Preparation method and application of oxidized low-density lipoprotein

Abstract

The invention relates to the technical field of biomedical detection, 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: low-density lipoprotein is subjected to high-frequency ultrasonic treatment under the conditions of 380-430 kHz and 30-45 W, the ultrasonic temperature is 25 DEG C, the ultrasonic time is 5-20 min, and the oxidized low-density lipoprotein is obtained. The method has the advantages of being rapid, efficient and environmentally friendly, the oxidation time is remarkably shortened, and meanwhile the oxidation effect is kept.

Description

Technical Field

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

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

[0003] Atherosclerosis (AS) remains one of the leading causes of cardiovascular disease worldwide and is 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, large amounts of low-density lipoproteins infiltrate the subendothelial layer and are converted into oxidized low-density lipoproteins. This process triggers the migration of monocytes into the subendothelium, where they differentiate into macrophages, actively engulf oxidatively modified low-density lipoproteins, 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] Oxidatively modified low-density lipoprotein is an important tool for in vitro experimental studies of atherosclerosis. In vitro models for studying atherosclerosis 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 method for preparing low-density lipoprotein, a method for preparing oxidized low-density lipoprotein, characterized in that it includes extracting normal human serum, centrifuging it in a high-speed centrifuge, adjusting the density after removing the upper layer of chylomicrons, centrifuging it twice in 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 catalytic oxidation with copper sulfate at a concentration of 1 to 10 μmol / L. The method of using divalent copper ions as a catalyst for oxidation to prepare oxidized low-density lipoprotein has significant disadvantages: (1) This method is time-consuming, usually requires a long incubation period, 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) Oxidized low-density lipoprotein (copper ion oxidized low-density lipoprotein) prepared by oxidation with divalent copper ions as a catalyst is relatively cytotoxic due to the possibility of copper ion residues. Therefore, there is an urgent need to develop efficient and environmentally friendly methods to produce oxidized low-density lipoprotein, which is crucial for advancing in vitro experimental models for the study of AS mechanisms. Summary of the invention

[0005] The present invention aims to provide a method for preparing oxidized low-density lipoprotein. The method obtains ultrasonic oxidized low-density lipoprotein by high-frequency ultrasonic treatment of low-density lipoprotein. Compared with copper ion oxidized low-density lipoprotein, ultrasonic oxidized low-density lipoprotein can reduce cytotoxicity and shorten incubation time. Lipidomics analysis shows that ultrasonic oxidized 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 copper ion oxidized low-density lipoprotein method.

[0006] The present invention is achieved by adopting the following technical solutions: A method for preparing oxidized low-density lipoprotein comprises the following steps: subjecting low-density lipoprotein to high-frequency ultrasonic treatment at 380-430kHz and 30-45W, with the ultrasonic temperature at 25°C and the ultrasonic time at 5-20min, to obtain oxidized low-density lipoprotein.

[0007] Preferably, in the above-mentioned method for preparing oxidized low-density lipoprotein, the ultrasonic time is 10 minutes.

[0008] In the above-mentioned method for preparing oxidized low-density lipoprotein, the method for preparing 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 (density = 1.21 g / mL); then, the serum is dispensed 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 at 4°C with phosphate buffered saline (PBS) containing 200 μM EDTA for 24 hours; thereafter, it is dialyzed again with fresh phosphate buffered saline at 4°C for 24 hours to remove EDTA and potassium bromide to obtain low-density lipoprotein.

[0009] The potassium bromide solution is a potassium bromide solution with a mass percentage concentration of 0.5%-39.7%.

[0010] The phosphate buffer can be a pH 6.8 sterile phosphate buffer, a pH 7.2 sterile phosphate buffer, or a pH 7.6 sterile phosphate buffer, which can be prepared according to the buffer solution (General Rule 8004 of the 2020 edition of the Chinese Pharmacopoeia), filtered, packaged, and sterilized.

[0011] In the above-mentioned method for preparing oxidized low-density lipoprotein, the low-density lipoprotein is saturated with nitrogen and then stored at 4°C.

[0012] In order to ensure sample stability and maintain biological activity, the oxidized low-density lipoprotein needs to be saturated with nitrogen and stored in a light-proof environment at 4°C.

