Application of IP6K1 and its product as drug target for increasing blood apolipoprotein A1

Abstract

This invention belongs to the fields of biotechnology and pharmaceutical technology, specifically relating to the application of IP6K1 and its products as drug targets for increasing blood apolipoprotein A1. IP6K1 and its product, inositol heptaphosphate, can interact with apolipoprotein A1 and the E3 ubiquitin ligase UBE4A, promoting the binding of apolipoprotein A1 to UBE4A, leading to ubiquitination and degradation of apolipoprotein A1. Knocking out IP6K1 can break the mechanism of UBE4A ubiquitination of apolipoprotein A1, promoting its secretion into the extracellular space, thereby exerting a protective effect against atherosclerosis. Therefore, IP6K1 and its product, inositol heptaphosphate, can serve as drug targets for increasing blood apolipoprotein A1 and also as potential therapeutic targets for treating atherosclerosis.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and pharmaceutical technology, specifically to the application of IP6K1 and its products as drug targets for increasing blood apolipoprotein A1. Background Technology

[0002] Inositol phosphate is a class of small molecule compounds that participate in various intracellular signaling pathways to transmit extracellular molecular signals such as hormones, neurotransmitters, and growth factors. Inositol triphosphate (InsP3) is a classic second messenger, its function being to release intracellularly stored calcium ions. InsP3 can be phosphorylated by a series of inositol kinases into inositol tetraphosphate (InsP4), inositol pentaphosphate (InsP5), inositol hexaphosphate (InsP6), and inositol heptaphosphate (InsP7). InsP7 has an intracellular concentration of approximately 5 μM and is also known as inositol pyrophosphate because it contains a pyrophosphate group. Mammalian cells contain three types of InsP7, named 1PP-InsP5, 3PP-InsP5, and 5PP-InsP5 according to the position of the pyrophosphate group on the inositol ring, with 5PP-InsP5 accounting for 90% of all InsP7. It functions by binding to target proteins, mediating protein-protein interactions, or altering protein conformation.

[0003] 5PP-InsP5 is synthesized by inositol hexaphosphate kinases (IP6Ks). Mammalian organisms express three IP6Ks: IP6K1, IP6K2, and IP6K3. These three IP6Ks possess the same kinase activity and can all utilize InsP6 as a substrate to generate 5PP-InsP5. Recent studies based on IP6KS knockout animals have gradually revealed the individual cellular biological functions and molecular mechanisms of IP6K1, IP6K2, and IP6K3. For example, IP6K1 regulates glucose and lipid metabolism, insulin secretion, growth and development, platelet aggregation, and immune inflammatory responses; IP6K2 plays an important regulatory role in cancer development and nerve cell function; and IP6K3 regulates cell morphology, motility, metabolism, and lifespan, and participates in the development of Alzheimer's disease. The distribution of these three IP6Ks subtypes within tissue cells is not entirely identical. IP6K1 is the most widely expressed subtype, present in almost all cells, catalyzing the production of 70% of 5PP-InsP5. IP6K1 / 5PP-InsP5 is widely involved in various cellular biological processes, such as mRNA transcription and translation, and protein stability. With in-depth research, the role of IP6K1 / 5PP-InsP5 in diseases is gradually being revealed. For example, inhibiting the biosynthesis of 5PP-InsP5 significantly improves insulin sensitivity, reduces inflammatory responses, inhibits platelet aggregation, increases ATP supply, and reduces myocardial ischemia-reperfusion injury. IP6K1 / 5PP-InsP5, as a potential therapeutic target for diabetes and cardiovascular diseases, is currently a hot research topic internationally.

[0004] Low levels of high-density lipoprotein (HDL) in plasma are a prominent feature of metabolic syndrome, contributing to atherosclerosis and associated with higher mortality rates. HDL removes cholesterol from arteries and transports it to the liver for metabolism and excretion. Therefore, enhancing HDL-mediated reverse cholesterol transport holds promise as an effective strategy for reducing the risk of coronary heart disease.

[0005] Apolipoprotein A1 (apoA-1) is a major structural and functional protein in HDL, accounting for approximately 70% of total HDL protein composition. apoA-1 forms nascent HDL by acquiring cholesterol and phospholipids. Existing literature has demonstrated that upregulating endogenous apoA-1 and overexpressing exogenous apoA-1 can enhance reverse cholesterol transport in animal models and reduce the size of atherosclerotic plaques. Clinical trials have shown that apoA-1 administration reduces the atherosclerotic burden in patients with coronary artery disease. Therefore, developing drug targets that can increase blood apolipoprotein A1 levels is of great significance for the treatment of atherosclerosis. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the purpose of this invention is to provide IP6K1 and its products as drug targets for increasing blood apolipoprotein A1.

[0007] Another object of the present invention is to provide the application of IP6K1 and its encoding gene as a drug target for increasing blood apolipoprotein A1.

[0008] Another object of the present invention is to provide the use of an inhibitor of IP6K1 or its encoding gene in the preparation of a medicament that increases the level of blood apolipoprotein A1.

[0009] Another object of the present invention is to provide the use of IP6K1 and its encoding gene in the preparation of medicaments for the prevention and / or treatment of cardiovascular diseases.

[0010] Another object of the present invention is to provide the application of inositol heptaphosphate as a drug target for increasing blood apolipoprotein A1.

[0011] Another object of the present invention is to provide the use of inositol heptaphosphate in the preparation of medicaments for the prevention and / or treatment of cardiovascular diseases.

[0012] To solve the above-mentioned technical problems, the present invention provides the following technical solution:

[0013] This invention, through extraction of plasma components from WT and IP6K1 KO mice, revealed a significant increase in apolipoprotein A1 in the blood of IP6K1 KO mice. Using immunoprecipitation, the inventors discovered that IP6K1, through its product 5PP-InsP5, binds to apolipoprotein A1 and the ubiquitin ligase UBE4A. The presence of 5PP-InsP5 mediates the formation of the apoA-1 / UBE4A complex and increases the ubiquitination of apoA1, ultimately leading to the intracellular degradation of apoA-1 protein. This result indicates that the presence of IP6K1 / 5PP-InsP5 can significantly inhibit the biosynthesis of apolipoprotein A1. Furthermore, using liver-specific IP6K1 knockout animals, it was confirmed that the blood apolipoprotein A1 levels in knockout animals were significantly higher than in the control group, and the increased apolipoprotein A1 mainly originated from the liver. The results show that both systemic and liver-specific IP6K1 knockout can increase the level of apolipoprotein A1 in the blood and enhance reverse cholesterol transport.

[0014] Finally, by establishing an atherosclerosis model, Oil Red O staining of the entire aorta and Oil Red O and HE staining of the aortic root were performed. The results showed that the plaque area of ​​IP6K1 liver-specific knockout mice was significantly lower than that of IP6K1 mice. flox / floxIn mice, further studies have confirmed that knocking out IP6K1 can increase the levels of apolipoprotein A1 and HDL in the blood by increasing the secretion of apolipoprotein A1 from the liver into the blood, thereby slowing the progression of atherosclerosis.

[0015] In summary, targeting IP6K1 or 5PP-InsP5 can provide a drug target for atherosclerosis by upregulating the protein level of blood apolipoprotein A1.

[0016] Therefore, this invention seeks protection for the use of IP6K1 and its products as drug targets for increasing blood apolipoprotein A1.

[0017] Specifically, this invention seeks protection for the use of IP6K1 and its encoding gene as a drug target for increasing blood apolipoprotein A1.

[0018] Preferably, the drug increases the blood apolipoprotein A1 content by inhibiting IP6K1 activity or reducing the transcription and / or expression of its encoding gene.

[0019] More preferably, the drug increases blood apolipoprotein A1 levels by systemic knockout of IP6K1 and / or liver-specific knockout of IP6K1.

[0020] The present invention also claims protection for the use of inhibitors of IP6K1 or its encoding gene in the preparation of medicaments that increase the level of blood apolipoprotein A1.

[0021] Preferably, the inhibitor of IP6K1 or its encoding gene is a substance capable of inhibiting IP6K1 activity or reducing the transcription and / or expression of its encoding gene, including interfering RNA, gRNA, nucleic acid aptamers, protein inhibitors, compound inhibitors, or combination inhibitors that target IP6K1 or its encoding gene.

[0022] This invention also seeks protection for the use of IP6K1 and its encoding gene in the preparation of medicaments for the prevention and / or treatment of cardiovascular diseases.

[0023] Specifically, the cardiovascular diseases include, but are not limited to, atherosclerosis and / or coronary heart disease.

[0024] Preferably, the drug increases reverse cholesterol transport and inhibits the formation of vascular plaques by inhibiting IP6K1 activity or reducing the transcription and / or expression of its encoding gene.

[0025] More preferably, the drug increases reverse cholesterol transport and inhibits vascular plaque formation by systemic knockout of IP6K1 and / or liver-specific knockout of IP6K1.

[0026] This invention also claims protection for the use of inositol heptaphosphate as a drug target for increasing blood apolipoprotein A1.

[0027] The present invention also claims protection for the use of inositol heptaphosphate in the preparation of medicaments for the prevention and / or treatment of cardiovascular diseases, including but not limited to atherosclerosis and / or coronary heart disease.

[0028] Preferably, the drug increases the blood apolipoprotein A1 content by reducing the biosynthesis of inositol heptaphosphate or reducing its expression level.

