A method for preparing oxidized low-density lipoprotein and its applications

Abstract

The present invention relates to the field of biomedical detection technology, and relates to a preparation method and application of oxidized low-density lipoprotein. The preparation method of the oxidized low-density lipoprotein comprises the following steps: subjecting low-density lipoprotein to high-frequency ultrasonic treatment under the conditions of 380-430 kHz and 30-45 W, with the ultrasonic temperature being 25 °C and the ultrasonic time being 5-20 min, thereby obtaining oxidized low-density lipoprotein. This method has the advantages of being fast, efficient and environmentally friendly, significantly reducing the oxidation time while maintaining the oxidation effect.

Description

Technical Field

[0001] The present invention relates to the field of biomedical detection technologies, and relates to a preparation method and application of oxidized low-density lipoprotein. Background Art

[0002] Low-density lipoprotein is a lipoprotein particle that transports cholesterol into peripheral tissue cells and can be oxidized into oxidized low-density lipoprotein. When low-density lipoprotein, especially oxidized modified low-density lipoprotein (oxLDL), is excessive, the cholesterol it carries accumulates on the arterial wall and is likely to cause arteriosclerosis over time.

[0003] Atherosclerosis (AS) remains one of the main causes of cardiovascular diseases globally, characterized by the formation of atherosclerotic or fibrous plaques in the vascular endothelium. The main pathological mechanism involves the transformation of macrophages into foam cells. During the formation of atherosclerotic plaques, a large amount of low-density lipoprotein infiltrates into the subendothelial layer and is transformed into oxidized low-density lipoprotein. This process triggers the migration of monocytes to the subendothelium, where they differentiate into macrophages, actively phagocytize oxidized modified low-density lipoprotein, and transform into foam cells. These foam cells accumulate beneath the arterial intima, ultimately leading to the formation of AS plaques, which may trigger serious clinical complications.

[0004] Oxidized modified low-density lipoprotein is an important tool for in vitro experimental studies of atherosclerosis. In vitro models for atherosclerosis research usually involve treating macrophages with oxidized modified low-density lipoprotein to generate foam cells. Traditional methods for oxidizing low-density lipoprotein rely on divalent copper ions. For example, Patent CN101787076A discloses a preparation method of low-density lipoprotein and a preparation method of oxidized low-density lipoprotein, which is characterized in that: it includes extracting normal human serum, centrifuging with a high-speed centrifuge, removing the upper layer of chylomicrons, adjusting the density, centrifuging twice with an ultracentrifuge, taking the upper layer of low-density lipoprotein, identifying it by agarose gel electrophoresis, and then oxidizing it with an oxidant to obtain oxidized low-density lipoprotein; the catalytic oxidation is carried out with copper sulfate as a catalyst, and the concentration is 1-10 μmol / L. This method of oxidizing and preparing oxidized low-density lipoprotein using divalent copper ions as a catalyst has significant disadvantages: (1) This method takes a long time, usually requires long-term incubation, and may cause protein denaturation or degradation. (2) The introduction of heavy metal copper ions will interfere with downstream analysis and cause environmental problems due to its toxicity and non-biodegradability. (3) The oxidized low-density lipoprotein (copper ion oxidized modified low-density lipoprotein) prepared by oxidizing with divalent copper ions as a catalyst may have copper ion residues and relatively high cytotoxicity. Therefore, there is an urgent need to develop efficient and environmentally friendly methods for producing oxidized modified low-density lipoprotein, which is crucial for promoting in vitro experimental models for studying the mechanism of AS. Summary of the Invention

[0005] The object of the present invention is to provide a method for preparing oxidized low-density lipoprotein. This method obtains ultrasonically oxidized modified low-density lipoprotein by subjecting low-density lipoprotein to high-frequency ultrasonic treatment. Compared with copper ion-oxidized modified low-density lipoprotein, ultrasonically oxidized modified low-density lipoprotein can reduce cytotoxicity and shorten the incubation time. Lipidomics analysis shows that ultrasonically oxidized modified low-density lipoprotein is close to natural low-density lipoprotein, which is helpful for atherosclerosis research. Ultrasonic oxidation avoids metal ions and provides an environmentally friendly alternative to the method of copper ion-oxidized modified low-density lipoprotein.

[0006] The present invention is realized by adopting the following technical scheme:

[0007] A method for preparing oxidized low-density lipoprotein, comprising the following steps: subjecting low-density lipoprotein to high-frequency ultrasonic treatment under the conditions of 380 - 430 kHz and 30 - 45 W, with the ultrasonic temperature being 25 °C and the ultrasonic time being 5 - 20 min, to obtain oxidized low-density lipoprotein.