[0013] In order to obtain the best experimental results, the oxidized low-density lipoprotein was used within 1-2 weeks after preparation.

[0014] The use of the oxidized low-density lipoprotein prepared by the above-mentioned method for preparing oxidized low-density lipoprotein in the preparation of oxidized low-density lipoprotein detection products.

[0015] The oxidized low-density lipoprotein prepared by the above-mentioned preparation method is used in the preparation of atherosclerosis experimental reagents.

[0016] The ultrasonic oxidation method of the present invention has the following beneficial effects: (1) The present invention provides a method for preparing oxidized low-density lipoprotein using high-frequency ultrasonic technology. The method has the advantages of being fast, efficient and environmentally friendly, significantly reducing the oxidation time while maintaining the oxidation effect.

[0017] (2) High-frequency ultrasonic cavitation can cause bubbles to collapse, releasing a large amount of energy and generating local high temperature and high pressure. Under these extreme conditions, water molecules produce highly reactive hydrogen and hydroxyl radicals that can quickly oxidize low-density lipoprotein. Our study demonstrated for the first time that high-frequency ultrasonic oxidation can quickly induce low-density lipoprotein oxidation. Traditional copper oxidation methods require at least 48 hours to induce low-density lipoprotein oxidation, while high-frequency ultrasound only takes no more than 20 minutes, significantly shortening the induction time.

[0018] (3) When copper sulfate is used as an oxidant to prepare oxidized low-density lipoprotein, copper ions are likely to remain in the product. There are reports that divalent copper ions may promote atherosclerosis. Therefore, copper ion oxidation modification of low-density lipoprotein may affect subsequent experiments due to the presence of trace amounts of copper ions. Ultrasonic oxidation modification of low-density lipoprotein prepared by high-frequency ultrasound does not introduce additional components, has low cytotoxicity, simple and reliable steps, and high efficiency.

[0019] (4) Comprehensive lipidomic and transcriptomic analyses revealed that ultrasound-oxidized LDL was very similar in composition to native LDL and caused much less molecular changes than copper-oxidized LDL. The method of preparing oxidized LDL using high-frequency ultrasound technology reduces cytotoxicity and retains the biological properties of oxidized LDL, making it a promising tool for preparing foam cells and studying the mechanisms of atherosclerosis. In addition, the results highlight the conditions and effects of ultrasound-oxidized 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 LDL in experimental studies of lipid metabolism. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The following are the appearance diagrams of low-density lipoprotein before and after oxidation treatment of Example 1, Example 2 and Comparative Example 1 of the present invention.

[0021] Figure 2 Agarose electrophoresis of low-density lipoprotein before and after oxidation treatment.

[0022] Figure 3 Figure 2 shows the lipid oxidation level (assessed by malondialdehyde (MDA) level) for each group.

[0023] Figure 4 The figure shows the cytotoxic effect on RAW264.7 macrophages (CCK-8 method was used to detect and evaluate cell survival rate).

[0024] Figure 5 This is the lipidomic analysis of lipoproteins in the non-oxidized low-density lipoprotein sample.

[0025] Figure 6 Lipidomic analysis of lipoproteins in a low-density lipoprotein sample oxidized by sonication.

[0026] Figure 7 Lipidomic analysis of lipoproteins in a low-density lipoprotein sample oxidized by copper ions.

[0027] Figure 8 This is a lipidomic concentration analysis diagram of ultrasonically oxidized low-density lipoprotein vs. non-oxidized low-density lipoprotein.

[0028] Fig. 9 This is a lipidomic concentration analysis diagram of ultrasound-oxidized low-density lipoprotein vs. copper-ion-oxidized low-density lipoprotein.

[0029] Fig.10 This is a photo of RAW264.7 cells stained with Oil Red O (the red scale represents 50 μm) showing the effect of unoxidized low-density lipoprotein control on RAW264.7 foam cell formation.

[0030] Fig.11 This is an experiment on the effect of copper ion-oxidized low-density lipoprotein (100 μg / mL) samples on the formation of RAW264.7 foam cells. The photos of RAW264.7 cells after Oil Red O staining (the red scale represents 50 μm).

[0031] Fig.12 This is an experiment on the effect of ultrasonically oxidized low-density lipoprotein (100 μg / mL) samples on the formation of RAW264.7 foam cells. The photograph of RAW264.7 cells after Oil Red O staining (the red scale represents 50 μm).

