Application of TTK gene and inhibitor of TTK in treating atherosclerosis
By studying TTK genes and inhibitors, they discovered their role in atherosclerosis, constructed relevant models and designed drugs, solved the problem of lack of effective targets in the existing technology, achieved significant reduction of plaques and provided new therapeutic strategies.
Patent Information
- Application Number
- CN202310712852.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-06-16
- Publication Date
- 2025-07-29
- Estimated Expiration
- 2043-06-16
AI Technical Summary
There is a lack of effective targets in the prior art to prevent and treat atherosclerosis. Although lipid-lowering treatment is effective, plaques are still progressing and new treatment strategies are urgently needed.
By studying the effects of TTK genes and TTK inhibitors, it was found that TTK knockout and inhibitors can significantly reduce atherosclerotic plaques, construct in vitro cell models and animal models, screen TTK as targets to design drugs, and use TTK inhibitors such as siRNA to interfere with TTK expression or design small molecule compounds to interfere with their function.
TTK knockout and inhibitors significantly reduce atherosclerotic plaques, providing new targets and strategies to provide theoretical basis and clinical basis for the preparation of drugs for preventing, relieving and treating atherosclerosis.
Smart Images
Figure CN116785436B_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of medicine and relates to the use of the TTK gene in treating atherosclerosis, specifically the use of the TTK gene as a drug target in screening drugs for preventing, alleviating and / or treating atherosclerosis, and the use of TTK inhibitors in preparing drugs for preventing, alleviating and / or treating atherosclerosis. Background Art
[0002] Cardiovascular and cerebrovascular diseases (CVDs) are systemic or systemic vascular disorders caused by numerous factors, including atherosclerosis and hypertension, and are associated with high morbidity and mortality. Atherosclerosis, the primary pathological basis of CVDs, is primarily caused by lipid deposition within the arterial wall, which forms atherosclerotic plaques that continuously expand, thickening and hardening the arterial wall and narrowing the lumen. Rupture of unstable atherosclerotic plaques, platelet aggregation, and thrombosis can lead to acute, fatal CVD events such as myocardial infarction and stroke.
[0003] Atherosclerotic plaque formation is a complex, chronic process. Various harmful factors, including ox-LDL, damage the vascular intima. Monocytes infiltrate the subintima and transform into macrophages. Both macrophages and smooth muscle cells in the vascular media participate in lipid phagocytosis, accumulating large amounts of lipid within the cells to form foam cells, which form the lipid core of the plaque. Foam cell accumulation leads to an enlarged lipid core and progression of atherosclerotic plaques. Lipid-lowering therapy is currently the mainstay of treatment. However, real-world studies have found that even among patients with targeted lipid control, 47.5% of carotid artery plaques continue to progress. Therefore, more effective therapeutic targets are urgently needed.
[0004] The TTK gene, located on chromosome 6q13-q21, encodes a serine / threonine and tyrosine protein kinase (TTK), also known as monopolar spindle 1 (Mps1). It is currently recognized as a dual-specificity kinase. TTK is a key component of the spindle assembly checkpoint (SAC). Elevated TTK levels have been found to be closely associated with tumorigenesis and poor prognosis, making TTK an important candidate target for anticancer therapy. However, the role of TTK in atherosclerosis has not been reported. Summary of the invention
[0005] To solve the defects and deficiencies of the above-mentioned prior art, the object of the present invention is to determine the relationship between the TTK gene and its inhibitors and atherosclerosis, and to provide a new use of the TTK gene and its inhibitors in the prevention, alleviation and / or treatment of atherosclerosis.
[0006] To achieve the above object, the present invention provides the following technical solutions;
[0007] The present invention first determines the relationship between the TTK gene and atherosclerosis, as follows: The present invention uses APOE - / - TTK WT gene knockout mice and APOE - / - TTK KO double gene knockout mice as experimental subjects, and induces an atherosclerotic mouse model through a high-fat diet. By studying the aortic tree and the plaque area at the aortic root, the results show that the knockout of the TTK gene can significantly reduce the area of the aortic tree and the plaque area at the aortic root.