[0008] Preferably, in the above method for preparing oxidized low-density lipoprotein, the ultrasonic time is 10 min.

[0009] In the above method for preparing oxidized low-density lipoprotein, the method for preparing low-density lipoprotein is as follows: centrifuging plasma at 288200×g for 7 hours using an ultracentrifuge to remove the upper layer containing very low-density lipoprotein; adjusting the density of the remaining serum to 1.063 g / mL with a potassium bromide solution (density = 1.21 g / mL); subsequently, aliquoting the serum into ultracentrifuge tubes and continuing to centrifuge at 288200×g for 17 hours to collect the upper layer containing low-density lipoprotein; dialyzing the collected low-density lipoprotein in a phosphate buffer solution (PBS) containing 200 μM EDTA at 4 °C for 24 hours; then, dialyzing again with fresh phosphate buffer solution at 4 °C for 24 hours to remove EDTA and potassium bromide, thus obtaining low-density lipoprotein.

[0010] The potassium bromide solution is a potassium bromide solution with a mass percentage concentration of 0.5% - 39.7%.

[0011] The phosphate buffer solution can be a pH 6.8 sterile phosphate buffer solution, a pH 7.2 sterile phosphate buffer solution, or a pH 7.6 sterile phosphate buffer solution. After being prepared according to the buffer solution (General Rules 8004 of the Chinese Pharmacopoeia 2020 Edition), it is filtered, aliquoted, and sterilized to obtain.

[0012] In the above method for preparing oxidized low-density lipoprotein, after the low-density lipoprotein is saturated with nitrogen, it is stored at 4 °C.

[0013] To ensure the stability of the sample and maintain its biological activity, the oxidized low-density lipoprotein needs to be saturated with nitrogen and stored in a light-proof environment at 4°C.

[0014] To obtain the best experimental results, the oxidized low-density lipoprotein should be used within 1-2 weeks after preparation.

[0015] Application of the oxidized low-density lipoprotein prepared by the above method for preparing oxidized low-density lipoprotein in the preparation of oxidized low-density lipoprotein detection products.

[0016] Application of the oxidized low-density lipoprotein prepared by the above method for preparing oxidized low-density lipoprotein in the preparation of experimental reagents for atherosclerosis.

[0017] The ultrasonic oxidation of the present invention has the following beneficial effects:

[0018] (1) The present invention provides a method for preparing oxidized low-density lipoprotein using high-frequency ultrasonic technology. This method has the advantages of being fast, efficient, and environmentally friendly, significantly reducing the oxidation time while maintaining the oxidation effect.

[0019] (2) High-frequency ultrasonic cavitation can cause bubbles to burst, releasing a large amount of energy and generating local high temperature and high pressure. Under these extreme conditions, water molecules will produce highly reactive hydrogen and hydroxyl radicals, which can rapidly oxidize low-density lipoprotein. Our research has demonstrated for the first time that high-frequency ultrasonic oxidation can rapidly induce the oxidation of low-density lipoprotein. The traditional copper oxidation method requires at least 48 hours to induce the oxidation of low-density lipoprotein, while high-frequency ultrasound only requires no more than 20 minutes, significantly shortening the induction time.

[0020] (3) When using copper sulfate as an oxidant to prepare oxidized low-density lipoprotein, copper ions are likely to remain in the product. Reports have shown that divalent copper ions may promote atherosclerosis. Therefore, the oxidation modification of low-density lipoprotein with copper ions may affect subsequent experiments due to the presence of trace amounts of copper ions. In contrast, the ultrasonic oxidation modification of low-density lipoprotein prepared by high-frequency ultrasound does not introduce additional components, has low cytotoxicity, a simple and reliable procedure, and high efficiency.

[0021] (4)Comprehensive lipidomics and transcriptomics analyses showed that ultrasonically oxidized modified low-density lipoprotein (LDL) is very similar in composition to native LDL and causes much smaller molecular changes than copper-oxidized LDL. The method of preparing oxidized LDL using high-frequency ultrasonic technology reduces cytotoxicity and preserves the biological properties of oxidized LDL, making it a promising tool for preparing foam cells and studying the mechanisms of atherosclerosis. In addition, the research results highlight the conditions and effects of ultrasonically oxidized modified LDL in promoting macrophage foam cell formation and lipid accumulation. The application of high-frequency ultrasonic oxidation may provide an efficient alternative for preparing oxidized modified LDL in experimental studies of lipid metabolism. Description of the Drawings

[0022] Figure 1 Appearance diagrams of LDL before and after oxidation treatment in Example 1, Example 2, and Comparative Example 1 of the present invention.