[0029] More preferably, the drug increases reverse cholesterol transport and inhibits vascular plaque formation by systemic knockout of IP6K1 and / or liver-specific knockout of IP6K1.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] This invention reveals that IP6K1 and its product, inositol heptaphosphate, can interact with apolipoprotein A1 and the E3 ubiquitin ligase UBE4A, promoting the binding of apolipoprotein A1 to UBE4A, leading to ubiquitination and degradation of apolipoprotein A1. Knocking out IP6K1 breaks the mechanism of UBE4A ubiquitination of apolipoprotein A1, promoting its secretion into the extracellular (intravascular) space, thereby increasing reverse cholesterol transport, inhibiting vascular plaque formation, and exerting a protective effect against cardiovascular diseases such as atherosclerosis. Therefore, IP6K1 and its product, inositol heptaphosphate, can serve as drug targets for increasing blood apolipoprotein A1 levels and as potential therapeutic targets for treating atherosclerosis. Attached Figure Description

[0032] Figure 1 Systemic knockout of IP6K1 increases plasma apolipoprotein A1 protein levels. Figure 1 A. Proteins of different molecular weights were separated from plasma samples of WT and IP6K1 KO mice using non-denaturing gels. The results showed that one protein was significantly increased, and the protein was confirmed to be apolipoprotein A1 by mass spectrometry analysis. Figure 1 B was used to further confirm the level of apolipoprotein A1 in the plasma of IP6K1 systemic knockout mice via SDS page.

[0033] Figure 2 The plasma levels of high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), and total cholesterol (CHO) in WT and IP6K1 KO mice were measured. The results showed that systemic knockout of IP6K1 increased plasma HDL-C levels.

[0034] Figure 3 Liver-specific knockout of IP6K1 increases plasma apolipoprotein A1 and high-density lipoprotein levels. Figure 3In IP6K1 liver-specific knockout mice, the plasma apolipoprotein A1 level was significantly higher than that in IP6K1 mice. flox / flox Group of mice; Figure 3 B is IP6K1 flox / flox And Alb-cre; IP6K1 flox / flox Plasma levels of high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), and total cholesterol (CHO) in mice.

[0035] Figure 4 Systemic knockout and liver-specific knockout of IP6K1 increased the level of apolipoprotein A1 protein in the liver. Figure 4 A shows the Western blot results of proteins extracted from liver tissues of WT and IP6K1 KO mice, respectively. Figure 4 B is for extracting IP6K1 respectively. flox / flox And Alb-cre; IP6K1 flox / flox Western blot results of mouse liver tissue proteins; the results showed that IP6K1 KO and Alb-cre; IP6K1 flox / flox The protein level of apolipoprotein A1 was significantly increased in mouse liver tissue.

[0036] Figure 5 Knockout or knockdown of IP6K1 in primary hepatocytes and the human normal hepatocyte line L02 cells increased apolipoprotein A1 protein levels. Figure 5 A represents Western blot analysis of protein levels in primary hepatocytes of WT and IP6K1 KO mice; Figure 5 B represents Western blot analysis of intracellular apolipoprotein A1 protein levels in L02 cells after shRNA transfection and IP6K1 knockdown.

[0037] Figure 6 Western blot analysis of intracellular apolipoprotein A1 protein levels in human normal hepatocyte cell line L02 after overexpression of myc-GFP and myc-IP6K1 showed that apolipoprotein A1 protein levels decreased after overexpression of IP6K1.

[0038] Figure 7 IP6K1, fed for 16 weeks after PCSK9 virus injection in WD patients. flox / flox And Alb-cre; IP6K1 flox / flox The levels of hepatic apolipoprotein A1, plasma apolipoprotein A1, and high-density lipoprotein in mice. Figure 7 A represents the level of liver apolipoprotein A1; Figure 7 B represents the plasma apolipoprotein A1 level; Figure 7C represents the plasma levels of high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), and total cholesterol (CHO).

[0039] Figure 8 IP6K1, fed for 16 weeks after PCSK9 virus injection in WD patients. flox / flox And Alb-cre; IP6K1 flox / flox Cholesterol efflux experiment was conducted using mouse plasma.

[0040] Figure 9 IP6K1, fed for 16 weeks after PCSK9 virus injection in WD patients. flox / flox And Alb-cre; IP6K1 flox / flox Frozen sections were prepared from the aortic root of mice to observe atherosclerotic lesions. Figure 9 A. Oil Red O staining of the inner surface of the aorta, and statistical results of plaque area and percentage of plaque area to vessel area; Figure 9 B is a section of the aortic root stained with Oil Red O and H&E, and the results of plaque area statistics are shown.

[0041] Figure 10 Western blot analysis results of apolipoprotein A1 protein levels in primary hepatocytes of IP6K1 KO mice overexpressing myc-gfp, mutant IP6K1 cells lacking kinase activity, and wild-type IP6K1 cells.

[0042] Figure 11 This study investigated the application of TNP / SC-919, a catalytic activity inhibitor of the IP6KS family, to cells and observed changes in intracellular apolipoprotein A1 protein levels. Figure 11 A represents Western blot analysis of the inhibition of apolipoprotein A1 protein levels in primary hepatocytes of WT mice by the IP6K kinase activity inhibitor TNP (3 μM). Figure 11 B is a Western blot analysis of intracellular apolipoprotein A1 protein levels after TNP (3 μM) was used to inhibit the production of 5PP-InsP5 in human L02 cells. Figure 11 C represents Western blot analysis of intracellular apolipoprotein A1 protein levels after treating human L02 cells with SC-919 (1 μM), an IP6K kinase activity inhibitor with stronger inhibitory efficiency.

[0043] Figure 12 Western blot analysis of apolipoprotein A1 protein levels in the liver and plasma of mice one week after injection of DMSO or TNP. Figure 12 A represents Western blot analysis of the liver apolipoprotein A1 protein level; Figure 12 B represents Western blot analysis of plasma apolipoprotein A1 protein levels.

[0044] Figure 13 Western blot analysis of apolipoprotein A1 protein levels in the liver and plasma of mice one week after injection of DMSO or SC-919. Figure 13 A represents Western blot analysis of the liver apolipoprotein A1 protein level; Figure 13 B represents Western blot analysis of plasma apolipoprotein A1 protein levels.

[0045] Figure 14 IP6K1 does not affect apolipoprotein A1 transcription levels. WT(IP6K1) was extracted. f / f ) and IP6K1 KO (Alb-cre; IP6K1 f / f The changes in apolipoprotein A1 transcription levels were observed by RT-PCR in mouse liver tissue RNA. It was found that knocking out IP6K1 did not change the mRNA level of apolipoprotein A1.

[0046] Figure 15 The results show the levels of intracellular ubiquitination of apolipoprotein A1 in the liver tissue of WT and IP6K1 KO mice, obtained through immunoprecipitation. Figure 15 A is a Western blot analysis of intracellular ubiquitination levels of apolipoprotein A1 after immunoprecipitation of primary hepatocytes from WT and IP6K1 mice. Figure 15 B represents Western blot analysis of intracellular ubiquitination levels after overexpression of myc-GFP and myc-IP6K1 in L02 cells, respectively.

[0047] Figure 16 To investigate the intracellular ubiquitination level of human L02 hepatocytes after treatment with the IP6K kinase activity inhibitors TNP and SC-919 to inhibit the production of 5-InsP7, the study focused on... Figure 16 A is a Western blot analysis of intracellular ubiquitination levels after TNP (3 μM) inhibited the production of 5-InsP7 in L02 cells. Figure 16 B represents the Western blot analysis of intracellular ubiquitination levels in L02 cells after SC-919 (1 μM) inhibited the production of 5-InsP7.

[0048] Figure 17 Immunoprecipitation of IgG and IP6K1 in liver tissue revealed a binding protein in the 100-150 kDa range, which mass spectrometry analysis identified as the E3 ubiquitin ligase UBE4A.

[0049] Figure 18 To isolate WT and IP6K1 KO primary hepatocytes, immunoprecipitation of apoA-1 protein was performed. A protein at around 130 kDa was found to bind to apoA-1, and the binding decreased after IP6K1 KO. Mass spectrometry analysis identified it as the E3 ubiquitin ligase UBE4A.

[0050] Figure 19 WB confirmed the combination of IP6K1 and UBE4A. Among them, Figure 19 A. Immunoprecipitation of IP6K1 and UBE4A in liver tissue to confirm their endogenous binding; Figure 19 B involves overexpressing myc-GFP, myc-IP6K1, and HA-UBE4A in 293A cells, respectively, and then immunoprecipitating the myc-tagged protein to confirm the exogenous binding of the two.

[0051] Figure 20 WB confirmed the binding of apoA-1 to UBE4A. Among other things, Figure 20 A involves overexpressing flag-GFP, flag-apoA-1, and HA-UBE4A in 293A cells, respectively, and then immunoprecipitating the flag tag protein to confirm the exogenous binding of the two. Figure 20 B involves overexpressing flag-GFP, flag-apoA-1, and HA-UBE4A in 293A cells, respectively, and then immunoprecipitating the HA-tagged protein to further confirm the exogenous binding of the two.

[0052] Figure 21 WB confirmed the binding of IP6K1 and apoA-1. Among them, Figure 21 A involves overexpressing myc-GFP, myc-IP6K1, and flag-apoA-1 in 293A cells, respectively, and immunoprecipitating the flag tag protein to confirm the exogenous binding of the two. Figure 21 In B, myc-GFP, myc-IP6K1, and flag-apoA-1 were overexpressed in 293A cells, and myc-tagged proteins were immunoprecipitated to further confirm their exogenous binding.

[0053] Figure 22 To reduce or overexpress UBE4A in human L02 cells, the protein level of intracellular apolipoprotein A1 was determined. Figure 22 A is a Western blot analysis of intracellular apolipoprotein A1 protein levels in L02 cells after UBE4A was knocked down with shRNA. Figure 22 B represents Western blot analysis of intracellular apolipoprotein A1 protein levels in L02 cells after overexpression of UBE4A.