[0032] Fig.13This is a statistical diagram of the Oil Red O-positive area in an experiment on the effects of oxidatively modified low-density lipoproteins from different sources on the formation of RAW264.7 foam cells.

[0033] Fig.14 This is an experiment to test the effect of oxidized low-density lipoprotein from different sources on the formation of RAW264.7 foam cells. The absorbance value of the isopropanol extract at 520nm (A520).

[0034] Fig.15 To experiment the effects of oxidatively modified low-density lipoproteins from different sources on the formation of RAW264.7 foam cells, the protein expression levels of CD36, SR-BI and SRA in RAW264.7 foam cells were detected by western blotting.

[0035] Fig.16 This is a statistical graph of CD36 optical density of Western blot bands to test the effects of oxidized low-density lipoproteins from different sources on the formation of RAW264.7 foam cells.

[0036] Fig.17 This is the SR-BI optical density statistical diagram of Western blot bands for the experiment on the effect of oxidized low-density lipoprotein from different sources on the formation of RAW264.7 foam cells.

[0037] Fig.18 This is a statistical graph of the SRA optical density of Western blot bands to test the effects of oxidatively modified low-density lipoproteins from different sources on the formation of RAW264.7 foam cells.

[0038] Fig.19 This is an experiment on the effect of unoxidized low-density lipoprotein control samples on BMDM foam cell formation. The bone marrow-derived macrophage (BMDM) foam cells were treated with the samples and stained with Oil Red O (the red bar represents 50 μm).

[0039] Fig. 20 This is an experiment on the effect of copper ion-oxidized low-density lipoprotein (100 μg / mL) samples on BMDM foam cell formation. The bone marrow-derived macrophage (BMDM) foam cells were treated with samples and then stained with Oil Red O (the red bar represents 50 μm).

[0040] Fig.21 This is an experiment on the effect of ultrasonically oxidized low-density lipoprotein (100 μg / mL) samples on BMDM foam cell formation. Bone marrow-derived macrophage (BMDM) foam cells were treated with samples and then stained with Oil Red O (the red bar represents 50 μm).

[0041] Fig. 22This is a statistical diagram of the Oil Red O positive area in an experiment on the effects of oxidatively modified low-density lipoprotein from different sources on BMDM foam cell formation.

[0042] Fig.23 This is a graph showing the absorbance value (A520) of the isopropanol extract at 520 nm for an experiment on the effects of oxidatively modified low-density lipoproteins from different sources on the formation of BMDM foam cells.

[0043] Fig.24 To test the effect of oxidatively modified low-density lipoprotein from different sources on the formation of BMDM foam cells, the expression levels of CD36, SR-BI and SRA proteins in BMDM foam cells were detected by Western blotting.

[0044] Fig.25 This is a statistical graph of CD36 optical density of Western blot bands for the experiment on the effect of oxidatively modified low-density lipoprotein from different sources on BMDM foam cell formation.

[0045] Fig.26 This is the SR-BI optical density statistical diagram of Western blot bands for the experiment on the effect of oxidative modified low-density lipoprotein from different sources on BMDM foam cell formation.

[0046] Fig. 27 This is a statistical graph of the SRA optical density of Western blot bands to test the effects of oxidatively modified low-density lipoproteins from different sources on BMDM foam cell formation. DETAILED DESCRIPTION

[0047] The present application will be further described below in conjunction with the accompanying drawings and specific implementation methods so that those skilled in the art can better understand the present application, but the present invention is not limited thereto.

[0048] Unless otherwise specified, the experimental materials and reagents used in the examples of the present invention are all consumables and reagents that can be obtained from conventional commercial channels.

[0049] All statistical analyses involved in the embodiments of the present invention were performed using GraphPad Prism software version 8.0.2. Data are presented as mean ± standard error of the mean (SEM). One-way analysis of variance (ANOVA) was used for comparison between groups. A p value less than 0.05 was considered statistically significant.

[0050] The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are calculated by weight.

[0051] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the methods of the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only.