[0023] Figure 2 Agarose electrophoresis diagrams of LDL before and after oxidation treatment.

[0024] Figure 3 Lipid oxidation level diagrams for each group (evaluated by malondialdehyde (MDA) level).

[0025] Figure 4 Cytotoxicity diagrams for RAW264.7 macrophages (cell viability was detected and evaluated using the CCK-8 method).

[0026] Figure 5 Lipidomics analysis diagrams of the lipoprotein of the non-oxidized LDL sample.

[0027] Figure 6 Lipidomics analysis diagrams of the lipoprotein of the ultrasonically oxidized LDL sample.

[0028] Figure 7 Lipidomics analysis diagrams of the lipoprotein of the copper-ion oxidized LDL sample.

[0029] Figure 8 Lipidomics concentration analysis diagrams of ultrasonically oxidized LDL vs. non-oxidized LDL.

[0030] Figure 9 Lipidomics concentration analysis diagrams of ultrasonically oxidized LDL vs. copper-ion oxidized LDL.

[0031] Figure 10Experiment on the effect of unoxidized low-density lipoprotein control on the formation of RAW264.7 foam cells, photograph of RAW264.7 cells after Oil Red O staining (the red scale bar represents 50 μm).

[0032] Figure 11 Experiment on the effect of copper ion-oxidized low-density lipoprotein (100 μg / mL) sample on the formation of RAW264.7 foam cells, photograph of RAW264.7 cells after Oil Red O staining (the red scale bar represents 50 μm).

[0033] Figure 12 Experiment on the effect of ultrasound-oxidized low-density lipoprotein (100 μg / mL) sample on the formation of RAW264.7 foam cells, photograph of RAW264.7 cells after Oil Red O staining (the red scale bar represents 50 μm).

[0034] Figure 13 Experiment on the effect of oxidized modified low-density lipoproteins from different sources on the formation of RAW264.7 foam cells, statistical chart of Oil Red O positive area.

[0035] Figure 14 Experiment on the effect of oxidized modified low-density lipoproteins from different sources on the formation of RAW264.7 foam cells, absorbance value (A520) of isopropanol extract at 520 nm.

[0036] Figure 15 Experiment on the effect of oxidized modified low-density lipoproteins from different sources on the formation of RAW264.7 foam cells, protein expression level diagram of CD36, SR-BI and SRA in RAW264.7 foam cells detected by western blot.

[0037] Figure 16 Experiment on the effect of oxidized modified low-density lipoproteins from different sources on the formation of RAW264.7 foam cells, statistical chart of optical density of CD36 in western blot bands.

[0038] Figure 17 Experiment on the effect of oxidized modified low-density lipoproteins from different sources on the formation of RAW264.7 foam cells, statistical chart of optical density of SR-BI in western blot bands.

[0039] Figure 18 Experiment on the effect of oxidized modified low-density lipoproteins from different sources on the formation of RAW264.7 foam cells, statistical chart of optical density of SRA in western blot bands.

[0040] Figure 19Experiment on the effect of unoxidized low-density lipoprotein control sample on the formation of BMDM foam cells. After the bone marrow-derived macrophage (BMDM) foam cells were treated with the sample, the photo after Oil Red O staining (the red bar indicates 50 μm).

[0041] Figure 20 Experiment on the effect of copper ion-oxidized low-density lipoprotein (100 μg / mL) sample on the formation of BMDM foam cells. After the bone marrow-derived macrophage (BMDM) foam cells were treated with the sample, the photo after Oil Red O staining (the red bar indicates 50 μm).

[0042] Figure 21 Experiment on the effect of ultrasound-oxidized low-density lipoprotein (100 μg / mL) sample on the formation of BMDM foam cells. After the bone marrow-derived macrophage (BMDM) foam cells were treated with the sample, the photo after Oil Red O staining (the red bar indicates 50 μm).

[0043] Figure 22 Experiment on the effect of oxidized modified low-density lipoproteins from different sources on the formation of BMDM foam cells. Statistical chart of Oil Red O positive area.

[0044] Figure 23 Experiment on the effect of oxidized modified low-density lipoproteins from different sources on the formation of BMDM foam cells. Absorbance value (A520) graph of isopropanol extract at 520 nm.

