Application of o-acetaminophen in the preparation of lipid-lowering related products

Abstract

The invention provides an application of o-acetaminophen in preparing a lipid-lowering related product, belonging to the field of biomedicine technology; specifically, the invention provides an application of o-acetaminophen in preparing a lipid-lowering related product; a lipid-lowering related product, wherein the active ingredient comprises o-acetaminophen; the invention discovers for the first time that o-acetaminophen has the effect of lowering blood lipids, and compared with the known atorvastatin calcium with lipid-lowering activity, the lipid-lowering activity of o-acetaminophen is stronger, and the invention provides a candidate for lipid-lowering drugs, having broad market prospects.

Description

Technical Field

[0001] The invention relates to application of o-acetaminophen in preparing blood lipid lowering related products, belonging to the technical field of biomedicine. Background Art

[0002] Blood lipids are a general term for neutral fats (triglycerides) and lipids (phospholipids, glycolipids, sterols, steroids, etc.) in plasma. Hyperlipidemia, also known as hyperlipidemia, is the most common dyslipidemia in humans. It refers to lipid metabolism disorders caused by one or more lipid substances in plasma being higher than normal, and its characteristic symptoms are abnormally elevated lipid levels in plasma. Hyperlipidemia can be caused by a variety of factors, such as unreasonable eating habits and living habits, abnormal lipid metabolism and inherited gene mutations, and is the main cause of diseases such as atherosclerosis and coronary heart disease. Commonly used therapeutic drugs for hyperlipidemia are statins, niacin and fibrates. Although currently approved lipid-lowering drugs (such as statins) can reduce low-density lipoprotein cholesterol (LDL-C) in the blood, about 20% of patients with high LDL-C levels do not respond to such treatments or show intolerance to statins. Therefore, it is very necessary to develop lipid-lowering drugs based on non-statins.

[0003] Zebrafish is a newly emerged vertebrate model organism in recent years, with advantages such as in vitro fertilization, short reproductive cycle, transparent embryos, and rapid growth and development. Zebrafish have a gastrointestinal organ system similar to that of humans, which contains cell types responsible for lipid absorption and processing. During embryonic development, zebrafish obtain lipids from the yolk sac and begin to absorb external food at 5 days post fertilization (dpf), eventually replacing yolk as the main energy source. It is reported that the zebrafish genome contains important genes for human lipid metabolism, including microsomal TG transporter (mttp), fatty acid transporter (slc27a), and acyl-CoA synthetase (acsl) gene families, as well as low-density lipoprotein receptor (ldlr), and the gene expression pattern is highly conserved with humans. Studies have shown that zebrafish also show a strong response to the classic high-fat feeding diet regimen used to induce hyperlipidemia, similar to other mammalian models of dyslipidemia, and high-fat feeding can rapidly promote hyperlipidemia in zebrafish larvae. Based on the above advantages, zebrafish has become a new research model for studying hyperlipidemia and related diseases.

[0004] 2-Acetamidophenol (2-AAP), also known as 2-acetaminophenol, is a synthetic compound that can be used as an intermediate in the synthesis of the new anti-tumor drug pyrimidine mustard in the pharmaceutical industry. It is reported that 2-acetaminophen can relieve acute kidney injury, is an effective anti-inflammatory agent in an in vivo arthritis model, and is also an inducer of apoptosis in human glioblastoma cell lines in vitro. However, the current research on 2-acetaminophen in lowering blood lipids is very limited, and no reports have been reported so far. Summary of the invention

[0005] In view of the deficiencies in the prior art, the present invention provides an application of o-acetaminophen in the preparation of lipid-lowering related products.

[0006] The technical solution of the present invention is as follows:

[0007] The application of o-acetaminophen in the preparation of lipid-lowering related products, wherein the chemical structural formula of the o-acetaminophen is as shown in Formula I:

[0008]

[0009] Preferably according to the present invention, the lipid-lowering related products include health foods, special medical foods, and medicines.

[0010] Preferably, according to the present invention, o-acetaminophen is used in the preparation of medicines for treating blood lipid diseases, coronary atherosclerosis and fatty liver.

[0011] Further preferably, the blood lipid disease includes hyperlipidemia, hypercholesterolemia, hypertriglyceridemia, and combined hyperlipidemia.

[0012] Preferably according to the present invention, the drug contains one or more pharmaceutically acceptable carriers or adjuvants.

[0013] More preferably, the auxiliary agent is at least one of a sustained-release agent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption enhancer, a surfactant or a lubricant.

[0014] Preferably according to the present invention, the dosage form of the drug is capsule, pill, tablet, oral liquid, granule, tincture or injection.

[0015] A blood lipid lowering product, the active ingredient of which includes o-acetaminophen, the chemical structural formula of the o-acetaminophen is shown in Formula I.

