Short peptide rapid screening method for inhibiting foaming of macrophages

Abstract

The invention discloses a method for screening fat-soluble oligopeptides for inhibiting foaming of macrophages. The method comprises the following steps: step 1, selecting fat-soluble oligopeptides from black bean protein hydrolysate to form a theoretical database; the method comprises the following steps: carrying out molecular docking on fat-soluble oligopeptides and key targets of macrophages, and taking primarily screened oligopeptides with higher molecular docking score; and 2, staining lipid droplets in the cells by using Nile red, and further screening the inhibition effect of the oligopeptide by using a fluorescence microplate reader. And re-screening oligopeptides with good inhibition effects according to selection. And 3, detecting CE / TC values of the cells so as to screen the oligopeptide with the best inhibition effect, and finally observing the screening result by oil red O staining. Compared with a traditional oil red O dyeing method, the screening method of the oligopeptide drugs has the advantages of reducing subjectivity, being low in cost and large in screening amount. The invention provides a feasible method for screening oligopeptide drugs.

Description

Technical Field

[0001] The invention relates to a method for quickly screening short peptides that inhibit macrophage foaming, and belongs to the technical field of biomedicine. Background Art

[0002] Macrophage foaming is an important sign of the formation of atherosclerotic cardiovascular diseases, and inhibiting macrophage foaming is an important target for the treatment of cardiovascular diseases. At present, there is no scientific and efficient screening method for drugs that inhibit macrophage foaming. The existing drug screening methods are mainly Oil Red O staining photography and cholesterol content detection, but these methods inevitably have the problems of large cell dosage, large consumables consumption, and high cost. In addition, the screening of basic characterization does not target the cell intervention mechanism. This creates certain difficulties for the exploration of drug intervention mechanisms.

[0003] Virtual screening is an economical and fast computer-aided drug design method. It has been widely used in the field of drug design. Molecular docking technology uses computer technology to study the interaction between small molecule compounds and protein receptors, and scores and sorts them according to the binding mode and affinity. Molecular docking can score and screen the compounds to be screened in a very short time. For the selection of targets, important targets that have an important regulatory effect on macrophage foaming should be selected. The ferroportin FPN can regulate the expression of cholesterol efflux proteins by regulating the expression of liver X receptor α (LXRα) to inhibit macrophage foaming. Therefore, FPN protein can be selected as a docking target. Due to the limited simulation conditions of virtual screening, the experimental results often fail to meet the expected requirements. Therefore, further experimental screening of the virtual screening structure can effectively improve the success rate of drug design.

[0004] Oil Red O is a common lipid dye that can stain lipids accumulated in cells and observe lipid distribution under a microscope to reflect the effect of drugs. However, cells stained with Oil Red O usually use 24-well plates or 12-well plates for sample preparation, which has the defects of large reagent consumption, strong selectivity in the observation area, and inability to conduct batch experiments. Nile red is a fluorescent dye that can specifically bind to lipids, and has a good linear relationship between fluorescence intensity and lipid content. It has the advantages of high throughput, low pollution, high efficiency and simplicity. Nile red staining has been used to quantitatively detect the intracellular lipid content of oil-producing algae. Lipid accumulation in foam cells is mainly in the form of cholesterol esters (CE), free cholesterol has normal cell function, and total cholesterol (TC) is the sum of free cholesterol and cholesterol esters. Therefore, the CE / TC value is often used to reflect the degree of cell foaming. Combined with Nile red staining, small molecules can be screened more accurately. Summary of the invention

[0005] In view of the above-mentioned deficiencies in the prior art, the present invention provides a method for screening short peptides that inhibit macrophage foaming, aiming to fill the gap in the current high-throughput screening method for drugs that target the inhibition of macrophage foaming, and to improve the shortcomings of traditional methods such as high consumption of consumables, large cell requirements, and strong subjectivity.

[0006] The first technical solution provided by the present invention is a method for screening short peptides that inhibit macrophage foaming, comprising the following steps:

[0007] S1, Molecular docking screening:

[0008] The short peptides in the black bean-derived fat-soluble short peptide database were docked with key proteins in macrophages by Lib-Dock molecular docking using Discovery Studio software, and scored to obtain the initial screening short peptides.