[0045] Figure 24 Experiment on the effect of oxidized modified low-density lipoproteins from different sources on the formation of BMDM foam cells. Graph of the protein expression levels of CD36, SR-BI, and SRA in BMDM foam cells detected by Western blotting.

[0046] Figure 25 Experiment on the effect of oxidized modified low-density lipoproteins from different sources on the formation of BMDM foam cells. Statistical chart of the optical density of CD36 in Western blot bands.

[0047] Figure 26 Experiment on the effect of oxidized modified low-density lipoproteins from different sources on the formation of BMDM foam cells. Statistical chart of the optical density of SR-BI in Western blot bands.

[0048] Figure 27 Experiment on the effect of oxidized modified low-density lipoproteins from different sources on the formation of BMDM foam cells. Statistical chart of the optical density of SRA in Western blot bands. Detailed implementation mode

[0049] The following further describes the present application in conjunction with the accompanying drawings and specific embodiments, so that those skilled in the art can better understand the present application, but it does not limit the present invention hereby.

[0050] In the embodiments of the present invention, the experimental materials and reagents used, unless otherwise specified, are all conventional consumables and reagents that can be obtained from commercial channels.

[0051] All statistical analyses involved in the embodiments of the present invention were performed using GraphPad Prism software version 8.0.2. The data are expressed as the mean ± standard error of the mean (SEM). One-way analysis of variance (ANOVA) was used for intergroup comparison. A p-value less than 0.05 was considered statistically significant.

[0052] The experimental methods without specific conditions noted in the following examples are generally carried out under conventional conditions or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0053] Unless otherwise defined, all professional and scientific terms used herein have the same meaning as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to the described content can be applied to the methods of the present invention. The preferred implementation methods and materials described herein are for illustrative purposes only.

[0054] Example 1 Isolation, purification and identification of low-density lipoprotein

[0055] 100 mL of frozen plasma extracted from healthy volunteers was thawed at 4°C. Using an ultracentrifuge (Beckman Coulter Inc., OPTiMAX PN-100), it was centrifuged at 288200×g for 7 hours to remove the upper layer containing very low density lipoprotein (VLDL). The density of the remaining serum was adjusted to 1.063 g / mL with potassium bromide solution (density = 1.21 g / mL). Subsequently, the serum was aliquoted into ultracentrifuge tubes. It was continued to be centrifuged at 288200×g for 17 hours, and the upper layer containing low-density lipoprotein was collected. The collected low-density lipoprotein was dialyzed against phosphate buffer (PBS) containing 200 μM EDTA at 4°C for 24 hours to remove potassium bromide. Then, it was dialyzed against fresh PBS at 4°C for another 24 hours to remove EDTA, and the dialysis fluid was changed every 4 hours. The dialyzed low-density lipoprotein was transferred to an EP tube, and its protein concentration was measured. After the low-density lipoprotein sample was saturated with nitrogen, it was stored at 4°C for further use within 1-2 weeks.

[0056] The acquisition of plasma samples in this study was approved by the Ethics Review Committee of the Second Affiliated Hospital of Shandong First Medical University (batch number: 2022-162), and the clinical trial registration number is ChiCTR2300077726.

[0057] Example 2: Preparation of Ultrasonic-Induced Oxidized Modified Low-Density Lipoprotein

[0058] The ultrasonic oxidized modified low-density lipoprotein was prepared by subjecting the dialyzed low-density lipoprotein obtained in Example 1 to high-frequency ultrasonic treatment at 400 kHz and an output power of 40 W for 10 min at a temperature of 25°C. After ultrasonic treatment, the sample was saturated with nitrogen to reduce further oxidative degradation. To ensure sample stability and maintain biological activity, the samples were stored in the dark at 4°C. To obtain the best experimental results, the ultrasonic oxidized modified low-density lipoprotein samples were used within 1-2 weeks after preparation to ensure the consistency and reproducibility of all experimental procedures.

[0059] Example 3: Preparation of Ultrasonic-Induced Oxidized Modified Low-Density Lipoprotein

[0060] The dialyzed low-density lipoprotein obtained in Example 1 was subjected to high-frequency ultrasonic treatment at 450 kHz and an output power of 45 W for 5 min at a temperature of 25°C. After ultrasonic treatment, the sample was saturated with nitrogen to reduce further oxidative degradation.

[0061] Example 4: Preparation of Ultrasonic-Induced Oxidized Modified Low-Density Lipoprotein

[0062] The dialyzed low-density lipoprotein obtained in Example 1 was subjected to high-frequency ultrasonic treatment at 386 kHz and an output power of 30 for 20 min at a temperature of 25°C. After ultrasonic treatment, the sample was saturated with nitrogen to reduce further oxidative degradation.