[0016] Preferably according to the present invention, the lipid-lowering related products include health foods, special medical foods, and medicines.

[0017] Preferably according to the present invention, the lipid-lowering related product is a medicine; the medicine contains one or more pharmaceutically acceptable carriers or adjuvants.

[0018] The beneficial effects of the present invention are as follows:

[0019] The present invention is the first to discover that acetaminophen has the effect of lowering blood lipids. The present invention uses zebrafish as experimental animals for detection and finds that acetaminophen can effectively reduce the increased lipid content in the blood vessels of zebrafish caused by high-fat feeding, has a significant blood lipid-lowering effect in the body and has little toxic and side effects. The present invention provides candidates for blood lipid-lowering drugs and has broad market prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a picture of lipid accumulation in blood vessels of zebrafish treated with 80 μM acetaminophen after staining with Oil Red O;

[0021] In the figure: 0 represents the blank control group, 0.2% egg yolk powder represents the modeling group, and 80 represents the 80 μM concentration of acetaminophen treatment group.

[0022] Figure 2 This is the IOD statistical bar graph of intravascular lipids after zebrafish were treated with 80 μM acetaminophen;

[0023] In the figure: 0 represents the blank control group, 0.2% egg yolk powder represents the model group, 80 represents the 80 μM concentration of acetaminophen treatment group; ## represents P<0.01 compared with the blank control group; ** represents P<0.01 compared with the model group.

[0024] Figure 3 This is a bar graph showing the lipid-lowering rate of zebrafish treated with 80 μM acetaminophen;

[0025] In the figure: 0.2% egg yolk powder represents the modeling group, 80 represents the 80 μM acetaminophen treatment group; ** represents P < 0.01 compared with the modeling group.

[0026] Figure 4 This is a statistical bar graph of TC content in zebrafish treated with 80 μM acetaminophen;

[0027] In the figure: 0 represents the blank control group, 0.2% egg yolk powder represents the model group, 80 represents the 80 μM concentration of acetaminophen treatment group; ## represents P<0.01 compared with the blank control group; ** represents P<0.01 compared with the model group.

[0028] Figure 5 This is a statistical bar graph of TG content in zebrafish treated with 80 μM acetaminophen;

[0029] In the figure: 0 represents the blank control group, 0.2% egg yolk powder represents the model group, 80 represents the 80 μM concentration of acetaminophen treatment group; ## represents P<0.01 compared with the blank control group; ** represents P<0.01 compared with the model group.

[0030] Figure 6 This is a statistical bar graph of LDL-C content after zebrafish were treated with 80 μM acetaminophen;

[0031] In the figure: 0 represents the blank control group, 0.2% egg yolk powder represents the model group, 80 represents the 80 μM concentration of acetaminophen treatment group; ## represents P<0.01 compared with the blank control group; ** represents P<0.01 compared with the model group.

[0032] Figure 7 This is the Veen diagram of differentially expressed genes in zebrafish treated with 80 μM concentration of acetaminophen;

[0033] In the figure: C represents the blank control group, M represents the modeling group, and AAP represents the 80 μM concentration of acetaminophen-treated group.

[0034] Figure 8 This is a bar graph of differentially expressed genes in zebrafish treated with 80 μM acetaminophen;

[0035] In the figure: in the M-vs-C group, C represents the blank control group, and M represents the model group; in the AAP-vs-M group, M represents the model group, and AAP represents the 80 μM concentration of acetaminophen-treated group.

[0036] Fig. 9 RNA-Seq was used to detect the GO map of gene enrichment in zebrafish treated with 80 μM acetaminophen;

[0037] In the figure: M represents the modeling group, and AAP represents the 80 μM concentration of acetaminophen-treated group.

[0038] Fig.10 RNA-Seq detected the gene enrichment KEGG map of zebrafish treated with 80 μM concentration of o-acetaminophen;

[0039] In the figure: M represents the modeling group, and AAP represents the 80 μM concentration of acetaminophen-treated group.

[0040] Fig.11 This is a bar graph of the expression levels of genes related to the ferroptosis pathway in zebrafish treated with 80 μM acetaminophen;

[0041] In the figure: EY indicates egg yolk powder feeding, 2-AAP indicates 80 μM acetaminophen treatment; ## indicates P<0.01 compared with the blank control group; ** indicates P<0.01 compared with the modeling group.

[0042] Fig.12 This is a bar graph of the expression levels of genes related to the glutathione metabolic pathway in zebrafish treated with 80 μM concentration of o-acetaminophen;

[0043] In the figure: EY indicates egg yolk powder feeding, 2-AAP indicates 80 μM acetaminophen treatment; ## indicates P<0.01 compared with the blank control group; ** indicates P<0.01 compared with the modeling group.