[0054] Figure 23 UBE4A promotes the ubiquitination modification of apoA-1. Among other things, Figure 23 A represents a Western blot analysis of intracellular ubiquitination levels in L02 cells after shRNA knockdown of UBE4A and treatment with MG132 for 4 hours. Figure 23B represents Western blot analysis of intracellular ubiquitination levels in L02 cells after overexpression of UBE4A and treatment with MG132 for 4 hours.

[0055] Figure 24 To facilitate the binding of apoA-1 and UBE4A to IP6K1. Among other things, Figure 24 A was obtained by overexpressing flag-apoA-1 and immunoprecipitating the flag tag protein after isolating primary hepatocytes from primary WT and IP6K1KO mice. The binding of apoA-1 to UBE4A was detected by Western blotting. Figure 24 B represents immunoprecipitation of UBE4A protein, and WB is used to detect the binding of apoA-1 to UBE4A.

[0056] Figure 25 5PP-InsP5 promotes the binding of apoA-1 to UBE4A. Among them, Figure 25 A. After L02 cells were treated with the IP6K family kinase activity inhibitor SC-919 to inhibit the production of 5-PPInsP5, the flag tag protein was immunoprecipitated, and the binding of apoA-1 to UBE4A was detected by Western blotting. Figure 25 B represents the immunoprecipitation of UBE4A protein after SC-919 inhibits the production of 5-PPInsP5 in L02 cells, and the Western blot is used to detect the binding of apoA-1 to UBE4A.

[0057] Figure 26 It combines 5PP-InsP5 with apoA-1 and UBE4A. Among them, Figure 26 A is to use pre-bound inositol heptaphosphate gel resin to precipitate HA-UBE4A and flag-apoA1 proteins overexpressed in 293A cells. Western blotting revealed that the product precipitated by inositol heptaphosphate resin contained UBE4A and apoA-1 proteins. Figure 26 B was prepared by using a gel resin pre-bound to inositol heptaphosphate to precipitate GST-UBE4A and GST-apoA1 proteins overexpressed in 293A cells and purified using GSH beads. Western blotting confirmed the direct binding of 5-PCP to UBE4A and apoA-1 proteins.

[0058] Figure 27 To overexpress GST-UBE4A and flag-apoA1 proteins in 293A cells, beads pre-bound with an equal amount of apoA-1 protein were added to an equal amount of UBE4A protein purified with GSH beads, and InsP6 and 5PP-InsP5 were added respectively. The binding of apoA-1 to UBE4A was verified by Western blotting.

[0059] Figure 28To overexpress GST-UBE4A and flag-apoA1 proteins in 293A cells, beads pre-bound with an equal amount of apoA-1 protein were added to an equal amount of UBE4A protein purified with GSH beads, and InsP6, 5-PCP, and CF2 were added respectively. The binding of apoA-1 to UBE4A was verified by Western blotting.

[0060] Figure 29 This is a diagram of the mechanism. Detailed Implementation

[0061] The present invention will be further described in detail below with reference to specific embodiments. The following embodiments are not intended to limit the present invention, but only to illustrate the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are generally performed under conventional conditions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0062] laboratory animals

[0063] All experimental procedures complied with the regulations of the Experimental Animal Management Committee of Tianjin Medical University. IP6K1 systemic knockout mice (IP6K1 - / - ) and IP6K1 flox / flox Mice were provided by the Solomon H. Snyder laboratory at Johns Hopkins University. Liver-specific cre knockout mice (Alb-cre) were provided by the laboratory of Professor Chang Yongsheng at Tianjin Medical University.

[0064] Statistical methods

[0065] Experimental results were analyzed using GraphPadPrism 6.0 software. All quantified experimental results in the study are expressed as mean ± standard error (Mean ± SEM), and the sample size (n) used in each study is shown in the legend. Unpaired two-tailed Student's t-tests were used for comparisons between two groups, and one-way or two-way ANOVA was used for comparisons between three or more groups. Further pairwise comparisons between groups were corrected using Bonferroni's posthoc test. In all experiments, a p-value < 0.05 was considered statistically significant, and the p-value for each experiment is given in the legend.

[0066] Experimental methods

[0067] I. Cell Culture

[0068] HEK293, 293T, and L02 cells were cultured in DMEM medium containing 10% serum (Thermo Fisher Scientific), while primary mouse hepatocytes were cultured in M199 medium containing 10% serum (Gibco). The cell culture incubator was set to a constant temperature of 37°C with 5% CO2 aeration.

[0069] 1. Thawing: Place the cryopreserved cells in a 37°C water bath for rapid thawing. During this process, quickly shake the cryovials by hand to allow the cells to pass through the 0°C critical point as soon as possible, preventing water from seeping into the cells and forming ice crystals that could cause damage. Add the thawed cell cryopreservation solution to 1 mL of culture medium, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, resuspend the cells in 10 mL of thawed culture medium, and add them along the wall of a 10 cm culture dish. For passage and thawing of primary mouse hepatocytes, collagen should be pre-coated to the bottom of the dish. After 15 minutes, the collagen should be removed and the cells re-coated to help the primary mouse hepatocytes adhere to the dish.

[0070] 2. Passaging: Once the cells have adhered and reached confluence, aspirate the culture medium and wash three times with PBS buffer to remove serum residue and dead cells. Add 1 mL of trypsin at 37°C to digest the cells, separating them from the bottom of the dish and from each other. After adding trypsin, transfer the cells to a microscope for observation. When the cells become rounded, pseudopodia retract, and they can be easily detached from the bottom of the dish by gentle shaking, promptly add culture medium containing serum to stop digestion and avoid over-digestion that could cause unnecessary damage to the cells. Centrifuge the cell digestion solution at 1000 rpm for 5 minutes, discard the supernatant, resuspend the cells in culture medium, and add them to culture dishes at a ratio of 1:3 or 1:4. Shake well and place in an incubator.

[0071] 3. Cryopreservation: After the cells are digested, centrifuge them, discard the supernatant, and resuspend them in cell cryopreservation solution (70% serum-free DMEM, 20% serum, 10% DMSO) at a ratio of 1:2 or 2:3. Aliquot the cells into cryopreservation tubes and place them in a cryopreservation box. Gradually cool the cells at -80°C to reduce the damage to the cells from ice crystals. After 24 hours, transfer the cell cryopreservation tubes to liquid nitrogen for long-term storage.

[0072] II. Isolation of Primary Mouse Hepatic Cells

[0073] 1. Anesthetize the mouse, disinfect with alcohol to expose the abdominal cavity, clear the field of vision, separate the inferior vena cava, tie a false knot, insert an indwelling venous catheter below the false knot, and fix the false knot.

[0074] 2. Inject 1-1.5 mL of heparin through the indwelling needle until the blood in the liver disappears, then slowly push in 50 mL of perfusion fluid, while simultaneously cutting open the portal vein and perfusing for about 3-4 minutes.

[0075] 3. Slowly perfuse the liver with 2 (30 mL) for 3-4 minutes until the liver shows a wet, striped, cloth-like texture. Turn the liver over and remove the gallbladder.

[0076] 4. Place the liver in a petri dish on an ice box and transfer it to the intercellular space. Then, repeatedly pipette the liver through a 400-mesh sieve and pre-cooled (4°C) serum-free 1640 until only the liver capsule remains.

[0077] 5. Take 20 μL of cell culture to observe cell viability, stain with 2 μL of trypan blue and observe under a microscope (preferably above 70%).

[0078] 6. Tilt the petri dish and let the cells settle for 5 minutes (liver parenchymal cells settle more). Carefully aspirate and discard the supernatant, and add 25 mL of pre-cooled culture medium. Gently pipette a few times to dissolve the cell clumps at the bottom, resuspend, and transfer to a 50 mL centrifuge tube.

[0079] Centrifuge at 50g for 2 min three times at 7.4℃, adding 25mL of culture medium each time, and finally resuspend with an appropriate amount of 10% FBS1640 and plate.

[0080] 8. Change the medium 2-6 hours after the cells adhere to the wall.

[0081] III. Protein Extraction and Quantification

[0082] (I) Extraction of total protein from tissues

[0083] 1. Take mouse liver tissue and place it in a 1mL glass grinding tube. Add 1mL of protein lysis buffer and place it in a grinding rod to grind and lyse the tissue manually.

[0084] 2. Use a 30g syringe needle to aspirate 30 times to fully lyse the tissue;

[0085] 3. Centrifuge the lysate at 4°C and 13,000 rpm for 15 minutes;

[0086] 4. Transfer the supernatant protein sample after centrifugation to a new 1.5 mL centrifuge tube, take 5 μL of protein sample into a 96-well plate, determine the protein concentration using the BCA method, and add SDS buffer to the remaining protein sample according to its volume.

[0087] 5. Place in a metal bath and heat at 100°C for 10 minutes to fully denature the protein. Remove the sample and place it on ice to cool. Store at -20°C.

[0088] (II) Extraction of cellular proteins (using a 10cm dish as an example)

[0089] 1. Discard the original cell culture medium, wash three times with clean PBS solution, discard the PBS as much as possible, add 1 mL of protein lysis buffer, place the dish on ice, wash the cell scraper twice with Milli-Q pure water, collect the cells, and use a pipette to aspirate the cell lysis buffer into a 1.5 mL centrifuge tube.

[0090] 2. Use a 30g syringe needle to aspirate and break down the material 30 times.

[0091] Centrifuge at 3.4℃, 13000rpm for 10 minutes;

[0092] 4. Transfer the protein supernatant sample after centrifugation to a new 1.5 mL centrifuge tube. Take 5 μL of protein sample into a 96-well plate. For protein samples whose protein concentration is determined by the BCA method, add different volumes of 5×SDS buffer according to their volume and mix well.

[0093] 5. Place in a metal bath and heat at 100°C for 10 minutes to fully denature the protein. Remove the sample and place it on ice to cool. Store at -20°C.