[0063] Comparative Example 1: Preparation of Copper Ion Oxidized Modified Low-Density Lipoprotein

[0064] The dialyzed low-density lipoprotein (LDL) obtained in Example 1 was transferred to a dialysis bag (8-12 kDa MWCO) and immersed in a PBS solution (pH 7.2) containing 10 μM copper sulfate, and dialyzed at 25°C in the dark for 24 hours to prepare copper ion oxidized modified low-density lipoprotein. Subsequently, the copper ion oxidized modified low-density lipoprotein was dialyzed in PBS containing 0.01% EDTA at 4°C for 24 hours to stop oxidation. Then it was dialyzed with PBS for another 24 hours to remove EDTA and residual copper sulfate, and the dialysis fluid was changed every 4 hours. The dialyzed oxidized modified low-density lipoprotein was transferred to an EP tube, sterile filtered through a 0.22 μm membrane, and its protein concentration was measured. The protein concentration was adjusted to 1 g / L, nitrogen was introduced, and the sample was stored at 4°C in the dark.

[0065] The appearance diagrams of low-density lipoprotein before and after oxidation treatment in Example 1, Example 2, and Comparative Example 1 are shown in Figure 1 . ThroughFigure 1 It can be seen that the lipoprotein sample obtained after ultracentrifugation is light yellow, and the appearance of copper ion-oxidized modified low-density lipoprotein after copper ion oxidation treatment and ultrasound-oxidized modified low-density lipoprotein after ultrasonic oxidation treatment is basically the same.

[0066] Determination of the oxidation level of oxidized modified low-density lipoprotein in Experimental Example 1

[0067] The oxidation degree of LDL was evaluated using Sudan Black B staining and agarose gel electrophoresis. LDL samples, including those before and after oxidation, were mixed with Sudan Black B at a ratio of 10:1 and incubated at 37 °C for 30 min. After centrifugation at 12,000×g for 5 min, the supernatant was analyzed by horizontal electrophoresis using 1% agarose gel, with a voltage of 120 V and a time of 30 min. The gel was photographed for further analysis. The results are shown in Figure 2 . The results showed that compared with LDL, the migration rate of ultrasound-oxidized modified low-density lipoprotein was faster, which was consistent with previous literature reports; compared with copper ion-oxidized modified low-density lipoprotein, the band of ultrasound-oxidized modified low-density lipoprotein was clearer, suggesting that there may be fewer operating steps and a relatively lower probability of protein degradation.

[0068] Experimental Example 2 Cell Counting Kit-8 (CCK-8) assay

[0069] RAW264.7 cells were seeded in a 96-well cell culture plate at a density of 10,000 cells per well and incubated overnight. The experiments included the following groups: (1) a blank group without cells; (2) a control group without added oxidized modified low-density lipoprotein; (3) an ultrasound-oxidized modified low-density lipoprotein group, with 100 μL of 50 μg / mL ultrasound-oxidized modified low-density lipoprotein added to each well; (4) a copper ion-oxidized modified low-density lipoprotein group, with 100 μL of 50 μg / mL copper ion-oxidized modified low-density lipoprotein added to each well.

[0070] Cells in all groups were incubated in a carbon dioxide incubator for 36 hours. After incubation, the old medium was removed, the cells were washed once with PBS, and each well was supplemented with 100 μL of fresh medium and 10 μL of CCK-8 solution. Subsequently, the plate was incubated for another 2 hours. The absorbance was measured at 450 nm using a microplate reader to determine cell viability. The absorbance values were recorded, and the cell viability was calculated using the following formula: Cell viability (%) = [(absorbance of the oxidized modified low-density lipoprotein group - absorbance of the blank group) / (absorbance of the control group - absorbance of the blank group)] × 100%.

[0071] The oxidation level of oxidized low-density lipoprotein was evaluated by detecting malondialdehyde (MDA). Both ultrasonically oxidized low-density lipoprotein and copper-ion oxidized low-density lipoprotein showed higher MDA levels after oxidation, with the level in the ultrasonically oxidized low-density lipoprotein group being higher, suggesting that ultrasonic oxidation is faster and more efficient than copper oxidation ( Figure 3 ). The cell viability results showed that the oxidation of ultrasonically oxidized low-density lipoprotein had lower cytotoxicity than that of copper-ion oxidized low-density lipoprotein, indicating that the ultrasonic treatment method may reduce the cytotoxicity of the sample ( Figure 4 ).

