a pharmaceutically acceptable carrier. Treating AAA with the drug can avoid the risks of surgery and is beneficial to controlling the progression of AAA and medical costs.

[0025] In the specific implementation process of the present invention, the abdominal aortic aneurysm is induced by hyperlipidemia and hypertension; the hyperlipidemia is induced by injecting the encoded PCSK9The adenovirus-associated virus (AAV) of the gene was simultaneously administered while feeding a high-fat diet containing 60% cholesterol to induce; the hypertension was induced by slow release of angiotensin II through an osmotic pump.

[0026] In the specific implementation process of the present invention, the prevention, treatment of abdominal aortic aneurysm or delaying the progression of abdominal aortic aneurysm is achieved by inhibiting the activation of macrophage inflammasome and / or reducing the release of macrophage IL1b.

[0027] The present invention also provides the application of KCNN4 as a biomarker in the preparation of detection products for abdominal aortic aneurysm. KCNN4 has an important regulatory role in the occurrence of AAA. The key genes PIEZO1, KCNN4, NLRP3, and IL-1β of the PIEZO-KCNN4-NLRP3 signaling axis are significantly up-regulated in macrophages in abdominal aortic aneurysm; KCNN4 is involved in the activation of macrophage inflammasome and the release of IL1b, and is involved in the occurrence of abdominal aortic aneurysm through the inflammatory effect of IL1b. Therefore, KCNN4 can be used as a new target for the prevention or treatment of AAA.

[0028] In the specific implementation process of the present invention, the KCNN4 is KCNN4 in macrophages.

[0029] The present invention also provides the application of KCNN4 in the construction of an animal model of abdominal aortic aneurysm.

[0030] To further illustrate the present invention, the following describes in detail the application of the potassium calcium-activated channel subfamily N member 4 inhibitor provided by the present invention in the preparation of drugs for preventing and treating abdominal aortic aneurysm and / or delaying the progression of abdominal aortic aneurysm in combination with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0031] Example 1 1. The expression of the PIEZO-KCNN4-NLRP3 signaling axis is significantly up-regulated in abdominal aortic aneurysm.

[0032] To study the changes in cell types and their functions during the development of abdominal aortic aneurysm, in this example, single-cell sequencing results of the aorta in control healthy mice and mouse aneurysm models in the public database (Gene Expression Omnibus, GEO) (GSE152583, GSE164678, GSE186865, GSE221789, and GSE237067) were collected, and these data were re-integrated for meta-analysis. The results of the meta-analysis identified 13 large cell lineages, including macrophages (MACs), T cells, B cells, neutrophils, large cells, fibroblasts, smooth muscle cells (SMCs), endothelial cells (ECs), etc. ( Figure 1 ). In the aneurysm tissue, the proportion of macrophages increased significantly, indicating the important role of inflammation in aneurysm.

[0033] Meanwhile, by analyzing the expression of key genes in the PIEZO-KCNN4-NLRP3 signaling axis in different cell lineages, it was found that the expressions of PIEZO1, KCNN4, NLRP3, and IL-1β were significantly up-regulated in macrophages in abdominal aortic aneurysm ( Figure 2 ), demonstrating the crucial role of this signaling axis in the development of abdominal aortic aneurysm.

[0034] 2. Knockout or inhibition of KCNN4 can reduce the activation of NLRP3 in macrophages.

[0035] The THP1 cell line, a commonly used human monocyte cell line, can well simulate the immune response of human mononuclear macrophages in vitro.