[0044] Fig.13 This is a photo of lipid accumulation in blood vessels of zebrafish treated with 80 μM acetaminophen and 0.4 μM atorvastatin calcium after staining with Oil Red O;

[0045] In the figure: 0 represents the blank control group, 0.2% egg yolk powder represents the modeling group, atorvastatin calcium represents the 0.4 μM atorvastatin calcium treatment group, and 80 represents the 80 μM concentration of acetaminophen treatment group.

[0046] Fig.14 This is the statistical bar graph of intravascular lipid IOD after zebrafish were treated with 80 μM acetaminophen and 0.4 μM atorvastatin calcium;

[0047] In the figure: 0 represents the blank control group, 0.2% egg yolk powder represents the model group, atorvastatin calcium represents the 0.4 μM atorvastatin calcium treatment group, 80 represents the 80 μM concentration of acetaminophen treatment group; ## represents P<0.01 compared with the blank control group; ** represents P<0.01 compared with the model group.

[0048] Fig.15 This is a bar graph showing the lipid-lowering rate of zebrafish treated with 80 μM acetaminophen and 0.4 μM atorvastatin calcium;

[0049] In the figure: 0.2% egg yolk powder represents the modeling group, atorvastatin calcium represents the 0.4 μM atorvastatin calcium treatment group, 80 represents the 80 μM concentration of acetaminophen treatment group; ** represents P < 0.01 compared with the modeling group.

[0050] Fig.16 This is a statistical bar graph of TC content in zebrafish treated with 80 μM acetaminophen and 0.4 μM atorvastatin calcium;

[0051] In the figure: 0 represents the blank control group, 0.2% egg yolk powder represents the model group, atorvastatin calcium represents the 0.4 μM atorvastatin calcium treatment group, 80 represents the 80 μM concentration of acetaminophen treatment group; ## represents P<0.01 compared with the blank control group; ** represents P<0.01 compared with the model group.

[0052] Fig.17This is a statistical bar graph of TG content in zebrafish treated with 80 μM acetaminophen and 0.4 μM atorvastatin calcium;

[0053] In the figure: 0 represents the blank control group, 0.2% egg yolk powder represents the model group, atorvastatin calcium represents the 0.4 μM atorvastatin calcium treatment group, 80 represents the 80 μM concentration of acetaminophen treatment group; ## represents P<0.01 compared with the blank control group; ** represents P<0.01 compared with the model group.

[0054] Fig.18 This is a statistical bar graph of LDL-C after zebrafish were treated with 80 μM acetaminophen and 0.4 μM atorvastatin calcium;

[0055] In the figure: 0 represents the blank control group, 0.2% egg yolk powder represents the model group, atorvastatin calcium represents the 0.4 μM atorvastatin calcium treatment group, 80 represents the 80 μM concentration of acetaminophen treatment group; ## represents P<0.01 compared with the blank control group; ** represents P<0.01 compared with the model group. DETAILED DESCRIPTION

[0056] The technical solution of the present invention is further described below in conjunction with the embodiments and the accompanying drawings of the specification, but the protection scope of the present invention is not limited thereto.

[0057] The contents without specific conditions in the examples were carried out under conventional conditions; the reagents or instruments used without indicating the manufacturer were all common commercially available products.

[0058] Drugs and reagents:

[0059] Zebrafish were provided by the Zebrafish Drug Screening Platform of the Institute of Biology, Shandong Academy of Sciences, and can also be purchased from the National Zebrafish Resource Center. Methylene blue was purchased from Sinopharm Chemical Reagent Co., Ltd.; egg yolk powder was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; paracetamol was purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; atorvastatin calcium was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; dimethyl sulfoxide (DMSO) was purchased from Shenggong Bioengineering Co., Ltd.; paraformaldehyde fixative (neutral, PFA) was purchased from Wuhan Sewell Biotechnology Co., Ltd.; Oil Red O was purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd.; phosphate buffered saline (PBS) was purchased from Wuhan Punosai Biotechnology Co., Ltd.; isopropanol was purchased from Sinopharm Chemical Reagent Co., Ltd.; nylon 66 (Nylon) filter membrane was purchased from Tianjin Jinteng Experimental Equipment Co., Ltd.; 1,2-propylene glycol was purchased from Sinopharm Chemical Reagent Co., Ltd.; Tween 20 was purchased from Sinopharm Chemical Reagent Co., Ltd.; methylcellulose was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; the composition of E3 culture medium is CaCl 2 0.4 mM, NaCl 5 mM, MgSO4 0.16 mM, KCl 0.17 mM, prepared with deionized water, and all compounds were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0060] Experimental instruments:

[0061] Zebrafish culture system (Beijing Aisheng Technology Co., Ltd.); HPG280BX illumination incubator (Harbin Donglian Electronic Technology Development Co., Ltd.); electronic analytical balance (Hemetler-Toledo Instrument Co., Ltd.); SB-5200DT ultrasonic cleaner (Ningbo Xinzhi Biotechnology Co., Ltd., Zhejiang, China); VORTEX-5 vortex mixer (Qilin Bell Instrument Manufacturing Co., Ltd.); Zeiss AXIO zoom V16 stereo fluorescence microscope (Carl Zeiss, Germany).