[0052] Example 1 Isolation, purification and identification of low-density lipoprotein 100 mL of frozen plasma extracted from healthy volunteers was thawed at 4°C. The upper layer containing very low-density lipoprotein (VLDL) was removed by centrifugation at 288200 × g for 7 hours using an ultracentrifuge (Beckman Coulter Inc., OPTiMAX PN-100). The density of the remaining serum was adjusted to 1.063 g / mL by potassium bromide solution (density = 1.21 g / mL). Subsequently, the serum was dispensed into ultracentrifuge tubes. Centrifugation was continued at 288200 × g for 17 hours, and the upper layer containing low-density lipoprotein was collected. The collected low-density lipoprotein was dialyzed for 24 hours at 4°C with phosphate buffered saline (PBS) containing 200 μM EDTA to remove potassium bromide. After that, the EDTA was removed by dialyzing again with new PBS at 4°C for 24 hours, and the dialysate was replaced every 4 hours. The dialyzed low-density lipoprotein was transferred to an EP tube, and its protein concentration was measured. After saturation with nitrogen, LDL samples were stored at 4 °C for further use within 1-2 weeks.

[0053] The acquisition of plasma samples for this study was approved by the Ethics Review Committee of the Second Affiliated Hospital of Shandong First Medical University (approval number: 2022-162), and the clinical trial registration number is ChiCTR2300077726.

[0054] Example 2: Preparation of Ultrasonic-Induced Oxidatively Modified Low-Density Lipoprotein Ultrasonic oxidation-modified low-density lipoprotein is prepared by subjecting the dialyzed low-density lipoprotein obtained in Example 1 to high-frequency ultrasonic treatment at 400kHz, an output power of 40W, a duration of 10min, and a temperature of 25°C. After ultrasonic treatment, the sample is saturated with nitrogen to reduce further oxidative degradation. In order to ensure sample stability and maintain biological activity, the samples are stored in a light-proof environment at 4°C. In order to obtain the best experimental results, the ultrasonic oxidation-modified low-density lipoprotein samples are used within 1-2 weeks after preparation to ensure the consistency and repeatability of all experimental procedures.

[0055] Example 3: Preparation of Ultrasonic-Induced Oxidatively Modified Low-Density Lipoprotein The dialyzed low-density lipoprotein obtained in Example 1 was subjected to high-frequency ultrasonic treatment at 450 kHz, an output power of 45 W, a duration of 5 min, and a temperature of 25° C. After the ultrasonic treatment, the sample was saturated with nitrogen to reduce further oxidative degradation.

[0056] Example 4: Preparation of Ultrasonic-Induced Oxidatively Modified Low-Density Lipoprotein The dialyzed low-density lipoprotein obtained in Example 1 was subjected to high-frequency ultrasonic treatment at 386 kHz, an output power of 30, a duration of 20 minutes, and a temperature of 25° C. After the ultrasonic treatment, the sample was saturated with nitrogen to reduce further oxidative degradation.

[0057] Comparative Example 1: Preparation of copper ion oxidatively modified low-density lipoprotein 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, dialyzed at 25°C for 24 hours in the dark to prepare copper ion oxidized modified low-density lipoprotein, and then 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. After that, it was dialyzed with PBS for another 24 hours to remove EDTA and residual copper sulfate, and the dialysate was replaced every 4 hours. The dialyzed oxidized modified low-density lipoprotein was transferred to an EP tube, aseptically filtered through a 0.22 μm membrane, and its protein concentration was determined. The protein concentration was adjusted to 1 g / L, nitrogen was introduced, and the sample was stored at 4°C in the dark.

[0058] The appearance of low density lipoprotein before and after oxidation treatment of Example 1, Example 2 and Comparative Example 1 is shown in Figure 1 .pass Figure 1 It can be seen that the lipoprotein samples obtained after ultracentrifugation are light yellow, and the appearance of the copper ion oxidized low-density lipoprotein after copper ion oxidation treatment and the ultrasonic oxidized low-density lipoprotein after ultrasonic oxidation treatment are basically the same.