[0036] The preparation method of the KCNN4 knockout cell line is as follows: Two specific sgRNAs were designed for KCNN4, specifically as follows: sgRNA-1: 5’-TTCGGCGTCTCAAGGCCCCCA-3’ (SEQ ID NO.2); sgRNA-2: 5’-CAGAGATGCTGTGGTTCGGG-3’ (SEQ ID NO.3); Its sequence was selected from the early exon region of the gene through NCBI database analysis to improve the knockout efficiency. After the sgRNA oligonucleotides were annealed to form double-stranded DNA, they were cloned into the pX330-P2A-EGFP or pX330-P2A-RFP vector (the vector was publicly available from the literature: Ran L, Ye T, Erbs E, Ehl S, Spassky N, Sumara I, Zhang Z, Ricci R. KCNN4 links PIEZO-dependent mechanotransduction to NLRP3 inflammasome activation. Sci Immunol. 2023 Dec 22;8(90):eadf4699. doi:10.1126 / sciimmunol.adf4699.) by T4 ligase respectively. This vector system integrated the Cas9 protein expression unit and the fluorescent reporter gene, facilitating subsequent screening. Two sgRNA expression plasmids (0.5 μg each) were co-transfected into the THP-1 cell line using the X-tremeGENE 9 transfection reagent. After 24 hours of transfection, the double-positive cell population of green fluorescent protein (GFP) and red fluorescent protein (RFP) was sorted by BD FACSAria III flow cytometer. This strategy could ensure the efficient enrichment of cells expressing both sgRNAs. The sorted cells were seeded into 96-well plates by gradient dilution to obtain single-cell clones through the limiting dilution method. The levels of IL-1β in the culture supernatants of wild-type (WT) and KCNN4 knockout (KO) THP-1 cells were detected by ELISA. The experimental procedure was as follows: After pre-stimulating with 1 μg / ml LPS for 3 hours, the cells were treated with 100 μM R837 for 1 hour in the presence or absence of 25 μM Yoda1. The results are shown in Figure 3 , #1, #2, and #3 are three different KCNN4 knockout THP1 monoclonal clones. This example found that knocking out KCNN4 in the THP1 monocyte-macrophage cell line by CRISPR-Cas9 technology could significantly reduce the activation of the inflammasome and the release of downstream IL1b ( Figure 3 , ** for P < 0.01).

[0037] Similarly, the use of the KCNN4 inhibitor TRAM34 could also reduce the release of macrophage IL1b ( Figure 4 , * for P < 0.05, ** for P < 0.01, *** for P < 0.001). Figure 4The experimental procedure was as follows: The level of IL-1β in the culture supernatant of THP-1 cells pre-stimulated with LPS was detected using ELISA. The experimental treatments were as follows: Treatment was carried out using 100 µM R837, 25 µM Yoda1, or a combination of 100 µM R837 and 25 µM Yoda1, and at the same time, TRAM34, 10 µM MCC950, or extracellular KCl was added and continued for 1 hour. This indicates that KCNN4 is indeed involved in the activation of the macrophage inflammasome and the release of IL1b, and thus participates in the occurrence of abdominal aortic aneurysm through the inflammatory effect of IL1b, and is therefore a new target for the treatment of abdominal aortic aneurysm.

[0038] Example 2 Mouse Model Experiment In this mouse model, hyperlipidemia (by AAV-PCSK9 and high-fat feeding) combined with angiotensin II (AngII) can induce abdominal aortic aneurysm in mice, and a mouse AAA model was constructed. The construction process is shown in Figure 5 . This model refers to the method described in the literature [Sawada H, Daugherty A, Lu HS. Expression of a PCSK9 Gain-of-Function Mutation in C57BL / 6J Mice to Facilitate Angiotensin II-Induced AAAs. Biomolecules. 2022 Jun 29;12(7):915. doi: 10.3390 / biom12070915.]. By injecting an adeno-associated virus (AAV) encoding the PCSK9 gene (rAAV-D377Y-mPCSK9, purchased from Vigene Biosciences) and at the same time giving a high-fat diet containing 60% cholesterol (Synerbio, XT108C), hypercholesterolemia in mice was induced. On this basis, angiotensin II (AngII) was slowly released through an osmotic pump to induce hypertension in mice. This model can induce abdominal aortic aneurysm in mice and is therefore commonly used in the mechanism research and drug development of abdominal aortic aneurysm.