[0008] From the above results, it can be seen that when atherosclerosis occurs, the knockout of the TTK gene significantly reduces the atherosclerotic plaque area. Therefore, TTK has the effect of promoting the occurrence and development of atherosclerosis, providing a theoretical basis and clinical foundation for the research of new targets and new strategies for the prevention and treatment of atherosclerosis.
[0009] The present invention also studies the relationship between TTK inhibitors and atherosclerosis, as follows: The present invention also uses APOE - / - gene knockout mice as experimental subjects, induces an atherosclerotic mouse model through a high-fat diet, and after intervening with a control vector and a TTK inhibitor respectively, studies the plaque area of the aortic tree. The results show that the TTK inhibitor can significantly reduce the plaque area of the aortic tree.
[0010] From the above results, it can be seen that when atherosclerosis occurs, the TTK inhibitor significantly reduces the atherosclerotic plaque area and alleviates atherosclerosis. Therefore, the TTK inhibitor has the effect of preventing, alleviating and / or treating atherosclerosis, providing a theoretical basis and clinical foundation for the research of preparing drugs for preventing, alleviating and / or treating atherosclerosis.
[0011] Therefore, constructing an in vitro cell model or animal model with overexpression of the TTK gene can be used to screen drugs for preventing, alleviating, and / or treating atherosclerosis; the TTK gene can also be used as a target gene in gene therapy to design and prepare drugs and / or biological reagents for preventing, alleviating, and / or treating atherosclerosis, and achieve the purpose of preventing, alleviating, and / or treating atherosclerosis through genetic engineering techniques. For example, using TTK as a target gene, designing double-stranded siRNA that can interfere with TTK expression, and after chemical synthesis, injecting it into the human body to silence the TTK gene through RNA interference to treat atherosclerosis; in addition, small molecule compound inhibitors can also be designed with TTK as the target, and molecules that can specifically inhibit TTK can be found through screening, thereby providing new therapeutic molecules for the treatment of atherosclerosis.
[0012] In view of the above functions of TTK, the present invention provides an application of using the TTK gene as a drug target in screening drugs for preventing, alleviating, and / or treating atherosclerosis.
[0013] Furthermore, the drug for preventing, alleviating, and / or treating atherosclerosis is a drug that inhibits the expression of the TTK gene.
[0014] A preferred technical solution of the present invention: The drug for preventing, alleviating, and / or treating atherosclerosis includes an inhibitor of TTK.
[0015] A preferred technical solution of the present invention: The inhibitor of TTK is one of siRNA of the TTK gene, an RNA interference vector of the TTK gene, or other inhibitors that can inhibit the expression or activity of the TTK gene.
[0016] In view of the above functions of the inhibitor of TTK, the present invention also provides an application of an inhibitor of TTK in preparing a drug for preventing, alleviating, and / or treating atherosclerosis.
[0017] A preferred technical solution of the present invention: The inhibitor of TTK is directly used as a drug for preventing, alleviating, and / or treating atherosclerosis.
[0018] A preferred technical solution of the present invention: The inhibitor of TTK is one of siRNA of the TTK gene, an RNA interference vector of the TTK gene, or other inhibitors that can inhibit the expression or activity of the TTK gene.
[0019] The present invention has the following advantages and effects compared with the prior art:
[0020] (1) The present invention discovers a new function of the TTK gene, that is, the TTK gene has the effect of promoting atherosclerosis.
[0021] (2) Based on the function of TTK in promoting atherosclerosis, it provides a target for the development of drugs for preventing, alleviating, and / or treating atherosclerosis.