[0059] Experimental Example 1 Determination of the oxidation level of oxidatively modified low-density lipoprotein Sudan Black B staining and agarose gel electrophoresis were used to assess the degree of LDL oxidation. LDL samples, including samples 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 at 120 V for 30 min. The gel was photographed for further analysis. Results are shown in Figure 2The results showed that compared with LDL, the migration rate of low-density lipoprotein modified by ultrasound oxidation was faster, which was consistent with previous literature reports; compared with low-density lipoprotein modified by copper ion oxidation, the band of low-density lipoprotein modified by ultrasound oxidation was clearer, suggesting that there may be fewer operation steps and the probability of protein degradation is relatively low.

[0060] Experimental Example 2 Cell Counting Kit-8 (CCK-8) Test RAW264.7 cells were seeded at a density of 10,000 cells per well in a 96-well cell culture plate and incubated overnight. The experiment included the following groups: (1) a blank group without cells; (2) a control group without the addition of oxidized low-density lipoprotein; (3) an ultrasonic oxidized low-density lipoprotein group, in which 100 μL of 50 μg / mL ultrasonic oxidized low-density lipoprotein was added to each well; (4) a copper ion oxidized low-density lipoprotein group, in which 100 μL of 50 μg / mL copper ion oxidized low-density lipoprotein was added to each well.

[0061] Cells in all groups were incubated in a carbon dioxide incubator for 36 hours. After incubation, the old culture medium was removed, the cells were washed once with PBS, and 100 μL of fresh culture medium and 10 μL of CCK-8 solution were added to each well. 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 cell viability was calculated by the following formula: Cell viability (%) = [(absorbance of oxidized low-density lipoprotein group - absorbance of blank group) / (absorbance of control group - absorbance of blank group)] × 100%.

[0062] Malondialdehyde (MDA) was detected to evaluate the oxidation level of oxidized low-density lipoprotein. Both ultrasound-oxidized low-density lipoprotein and copper-oxidized low-density lipoprotein showed higher MDA levels after oxidation, among which the ultrasound-oxidized low-density lipoprotein group had a higher level, indicating that ultrasound oxidation is faster and more efficient than copper oxidation ( Figure 3 ). The cell viability results showed that the oxidation of low-density lipoprotein modified by ultrasound oxidation had lower cytotoxicity, which was better than that of low-density lipoprotein modified by copper ion oxidation, which indicated that the use of ultrasonic treatment method may reduce the cytotoxicity of samples ( Figure 4 ).

[0063] Experimental Example 3 Lipidomics of LDL Samples The methyl tert-butyl ether (MTBE) method was used to extract lipids from LDL samples of each group. First, an appropriate amount of internal standard lipid standard was added to the sample, and then it was homogenized with 200µL water and 240µL methanol. After that, 800µL MTBE was added, and the mixture was ultrasonically treated at 4°C for 20 minutes, and then allowed to stand at room temperature for 30 minutes. The solution was centrifuged at 14000×g for 15 minutes at 10°C, and the upper organic solvent 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 BEHC18 column and a Thermo Q Exactive Focus mass spectrometer (Thermo Fisher Scientific). The results are shown in Figure 5-Figure 9 .

[0064] like Figure 5 , Figure 6 , Figure 7 As shown, a total of 35 lipid classes were identified in all LDL samples. When comparing ultrasound-oxidized LDL to untreated N-LDL, the top five abundant lipid classes were similar in both groups. These included cholesterol esters (ChE), triglycerides (TG), diacylglycerols (DG), sphingomyelin (SM), and phosphatidylcholine (PC). Specifically, cholesterol esters accounted for 29.17% of N-LDL and 26.83% of ultrasound-oxidized LDL, and triglycerides accounted for 24.73% of N-LDL and 27.68% of ultrasound-oxidized LDL. The proportion of diacylglycerols was 12.99% in N-LDL and 10.53% in ultrasound-oxidized LDL, while the proportion of sphingomyelin decreased from 5.30% in N-LDL to 4.48% in ultrasound-oxidized LDL.

[0065] The proportion of phosphatidylcholine in ultrasound-oxidized LDL was significantly reduced to 1.73%, while it was 17.15% in N-LDL. This suggests 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, the lipid composition of copper-oxidized LDL was significantly different. The top five lipid species in copper-oxidized LDL included yeast sterol (Zye, 24.81%), diacylglycerol (DG, 17.11%), cholesterol ester (ChE, 14.81%), triglyceride (TG, 14.14%) and ceramide (Cer, 6.95%), which were significantly different from N-LDL and ultrasound-oxidized LDL. These findings highlight the unique lipidome characteristics induced by copper oxidation, which may reflect a more extensive or stronger oxidative modification than ultrasound treatment.