[0072] Experimental Example 3 Lipidomics of LDL Samples

[0073] Lipids were extracted from LDL samples of each group using the methyl tert-butyl ether (MTBE) method. First, an appropriate amount of internal standard lipid standard was added to the sample, and then it was homogenized with 200 µL of water and 240 µL of methanol. Subsequently, 800 µL of MTBE was added, and the mixture was sonicated for 20 min at 4 °C and then left standing for 30 min at room temperature. The solution was centrifuged at 14000×g for 15 min at 10 °C, and the upper organic solvent layer was collected and dried with nitrogen. The samples were analyzed by LC-MS / ESI-MSn using a Thermo Ultimate 3000 system equipped with an ACQUITY UPLC BEH C18 column and a Thermo Q Exactive Focus mass spectrometer (Thermo Fisher Scientific). The results are shown in Figures 5 - 9 .

[0074] As Figure 5 , Figure 6 , Figure 7 shown, 35 lipid classes were identified in all LDL samples. When comparing ultrasonically oxidized low-density lipoprotein with untreated N-LDL, the top five most abundant lipid species in both groups were similar. These included cholesterol esters (ChE), triglycerides (TG), diacylglycerols (DG), sphingomyelins (SM), and phosphatidylcholines (PC). Specifically, cholesterol esters accounted for 29.17% of N-LDL and 26.83% of ultrasonically oxidized low-density lipoprotein, triglycerides accounted for 24.73% of N-LDL and 27.68% of ultrasonically oxidized low-density lipoprotein. The proportion of diacylglycerol was 12.99% in N-LDL and 10.53% in ultrasonically oxidized low-density lipoprotein, while the proportion of sphingomyelin decreased from 5.30% in N-LDL to 4.48% in ultrasonically oxidized low-density lipoprotein.

[0075] The proportion of phosphatidylcholine in ultrasound-oxidized modified low-density lipoprotein decreased significantly to 1.73%, while it was 17.15% in N-LDL. This indicates that high-frequency ultrasound treatment mainly affects phospholipid metabolism, especially phosphatidylcholine, which plays a key role in membrane stability and lipid bilayer structure. In contrast, there are significant differences in the lipid composition of copper-ion oxidized modified low-density lipoprotein. The top five lipid species in copper-ion oxidized modified low-density lipoprotein include zymosterol (Zye, 24.81%), diglyceride (DG, 17.11%), cholesteryl ester (ChE, 14.81%), triglyceride (TG, 14.14%), and ceramide (Cer, 6.95%), and these components are significantly different from N-LDL and ultrasound-oxidized modified low-density lipoprotein. These findings highlight the unique lipidomic characteristics induced by copper oxidation, which may reflect a more extensive or stronger oxidation modification effect than ultrasound treatment.

[0076] Lipid concentration analysis showed Figure 8 、 Figure 9 。 Figure 8 、 Figure 9 showed that the level of phosphatidylglycerol (PG) increased significantly, while the levels of cholesteryl ester (ChE), triglyceride, phosphatidylcholine (PC), hexosylceramide (Hex2Cer), sterol ester (StE), and sterol lipid (ST) decreased. Previous lipidomic studies on oxidized modified low-density lipoprotein reported that the contents of phospholipid (PL), fatty acyl (FA), glycerolipid (GL), sphingolipid (SP), and sterol lipid (ST) increased in high-concentration oxidized modified low-density lipoprotein. However, our results showed that both copper oxidation and high-frequency ultrasound led to LDL oxidation, and the levels of PC, phosphatidylinositol (PI), and ST decreased compared with untreated LDL. This suggests that the degree of oxidation in previous studies may be more extensive, resulting in differences in the contents of phospholipid and sterol ester. It is necessary to further explore the specific mechanisms of lipoprotein modification and compositional changes induced by high-frequency ultrasound.

[0077] In summary, lipidomics analysis also showed that the effect of sonication on LDL composition was less than that on untreated low-density lipoproteins, indicating that high-frequency ultrasonic oxidation might have a smaller impact on lipoproteins. In the foam cells of the copper-oxidized LDL group, 28 components were altered, such as zymosterol and hexosylceramide, while in the sonication-oxidized LDL group, only 22 components changed. This indicates that sonication-oxidized modified LDL is closer to N-LDL. The above results suggest that the treatment of LDL by high-frequency ultrasonic oxidation might provide a more precise and controllable method than the traditional copper ion-induced oxidation. It can induce oxidation in a way that less changes the lipid composition, which can provide valuable insights for future studies on the role of oxidized modified LDL in various disease processes, including atherosclerosis, and may provide an alternative method for studying the cellular and molecular effects of oxidized LDL without introducing the extensive oxidative modifications caused by copper oxidation.

