Application of reagent for inhibiting expression of CCDC92 in preparation of medicine for treating hyperlipidemia and atherosclerosis
By inhibiting reagents expressed in CCDC92, preparing drugs used to treat hyperlipidemia and atherosclerosis, solve the problems of adverse reactions and lack of LDL-C levels in existing technologies, and realize the reduction of lipid drip formation and activity The effect of oxygen and apoptosis provides new ideas for the treatment of hyperlipidemia and atherosclerosis.
Patent Information
- Application Number
- CN202411324281.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-23
- Publication Date
- 2025-01-28
AI Technical Summary
The prior art has shortcomings in reducing plasma LDL-C levels, especially the adverse reactions of statins affect patient compliance and treatment persistence, making it difficult to reduce LDL-C levels below the target value.
By inhibiting reagents expressing CCDC92, drugs used to treat hypertrophic and atherosclerotic drugs, inhibit the formation of intracellular droplets, activate the signaling of adenolate activated protein kinase, and increase the expression of carnitine palmicolylase 1. Reduce active oxygen generating and mitochondrial film damage to inhibit cell apoptosis.
Effectively suppress the mechanism of CCDC92 in hyperlipidemia and atherosclerosis, reduce the formation of intracellular lipid dripping, activated oxygen production and apoptosis, and provide a new effective way to treat hyperlipidemia and atherosclerosis Essence
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Figure CN119367541A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biomedical technology, and in particular to application of an agent for inhibiting CCDC92 expression in preparing a medicine for treating hyperlipidemia and atherosclerosis. Background Art
[0002] Cardiovascular disease is one of the leading causes of morbidity and mortality worldwide. Atherosclerosis (As) and the resulting atherosclerotic cardiovascular disease (ASCVD) are the leading causes of human health and mortality. Atherosclerosis is a lipid-driven chronic inflammatory disease that usually forms plaques in large and medium-sized arteries and is the main cause of ischemic heart disease and stroke. Decades of in-depth research have shown that the pathogenesis of atherosclerosis is complex, and its main components are lipid accumulation and chronic inflammation of the arterial wall. Atherosclerosis is usually associated with changes in lipid metabolism and hypercholesterolemia.
[0003] Dyslipidemia, especially elevated low-density lipoprotein cholesterol (LDL-C) levels, is considered a pathogenic risk factor for ASCVD. In recent decades, humans have conducted extensive and in-depth research on the relationship and mechanism between dyslipidemia and the occurrence and development of atherosclerosis, and have made great progress. The earliest case study of hyperlipidemia dates back to the early 19th century, when American scientist Ancel Keys initiated an epidemiological study on heart attacks, and the results showed that increased serum total cholesterol (TC) was positively correlated with the occurrence of atherosclerosis. Since then, scientists have conducted in-depth research on the pathogenesis of hyperlipidemia and lipid-lowering drugs. Lipid-lowering drugs are drugs that reduce plasma triglycerides (TG) or TC levels by preventing bile acid or cholesterol from being absorbed from the intestine, promoting bile acid or cholesterol to be excreted with feces, activating lipoprotein metabolism and promoting the hydrolysis of TG. The drugs commonly used to treat hypercholesterolemia are statins, which can have a good inhibitory effect on 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme in the cholesterol biosynthesis pathway. Although statins are widely used to reduce cardiovascular events and are generally safe and effective, they still have some adverse reactions, such as myopathy or myalgia, asymptomatic increase in liver transaminases, and hepatotoxicity. Although these adverse reactions are rarely serious or life-threatening, they do reduce patient compliance or persistence of treatment, thereby undermining the expected therapeutic benefit. Therefore, statins alone are often not enough to reduce plasma LDL-C levels to below the target value.
[0004] Therefore, the prior art still needs to be improved and developed. Summary of the invention
[0005] In view of the above-mentioned deficiencies in the prior art, the purpose of the present invention is to provide an agent for inhibiting the expression of CCDC92 for use in the preparation of a drug for treating hyperlipidemia and atherosclerosis, aiming to use CCDC92 as a therapeutic target for hyperlipidemia and atherosclerosis, and to provide a new and effective approach for the clinical treatment of hyperlipidemia and atherosclerosis.