[0066] Lipid concentration analysis showed Figure 8 , Fig. 9 . Figure 8 , Fig. 9 The results showed that phosphatidylglycerol (PG) levels were significantly increased, while cholesterol ester (ChE), triglyceride, phosphatidylcholine (PC), hexosylceramide (Hex2Cer), sterol ester (StE), and sterol lipid (ST) levels were decreased. Previous lipidomics studies on oxidatively modified LDL reported that the contents of phospholipids (PL), fatty acyl (FA), glycerolipids (GL), sphingolipids (SP), and sterol lipids (ST) were increased in high concentrations of oxidatively modified LDL. However, our results showed that both copper oxidation and high-frequency ultrasound caused 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 phospholipids and sterol esters. It is necessary to further explore the specific mechanisms of lipoprotein modification and composition changes induced by high-frequency ultrasound.

[0067] In summary, lipidomics analysis also showed that ultrasound treatment had less effect on LDL composition than untreated LDL, suggesting that high-frequency ultrasound oxidation may have less effect on lipoproteins. In the foam cells of the copper-oxidized LDL group, 28 components were altered, such as yeast sterol and hexosylceramide, while only 22 components were changed in the ultrasound-oxidized LDL group. This suggests that ultrasound-oxidized LDL is closer to N-LDL. The above results suggest that high-frequency ultrasound oxidation of LDL may provide a more precise and controllable method than traditional copper ion-induced oxidation. The ability to induce oxidation in a way that changes lipid composition less may provide valuable insights into the future study of the role of oxidatively modified LDL in a variety of 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.

[0068] One of the main research uses of oxidized low-density lipoprotein is to induce foam cell formation in in vitro studies. Therefore, the two most commonly used cell types in this field, RAW264.7 and primary cultured bone marrow-derived macrophages (BMDMs), were used to compare the effects of ultrasound-induced oxidative modification of low-density lipoprotein and copper-ion-induced oxidative modification of low-density lipoprotein on inducing foam cells.

[0069] Experimental Example 4 Oxidative modification of low-density lipoprotein induces foam cells RAW264.7 cell culture: RAW264.7 cells were resuspended in 1 mL of pre-warmed culture 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 culture medium was changed regularly. Cells were passaged when the cell density reached 80-90% confluence.

[0070] BMDMs culture: BMDMs were isolated from the tibia and femur of male C57BL / 6J mice and cultured in sterile culture dishes containing complete macrophage 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 on cell culture plates on the fifth day. The cells were cultured at 37°C in a 5% carbon dioxide environment.

[0071] After the RAW264.7 or BMDMs cells in the culture flask adhered to the wall, they were resuspended in DMEM and then plated at 5 × 10 cells per well. 4 The cells were inoculated into 24-well plates at a density of 1 mL DMEM and incubated at 37°C for 2 hours in 5% carbon dioxide. After culturing in serum-free high-glucose DMEM medium for 12 hours, 100 μg / mL ultrasonic oxidized low-density lipoprotein or copper ion oxidized low-density lipoprotein was added to each well. The plate was then returned to 5% carbon dioxide and two culture dishes were cultured at 37°C for 36 hours. The control wells containing only RAW264.7 or BMDM were used as blank controls. After culture, the cell morphology was observed under a microscope.

[0072] Oil Red O staining method for fat accumulation: Staining was performed using Oil Red O staining according to the instructions of the commercially available kit. Briefly, after removing the culture 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, they were stained with Oil Red O staining solution for 30 min in the dark. After washing three times with PBS, they were counterstained with hematoxylin for 30 seconds and rinsed with distilled water to remove excess dye. Intracellular lipid accumulation was observed under an optical microscope, which appeared as bright red lipid droplets. The cells were destained with 300 μL of isopropanol for 10 min, and the absorbance was measured at 520 nm to quantify the intracellular lipophilic dye content, which reflects the lipid content.

[0073] Results showed that macrophages were found to extensively take up and accumulate lipids to form foam cells, which appeared as red granules in the cytoplasm when stained with Oil Red O. We observed a significant increase in these red granules in macrophage cell lines treated with oxidatively modified low-density lipoprotein, indicating enhanced lipid accumulation (see Fig.10 , Fig.11 , Fig.12 and Fig.13 ).