[0022] (3) The present invention has found that an inhibitor of TTK can be used to prepare a drug for preventing, alleviating, and / or treating atherosclerosis. Brief Description of the Drawings
[0023] Figure 1 It is the Oil Red O staining map of the aortic tree of APOE - / - TTK WT mice and APOE - / - TTK KO mice in Example 1;
[0024] Figure 2 It is the Oil Red O staining map of the aortic tree of APOE - / - TTK WT mice and APOE - / - TTK KO mice in Example 1;
[0025] Figure 3 It is the HE and Oil Red O staining map of the aortic root of APOE - / - TTK WT mice and APOE - / - TTK KO mice in Example 2;
[0026] Figure 4 It is the HE and Oil Red O staining map of the aortic root of APOE - / - TTK WT mice and APOE - / - TTK KO mice in Example 2;
[0027] Figure 5 It is the statistical chart of blood lipid detection of APOE - / - TTK WT mice and APOE - / - TTK KO mice in Example 3; Among them, Figures A, B, C, and D are the content comparison charts of total cholesterol, triglycerides, low-density lipoprotein cholesterol (LDL-c), and high-density lipoprotein cholesterol (HDL-c), respectively;
[0028] Figure 6 It is the Oil Red O staining map of the aortic tree of mice treated with control vector and BAY 1217389 respectively in Example 4;
[0029] Figure 7The aortic tree of mice treated with the control vector and BAY 1217389 respectively in Example 4
[0030] Statistical chart of plaque area;
[0031] Figure 8 It is a statistical chart of blood lipid detection of mice treated with the control vector and BAY 1217389 respectively in Example 5, where Figures A, B, C, and D respectively represent the content comparison charts of total cholesterol, triglycerides, low-density lipoprotein cholesterol (LDL-c), and high-density lipoprotein cholesterol (HDL-c). Detailed implementation manners
[0032] The technical solutions of the present invention will be further described in detail below in conjunction with specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but do not limit the present invention in any form. It should be noted that those of ordinary skill in the art can make several deformations and improvements without departing from the concept of the present invention. These all belong to the protection scope of the present invention.
[0033] The terms used in the following embodiments, unless otherwise specified, have the same meanings as those commonly understood by those skilled in the art. The experimental methods without specifying specific conditions are usually completed according to the conventional protocols in the art or the protocols recommended by the manufacturers. The reagents and materials without special instructions can be purchased on the market.
[0034] Experimental animals and breeding in the following embodiments: APOE - / - Mice were purchased from Jackson lab; TTK f / f Mice were purchased from Jiangsu Gather Biopharm Co., Ltd.; Ubc CreERT2 Mice were purchased from Jackson lab; Inducible systemic knockout mice of TTK (TTK f / f ; Ubc CreERT2 Abbreviated as TTK KO ) were obtained by crossing TTK f / f mice and Ubc CreERT2 mice; APOE - / - TTK WT mice were obtained by crossing APOE - / - mice and TTK f / f mice; APOE - / - TTK KO double knockout mice were obtained by crossing APOE - / - mice and TTK KO mice.
[0035] Experimental animal feed formula: High-fat diet (HFD, purchased from Beijing Huafukang Biotechnology Co., Ltd., formulated according to the AIN-76A Western diets formula, calorie percentage: protein 15.8%, fat 40%, carbohydrate 44.2%).
[0036] Administration of animal jelly: Stir Splenda powder evenly after adding it to double-distilled water to prepare a 20%
[0037] (wt / vol) Splenda solution; Add gelatin to the prepared Splenda solution to prepare a 14% (wt / vol) gelatin stock solution, stir and heat to 55 - 60 °C until the solution becomes clear. Add the calculated dose of the drug solution (1 mg / kg) and the gelatin stock solution to each well in the jelly mold, with a total volume of 200 μl, mix well, and place it at -20 °C for about 3 hours to solidify into jelly. Administer the control jelly and the TTK inhibitor drug jelly to the mice on the 1st and 2nd days of each week respectively, keeping the mice as undisturbed as possible, and wait for the mice to finish eating the jelly, twice a day; Adopt the intermittent administration method, administer the drug for two days and then rest for five days each week, for a total of four weeks.