[0006] The technical solution of the present invention is as follows:
[0007] The present invention provides application of a reagent for inhibiting CCDC92 expression in preparing a medicine for treating hyperlipidemia and atherosclerosis.
[0008] In a preferred technical solution, the drug inhibits the formation of intracellular lipid droplets.
[0009] In a preferred technical solution, the drug inhibits lipid droplet formation by activating the adenylate-activated protein kinase signaling pathway.
[0010] In a preferred technical solution, the drug inhibits lipid droplet formation by increasing the expression of carnitine palmitoyltransferase 1.
[0011] In a preferred technical solution, the drug inhibits lipid droplet formation by increasing the expression of carnitine palmitoyltransferase 1A.
[0012] In a preferred technical solution, the drug reduces the generation of reactive oxygen species in cells.
[0013] In a preferred technical solution, the drug reduces the damage of mitochondrial membrane potential to cells.
[0014] In a preferred technical solution, the drug reduces cell apoptosis.
[0015] Beneficial effects: The present invention is the first to discover that CCDC92 is related to cardiovascular diseases, and experimentally verifies the mechanism of action of CCDC92 in the occurrence and development of hyperlipidemia and atherosclerosis, providing a new and effective approach for the clinical treatment of hyperlipidemia and atherosclerosis. BRIEF DESCRIPTION OF THE DRAWINGS
[0016] Figure 1 is a graph of the positive correlation between coronary artery disease risk alleles and the disease or trait.
[0017] Figure 2 It is a diagram of the expression level of CCDC92 in various tissues and organs of humans and mice; among them, A is a schematic diagram of the expression of CCDC92 in various parts of the human body, B is a statistical diagram of the RNA expression of CCDC92 in various tissues and organs of humans, C is a WB detection result diagram of the protein expression of CCDC92 in various tissues and organs of mice, and D is a comparison diagram of the WB detection results of CCDC92 expression in the liver tissues of wild-type mice and atherosclerotic mice.
[0018] Figure 3 These are the relevant experimental result graphs after knocking down CCDC92 in the HepG2 cell line using siRNA in Example 1; wherein A is a WB detection result graph, B is an RNA detection result graph, C is a cell lipid droplet induced oil red staining result graph before knocking down CCDC92, and D is a cell lipid droplet induced oil red staining result graph after knocking down CCDC92.
[0019] Figure 4 These are the relevant experimental result graphs after knocking down CCDC92 in the AML-12 cell line using siRNA in Example 1; wherein A is a WB detection result graph, B is an RNA detection result graph, C is a cell lipid droplet induced oil red staining result graph before knocking down CCDC92, and D is a cell lipid droplet induced oil red staining result graph after knocking down CCDC92.
[0020] Figure 5 It is a graph of the experimental results of obtaining a stable knockdown cell line of CCDC92 using Crispr / Cas9 technology in Example 1; wherein A is the sgRNA sequence, B is a gene knockout flow chart, C is a graph of the monoallelic knockout sequencing results, and D is a graph of the WB detection results of the cell clone knockout situation.
[0021] Figure 6 It is a diagram of cell lipid droplet staining and paper detection results of the CCDC92 stable knockdown cell line in Example 1; wherein A is a diagram of cell lipid droplet induced oil red staining and fluorescent staining results, and B is a diagram of cell LDL-C and TC detection results.
[0022] Figure 7 It is the result graph of intracellular ROS detection, cell mitochondrial membrane potential detection, flow cytometric analysis and cell apoptosis percentage of the CCDC92 stable knockdown cell line in Example 1; wherein A is a graph of intracellular ROS detection results, B is a graph of cell mitochondrial membrane potential detection results, C is a graph of flow cytometric analysis results, and D is a graph of cell apoptosis percentage calculated by flow cytometric analysis results.