[0078] One of the most important research uses of oxidized LDL is to induce foam cell formation in in vitro studies. Therefore, two of the most commonly used cell types in this field of research, RAW264.7 and primary cultured bone marrow-derived macrophages (BMDMs), were used to compare the effects of sonication-oxidized modified LDL and copper ion-oxidized modified LDL on inducing foam cells.

[0079] Experimental Example 4 Foam cell induction by oxidized modified LDL

[0080] RAW264.7 cell culture: RAW264.7 cells were resuspended in 1 mL of pre-warmed medium and transferred to a culture flask containing DMEM, 10% fetal bovine serum, and 1% penicillin-streptomycin. The culture flask was placed in an incubator at 37 °C and 5% carbon dioxide, and the medium was changed regularly. Subculture was performed when the cell density reached 80 - 90% confluence.

[0081] BMDMs culture: BMDMs were isolated from the tibias and femurs of male C57BL / 6J mice and cultured in a sterile petri dish containing macrophage complete medium, which consisted of DMEM, 10% heat-inactivated fetal bovine serum (FBS), and 25 ng / mL M-CSF. The medium was changed on the third day after isolation, and the cells were seeded onto a cell culture plate on the fifth day. The cells were cultured at 37 °C in an environment of 5% carbon dioxide.

[0082] After the RAW264.7 or BMDMs cells in the culture flask adhered, they were resuspended with DMEM and then seeded at 5×10 per well 4Cells were seeded at a density of [number of cells] into 24-well plates with 1 mL of DMEM and incubated in 5% carbon dioxide at 37 °C for 2 hours. After culturing in serum-free high-glucose DMEM medium for 12 hours, 100 μg / mL of ultrasonically oxidized modified low-density lipoprotein or copper ion-oxidized modified low-density lipoprotein was added to each well. Then the plates were returned to 5% carbon dioxide and continued to be cultured at 37 °C for 36 hours in 2 culture dishes. Control wells containing only RAW264.7 or BMDM served as blank controls. After culturing, cell morphology was observed under a microscope.

[0083] Oil Red O staining for determination of lipid accumulation: Staining was performed using the Oil Red O staining method according to the instructions of a commercially available kit. Briefly, after removing the medium, the cells were washed twice with pre-cooled PBS and fixed with 4% paraformaldehyde solution for 30 min. After fixation, the cells were washed twice with PBS and differentiated with 60% isopropanol. Subsequently, the cells were stained with Oil Red O staining solution in the dark for 30 min. After washing three times with PBS, counterstaining was performed with hematoxylin for 30 seconds, and rinsed with distilled water to remove excess dye. Intracellular lipid accumulation was observed under an optical microscope as bright red lipid droplets. The cells were decolorized with 300 μL of isopropanol for 10 min, and the absorbance was measured at 520 nm to quantify the content of lipophilic dyes in the cells, reflecting the lipid content.

[0084] The results showed that macrophages were found to widely uptake and accumulate lipids to form foam cells, which appeared as red granules in the cytoplasm when stained with Oil Red O. We observed that in macrophage cell lines treated with oxidized modified low-density lipoprotein, these red granules were significantly increased, indicating enhanced lipid accumulation (see Figure 10 , Figure 11 , Figure 12 and Figure 13 ).

[0085] To verify this finding, we fixed the macrophages, extracted the lipids with isopropanol, and measured the absorbance at 520 nm (A520). The results showed that the A520 values of the two oxidized modified low-density lipoprotein treatment groups were significantly higher compared to the blank control group (see Figure 14 ). These results indicate that ultrasonically oxidized modified low-density lipoprotein, like oxidized modified low-density lipoprotein generated by other methods, can effectively induce macrophages to transform into foam cells, characterized by increased lipid uptake and accumulation.

[0086] Experimental Example 6 Western Blot Detection

[0087] Each group of cells was lysed with pre - cooled RIPA buffer at 4°C for 30 min to extract total cellular proteins. After quantifying the protein concentration using the BCA method, the proteins were denatured by heating at 95°C for 10 min. Subsequently, the proteins were separated by 10% SDS - PAGE and transferred to PVDF membranes. The membranes were blocked with Tris - buffered saline (TBST) containing 5% non - fat milk at room temperature for 2 h. After blocking, the membranes were incubated with primary antibodies overnight at 4°C. After washing three times with TBST, the membranes were incubated with the corresponding secondary antibodies for 1 h at room temperature. After washing three times again with TBST, protein bands were detected using enhanced chemiluminescence (ECL) method, and the band density was quantitatively analyzed using ImageJ software 1.53k.