[0062] Example 1

[0063] Hypolipidemic activity of 80 μM acetaminophen in zebrafish

[0064] 1. Zebrafish sample pretreatment:

[0065] Healthy, sexually mature wild-type AB zebrafish were placed in a mating tank at a ratio of 1:1 between male and female, with a partition placed in the middle and placed in a dark environment. The partition was removed before the lights were turned on the next day, and light stimulation was used to stimulate ovulation. The fish eggs were collected within 2 hours, and the fertilized eggs were disinfected and cleaned. The fertilized eggs were then transferred to clean zebrafish embryo culture water, and 0.2 ppm methylene blue was added. The clean fish culture water was replaced every day at 28°C and a 14-hour light / 10-hour dark cycle. The fish were incubated until a specific time, and healthy zebrafish fry were selected for the experiment.

[0066] 2. Acetaminophen treatment:

[0067] 5dpf zebrafish were selected and randomly divided into a blank control group, a modeling group, and an acetaminophen treatment group, with 3 parallel wells in each group and 20 fish in each well. The blank control group was not treated, and the zebrafish in the modeling group and the acetaminophen treatment group were fed with 0.2% egg yolk powder for 48 hours to establish a zebrafish hyperlipidemia model. Acetaminophen was prepared as a 100mM stock solution with DMSO and diluted with E3 when used. 3mL E3 was added to the blank control group, and Oil Red O staining was performed after 48 hours of culture in a 28℃ constant temperature incubator; 3mL E3 was added to the modeling group, and Oil Red O staining was performed after 48 hours of culture in a 28℃ constant temperature incubator; 3mL E3 was added to the acetaminophen treatment group, and acetaminophen with a final concentration of 80μM was added to the 3mL E3, and Oil Red O staining was performed after 48 hours of culture in a 28℃ constant temperature incubator. The Oil Red O staining method is as follows:

[0068] ① Fixation: Remove the zebrafish treatment solution, add PFA, and fix at room temperature for 4 hours or at 4°C overnight.

[0069] ② Dehydration: Dehydrate with 25%, 50%, 75% and 100% 1,2-propylene glycol / PBS for 10 min in sequence.

[0070] ③ Staining: Stain with 0.6% Oil Red O staining solution at 28℃ for 4h.

[0071] ④ Rehydration: Decolorize with 100%, 75%, 50% and 25% 1,2-propylene glycol / PBS for 10 min in sequence.

[0072] ⑤ Elution: Elute with PBS solution containing 0.1% Tween 20 for 15 min × 4 times.

[0073] 3. Measurement of zebrafish lipid-lowering indexes:

[0074] The lipid staining of the zebrafish tail vein was observed under a Zeiss fluorescence microscope to qualitatively determine whether the hyperlipidemia model was successfully established. The side view was used to take pictures, and the cumulative optical density (IOD) of the lipids in the tail blood vessels of each group of zebrafish was counted using Imagepro Plus 5.1. The lipid-lowering rate was calculated and statistically analyzed. Lipid-lowering rate (%) = (lipid staining intensity of the modeling group - lipid staining intensity of the acetaminophen-treated group) / lipid staining intensity of the modeling group × 100%. GraphPad Prism 8.0.2 software was used for data processing.

[0075] The experimental results are as follows Figure 1 , Figure 2 , Figure 3 As shown, compared with the blank control group, the zebrafish in the modeling group had lipid accumulation in the blood vessels, and the IOD of the tail blood vessels was significantly increased, and there were significant differences, which indicated that egg yolk powder feeding could increase the lipid content in the zebrafish blood vessels, and the modeling was successful; compared with the modeling group, 80μM acetaminophen could significantly reduce the IOD of the zebrafish tail blood vessels, and the lipid-lowering rate was significantly increased, and there were significant differences, indicating that 80μM acetaminophen could effectively improve the increased blood lipid content in zebrafish caused by egg yolk powder feeding.