[0023] Figure 8 This is a graph of differential gene enrichment analysis of transcriptome sequencing of the CCDC92 stable knockdown cell line in Example 1.
[0024] Fig. 9 This is a graph showing the WB detection results of the APMK signaling pathway protein in the CCDC92 stable knockdown cell line in Example 1. DETAILED DESCRIPTION
[0025] The present invention provides an application of an agent for inhibiting CCDC92 expression in the preparation of a drug for treating hyperlipidemia and atherosclerosis. To make the purpose, technical scheme and effect of the present invention clearer and more specific, the present invention is further described in detail below.
[0026] CCDC (coiled-coil domain-containing) proteins basically contain a coiled-coil domain, which is a highly conserved supercoiled protein motif with one or more α-helical peptides that are intertwined in a supercoiled manner. CCDC proteins are expressed in a wide range of tissues, and their cellular localization is diverse, thus exhibiting functional roles in most physiological processes. At the cellular level, they are involved in chromosome segregation, centriole structure, DNA recognition and cleavage, protein scaffolds, mediating vesicle tethering and separating functional domains in proteins (by acting as molecular spacers), etc. CCDC proteins are involved in various processes from the organismal to the molecular level. They are involved in regulating the expression and function of a variety of proteins, such as DNA integrity, cell proliferation, embryonic development, immune response, and hematopoiesis. In addition, these proteins have been shown to be associated with migration, proliferation, and metastasis of different cancers. The study of CCDC proteins continues to be an active research field due to their indispensable functions.
[0027] CCDC92 was found to be a previously unknown protein that affects adipocyte differentiation. However, whether it is related to blood lipids and its related molecular mechanisms have not been systematically studied so far.
[0028] Based on this, an embodiment of the present invention provides the use of an agent for inhibiting the expression of CCDC92 in the preparation of a drug for treating hyperlipidemia and atherosclerosis.
[0029] This example first discovered that CCDC92 may be related to cardiovascular disease through big data mining; then used WB technology to detect the expression of CCDC92 in the livers of wild-type mice and atherosclerotic mice, and found that the expression of CCDC92 in the livers of atherosclerotic mice was significantly increased; further knocked down the CCDC92 gene in mouse liver cells through CRISPR / Cas9 technology, detected various indicators such as lipid droplet formation, and analyzed the potential functions of CCDC92; finally, transcriptomics sequencing was used to detect related signal pathways. This example experimentally verified the mechanism of action of CCDC92 in the occurrence and development of hyperlipidemia and atherosclerosis, and provided a new and effective way for the clinical treatment of hyperlipidemia and atherosclerosis.
[0030] In one embodiment, the drug inhibits the formation of intracellular lipid droplets.
[0031] In one embodiment, the drug inhibits lipid droplet formation by activating the AMP-activated protein kinase signaling pathway.
[0032] In one embodiment, the medicament inhibits lipid droplet formation by increasing the expression of carnitine palmitoyltransferase 1.
[0033] In one embodiment, the medicament inhibits lipid droplet formation by increasing the expression of carnitine palmitoyltransferase 1A.
[0034] In one embodiment, the medicament reduces the generation of reactive oxygen species within a cell.
[0035] In one embodiment, the drug reduces damage to the mitochondrial membrane potential of the cell.
[0036] In one embodiment, the medicament reduces apoptosis.
[0037] The present invention will be further described below by means of specific examples.
[0038] Example 1
[0039] (1) Discovery of the relationship between CCDC92 and atherosclerosis
[0040] This example first investigates the relationship between coronary artery disease (CAD) risk alleles and diseases or traits through literature, and finds that CCDC92 may be related to cardiovascular disease. The results are as follows Figure 1 However, there is no report on the mechanism of action of CCDC92 and cardiovascular disease. Then, through website analysis, it was found that CCDC92 is expressed in various organs of the body. The results are as follows Figure 2 As shown in A and B. Further, proteins from various tissues and organs of mice were extracted for WB detection, and the results are shown in Figure 2 As shown in Figure C, CCDC92 is expressed in all tissues and organs of mice. Furthermore, liver tissues of wild-type mice and APOE knockout mice that developed atherosclerosis after high-fat induction were collected for WB detection. The results are shown in Figure 3. Figure 2 As shown in D, CCDC92 was found to be significantly overexpressed in the liver of mice with atherosclerosis. The above results indicate that CCDC92 may be involved in the occurrence and development of atherosclerosis.