[0088] Macrophages take up oxidized modified low - density lipoproteins through receptors such as CD36 and SRA, both of which are scavenger receptors involved in the endocytosis of modified lipoproteins. SR - BI plays a key role in cholesterol efflux and contributes to lipid homeostasis. During foam cell formation, RAW264.7 macrophages take up oxidized modified low - density lipoproteins through CD36 and SRA, leading to up - regulation of these receptors. As Figure 10 shown, compared with the control group, the mRNA levels of CD36, SRA, and SR - BI were significantly increased. In addition, the protein levels of CD36, SRA, and SR - BI also showed significant up - regulation (see Figure 15 , Figure 16 , Figure 17 , Figure 18 ). These results further confirmed the positive role of these key receptors in macrophage lipid accumulation during foam cell formation.

[0089] Evaluation of bone marrow - derived macrophages in Experimental Example 7

[0090] Primary cultured BMDMs can better mimic the in - vivo physiological environment than immortalized cell lines. This part evaluated the effects of sonochemically oxidized modified low - density lipoproteins on lipid accumulation and the expression of related lipid - transport proteins in BMDMs. As Figure 19 , Figure 20 , Figure 21 , Figure 22 shown, after co - incubating with sonochemically oxidized modified low - density lipoproteins or copper - oxidized modified low - density lipoproteins for 36 h, enhanced lipid accumulation was observed. To verify the results, BMDM foam cells were fixed and lipids were extracted for quantitative analysis. The results showed that the A520 value of the sonochemically oxidized modified low - density lipoprotein treatment group was significantly higher than that of the control group and the copper - oxidized modified low - density lipoprotein treatment group ( Figure 23 ). These findings indicate that sonochemically oxidized modified low - density lipoproteins, like oxidized modified low - density lipoproteins prepared by other methods, effectively induce the transformation of BMDMs into foam cells, characterized by increased lipid uptake and accumulation.

[0091] To further explore the potential mechanisms underlying foam cell formation, the expression of key cholesterol transporters in BMDM foam cells induced by sonochemically oxidized low-density lipoprotein was evaluated. Western blot analysis showed that the expression of CD36, scavenger receptor A (SRA), and SR-BI was significantly upregulated in foam cells treated with sonochemically oxidized low-density lipoprotein compared with the control group ( Figure 24 , Figure 25 , Figure 26 , Figure 27 ). These transporters are crucial for the internalization of modified lipoproteins and the efflux of cholesterol in foam cells. The upregulation of CD36, SRA, and SR-BI indicates that sonochemically oxidized low-density lipoprotein stimulates lipid accumulation in BMDMs through mechanisms similar to those observed for oxidized low-density lipoproteins from other sources.

[0092] The preferred embodiments of the present invention disclosed above are only used to help illustrate the present invention. To better highlight the innovative features of the present invention, so that those skilled in the art can well understand and utilize the present invention. The remaining technical features are not described in detail here, nor is the invention limited to the specific embodiments described.

Claims

1. A method for preparing oxidized low-density lipoprotein, characterized in that: The method comprises the following steps: subjecting low-density lipoprotein to high-frequency ultrasonic treatment under the conditions of 380-430kHz and 30-45W, the ultrasonic temperature is 25°C, and the ultrasonic time is 5-20min, so as to obtain oxidized low-density lipoprotein.

2. The method for preparing oxidized low-density lipoprotein according to claim 1, characterized in that: The ultrasonic time is 10 min.

3. The method for preparing oxidized low-density lipoprotein according to claim 1, characterized in that: The preparation method of the low-density lipoprotein is as follows: the plasma is centrifuged at 288200×g for 7 hours using an ultracentrifuge to remove the upper layer containing very low-density lipoprotein; the density of the remaining serum is adjusted to 1.063 g / mL by potassium bromide solution; then, the serum is divided into ultracentrifuge tubes, and centrifuged at 288200×g for 17 hours to collect the upper layer containing low-density lipoprotein; the collected low-density lipoprotein is dialyzed with phosphate buffer containing 200 μM EDTA at 4°C for 24 hours; thereafter, it is dialyzed again with fresh phosphate buffer at 4°C for 24 hours to remove EDTA and potassium bromide to obtain low-density lipoprotein.

Images