[0076] Example 2

[0077] 80 μM acetaminophen improves lipid metabolism activity in zebrafish

[0078] 1. Zebrafish sample pretreatment is the same as in Example 1

[0079] 2. Acetaminophen treatment:

[0080] Zebrafish at 5 dpf after fertilization were selected and randomly divided into a blank control group, a modeling group, and an acetaminophen treatment group, with 3 parallel wells in each group and 20 fish in each well. The blank control group was not treated, and the zebrafish in the modeling group and the acetaminophen treatment group were fed with 0.2% egg yolk powder for 48 hours to establish a zebrafish hyperlipidemia model. Acetaminophen was prepared as a 100mM stock solution with DMSO and diluted with E3 when used. 3mL E3 was added to the blank control group and cultured in a 28℃ constant temperature incubator for 48 hours; 3mL E3 was added to the modeling group and cultured in a 28℃ constant temperature incubator for 48 hours; 3mL E3 was added to the acetaminophen treatment group, and acetaminophen with a final concentration of 80μM was added to the 3mL E3, and cultured in a 28℃ constant temperature incubator for 48 hours. Remove the treatment solution from each group, wash the zebrafish in each group three times with PBS, transfer them to a 1.5 mL centrifuge tube, drain the residual liquid in the tube, weigh accurately, add homogenization medium PBS at a ratio of weight (g): volume (mL) = 1:9, use a tissue cell disruptor at 10000-15000 rpm to grind into 10% tissue homogenate, centrifuge the homogenate at 3000 rpm at 4°C for 15 min, take the supernatant and measure it immediately or freeze it in a -80°C refrigerator for later use.

[0081] (1) BCA protein concentration determination

[0082] According to the instructions of the BCA protein detection kit, prepare BCA working solution and protein standards, and add each solution to a 96-well plate as a standard well. Take 2μL of each group of samples and 18μL of PBS and add them to a 96-well plate, and then add 200μL of BCA working solution to each well as a sample well. Each of the above wells is set up with 3 replicates. Place the 96-well plate in a constant temperature incubator (37°C) for 30 minutes, use an ELISA reader to measure A562, record and analyze the data of each well, and calculate the protein concentration of the sample according to the obtained standard curve formula.

[0083] (2) Determination of total cholesterol (T-CHO) content

[0084] According to the instructions of the T-CHO test kit, each solution was added to a 96-well plate, mixed, and 3 replicate wells were set up for each group. The 96-well plate was placed in a constant temperature incubator (37°C) for 10 minutes, and A510 was measured using an ELISA instrument. The data of each well was recorded and analyzed, and the T-CHO content of each group of samples was calculated according to the instructions.

[0085] (3) Determination of triglyceride (TG) content

[0086] According to the TG test kit instructions, each solution was added to a 96-well plate, mixed, and three replicate wells were set up for each group. The 96-well plate was placed in a constant temperature incubator (37°C) for 10 minutes, and A510 was measured using an ELISA instrument. The data of each well was recorded and analyzed, and the TG content of each group of samples was calculated according to the instructions.

[0087] (4) Determination of low-density lipoprotein (LDL-C) content

[0088] According to the instructions of the LDL-C test kit, add each solution to a 96-well plate and mix well. Each group has 3 replicate wells. Place the 96-well plate in a constant temperature incubator (37°C) and incubate for 5 minutes. Use an enzyme marker to measure A546, which is recorded as A1. Add 60μLR2 to each well, mix well, and then place the 96-well plate in a constant temperature incubator (37°C) and incubate for 5 minutes. Use an enzyme marker to measure A546, which is recorded as A2. Calculate the LDL-C content of each group of samples according to the instructions.

[0089] 3. Detection of factors related to zebrafish lipid content.

[0090] The experimental results are as follows Figure 4 , Figure 5 , Figure 6 As shown, compared with the blank control group, the TC, TG, and LDL-C contents of zebrafish in the modeling group were significantly increased, and there were significant differences, which indicated that egg yolk powder feeding could increase the TC, TG, and LDL-C contents in zebrafish blood vessels, and the modeling was successful; compared with the modeling group, 80 μM acetaminophen could significantly reduce the TC, TG, and LDL-C contents in zebrafish, and there were significant differences, indicating that 80 μM acetaminophen could effectively improve the increased contents of lipid metabolism-related factors in zebrafish caused by egg yolk powder feeding.

[0091] Example 3

[0092] RNA-Seq sequencing detects differential gene changes in zebrafish

[0093] 1. Zebrafish sample pretreatment is the same as in Example 1

[0094] 2. Acetaminophen treatment:

[0095] Zebrafish at 5 dpf after fertilization were selected and randomly divided into a blank control group, a modeling group, and an acetaminophen treatment group, with 3 parallel wells in each group and 20 fish in each well. The blank control group was not treated, and the zebrafish in the modeling group and the acetaminophen treatment group were fed with 0.2% egg yolk powder for 48 hours to establish a zebrafish hyperlipidemia model. Acetaminophen was prepared into a 100mM stock solution with DMSO and diluted with E3 when used. 3mL E3 was added to the blank control group and cultured in a 28℃ constant temperature incubator for 48 hours; 3mL E3 was added to the modeling group and cultured in a 28℃ constant temperature incubator for 48 hours; 3mL E3 was added to the acetaminophen treatment group, and acetaminophen with a final concentration of 80μM was added to the 3mL E3, and cultured in a 28℃ constant temperature incubator for 48 hours. The treatment solution of each group was removed, and the zebrafish of each group were washed 3 times with enzyme-free water and transferred to a 1.5mL centrifuge tube. The entire RNA-Seq sequencing and analysis process was completed by Ouyi Biotechnology Co., Ltd. (Shanghai, China).