[0041] (2) CCDC92 knockdown reduced lipid droplet formation in human and mouse liver cell lines
[0042] In order to verify the role of CCDC92, this example uses small molecule RNA (siRNA) interference technology to downregulate the expression of human (HepG2) and mouse (AML-12) liver cell lines, respectively. Through in vitro OA / PA induction experiments, the number and size of lipid droplets formed are detected using the oil red staining method. The results are as follows Figure 3 and Figure 4 shown. Figure 3In the figure, A is WB detection of siRNA knockdown in HepG2 cell line; B is RNA detection of siRNA knockdown in HepG2 cell line; C and D are the results of lipid droplet-induced oil red staining in HepG2 cell line before and after CCDC92 knockdown, respectively. Figure 4 In the figure, A is the WB detection of siRNA knockdown in AML-12 cell line; B is the RNA detection of siRNA knockdown in AML-12 cell line; C and D are the results of lipid droplet induced oil red staining in AML-12 cell line before and after CCDC92 knockdown, respectively. Figure 3 and Figure 4 It can be seen that the formation of cellular lipid droplets was significantly reduced after CCDC92 knockdown.
[0043] (3) Construction of stable knockdown cell lines
[0044] In this example, the Crispr / Cas9 system was used to knock down CCDC92 in the mouse liver cell line AML-12. Finally, several cell clones were screened, and the genome of the cell clones was extracted for PCR identification. The sequencing results showed that the single allele was knocked out, and the expression level of CCDC92 was indeed reduced after WB detection. The results are as follows Figure 5 shown. Figure 5 In the figure, A is the sgRNA sequence used; B is the gene knockout flowchart; C is the single allele knockout sequencing result; D is the knockout status of cell clones detected by WB.
[0045] (4) CCDC92 knockdown inhibited lipid droplet formation
[0046] The CCDC92 stable knockdown cell line obtained in this example was induced in vitro by OA / PA and detected by oil red staining and fluorescent staining, respectively. Figure 6 shown. Figure 6 In the figure, A is the results of cell lipid droplet oil red staining and fluorescence staining, and B is the results of cell LDL-C and TC detection. Figure 6 It can be seen that the formation of lipid droplets was significantly reduced, and the expression of intracellular LDL-C and TC was also significantly reduced.
[0047] (5) Knockdown of CCDC92 changes mitochondrial membrane potential and inhibits cell apoptosis
[0048] ROS has long been regarded as a macromolecule that damages organisms. Therefore, the intracellular reactive oxygen species (ROS) of the CCDC92 stable knockdown cell line obtained in this example were detected. The results are as follows: Figure 7 As shown in A. Figure 7As shown in Figure A, after CCDC92 was knocked down, the generation of ROS in the cell was reduced. In the body, one of the main sources of ROS is produced by the respiratory chain of the mitochondrial inner membrane. Therefore, the CCDC92 stable knockdown cell line obtained in this example was further tested using a mitochondrial membrane potential detection kit. The results are as follows: Figure 7 As shown in B. Figure 7 In B, red fluorescence marks live cells that maintain mitochondrial membrane potential, while green fluorescence marks cells that have undergone apoptosis or necrosis. Figure 7 As shown in Figure B, the green fluorescence decreased and the mitochondrial membrane potential decreased after CCDC92 knockdown, indicating that the reduction of CCDC92 inhibited the damage of mitochondrial membrane potential to cells. Furthermore, cell apoptosis was analyzed by flow cytometry. The results of cell apoptosis detection are shown in Figure 2. Figure 7 As shown in C, the percentage of cell apoptosis is as follows Figure 7 As shown in D. Figure 7 As shown in C and D, cell apoptosis was reduced after CCDC92 knockdown.