[0094] (III) Protein concentration determination by BCA method

[0095] 1. First, dilute the 2000 μg / mL protein standard with PBS to create protein standards with concentration gradients of 0, 125, 250, 500, 750, 1000, 1500, and 2000 (μg / mL);

[0096] 2. Add the protein standards and the protein samples to be tested in the concentration gradient to the 96-well microplate, 5 μL to each well, and then add 200 μL of BCA working solution (solution A:solution B = 50:1) to each well.

[0097] Place in an incubator at 3.37℃ for 30 minutes;

[0098] 4. Use a multi-functional microplate reader to read the absorbance (OD) values ​​of the standards and protein samples at a wavelength of 562 nm. Create a standard curve using Excel software to calculate the concentration of the protein sample to be tested.

[0099] 5. Based on the measured final sample concentration and protein loading mass, calculate the final loading volume for polypropylene gel electrophoresis experiments.

[0100] IV. Protein Immunoblot Experiment

[0101] (I) Protein electrophoresis and transfer

[0102] 1. Based on the different molecular weights and loading volumes of proteins, prepare SDS-polyacrylamide gels (SDS-PAGE) of different concentrations and insert them into upper gel combs with different numbers of wells;

[0103] 2. Perform protein gel electrophoresis using the Bio-Rad mini system. Fix the gel with the clamp, place the clamp and gel into the electrophoresis tank according to the positive and negative electrodes, pour the electrophoresis solution into the appropriate position in the tank, remove the upper gel comb, and add the quantitative protein sample and 3 μL of protein marker in sequence according to the protein loading order. Run the system at a constant voltage of 90-120V until the bromophenol blue reaches the lower edge of the separating gel.

[0104] 3. After electrophoresis, cut the gel to the appropriate size. Place the sponge pad, NC membrane, and gel in sequence on the black side of the transfer clamp, removing any air bubbles between the gel and the NC membrane. Then, clamp the transfer clamp and place the gel into the transfer apparatus. Transfer at 250mA for 120 minutes. After the transfer is complete, remove the NC membrane.

[0105] (II) Non-denaturing gel electrophoresis

[0106] 1. Centrifuge mouse blood samples at 5000 rpm and 4℃ for 5 minutes, and use the supernatant as the target sample;

[0107] 2. BCA method for determining sample protein concentration;

[0108] 3. Add 2×sample buffer to the protein sample proportionally;

[0109] 4. Prepare native gels of different concentrations according to different protein loading volumes, and insert stacking gel combs of different well sizes;

[0110] 5. Perform protein gel electrophoresis using the Bio-Rad mini system. Fix the gel with the clamp, place the clamp and gel into the electrophoresis tank according to the positive and negative electrodes, pour the SDS-free electrophoresis solution into the appropriate position in the tank, remove the upper gel comb, add the quantitative protein sample and 3 μL of protein marker in the order of protein loading, run the gel at a constant voltage of 150V until the bromophenol blue reaches the bottom of the separating gel.

[0111] 6. After electrophoresis, cut the gel to the appropriate size. Place the sponge pad, PVDF membrane, and gel in sequence on the black side of the transfer clamp, removing any air bubbles between the gel and the PVDF membrane. Then, clamp the transfer clamp and place the membrane into the transfer apparatus. Transfer at 250mA for 150 minutes. After the transfer is complete, remove the PVDF membrane.

[0112] (III) Immunohistochemistry

[0113] 1. After removing the NC or PVDF membrane, place it in 5% skim milk powder or 5% BSA blocking solution and incubate it on a horizontal shaker at room temperature for 30 minutes. Wash away the residual blocking solution with TBST washing solution.

[0114] 2. After filtering 5% BSA through a 0.22 μm filter membrane, dilute the primary antibody at a ratio of 1:1000. Place the NC membrane containing the target protein into the primary antibody containing the anti-target protein and incubate overnight at 4°C on a shaker.

[0115] 3. Remove the NC membrane and place it in TBST washing solution. Place it on a shaker and wash the membrane for 10 minutes at medium-fast speed. Repeat this washing process 3 times.

[0116] 4. Dilute the secondary antibody with 5% skim milk powder at a ratio of 1:5000, put the NC membrane into the corresponding secondary antibody, and incubate it on a horizontal shaker at room temperature for 1 hour for binding.

[0117] 5. Remove the NC membrane and place it in TBST washing solution. Wash the membrane quickly in a horizontal shaker for 10 minutes, repeating 3 times.

[0118] (iv) Chemiluminescence and results analysis

[0119] 1. Mix chemiluminescent solution A and solution B in a 1:1 ratio. After the chemiluminescent solution is evenly applied to the protein surface of the imprint membrane, place the NC membrane neatly in the fully automated chemiluminescence imaging system for exposure and imaging.

[0120] 2. Collect the strips with different exposure times and analyze the grayscale value and net optical density value of the target strip using ImageJ software.

[0121] V. Immunoprecipitation test

[0122] (I) Plasmid transfection (taking a 10cm dish as an example)

[0123] 1. Transfect HEK293A cells after their cell density reaches 70-80%;

[0124] 2. Take 500 μL of serum-free DMEM medium and add 15 μL of transfection reagent. 3000, mix thoroughly and incubate for 15 minutes;

[0125] 3. Take 500 μL of DMEM medium, add 20 μL of P3000, 10 μg of plasmid A and 10 μg of plasmid B, mix thoroughly and incubate for 5 minutes;

[0126] 4.5 minutes later, will contain Mix 3000 DMEM medium and plasmid-containing DMEM medium at a 1:1 ratio to form 1 mL of plasmid-lipid complex, and incubate for 10-15 minutes.

[0127] 5. Add 1 mL of plasmid-lipid complex dropwise to the cell culture dish, incubate at 37°C in a 5% CO2 cell culture incubator for 4-6 hours, then change the medium.

[0128] 6.48 hours later, cells were collected, and whole-cell proteins were extracted and detected by adding cell lysis buffer.

[0129] (II) Immunoprecipitation

[0130] 1. Remove the transfected HEK293A cells, discard the original cell culture medium, wash 3 times with clean PBS, discard the residual PBS in the dish with a pipette, add 1 mL of immunoprecipitation lysis buffer to each 10 cm dish, and place on ice;

[0131] 2. Use a cell scraper rinsed with Milli-Q purified water to scrape the cells into a 1.5 mL centrifuge tube;

[0132] 3. Use a 30g syringe needle to aspirate 30 times to thoroughly break down the material;

[0133] 4. After suction is complete, centrifuge at 4℃ and 13000 rpm for 10 minutes;

[0134] 5. After centrifugation, take 5 μL of the supernatant and place it into a 96-well plate. Determine the protein concentration using the BCA method.

[0135] 6. Add protein A / G agarose beads to the cell lysis buffer, suspend at 4°C for 2 hours, centrifuge to discard protein A / G agarose beads bound to non-specific proteins, and collect the supernatant for immunoprecipitation;

[0136] 7. Take a portion of the protein sample as input, and divide the remainder into two portions. Add control IgG antibody and target protein antibody to each portion, and suspend at 4°C overnight.

[0137] 8. Add 35 μL of protein A / G agarose beads to each of the samples containing IgG antibodies and the samples containing the target protein antibodies, and slowly suspend at 4°C for 1.5 hours;

[0138] Centrifuge at 3000 rpm for 5 minutes at 4°C;

[0139] 10. Discard the supernatant protein lysis buffer, add 300 μL of cell lysis buffer, and wash the agarose beads;

[0140] Centrifuge at 3000 rpm and 4°C for 5 minutes, then replace with fresh cell lysis buffer and wash a total of 3 times.

[0141] 12. After discarding the cell lysis buffer, collect the protein A / G agarose beads from the immunosettling sample, add 70 μL of 1×SDS, and incubate in a 100°C metal bath for 10 minutes to denature the protein and elute it into the SDS buffer. Remove the sample, place it on ice to cool, and store it at -20°C or perform a protein immunoblotting experiment.

[0142] VI. Plasmid Transformation and Amplification

[0143] 1. Depending on the plasmid concentration, add 200-500 ng of plasmid to 20 μL of Trans5α competent cells (E. coli) and mix gently. Set up a negative control containing only competent cells.

[0144] 2. Place the two groups of competent cells (1.5 mL centrifuge tubes) on ice and incubate for 2 minutes;

[0145] 3. Transfer to a 42℃ water bath and heat shock for 75 seconds (moving the centrifuge tube smoothly);

[0146] 4. Place on ice and cool for 2 minutes (moving the centrifuge tubes gently);

[0147] 5. Add 500 μL of antibiotic-free LB medium to each of the two groups of competent cells;

[0148] 6. Place a 1.5 mL centrifuge tube in a 37°C constant temperature shaker and shake at 220 rpm for 1 hour to revive E. coli;

[0149] 7. Take 50-150 μL of the suspended LB medium and spread it evenly in an antibiotic-containing LA culture dish using a sterile triangular glass scraper;

[0150] 8. Place the LA culture dish in a 37°C incubator. After the suspension in the dish dries, invert the dish and incubate overnight.

[0151] 9. Once the colonies have grown to a suitable size, take 5 mL of LB medium (containing 5 μL of antibiotic) and place it in a 50 mL centrifuge tube. Select single colonies and transfer them to the 50 mL centrifuge tube. Set up a negative control. Tilt the 15 mL centrifuge tube at 60° and place it on a 37°C constant temperature shaker. Loosen the cap so that it will not fall off. Amplify at 220 rpm for 8-10 hours.

[0152] 10. Add 400 μL of the amplified bacterial culture to a conical flask containing 200 mL of LB medium (containing 200 μL of antibiotics) at a ratio of 1:500. Place the conical flask in a 37°C constant temperature shaker and amplify at 220 rpm for 12-16 hours.