[0039] The mice were divided into 3 groups, namely the normal saline group (control group, Control), the TRAM34 group (Tram34), and the Senicapoc group. At the same time as the angiotensin II slow-release pump was implanted, the control group of mice began to receive intraperitoneal injection of normal saline, the TRAM34 group of mice began to receive intraperitoneal injection of TRAM34 (15 mg / kg / d), and the Senicapoc group of mice began to receive intraperitoneal injection of Senicapoc (15 mg / kg / d). After 4 weeks of angiotensin II treatment, the mice were euthanized and dissected, and the maximum diameter of the abdominal aorta and the incidence of aneurysm in the mice were measured.

[0040] The results are as follows: 1. Both Tram34 and Senicapoc can effectively reduce the rupture of AAA and decrease the mortality rate of AAA in mice.

[0041] The results are shown in Figure 6 , and the results show that, compared with the control group, intraperitoneal injection once a day in the TRAM34 group or the Senicapoc group can effectively reduce the mortality rate of mice in the AAA model. In the control group, 67.7% of the mice died of abdominal aortic rupture during the experiment, while the mortality rates in the Tram34 group and the Senicapoc group decreased to 27.7% and 30% respectively. This indicates that the KCNN4 inhibitor Tram34 or Senicapoc can effectively reduce the rupture of AAA and decrease the mortality rate of AAA in mice.

[0042] 2. Both Tram34 and Senicapoc can reduce the incidence rate of AAA.

[0043] See Figure 7 and Figure 8 , and the results show that, compared with the control group, intraperitoneal injection once a day of Tram34 or Senicapoc can effectively reduce the incidence rate of AAA in mice. Injecting Tram34 or Senicapoc can decrease the incidence rate of AAA in mice from 80% to 53.3% and 40%. Both Tram34 and Senicapoc can reduce the maximum diameter of the abdominal aorta, indicating that they can protect the aortic structure and prevent the formation of aneurysms. This further shows that these two drugs can significantly protect the aorta and prevent the destruction of the aortic structure caused by hyperlipidemia and hypertension.

[0044] 3. Tram34 or Senicapoc has no obvious hepatotoxicity.

[0045] See Figures 9 - 11 , where ns indicates that the results have no significant difference. After injecting Tram34 or Senicapoc for 4 weeks, the serum of mice was collected to detect alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in it, and no significant difference was found compared with the control group. At the same time, there was no obvious difference in the body weights of the three groups of mice. This result proves the safety of Tram34 or Senicapoc.

[0046] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments based on these embodiments without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Use of a potassium-calcium activated channel subfamily N member 4 inhibitor in the preparation of a drug for preventing and treating abdominal aortic aneurysm and / or delaying the progression of abdominal aortic aneurysm.

2. The use according to claim 1, characterized in that: The potassium-calcium activated channel subfamily N member 4 inhibitors include TRAM34 and / or Senicapoc; The chemical structure of the TRAM34 is shown in Formula I; Formula I; The chemical structural formula of the Senicapoc is shown in Formula II; Formula II.

3. The use according to claim 1, characterized in that: The potassium-calcium activated channel subfamily N member 4 inhibitor is the only active ingredient of the drug.

4. Use of reagents for knocking out or knocking down potassium-calcium activated channel subfamily N member 4 in macrophages in the preparation of drugs for preventing, treating or delaying the progression of abdominal aortic aneurysm.

5. The use according to any one of claims 1 to 4, characterized in that: The dosage form of the drug includes injection.

6. The use according to any one of claims 1 to 4, characterized in that: The abdominal aortic aneurysm was induced by hyperlipidemia and hypertension.

7. The use according to any one of claims 1 to 4, characterized in that: The prevention, treatment or delay of the progression of abdominal aortic aneurysm is achieved by inhibiting the activation of macrophage inflammasomes and / or reducing the release of macrophage IL1b.

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