[0096] 3. RNA-Seq sequencing results:

[0097] The statistics of the number of common and unique differentially expressed genes between different comparison groups are as follows Figure 7 As shown, there were 716 differentially expressed genes between the blank control group and the 0.2% egg yolk powder fed modeling group, and there were 459 differentially expressed genes between the modeling group and the 80 μM acetaminophen administration group, of which 173 were common differentially expressed genes. Figure 8 The figure is a histogram of differential gene expression, with red representing up-regulated differential genes and blue representing down-regulated differential genes. The results showed that compared with the blank control group, the modeling group had 738 up-regulated differential genes and 151 down-regulated differential genes. Compared with the modeling group, the drug-treated group had 355 up-regulated differential genes and 227 down-regulated differential genes. The results of GO enrichment analysis are shown in Figure 2. Fig. 9 As shown in the figure, the GO functional enrichment analysis results were sorted from small to large by P value, and the TOP10 in each category were screened out. The biological process part (BP) was highly enriched in hydrogen peroxide decomposition metabolism, cell response to exogenous stimuli, etc.; the cell component part (CC) was highly enriched in the extracellular region, hemoglobin complex, etc.; the molecular function part (MF) was highly enriched in ATPase-coupled transmembrane transporter activity, oxidoreductase activity, etc. The KEGG pathway enrichment analysis results were sorted from small to large by P value, and the TOP20 pathways were selected, such as Fig.10 As shown, the bubble size indicates the number of differentially expressed genes in the pathway. The larger the number, the larger the bubble. The color of the bubble indicates the P value of the pathway. The smaller the P value, the redder the bubble, and the more significant the pathway enrichment. The results show that acetaminophen may improve atherosclerosis through signaling pathways such as ferroptosis and glutathione metabolism.

[0098] Example 4

[0099] RT-qPCR detection of changes in related gene expression levels

[0100] 1. Zebrafish sample pretreatment is the same as in Example 1

[0101] 2. Acetaminophen treatment:

[0102] Zebrafish at 5 dpf after fertilization were selected and randomly divided into a blank control group, a modeling group, and an acetaminophen treatment group, with 3 parallel wells in each group and 20 fish in each well. The blank control group was not treated, and the zebrafish in the modeling group and the acetaminophen treatment group were fed with 0.2% egg yolk powder for 48 hours to establish a zebrafish hyperlipidemia model. Acetaminophen was prepared into a 100mM stock solution with DMSO and diluted with E3 when used. 3mL E3 was added to the blank control group and cultured in a 28℃ constant temperature incubator for 48 hours; 3mL E3 was added to the modeling group and cultured in a 28℃ constant temperature incubator for 48 hours; 3mL E3 was added to the acetaminophen treatment group, and acetaminophen with a final concentration of 80μM was added to the 3mL E3, and cultured in a 28℃ constant temperature incubator for 48 hours. The treatment solution of each group was removed, and the zebrafish of each group were washed 3 times with enzyme-free water and transferred to a 1.5mL centrifuge tube. According to the instructions, RNA extraction kit was used to extract total RNA from each group of zebrafish samples, and reverse transcription was performed using a reverse transcription kit and gradient PCR instrument to obtain cDNA from each group of zebrafish samples. Quantitative real-time RT-qPCR was performed on a 96-well plate. The operating program was: 95°C pre-denaturation for 1 minute, each cycle of denaturation at 95°C for 20 seconds, annealing at 55°C for 20 seconds, and extension at 72°C for 30 seconds, for a total of 45 cycles. Rpl13a was used as an internal reference gene. The gene primer sequences used in this article are shown in Table 1.

[0103] Table 1

[0104]

[0105]

[0106] 3. Changes in related gene expression levels:

[0107] Ferroptosis is a new type of cell death driven by iron-dependent lipid peroxidation. Glutathione peroxidase 4 (GPX4) is currently considered to be a key resistance factor to ferroptosis. The enzyme uses the prototype glutathione (GSH) in a selenium-dependent manner to catalyze the reduction reaction of lipid peroxides. Studies have found that inhibiting the ferroptosis pathway can significantly promote lipid metabolism and alleviate lipid accumulation in blood vessels.