[0153] 11. Extract plasmids using an endotoxin-free large-scale extraction kit.

[0154] VII. Plasmid Extraction

[0155] 1. Add 200 mL of overnight cultured bacterial solution to 50 mL centrifuge tubes, centrifuge at 8000 rpm for 3 minutes at room temperature, and collect the bacteria;

[0156] 2. Absorb as much water as possible from the top of the bottle; use clean absorbent paper to absorb any water droplets on the bottle walls.

[0157] 3. Add 8 mL of solution P1 from the kit to the centrifuge tube containing the bacterial cell pellet, and vortex thoroughly to suspend the bacterial cell pellet.

[0158] 4. After resuscitation, add 8 mL of solution P2 from the kit to the centrifuge tube, and immediately and gently invert it 6-8 times to fully lyse the cells. Let it stand at room temperature for 5 minutes.

[0159] 5. Add 8 mL of solution P4 from the kit to the centrifuge tube, and immediately and gently invert the tube 6-8 times to mix thoroughly until a white, dispersed flocculent precipitate appears. Then, let it stand at room temperature for 10 minutes, and centrifuge at 8000 rpm for 10 minutes to allow the white precipitate to settle to the bottom of the tube.

[0160] 6. After centrifugation, carefully pour all the solution into the filter CS1 included in the kit, slowly push the push handle to filter, and collect the filtrate in a clean 50mL centrifuge tube;

[0161] 7. Add 2.5 mL of equilibration buffer BL to the CP6 adsorption column contained in the kit to equilibrate the column, centrifuge at 8000 rpm for 2 minutes, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

[0162] 8. Add 0.3 times the volume of isopropanol to the filtrate collected in step 6, mix by inverting the container, and then transfer it to the adsorption column CP6 in several portions.

[0163] 9. Centrifuge at 8000 rpm for 2 minutes at room temperature, discard the waste liquid in the collection tube, and put the CP6 adsorption column back into the collection tube;

[0164] 10. Add 10 mL of the wash buffer contained in the kit to the CP6 adsorption column, centrifuge at 8000 rpm for 2 minutes, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

[0165] 11. Repeat step 10;

[0166] 12. Add 3 mL of anhydrous ethanol to the CP6 adsorption column, centrifuge at 8000 rpm for 2 minutes at room temperature, and discard the waste liquid;

[0167] 13. Place the adsorption column CP6 back into the collection tube and centrifuge at 8000 rpm for 5 minutes to remove residual wash solution from the adsorption column.

[0168] 14. After centrifugation, open the CP6 adsorption column and place it at room temperature for several minutes to thoroughly dry any residual washing solution in the adsorption column.

[0169] 15. Place the CP6 adsorption column in a clean 50mL centrifuge tube, add 1mL of high-pressure water dropwise to the middle of the adsorption membrane, let it stand at room temperature for 5 minutes, and then centrifuge at 8000rpm for 2 minutes at room temperature.

[0170] 16. To increase elution efficiency, add 1 mL of solution from the bottom of the tube back to the middle of the adsorption membrane, let it stand at room temperature for 5 minutes, and then centrifuge at 8000 rpm for 2 minutes at room temperature.

[0171] 17. Transfer all the eluent from the bottom of the 50mL centrifuge tube into a clean 1.5mL centrifuge tube and store at -20℃.

[0172] VIII. Construction of an Atherosclerosis Model

[0173] Select 6-8 week old male mice, and inject each mouse via the tail vein with 5×10 11 AAV8-PCSK9 was injected. After 12 weeks of Western diet feeding, specimens from the base of the ascending aorta to the bifurcation of the iliac artery were taken for Oil Red O staining. Frozen sections were prepared from the root of the aorta to observe the atherosclerotic lesions.

[0174] IX. Staining-related experiments

[0175] 1. Oil Red O Staining: Dissolve Oil Red O in isopropanol to prepare a 0.5% stock solution. Mix the stock solution with double-distilled water at a ratio of 3:2 to prepare the Oil Red O working solution. Filter and store at 4°C. Fix the cultured cell samples with 4% paraformaldehyde for 20 minutes. Then wash three times with PBS buffer, 5 minutes each time. Before staining, you can choose to rinse with 60% isopropanol for 30 seconds. Add Oil Red O staining solution and stain in the dark for 30 minutes. After staining, you can choose to rinse with 60% isopropanol to remove excess Oil Red O staining solution. After washing three times with PBS buffer, take pictures or mount with glycerol.

[0176] 2. HE staining: After warming the sections, wash them three times with PBS for 5 minutes each time. Fix them with 4% paraformaldehyde for 20-30 minutes, then wash them three times with PBS for 5 minutes each time. Next, perform the nuclear staining step: Immerse the sections in hematoxylin staining solution for 2-3 minutes, then rinse with tap water for 5 minutes. Observe the nuclear staining under a microscope. If the color is too dark, differentiate with 0.5% hydrochloric acid alcohol differentiation solution for 1-5 seconds, then rinse with tap water. If the color is too light, repeat the staining step. After completing the nuclear staining step, immerse the sections in eosin staining solution for 1-2 minutes, then rinse with tap water. Observe the color under a microscope. If the red is too dark, increase the soaking time in 75% or 85% alcohol. If the color is too light, repeat the above staining. Dehydrate the stained sections in 75%, 85%, 95%, 100% (1), and 100% (2) alcohol. The 75% and 85% alcohol should be immersed quickly. After dehydration, place the slides in xylene for 20 minutes to clear them, then blow-dry or air-dry the stem cells, and finally mount them with neutral resin.

[0177] 3. Oil Red O staining of the inner surface and root of the aorta: After blood perfusion, the mouse aorta was isolated, and the heart was cut off along the root of the aorta, fixed, and embedded for frozen section preparation. After the aorta was cleaned in PBS, it was gently cut open along one side under a stereomicroscope, and then fixed with microneedles in a wax dish containing PBS. The method of Oil Red O staining was the same as above.

[0178] 10. Extraction of rat tail DNA

[0179] A 0.5 cm section of mouse tail was cut from each mouse and placed into a 1.5 mL centrifuge tube, which was then labeled. 0.5 mL of mouse tail lysis buffer and 2.5 μL of proteinase K were added to each EP tube and incubated overnight at 56°C. The next day, the samples were centrifuged at 12000 rpm for 10 minutes. The supernatant was transferred to a new 2 mL EP tube, 1 mL of anhydrous ethanol was added, the tube was tightly capped, and the mixture was inverted until a flocculent precipitate appeared. The tube was centrifuged at 12000 rpm for 10 minutes, and the supernatant was discarded. 1 mL of 80% ethanol was added, and the precipitate was washed by inverting. The tube was centrifuged at 10000 rpm for 10 minutes, the supernatant was discarded, and the precipitate was collected. The washing steps were repeated once. The EP tubes were drained and allowed to air dry at room temperature for 5-10 minutes. After the alcohol had completely evaporated, 200 μL of autoclaved water was added to each tube, and the tubes were incubated at 37°C for 1-2 hours. After the DNA precipitate had completely dissolved, the mouse tails were stored at 4°C or directly subjected to PCR identification.

[0180] XI. RNA-related experiments

[0181] (I) RNA extraction

[0182] 1. Homogenization: Place the tissue in a glass grinding tube, add 1 mL of Trizol, and homogenize thoroughly. Transfer the homogenized sample to a centrifuge tube and incubate at room temperature for 5 minutes.

[0183] 2. For every 1 mL of Trizol Up used, add 0.2 mL of chloroform, shake vigorously for 30 seconds, and incubate at room temperature for 3 minutes;

[0184] 3. Centrifuge at 10000×g, 4℃ for 15 minutes. At this point, the sample separates into three layers: a colorless aqueous phase (upper layer), a middle layer, and several pink layers (lower layer); RNA is in the aqueous phase.

[0185] 4. Transfer the colorless aqueous phase to a new centrifuge tube, add an equal volume of anhydrous ethanol, and gently invert to mix.

[0186] 5. Add the obtained solution and precipitate together into a centrifuge column, centrifuge at 12000g at room temperature for 30 seconds, and discard the waste liquid;

[0187] 6. Add 500 μL CB9, centrifuge at 12000×g for 30 seconds at room temperature, and discard the waste liquid;

[0188] 7. Repeat step 6 once;

[0189] 8. Add 500 μL of WB9, centrifuge at 12000g for 30 seconds at room temperature, and discard the waste liquid;

[0190] 9. Repeat step 8 once;

[0191] 10. Centrifuge at 12000×g for 2 minutes at room temperature to completely remove residual ethanol;

[0192] 11. Place the centrifuge column in an RNase-free tube, add 30 μL of RNase-free water to the center of the centrifuge column, let it stand at room temperature for 2 minutes, centrifuge at 12000×g for 1 minute, and elute the RNA;

[0193] 12. Store the RNA at -80°C;

[0194] (II) RNA Reversal and Quantification

[0195] 1. RNA concentration measurement

[0196] After turning on Nanodrop, select the RNA option, set up a blank control with Milli Q water, then drop 1 μL of RNA solution onto the probe and record the measured concentration value, 260 / 280 ratio, and 260 / 230 ratio.

[0197] (2) RNA reverse transcription

[0198] Take 1-2 μg of total RNA and prepare a 20 μL reaction system according to the Thermo Fisher Reverse Transcription Kit instructions: 4 μL 5×Reaction Buffer, 1 μL Random Hexamer Primer, 2 μL dNTP Mix, 1 μL RevertAid Reverse Transcriptase, and 1 μL Ribo LockRNase Inhibitor.

[0199] Reaction conditions: 37-42℃ for 60 minutes, 95℃ for 5 minutes; after the reaction, dilute with water to remove RNase at a ratio of 1:10 and store at -20℃ for later use.