[0074] To verify this finding, we fixed the macrophages, extracted lipids with isopropanol, and measured the absorbance at 520 nm (A520). The results showed that the A520 values ​​of the two oxidized LDL-treated groups were significantly higher than those of the blank control group (see Fig.14 ). These results indicate that ultrasound-oxidized LDL, like oxidized LDL generated by other methods, can effectively induce the transformation of macrophages into foam cells, which is characterized by increased lipid uptake and accumulation.

[0075] Experimental Example 6: Immunoblotting Each group of cells was lysed with pre-cooled RIPA buffer at 4°C for 30 min to extract total cellular protein. After quantifying protein concentration using the BCA method, the protein was denatured by heating at 95°C for 10 min. Subsequently, the protein was separated by 10% SDS-PAGE and transferred to a PVDF membrane. The membrane was blocked with Tris-buffered saline (TBST) containing 5% skim milk at room temperature for 2 h. After blocking, the membrane was incubated with the primary antibody overnight at 4°C. After three TBST washes, the membrane was incubated with the corresponding secondary antibody at room temperature for 1 h. After washing with TBST three times again, the protein bands were detected by enhanced chemiluminescence (ECL) and the band density was quantitatively analyzed using ImageJ software 1.53k.

[0076] Macrophages take up oxidized modified LDL via 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 LDL via CD36 and SRA, leading to the upregulation of these receptors. Fig.10 As shown in Figure 2, the mRNA levels of CD36, SRA, and SR-BI were significantly increased compared with the control group. In addition, the protein levels of CD36, SRA, and SR-BI also showed significant upregulation (see Figure 2). Fig.15 , Fig.16 , Fig.17 , Fig.18 ). These results further confirm the active role of these key receptors in macrophage lipid accumulation during foam cell formation.

[0077] Experimental Example 7 Evaluation of bone marrow-derived macrophages Primary cultured BMDMs can better simulate the in vivo physiological environment than immortalized cell lines. This part evaluates the effect of ultrasound-induced oxidation modification of low-density lipoprotein on lipid accumulation and expression of related lipid transport proteins in BMDMs. Fig.19 , Fig. 20 , Fig.21 , Fig. 22 As shown, enhanced lipid accumulation was observed after 36 hours of co-incubation with ultrasound-oxidized low-density lipoprotein or copper-ion-oxidized low-density lipoprotein. To verify the results, BMDMs foam cells were fixed and lipids were extracted for quantitative analysis. The results showed that the A520 value of the ultrasound-oxidized low-density lipoprotein treatment group was significantly higher than that of the control group and the copper-ion-oxidized low-density lipoprotein treatment group ( Fig.23 ). These findings suggest that ultrasound-oxidized LDL, like oxidized LDL prepared by other methods, effectively induces BMDM transformation into foam cells, which is characterized by increased lipid uptake and accumulation.

[0078] To further explore the potential mechanism of foam cell formation, the expression of key cholesterol transport proteins in BMDM foam cells induced by ultrasound-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 ultrasound-oxidized low-density lipoprotein compared with the control group ( Fig.24 , Fig.25 , Fig.26 , Fig. 27 ). These transporters are essential for the internalization of modified lipoproteins and the efflux of cholesterol in foam cells. The upregulation of CD36, SRA, and SR-BI suggests that ultrasound-induced oxidative LDL stimulates BMDM to accumulate lipids through a mechanism similar to that observed for oxidative LDL from other sources.

[0079] The preferred embodiments of the present invention disclosed above are only used to help explain the present invention. The innovative features of the present invention are better highlighted, so that those skilled in the art can better understand and use the present invention. The remaining technical features are not described in detail here, nor are 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.

4. Use of oxidized low-density lipoprotein prepared by the method for preparing oxidized low-density lipoprotein according to any one of claims 1 to 3 in preparing an oxidized low-density lipoprotein detection product.

5. Use of the oxidized low-density lipoprotein prepared by the method for preparing oxidized low-density lipoprotein according to any one of claims 1 to 3 in preparing an atherosclerosis experimental reagent.

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