[0108] The expression levels of mRNA of genes related to ferroptosis and glutathione metabolism signaling pathway were detected by RT-qPCR. Fig.11 , Fig.12The results showed that compared with the blank control group, the mRNA expression levels of tfr1a and tf in the modeling group were significantly increased, and compared with the modeling group, the mRNA expression levels of tfr1a and tf in the acetaminophen administration group were significantly decreased; compared with the blank control group, the mRNA expression levels of fth, fpn1, gclm, gpx4a, gpx1b, gclc, gss, and ggt1a in the modeling group were significantly decreased, and compared with the modeling group, the mRNA expression levels of fth, fpn1, gclm, gpx4a, gpx1b, gclc, gss, and ggt1a in the acetaminophen administration group were significantly increased.

[0109] Experimental Example 1

[0110] Comparison of the hypolipidemic activity of acetaminophen and atorvastatin calcium in zebrafish

[0111] 1. The pretreatment of zebrafish samples was the same as in Example 1.

[0112] 2. Treatment of zebrafish with acetaminophen and atorvastatin calcium:

[0113] Zebrafish at 5 dpf after fertilization were selected and randomly divided into blank control group, model group, atorvastatin calcium group and acetaminophen treatment group, with 3 parallel wells in each group and 20 fish in each well. The blank control group was not treated, and the zebrafish in the model group, atorvastatin calcium group and acetaminophen treatment group were fed with 0.2% egg yolk powder to establish the zebrafish hyperlipidemia model. Acetaminophen was prepared into a 100mM stock solution with DMSO and diluted with E3 when used. The blank control group was added with 3mL E3, and the cells were cultured in a 28℃ constant temperature incubator for 48 hours before Oil Red O staining; the modeling group was added with 3mL E3, and the cells were cultured in a 28℃ constant temperature incubator for 48 hours before Oil Red O staining; the atorvastatin calcium group was added with 3mL E3 with a final concentration of 0.4μM atorvastatin calcium, and the cells were cultured in a 28℃ constant temperature incubator for 48 hours before Oil Red O staining; the acetaminophen treatment group was added with 3mL E3 with a final concentration of 80μM acetaminophen, and the cells were cultured in a 28℃ constant temperature incubator for 48 hours before Oil Red O staining. The Oil Red O staining method is as follows:

[0114] ① Fixation: Remove the zebrafish treatment solution, add PFA, and fix at room temperature for 4 hours or at 4°C overnight.

[0115] ② Dehydration: Dehydrate with 25%, 50%, 75% and 100% 1,2-propylene glycol / PBS for 10 min in sequence.

[0116] ③ Staining: Stain with 0.6% Oil Red O staining solution at 28℃ for 4h.

[0117] ④ Rehydration: Decolorize with 100%, 75%, 50% and 25% 1,2-propylene glycol / PBS for 10 min in sequence.

[0118] ⑤ Elution: Elute with PBS solution containing 0.1% Tween 20 for 15 min × 4 times.

[0119] 3. The measurement of zebrafish blood lipid lowering index is the same as in Example 1.

[0120] The experimental results are as follows Fig.13 , Fig.14 , Fig.15 As shown, compared with the blank control group, the zebrafish in the modeling group had lipid accumulation in the body blood vessels, and the IOD of the tail blood vessels was significantly increased, and there were significant differences, which indicated that egg yolk powder feeding could increase the lipid content in the zebrafish blood vessels, and the modeling was successful; compared with the modeling group, atorvastatin calcium and acetaminophen could significantly reduce the IOD of the zebrafish tail blood vessels, and the lipid-lowering rate was significantly increased, and there were significant differences, among which the IOD of the acetaminophen-treated group was significantly lower than that of the atorvastatin calcium group, and the lipid-lowering rate of the acetaminophen-treated group was significantly higher than that of the atorvastatin calcium group, indicating that compared with the known atorvastatin calcium with lipid-lowering activity, acetaminophen has stronger lipid-lowering activity.

[0121] Experimental Example 2

[0122] Comparison of the effects of acetaminophen and atorvastatin calcium on improving lipid metabolism

[0123] 1. The pretreatment of zebrafish samples was the same as in Example 1.

[0124] 2. Treatment of zebrafish with acetaminophen and atorvastatin calcium:

[0125] Zebrafish at 5 dpf after fertilization were selected and randomly divided into blank control group, model group, atorvastatin calcium group and acetaminophen treatment group, with 3 parallel wells in each group and 20 fish in each well. The blank control group was not treated, and the zebrafish in the model group, atorvastatin calcium group and acetaminophen treatment group were fed with 0.2% egg yolk powder to establish the zebrafish hyperlipidemia model. Acetaminophen was prepared into a 100mM stock solution with DMSO and diluted with E3 when used. The blank control group was added with 3 mL of E3 and cultured in a constant temperature incubator at 28°C for 48 hours; the modeling group was added with 3 mL of E3 and cultured in a constant temperature incubator at 28°C for 48 hours; the atorvastatin calcium group was added with 3 mL of E3 with a final concentration of 0.4 μM atorvastatin calcium, and cultured in a constant temperature incubator at 28°C for 48 hours; the acetaminophen treatment group was added with 3 mL of E3 with a final concentration of 80 μM acetaminophen, and cultured in a constant temperature incubator at 28°C for 48 hours. Remove the treatment solution from each group, wash the zebrafish in each group three times with PBS, transfer them to a 1.5 mL centrifuge tube, drain the residual liquid in the tube, weigh accurately, add homogenization medium PBS at a ratio of weight (g): volume (mL) = 1:9, use a tissue cell disruptor at 10000-15000 rpm to grind into 10% tissue homogenate, centrifuge the homogenate at 3000 rpm at 4°C for 15 min, take the supernatant and measure it immediately or freeze it in a -80°C refrigerator for later use.