[0200] XII. Real-time PCR Analysis

[0201] Prepare a 20 μL reaction system from the extracted cDNA according to the instructions: 2 μL template cDNA, 0.5 μL forward primer (10 μM), 0.5 μL reverse primer (10 μM), 10 μL 2×TransStart Tip Green qPCR Super Mix, and add ddH2O to 20 μL.

[0202] PCR three-step method: 95℃ for 5 minutes; 95℃ for 10 seconds, 60℃ for 10 seconds, 72℃ for 20 seconds, for a total of 40 cycles; 72℃ for 5 minutes; 95℃ for 5 minutes.

[0203] XIII. Construction of Recombinant Plasmids

[0204] 1. Obtaining the target gene fragment: PCR primers were designed based on the target protein gene sequence provided by NCBI, and ordered from Sangon Biotech. Using a cDNA library as a template, PCR products were obtained using high-fidelity DNA polymerase (Thermo Fisher Scientific). The PCR program required designing the annealing temperature according to the primer TM value gradient and resetting the extension time according to the size of the target fragment. DNA loading buffer was added to the obtained PCR products, and DNA fragments of different molecular weights were separated on agarose gel using DNA gel electrophoresis. The separated agarose gel was observed in a UV transmission chamber, and the indication positions of the DNA markers were compared. The target gene fragment was recovered and its concentration was determined.

[0205] 2. Tag Sequence Ligation: Smaller molecular weight tags such as flag and myc can be directly ligated to the target gene using primer design. This primer design step requires the inclusion of a homologous fragment of the target gene, the tag sequence, and a homologous fragment of the vector. Larger molecular weight tags such as GST require a first-step PCR step. The GST fragment is obtained separately and then ligated to the target gene fragment via homologous recombination, ultimately yielding a tag-target gene fragment with vector homology information. Furthermore, the ligation of large molecular weight tags to the target gene should be carefully designed with a ligation sequence to ensure that the folding of the tertiary structure does not interfere with each other during protein expression. Generally, five consecutive glycine residues are chosen as the ligation sequence.

[0206] 3. Vector Dissection: Select the vector's restriction enzyme site, which must be consistent with the homologous sequence site designed for the ligation primers. Use the site-specific enzyme to cleave the vector from a circular to a linear form. Separate the digestion products by agarose gel electrophoresis, recover the target fragment, and determine the DNA concentration. Because linear DNA faces greater resistance in the small pores of agarose gel, circular DNA tends to pass through faster. If two DNA bands are observed at similar positions relative to the target molecular weight, the band with the larger molecular weight is the linearly cleaved vector fragment.

[0207] 4. Homologous recombination of vector and target gene: A high-fidelity DNA ligase (Takara Bio) is used to ligate the target gene, tag, and linear vector. Based on the homologous sequences between the fragments, the products can be ligated into a complete circular DNA plasmid. After obtaining the ligation product, it is transformed into competent cells and cultured overnight on LA plates. The next day, single colonies are picked for PCR verification. Successfully verified colonies are further expanded, plasmids are extracted, and DNA sequencing is performed (Sangon Biotech). Successfully sequenced plasmids will be used for cell transfection and protein overexpression.

[0208] XIV. Small Molecule Synthesis

[0209] All small molecule compounds used in this experiment were provided by collaborating laboratories, such as 5-PCF2Am-InsP5, 5-InsP7, and 5-PCP-InsP5. All small molecules were purified by ion-exchange chromatography, characterized and analyzed using 1H, 31P, and 13C nuclear magnetic resonance (NMR) spectroscopy, and finally quantified by total phosphate analysis.

[0210] XV. Synthesis of Inositol Heptaphosphate Affinity Reagent

[0211] The 5-PCP-InsP5 resin used in the immunoprecipitation and in vitro binding experiments was synthesized by a collaborating laboratory, using the methods described in the references [Ghosh, S. et al. Inositol hexakisphosphate kinase 1 maintains hemostasis in mice by regulating platelet polyphosphate levels. Blood 122, 1478-1486] and [Morrison BH, Bauer JA, Karvakolanu DV, Lindner DJ. Inositol hexakisphosphate kinase 2 mediates growth suppressive and apoptotic effects of interferon-b in ovarian carcinoma cells. J. Biol. Chem. 2001; 276: 24965-24970.].

[0212] XVI. In vitro binding experiment

[0213] (a) Combination of apoA-1 and UBE4A

[0214] 1. Transfer HEK293 to a 10cm culture dish and incubate at 37℃;

[0215] 2. When the cell density reaches 80%, use lipo3000 to transfect flag-apoA-1 and GST-UBE4A into HEK293 cells;

[0216] 3. After 48 hours, collect the cells, add 1 mL of lysis buffer to fully lyse the cells, and scrape them into a 1.5 mL EP tube;

[0217] 4. After fully aspirating with a 30g syringe needle, centrifuge at 13000rpm at 4℃ for 10 minutes, collect the supernatant, add pre-washed protein A / G agarose beads, and pre-precipitate on a shaker at 4℃ for 1.5 hours;

[0218] 5. Centrifuge to remove agarose beads, add 5 μL of flag antibody to flag-apoA-1, add 20 μL of Glutathione Sepharose 4B beads to GST-UBE4A, and incubate overnight at 4°C on a shaker.

[0219] 6. Centrifuge the GST group samples to retain the beads, add 1 mL of GSH glutathione elution buffer, and incubate at 4°C for 12 hours on a shaker.

[0220] 7. Add 30 μL of pre-washed protein A / G agarose beads to the flag sample and incubate at 4°C in a shaker for 2 hours;

[0221] 8. Add the eluent obtained in step 6 to the complex obtained in step 7, and divide it evenly into 4 portions. Add H2O or 5mM of 1PP-InsP5, 3PP-InsP5, 5PP-InsP5, 5PCP, CF2, InsP3, InsP4, InsP5, InsP6 to each portion, and incubate on a shaker at 4°C for 6-8 hours.

[0222] 9. Centrifuge the obtained sample to obtain agarose beads, wash 3 times with lysis buffer, aspirate dry, add 1×SDS loading buffer, and heat in a metal bath at 100℃ for 10 minutes;

[0223] 10. Perform protein polyacrylamide gel electrophoresis.

[0224] XVII. Preparation of Competent States

[0225] 1. Seed HST08 competent cells on LB culture plates;

[0226] 2. In a clean bench, pick a single colony and add it to 100 mL of antibiotic-free LB solution. Shake at 37°C and 220 rpm until the colony becomes cloudy.

[0227] 3. Collect bacterial cells by centrifuging at 4000 rpm for 15 minutes in 50 mL centrifuge tubes at 4℃;

[0228] 4. The bacterial cells were resuspended in 10 mL of pre-cooled sterile 0.1 M calcium chloride solution;

[0229] 5. Place on ice for 30 minutes, then centrifuge at 4000 rpm for 10 minutes at 4℃;

[0230] 6. Resuspend the precipitate in 2 mL of pre-cooled 0.1 M calcium chloride solution, add 10% sterile glycerol, dispense into aliquots, freeze quickly on dry ice, and store at -80°C.

[0231] Example 1: Systemic knockout of IP6K1 upregulates plasma apolipoprotein A1 and HDL protein levels.

[0232] (i) Systemic knockout of IP6K1 increases plasma apolipoprotein A1 protein levels.

[0233] To investigate the biological functions of IP6K1 and its product 5PP-InsP5, plasma was first extracted from three groups of 6-8 week old male WT and IP6K1 KO mice. Proteins were separated by native pAGE, and changes in protein levels after IP6K1 knockout were observed using Coomassie Brilliant Blue (Bradford assay). The results showed a significant increase in a protein at 25 kDa after IP6K1 knockout. Figure 1 A) The protein was identified as apolipoprotein A1 by mass spectrometry. To further confirm the binding of the two, the protein was separated by polypropylene gel electrophoresis (SDS page), yielding results consistent with the above experiments, namely, apoA-1 protein levels were significantly upregulated in the plasma of IP6K1 knockout mice. Figure 1 B).

[0234] (ii) Systemic knockout of IP6K1 increases plasma high-density lipoprotein (HDL) levels

[0235] Apolipoprotein A1 (apoA-1) is the main structural and functional protein of high-density lipoprotein (HDL), accounting for approximately 70% of total HDL protein. Newly synthesized apoA-1 acquires cholesterol and phospholipids to form nascent HDL. HDL, a beneficial protein, can slow the progression of atherosclerosis by promoting the reverse transport of cholesterol from macrophages, reducing foam cell formation, and thus mitigating cholesterol efflux.

[0236] To further investigate whether changes in apoA-1 levels affect plasma high-density lipoprotein (HDL) levels, five groups of WT and IP6K1 KO male mice were selected, and plasma samples were obtained. HDL-C, LDL-C, and total cholesterol levels in the plasma were measured. Results indicated that systemic IP6K1 knockout significantly upregulated HDL levels. Figure 2 Example 2: Liver-specific knockout of IP6K1 upregulates hepatic apolipoprotein A1 and HDL protein levels.

[0237] (I) Liver-specific knockout of IP6K1 increases plasma apolipoprotein A1 protein levels.

[0238] Approximately 80% of plasma apoA-1 is secreted by the liver. To further confirm the regulatory role of IP6K1 on apolipoprotein A1, a liver-specific IP6K1 knockout assay (Alb-cre; IP6K1) was constructed. f / f Mice were tested, and the IP6K1 levels in three groups of 6-8 week old male mice were measured. f / f and Alb-cre; IP6K1 f / fWestern blot results of mouse plasma apolipoprotein A1 protein levels indicated: Alb-cre; IP6K1 f / f The protein level of mouse apolipoprotein A1 was significantly higher than that of IP6K1. f / f mice ( Figure 3 A) suggests that liver-specific knockout of IP6K1 can significantly upregulate apolipoprotein A1 levels.