[0049] (6) Multi-omics sequencing and related signal pathways
[0050] After CCDC92 knockdown, RNAseq was performed, and the results were as follows: Figure 8 As shown. Figure 8 It can be seen that lipid metabolism and cardiovascular disease-related pathways are significantly enriched. Further, the effect of CCDC92 on lipid-related signaling pathways was detected by WB. The results are as follows Fig. 9 As shown. Fig. 9 It can be seen that after CCDC92 knockdown, the adenylate-activated protein kinase (APMK) signaling pathway is activated and carnitine palmitoyltransferase 1 (CPT1A) increases. Carnitine palmitoyltransferase 1 (CPT1) constitutes the rate-limiting step of fatty acid oxidation by transporting long-chain fatty acids to mitochondria. CPT1A is a subtype of the CPT1 transport system that controls the entry of fatty acids into mitochondria for oxidation. After CCDC92 knockdown, fatty acid oxidation is promoted by increasing the expression of CPT1A, reducing the accumulation of fatty acids and inhibiting lipid droplet formation.
[0051] In summary, the present invention found that the expression of CCDC92 in the liver of APOE knockout mice after a high-fat diet increased significantly. It shows that CCDC92 is involved in the occurrence and development of atherosclerosis. Further, the present invention transfected CCDC92 siRNA in human liver cell lines and used OA / PA induction, and found that the formation of cellular lipid droplets was reduced and the number of apoptotic cells was reduced. In order to further study the mechanism of action of CCDC92, the present invention stably knocked down CCDC92 in mouse liver cell lines, and also found that when OA / PA was used for induction, the formation of lipid droplets was reduced and the formation of intracellular ROS was reduced. The RNAseq sequencing results showed that after CCDC92 was knocked down, the genes related to lipid metabolism and cardiovascular disease in cells changed significantly. Further analysis found that after CCDC92 was knocked down, the expression of CPT1A increased, and CPT1A is the rate-limiting enzyme for fatty acid oxidation. Therefore, after CCDC92 was knocked down, by increasing the expression of CPT1A, fatty acid oxidation was promoted, lipid accumulation was reduced, lipid droplet formation was inhibited, intracellular ROS was reduced, and apoptosis was reduced, thereby affecting blood lipids and the progression of atherosclerosis. Therefore, CCDC92 can be used as a potential target for the treatment of hyperlipidemia and atherosclerosis, and reagents that inhibit the expression of CCDC92 can be used to prepare drugs for the treatment of hyperlipidemia and atherosclerosis, providing a new idea and strategy for the treatment of hyperlipidemia and atherosclerosis.
[0052] It should be understood that the application of the present invention is not limited to the above examples. For ordinary technicians in this field, improvements or changes can be made based on the above description. All these improvements and changes should fall within the scope of protection of the claims attached to the present invention.
Claims
1. Use of an agent that inhibits CCDC92 expression in the preparation of a drug for treating hyperlipidemia and atherosclerosis.
2. The use according to claim 1, characterized in that: The drug inhibits the formation of intracellular lipid droplets.
3. The use according to claim 2, characterized in that: The drug inhibits lipid droplet formation by activating the AMP-activated protein kinase signaling pathway.
4. The use according to claim 3, characterized in that: The drug inhibits lipid droplet formation by increasing the expression of carnitine palmitoyltransferase 1.
5. The use according to claim 4, characterized in that: The drug inhibits lipid droplet formation by increasing the expression of carnitine palmitoyltransferase 1A.
6. The use according to claim 1, characterized in that: The drug reduces the generation of reactive oxygen species within cells.
7. The use according to claim 1, characterized in that: The drug reduces damage to cells caused by mitochondrial membrane potential.
8. The use according to claim 1, characterized in that: The drug reduces cell apoptosis.