[0038] Animal rearing conditions: All experimental mice were reared in the SPF-class animal house of Tongji Medical College, Huazhong University of Science and Technology. The lighting was alternated every 12 hours, the temperature was 24 ± 2 °C, the humidity was 40% - 70%, and the mice had free access to water and food.
[0039] Oil red O staining solution: Dissolve 0.5 g of oil red O in 100 mL of 100% isopropanol to prepare an oil red O stock solution and store it in a 4 °C refrigerator. Before use, dilute the stock solution and distilled water in a ratio of 3:2 to prepare an oil red O working solution and filter it. The oil red O working solution normally appears wine-red and has no precipitation.
[0040] Obtaining a mouse atherosclerosis model (AS):
[0041] Grouping of experimental animals: Select 8-week-old, male mice weighing 19 - 25 g, APOE - / - TTK WT mice and APOE - / - TTK KO mice, fed with a high-fat diet (HFD), APOE - / - TTK WT mouse group, APOE - / - TTK KO There are two groups of mice in total.
[0042] Example 1:
[0043] For 8-week-old, male APOE mice weighing 19 - 25 g - / - TTK WTMice and APOE - / - TTK KO Mice were fed a high-fat diet (HFD) for 16 weeks, euthanized, and specimens were collected. The aorta was removed from the root to the bifurcation of the common iliac artery. The adventitia and fat were separated, fixed with 4% paraformaldehyde overnight, and then stained with Oil Red O. The staining results are as Figure 1 shown Figure 1 in the Oil Red O staining of the middle aortic tree. The aortic arch is the most obvious site of atherosclerotic plaques; the plaque areas in the aortic tree regions of the two groups of mice were statistically analyzed, and the statistical results are as Figure 2 shown. It can be seen from Figure 1 and Figure 2 that in the atherosclerotic model with TTK deficiency (APOE - / - TTK KO ), the plaque area in the aortic tree region of mice was significantly reduced compared with that of the control (APOE - / - TTK WT ) mice.
[0044] Example 2
[0045] Male APOE - / - TTK WT mice and APOE - / - TTK KO mice at 8 weeks of age and weighing 19 - 25 g were fed a high-fat diet (HFD) for 16 weeks, euthanized, and specimens were collected. Their heart tissues were fixed with 4% paraformaldehyde overnight and embedded with OCT. 10-μm frozen sections were made from the aortic root, and then stained with Oil Red O and Oil Red-Hematoxylin (HE). The staining results are as Figure 3 shown, and the plaque areas at the aortic root of the two groups of mice were statistically analyzed, and the statistical results are as Figure 4 shown. It can be seen from Figure 3 and Figure 4 that in the atherosclerotic model with TTK deficiency (APOE - / - TTK KO ), the plaque area at the aortic root of mice was significantly reduced compared with that of the control (APOE - / - TTK WT ) mice.