[0239] (ii) Liver-specific knockout of IP6K1 increases plasma high-density lipoprotein (HDL) levels.

[0240] To further explore IP6K1 f / f and Alb-cre; IP6K1 f / f Does high-density lipoprotein (HDL) in mouse plasma also increase with increasing apolipoprotein A1? IP6K1 was measured separately. f / f and Alb-cre; IP6K1 f / f HDL-C, LDL-C, and total cholesterol in mouse plasma. Results suggest that liver-specific knockout of IP6K1 can also significantly upregulate plasma high-density lipoprotein (HDL) levels. Figure 3 B).

[0241] Example 3: IP6K1 negatively regulates the level of intracellular apolipoprotein A1 protein.

[0242] (I) Systemic knockout of IP6K1 increases apolipoprotein A1 protein levels in liver tissue.

[0243] To further investigate whether the increase in plasma apolipoprotein A1 originates from the liver, the changes in apolipoprotein A1 protein levels in the liver tissues of WT and IP6K1KO mice were examined. Western blot results showed that the protein level of apolipoprotein A1 in the liver tissues of IP6K1KO mice was significantly higher than that in WT mice. Figure 4 A) suggests that knocking out IP6K1 can upregulate the level of apolipoprotein A1 in liver tissue.

[0244] (ii) Liver-specific knockout of IP6K1 increases the level of apolipoprotein A1 protein in liver tissue.

[0245] To further confirm that the increased apolipoprotein A1 in plasma originated from hepatic secretion, IP6K1 levels were measured in three groups of 6-8 week old males. f / f and Alb-cre; IP6K1 f / f Western blot analysis of apolipoprotein A1 protein levels in mouse liver tissue showed that the protein content of apolipoprotein A1 expressed in the liver of hepatocyte-specifically knocked-out IP6K1 mice was higher than that in the control group (IP6K1). flox / flox ) mice ( Figure 4 B).

[0246] (iii) Increased apolipoprotein A1 protein levels in primary hepatocytes with IP6K1 knockout

[0247] To eliminate interference from other cell types in the liver, primary hepatocytes from WT and IP6K1 KO mice were isolated, and the protein level of apolipoprotein A1 in the liver tissue was detected. Western blot results showed that the apolipoprotein A1 protein content expressed in primary hepatocytes from IP6K1 knockout mice was higher than that in primary hepatocytes from the control group. Figure 5 A) This further demonstrates that the knockout of IP6K1 can significantly increase the expression of apolipoprotein A1 protein.

[0248] (iv) Knockout of IP6K1 in human L02 cells increases the level of apolipoprotein A1.

[0249] The above results demonstrate that IP6K1 negatively regulates apolipoprotein A1 in mice. Next, to investigate whether IP6K1 plays the same regulatory role in human hepatocytes, IP6K1 was knocked out in normal human hepatocytes using shRNA virus, and the protein level of apolipoprotein A1 in the cells was observed. The results showed that in normal human hepatocytes L02, knocking out IP6K1 significantly increased the protein level of apolipoprotein A1. Figure 5 B).

[0250] (v) Overexpression of IP6K1 reduces the protein level of apolipoprotein A1 in L02 cells.

[0251] To further verify whether overexpression of IP6K1 can also regulate apolipoprotein A1, the protein level of apolipoprotein A1 in the human hepatocyte cell line L02 was measured after overexpression of myc-GFP and myc-IP6K1 tag proteins, respectively. Western blot results showed that overexpression of IP6K1 could downregulate the intracellular protein level of apolipoprotein A1. Figure 6 ).

[0252] Example 4: IP6K1 liver-specific knockout mice can effectively alleviate the progression of atherosclerosis.

[0253] (I) Apolipoprotein A1 was still upregulated after IP6K1 liver-specific knockout mice were used to establish an atherosclerosis model.

[0254] Studies have reported that injection of exogenous apolipoprotein A1 or apolipoprotein A1 analogues can effectively reduce the size of atherosclerotic plaques in mice. Clinical studies have shown that supplementing patients with apolipoprotein A1 can effectively reduce the risk of atherosclerosis caused by coronary heart disease. Therefore, to further investigate whether apolipoprotein A1 still increases after establishing an atherosclerosis model and whether the increased apolipoprotein A1 and HDL can exert an anti-atherosclerotic effect, six groups of patients with IP6K1 antibodies for 6-8 weeks were selected. f / f and Alb-cre; IP6K1 f / f Mice were fed a Western blot of PCSK9 virus Western Diet (TD88137) via tail vein injection for 16 weeks. The protein levels of apolipoprotein A1 and HDL in mouse plasma and apolipoprotein A1 in mouse liver were measured. Western blot results showed that after establishing the atherosclerosis model, Alb-cre and IP6K1 levels were significantly increased. f / f apolipoprotein A1 in mouse plasma Figure 7 B), High-density lipoprotein (HDL) level ( Figure 7 C), the protein level of apolipoprotein A1 in mouse liver ( Figure 7 A) There is still a significant increase.

[0255] (II) Increased plasma-mediated cholesterol efflux in IP6K1 liver-specific knockout mice

[0256] Apolipoprotein A1 functions primarily by interacting with the abca1 protein on macrophages to acquire phospholipids and cholesterol, and by activating lecithin-cholesterol acyltransferase (LCAT) to achieve reverse cholesterol transport. Therefore, a kit was used to label cholesterol in J774A macrophages, and the cholesterol was then transported using IP6K1. f / f and Alb-cre; IP6K1 f / f Mouse plasma was used as an acceptor to measure cholesterol efflux capacity. The results showed that mice with IP6K1 liver-specific knockout had increased plasma-mediated cholesterol efflux capacity. Figure 8 This further illustrates that increased apoA-1 can alleviate the progression of atherosclerosis by enhancing the cholesterol efflux capacity of macrophages.

[0257] (III) The area of ​​atherosclerotic plaques was significantly reduced in mice with IP6K1 liver-specific knockout.

[0258] To investigate whether increased apolipoprotein A1 and HDL can exert an anti-atherosclerotic effect, specimens from the base of the ascending aorta to the bifurcation of the iliac artery were stained with Oil Red O. Frozen sections were prepared from the aortic root to observe the atherosclerotic lesions. Oil Red O staining results of the entire aorta indicated Alb-cre; IP6K1f / f Mice compared to IP6K1 f / f The area of ​​atherosclerotic lesions in mice was significantly reduced. Figure 9 A). Oil Red O and HE staining results for aortic root plaques also showed Alb-cre; IP6K1 f / f The area of ​​atherosclerotic plaques in mice was significantly reduced. Figure 9 B).

[0259] Example 5: The product 5PP-InsP5 of IP6K1 plays a decisive role in regulating the level of apolipoprotein A1 protein.

[0260] (a) The negative regulation of apolipoprotein A1 by IP6K1 requires its kinase activity.

[0261] To investigate the role of the IP6K1 product 5PP-InsP5 in this process, primary liver cells from IP6K1 KO mice were isolated and overexpressed with myc-gfp, IP6K1 with kinase-deficient mutation, and wild-type IP6K1. The results suggested that only wild-type IP6K1 could negatively regulate apolipoprotein A1 protein in cells (…). Figure 10 ).

[0262] (ii) Inhibiting the production of IP6K1 product 5PP-InsP5 can increase the protein level of intracellular apolipoprotein A1.

[0263] To further investigate the regulatory effect of IP6K1 product 5PP-InsP5 on apolipoprotein A1 protein levels, cells were treated with TNP / SC-919, a catalytic activity inhibitor of the IP6KS family, and changes in intracellular apolipoprotein A1 protein levels were observed. The results suggest that regardless of the level of apolipoprotein A1 in primary WT mouse hepatocytes (… Figure 11 A) or human L02 cells ( Figure 11 B Figure 11 C) Inhibition of 5PP-InsP5 production can upregulate the protein level of intracellular apolipoprotein A1.

[0264] (iii) Inhibition of the production of IP6K1 product 5PP-InsP5 can increase the protein level of apolipoprotein A1 in plasma and tissues.

[0265] To investigate whether the inhibition of IP6K1 product 5PP-InsP5 can regulate apolipoprotein A1 protein levels similarly to IP6K1 gene knockout, six groups of 6-8 week old WT male mice were selected and injected with the IP6K1 kinase activity inhibitor TNP / SC-919 and DMSO as controls, respectively. Samples were collected one week later. Western blot results showed that after injection of TNP / SC-919, the plasma of mice (… Figure 12 B Figure 13B) and liver ( Figure 12 A, Figure 13 The levels of apolipoprotein A1 in A) were significantly upregulated compared to the control group.

[0266] Example 6: IP6K1 regulates the ubiquitination and degradation of apolipoprotein A1 via 5PP-InsP5

[0267] (a) IP6K1 does not regulate the transcriptional level of apolipoprotein A1.

[0268] To investigate the molecular mechanism by which IP6K1 regulates apolipoprotein A1 protein levels, we first extracted WT(IP6K1) at the transcriptional level. f / f ) and IP6K1 KO (Alb-cre; IP6K1 f / f RNA from mouse liver tissue was analyzed, and WT(IP6K1) was detected using qPCR. f / f ) and IP6K1 KO (Alb-cre; IP6K1 f / f The expression of apolipoprotein A1 mRNA in mouse liver tissue was investigated, and the results showed that knocking out IP6K1 did not affect the transcriptional level of apolipoprotein A1. Figure 14 This result suggests that IP6K1 may regulate apolipoprotein A1 at the protein level.

[0269] (ii) IP6K1 knockout reduces ubiquitination of apolipoprotein A1.