[0126] (1) BCA protein concentration determination

[0127] According to the instructions of the BCA protein detection kit, prepare BCA working solution and protein standards, and add each solution to a 96-well plate as a standard well. Take 2μL of each group of samples and 18μL of PBS and add them to a 96-well plate, and then add 200μL of BCA working solution to each well as a sample well. Each of the above wells is set up with 3 replicates. Place the 96-well plate in a constant temperature incubator (37°C) for 30 minutes, use an ELISA reader to measure A562, record and analyze the data of each well, and calculate the protein concentration of the sample according to the obtained standard curve formula.

[0128] (2) Determination of T-CHO content

[0129] According to the instructions of the T-CHO test kit, each solution was added to a 96-well plate, mixed, and 3 replicate wells were set up for each group. The 96-well plate was placed in a constant temperature incubator (37°C) for 10 minutes, and A510 was measured using an ELISA instrument. The data of each well was recorded and analyzed, and the T-CHO content of each group of samples was calculated according to the instructions.

[0130] (3) TG content determination

[0131] According to the TG test kit instructions, each solution was added to a 96-well plate, mixed, and three replicate wells were set up for each group. The 96-well plate was placed in a constant temperature incubator (37°C) for 10 minutes, and A510 was measured using an ELISA instrument. The data of each well was recorded and analyzed, and the TG content of each group of samples was calculated according to the instructions.

[0132] (4) Determination of LDL-C content

[0133] According to the instructions of the LDL-C test kit, add each solution to a 96-well plate and mix well. Each group has 3 replicate wells. Place the 96-well plate in a constant temperature incubator (37°C) and incubate for 5 minutes. Use an enzyme marker to measure A546, which is recorded as A1. Add 60μLR2 to each well, mix well, and then place the 96-well plate in a constant temperature incubator (37°C) and incubate for 5 minutes. Use an enzyme marker to measure A546, which is recorded as A2. Calculate the LDL-C content of each group of samples according to the instructions.

[0134] 3. Detection of zebrafish lipid content-related factors is the same as in Example 2

[0135] The experimental results are as follows Fig.16 , Fig.17 , Fig.18 As shown, compared with the blank control group, the TC, TG, and LDL-C contents of zebrafish in the modeling group were significantly increased, and there were significant differences, which indicated that egg yolk powder feeding could increase the TC, TG, and LDL-C contents in zebrafish blood vessels, and the modeling was successful; compared with the modeling group, atorvastatin calcium and acetaminophen could significantly reduce the TC, TG, and LDL-C contents in zebrafish, and there were significant differences, among which the TC and TG contents of the acetaminophen-treated group were significantly lower than those of the atorvastatin calcium-treated group, and the LDL-C content was comparable to that of the atorvastatin calcium group, which indicated that compared with the known atorvastatin calcium with lipid-lowering activity, acetaminophen had stronger lipid-lowering activity.

[0136] The present invention discovers for the first time that acetaminophen has a lipid-lowering effect, and compared with the known atorvastatin calcium with lipid-lowering activity, acetaminophen has a stronger lipid-lowering activity. The present invention provides a candidate for lipid-lowering drugs and has broad market prospects.

Claims

1. The use of o-acetaminophen in the preparation of a drug for treating hyperlipidemia, wherein the chemical structural formula of the o-acetaminophen is as shown in Formula I: Formula Ⅰ.

2. The use as claimed in claim 1, wherein acetaminophen is used in the preparation of a drug for treating hypercholesterolemia, hypertriglyceridemia, and mixed hyperlipidemia.

3. The use according to any one of claims 1 to 2, characterized in that: The medicine contains one or more pharmaceutically acceptable carriers or adjuvants.

4. The use according to claim 3, characterized in that The auxiliary agent is at least one of a sustained-release agent, a filler, a binder, a wetting agent, a disintegrant, a surfactant or a lubricant.

5. The use according to any one of claims 1 to 2, characterized in that: The dosage form of the medicine is capsule, pill, tablet, oral liquid, granule, tincture or injection.

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