[0046] Example 3
[0047] Male APOE - / - TTK WT mice and APOE - / - TTK KOMice were fed a high-fat diet (HFD) for 16 weeks and then euthanized, and blood samples were collected (fasting from water and food for 12 h before blood collection). After standing at room temperature for 30 min, the samples were centrifuged at 3000 rpm / min for 15 min, and the upper-layer serum was collected. Total cholesterol assay kit (Nanjing Jiancheng Bioengineering Institute, A111-1-1), triglyceride assay kit (Nanjing Jiancheng Bioengineering Institute, A110-1-1), low-density lipoprotein cholesterol assay kit (Nanjing Jiancheng Bioengineering Institute, A113-1-1), and high-density lipoprotein cholesterol assay kit (Nanjing Jiancheng Bioengineering Institute, A112-1-1) were used to measure the contents of total cholesterol (Total cholesterol), triglycerides (Tirglycerides), low-density lipoprotein cholesterol (LDL-c), and high-density lipoprotein cholesterol (HDL-c) in the sera of the mice in each group according to the instructions through a microplate spectrophotometer, and the data were statistically analyzed. The results are as Figure 5 shown, Figure 5 where A, B, C, and D in Figure 5 are the comparative graphs of the contents of total cholesterol (Total cholesterol), triglycerides (Tirglycerides), low-density lipoprotein cholesterol (LDL-c), and high-density lipoprotein cholesterol (HDL-c) respectively. It can be seen from - / - that there is no significant difference in blood lipids between the atherosclerotic model (APOE KO ) mice lacking TTK and the control (APOE - / - ) mice. WT
[0048] Example 4
[0049] Male APOE - / - mice at 8 weeks of age and weighing 19 - 25 g were fed a high-fat diet (HFD) for 16 weeks to form atherosclerotic plaques, and then randomly divided into 2 groups and continued to be fed a high-fat diet, namely the control vehicle group (Vehicle) and the TTK inhibitor group (the inhibitor used was BAY 1217389). After 4 weeks, the mice were euthanized and specimens were collected. The aorta was removed from the root to the bifurcation of the common iliac artery, the adventitia and fat were separated, fixed with 4% paraformaldehyde overnight, and then stained with Oil Red O. The staining results are as Figure 6 shown; and the plaque areas in the aortic tree region of the 2 groups of mice were statistically analyzed, and the statistical results are as Figure 7 shown. It can be seen from Figure 6 and Figure 7 that the atherosclerotic model mice in the TTK inhibitor group (BAY 1217389) had significantly reduced plaque areas in the aortic tree region compared with the mice in the vehicle group (Vehicle).
[0050] Example 5
[0051] 8-week-old male APOE - / - Mice were fed a high-fat diet (HFD) for 16 weeks. After atherosclerotic plaques formed, they were randomly divided into two groups and continued to be fed a high-fat diet, namely the control vehicle group (Vehicle) and the TTK inhibitor group (BAY 1217389 was selected as the inhibitor). After 4 weeks, the mice were euthanized and blood samples were collected from the mice (no water or food was allowed for 12 hours before blood collection). After standing at room temperature for 30 minutes, the mice were centrifuged at 3000 rpm / min for 15 minutes, and the upper serum was collected. The above-mentioned kit was used according to the instructions to measure the total cholesterol (Total cholesterol), triglycerides (Tirglycerides), low-density lipoprotein cholesterol (LDL-c) and high-density lipoprotein cholesterol (HDL-c) in the serum of the mice in the above-mentioned groups using a microplate spectrophotometer, and the data were statistically analyzed. Results Figure 8 As shown, Figure 8 A, B, C, and D are the comparison results of total cholesterol (Totalcholesterol), triglycerides (Tirglycerides), low-density lipoprotein cholesterol (LDL-c) and high-density lipoprotein cholesterol (HDL-c). Figure 8 It can be seen that there is no significant difference in blood lipids between the atherosclerosis model mice in the TTK inhibitor group (BAY 1217389) and the vehicle group (Vehicle) mice.
[0052] The results of the above examples demonstrate that TTK can significantly promote the formation of aortic plaques and the development of atherosclerosis. Knocking out the TTK gene or using its inhibitors can reduce the severity of atherosclerotic lesions and, therefore, can be used to prepare drugs for preventing, alleviating, and / or treating atherosclerosis.
[0053] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. It will be clear to those skilled in the art that various modifications to the above embodiments can be made without departing from the overall spirit and concept of the present invention, including changes, modifications, substitutions, combinations, simplifications, etc., all of which should be equivalent replacement methods and are included in the scope of protection of the present invention. The protection scheme of the present invention shall be subject to the claims attached to the present invention.
Claims
1. Use of an inhibitor of TTK in the preparation of a medicament for preventing, alleviating, and / or treating atherosclerosis, wherein the inhibitor of TTK is BAY 1217389.
2. Use of a TTK inhibitor according to claim 1 in the preparation of a medicament for preventing, alleviating and / or treating atherosclerosis, characterized in that: The inhibitor of TTK is directly used as a medicament for preventing, alleviating, and / or treating atherosclerosis.
Citation Information
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