[0270] To investigate whether IP6K1 regulates the ubiquitination and degradation of apolipoprotein A1, apolipoprotein A1 was immunoprecipitated in the liver tissues of WT and IP6K1 KO mice, and its ubiquitination level was detected. Western blot results showed that the ubiquitination modification of apolipoprotein A1 in IP6K1 KO mice was reduced compared with that in WT mice. Figure 15 A). However, after overexpression of IP6K1 in human L02 hepatocytes and inhibition of proteasome degradation by MG132, the ubiquitination level of apolipoprotein A1 protein was significantly increased. Figure 15 B).

[0271] (iii) Inhibiting the production of 5PP-InsP5 can reduce the ubiquitination modification of apolipoprotein A1.

[0272] To investigate the regulatory effect of IP6K1 product 5PP-InsP5 on the ubiquitination modification of apolipoprotein A1, human hepatocytes L02 were treated with TNP and SC-919, inhibitors of the IP6K family, and proteasome degradation was inhibited with MG132. Apolipoprotein A1 was immunoprecipitated, and its ubiquitination modification was observed. Western blot results showed that inhibiting the production of 5PP-InsP5 effectively reduced the ubiquitination level of apolipoprotein A1. Figure 16 ).

[0273] Example 7: UBE4A can interact with IP6K1 and apoA-1

[0274] To investigate how IP6K1 regulates the ubiquitination and degradation of apoA-1 protein, IP6K1 was immunoprecipitated in liver tissue. Proteomics and mass spectrometry were then used to identify proteins that might influence the ubiquitination and degradation of apoA-1. Silver staining images revealed that both IP6K1 and apoA-1 can bind to an E3 ubiquitin ligase, UBE4A, which is approximately 130 kDa in size. Figure 17 , Figure 18 ).

[0275] To further confirm the binding of IP6K1 and UBE4A, immunoprecipitation of IP6K1 and UBE4A in liver tissue confirmed their endogenous binding. Figure 19 A). To verify the exogenous binding of the two, myc-GFP, myc-IP6K1, and HA-UBE4A were overexpressed in 293A cells, and the myc-tagged protein was immunoprecipitated, confirming the exogenous binding of the two. Figure 19 B).

[0276] To further confirm the binding of apoA-1 to UBE4A, flag-GFP, flag-apoA-1, and HA-UBE4A were overexpressed in 293A cells, and flag was immunoprecipitated. Figure 20 A) and HA-tagged proteins ( Figure 20 B) further confirmed the exogenous binding of the two.

[0277] To further confirm the binding of IP6K1 and apoA-1, myc-GFP, myc-IP6K1, flag-apoA-1, and immunoprecipitated flag were overexpressed in 293A cells. Figure 21 A) and myc-tagged protein ( Figure 21 B) further confirmed the exogenous binding of the two.

[0278] Example 8: UBE4A regulates the ubiquitination and degradation of apolipoprotein A1

[0279] (a) UBE4A negatively regulates the protein level of apolipoprotein A1.

[0280] To confirm the effect of UBE4A on the ubiquitination and degradation of apolipoprotein A1, UBE4A was knocked down in the human normal hepatocyte L02 cell line using shRNA, and a stably knocked-down cell line was constructed using POO screening. Western blot analysis was performed to detect changes in apolipoprotein A1 protein expression in this cell line. The results indicated that knockdown of UBE4A increased apolipoprotein A1 protein expression. Figure 22 A). To further investigate whether overexpression of UBE4A also regulates apolipoprotein A1, UBE4A was overexpressed in human L02 hepatocytes, and changes in intracellular apolipoprotein A1 protein levels were observed. The results showed that overexpression of UBE4A downregulated intracellular apolipoprotein A1 protein levels. Figure 22 B).

[0281] (ii) UBE4A negatively regulates the ubiquitination level of apolipoprotein A1.

[0282] To further investigate whether the E3 ubiquitin ligase UBE4A is involved in the ubiquitination modification of apolipoprotein A1, UBE4A was knocked down using shRNA in L02 cells, and MG132 treatment was used to inhibit the proteasome degradation pathway. Changes in intracellular apolipoprotein A1 ubiquitination levels were observed. The results indicated that knocking down UBE4A significantly reduced apolipoprotein A1 ubiquitination levels. Figure 23 A). To further investigate whether overexpression of UBE4A also regulates the ubiquitination level of apolipoprotein A1, UBE4A was overexpressed in L02 cells and treated with MG132 to inhibit the proteasome degradation pathway. Changes in intracellular apolipoprotein A1 ubiquitination levels were observed. The results suggested that overexpression of UBE4A can upregulate the ubiquitination modification of apolipoprotein A1 protein. Figure 23 B).

[0283] Example 9: 5PP-InsP5 promotes the interaction between UBE4A and apoA-1

[0284] (i) Knocking out IP6K1 can reduce the interaction between UBE4A and apoA-1.

[0285] To further investigate how IP6K1 mediates the regulation of apoA-1 by UBE4A, primary hepatocytes from WT and IP6K1 KO mice were used, and the flag-apoA-1 tag plasmid was overexpressed. Immunoprecipitation of the flag showed that the binding of apoA-1 to UBE4A was reduced after IP6K1 KO. Figure 24A). Similarly, reverse immunoprecipitation of UBE4A also revealed that the apolipoprotein A1 precipitated by UBE4A in liver tissue samples from IP6K1 KO mice was significantly less than that in liver tissue samples from WT mice ( Figure 24 B). The results suggest that knocking out IP6K1 can reduce the binding between UBE4A and apoA-1.

[0286] (ii) Inhibiting the production of 5PP-InsP5 reduces the interaction between UBE4A and apoA-1.

[0287] To further investigate the effect of IP6K1 product 5PP-InsP5 on the interaction between UBE4A and apoA-1, apoA-1 was overexpressed in L02 cells, and the cells were treated with the IP6K family inhibitor SC-919. Immunoprecipitation of the flags revealed that the binding of apoA-1 to UBE4A was reduced in the SC-919-treated group compared to the DMSO group. Figure 25 A). Furthermore, a reverse pull-down experiment was performed using an antibody against UBE4A, and it was also found that after SC-919 treatment of cells, the binding of apoA-1 to UBE4A was significantly reduced. Figure 25 B). The above results indicate that IP6K1 can promote the tight binding of apoA-1 and UBE4A through its product 5PP-InsP5, thereby increasing its degradation.

[0288] (iii) 5-PPInsP5 can bind directly to UBE4A and apoA-1.

[0289] To investigate whether the IP6K1 product 5-PPInsP5 binds to UBE4A and apoA-1 and regulates their interaction, HA-UBE4A and flag-apoA1 proteins overexpressed in 293A cells were precipitated using a gel resin pre-bound with inositol heptaphosphate. The precipitate contained both UBE4A and apoA-1 proteins, suggesting that inositol heptaphosphate can bind to both UBE4A and apoA-1 proteins. Figure 26 A).

[0290] To further verify whether UBE4A and apoA-1 proteins directly bind to the IP6K1 product 5-PPInsP5, plasmids GST-UBE4A and GST-apoA-1 were constructed, and the proteins were purified by GSH elution. The purified proteins were then immunoprecipitated using gel resin pre-bound with inositol heptaphosphate. The results indicated that UBE4A and apoA-1 proteins can directly bind to the IP6K1 product 5-PPInsP5. Figure 26 B).

[0291] (iv) 5-PPInsP5 can promote the interaction between UBE4A and apoA-1 proteins.

[0292] To further confirm the regulatory effect of 5-PPInsP5 on the interaction between UBE4A and apoA-1 proteins, an in vitro binding assay was performed to detect whether 5-PPInsP5 mediates the binding between purified UBE4A and apoA-1 proteins. Western blot results showed that, compared with InsP6, 5-PPInsP5 significantly promoted the binding between UBE4A and apoA-1 proteins. Figure 27 ).

[0293] Two non-hydrolyzable analogs of 5-PPInsP5, 5-PCP and CF2, are known. While their physicochemical and biochemical properties are similar, neither can transfer their β-phosphate groups to the protein substrate, thus preventing substrate phosphorylation. To further investigate whether 5PCP and CF2 can regulate the interaction between UBE4A and apoA-1 proteins, in vitro binding experiments were also performed. The results showed that the treatment group with the addition of the small molecule compounds 5-PCP and CF2 showed a significant increase in the binding of UBE4A and apoA-1 proteins compared to the InsP6 treatment group. Figure 28 The 5-PPInsP5-mediated binding of UBE4A to apoA-1 increases pyrophosphorylation that does not require a protein substrate. Figure 29 ).

[0294] In summary, the results of the embodiments of this invention demonstrate that IP6K1 and its product, inositol heptaphosphate, can interact with apolipoprotein A1 and the E3 ubiquitin ligase UBE4A, promoting the binding of apolipoprotein A1 to UBE4A, leading to ubiquitination and degradation of apolipoprotein A1. Knocking out IP6K1 can break the mechanism of UBE4A ubiquitination of apolipoprotein A1, promoting its secretion into the extracellular (intravascular) space, thereby increasing reverse cholesterol transport, inhibiting vascular plaque formation, and exerting a protective effect against cardiovascular diseases (such as atherosclerosis). Therefore, IP6K1 and its product, inositol heptaphosphate, can serve as a drug target for increasing blood apolipoprotein A1 levels and as a potential therapeutic target for treating atherosclerosis.

[0295] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. The use of TNP / SC-919 in the preparation of medicaments for the prevention and / or treatment of cardiovascular diseases, characterized in that, The cardiovascular disease is atherosclerosis, and TNP / SC-919 is an inhibitor of IP6K1 kinase activity. It increases the level of apolipoprotein A1 protein in plasma and tissues by inhibiting the production of IP6K1 product 5PP-InsP5.

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