[0009] S2, Nile red staining screening:

[0010] The short peptides screened in step S1 are mixed with foam cells, and the lipid content of the foam cells is detected by Nile red to obtain the short peptides screened again;

[0011] S3, CE / TC quantitative screening and Oil Red O staining verification:

[0012] The rescreened short peptides in step S2 were subjected to CE / TC detection and Oil Red O staining to eliminate the false positives caused by uneven cell plating in the Nile red screening experiment, thereby ultimately determining the effect of the screened drug and obtaining short peptides that inhibit macrophage foaming.

[0013] In some embodiments, step S1 specifically includes the following steps:

[0014] S11 database preparation: 80 to 200 fat-soluble short peptides with 2-9 amino acid residues and GRAVY value greater than 0 were selected from black bean protein hydrolysate as the database to be screened;

[0015] S12 Preparation of lipid-soluble short peptide structure: Use Pymol to create the peptide structure of the database in step S11;

[0016] S13 Docking protein receptor selection: FPN, a key protein that regulates macrophage foaming, was selected for docking;

[0017] S14 Molecular docking: a. Receptor protein preparation: remove small molecule ligands, add hydrogen, define and edit the protein docking sites in step S13; b. Ligand preparation: import the peptide structure in step S12, generate configuration, and perform energy minimization;

[0018] S15 virtual screening: perform batch docking calculations on the ligands and receptors in step S14, and select 20 to 50 short peptides with higher docking result scores as the initial screening short peptides.

[0019] In some embodiments, step S2 specifically includes the following steps:

[0020] S21 cell culture: THP-1 cells were inoculated with complete medium and cultured until the cell density reached 80-90%;

[0021] S22 cell induction: 600-800 r / min. Collect the cells cultured in step S21 and adjust the cell density to 6x10 with complete medium. 5 ~10 6 / mL uniform cell suspension, and add phorbol ester to the complete culture medium with a final concentration of 100ng / mL, and culture the cell suspension for 24h to 72h to induce macrophages, until the cells adhere to the wall and extend pseudopodia;

[0022] S23 drug addition culture: adding the short peptide initially screened in step S1 to the cells cultured in step S22 and co-culturing;

[0023] S24 Nile red staining: remove the cells and remove the culture medium, wash twice with PBS solution, add 4% paraformaldehyde to fix for 10-20 minutes, wash twice with PBS solution, stain with Nile red reagent for 10-20 minutes, and wash twice with PBS;

[0024] S25 numerical detection: The fluorescence microplate reader Ex / Em=552 / 636nm detects the degree of lipid accumulation in the numerical response cells to obtain the multiple screening short peptides.

[0025] In some embodiments, step S3 specifically includes the following steps:

[0026] S31 Cell culture and induction: Foam cells were inoculated with complete culture medium and cultured until the cell density reached 80-90%; the cells cultured in step S21 were collected at 600-800 r / min and the cell density was adjusted to 6×10 5 ~10 6 / mL uniform cell suspension, and add phorbol ester to the complete culture medium with a final concentration of 100ng / mL, and culture the cell suspension for 24h to 72h to induce macrophages, until the cells adhere to the wall and extend pseudopodia;

[0027] S32 cell lysis: About 5 million cells were added with 100-150 μL lysis buffer and lysed on ice for 20-30 min, 10,000-12,000 r / min, centrifuged for 5-10 min, and the supernatant was collected for testing;

[0028] S33 TC, FC detection: 180 μL of TC and FC reagent working solutions were mixed with 20 μL of cell supernatant from step S32, and incubated in a 37°C water bath for 20 minutes. Detect the OD value at 550 nm using an ELISA reader;

[0029] S34 calculation: using the OD value of step S33 to calculate TC, FC, and CE according to the standard curve, a short peptide that inhibits macrophage foaming is obtained.

[0030] The second technical solution provided by the present invention is the use of the method described in the first technical solution in preparing a product for inhibiting macrophage foaming, wherein the product contains one or two short peptides of FLR and LPSVL.

[0031] The third technical solution provided by the present invention is the use of the method described in the first technical solution in preparing a product for preventing, alleviating and / or treating atherosclerosis, wherein the product contains one or two short peptides of FLR and LPSVL.

[0032] In certain embodiments, the product is a medicine or a food.

[0033] In certain embodiments, the drug further contains a drug carrier and / or a pharmaceutical excipient.

[0034] In certain embodiments, the drug carrier comprises microcapsules, microspheres, nanoparticles and liposomes.

[0035] In certain embodiments, the pharmaceutical excipient comprises an excipient and an additive.

[0036] In certain embodiments, the pharmaceutical excipients include one or more of a stabilizer, a flavoring agent, a coloring agent, a filler, a binder, a wetting agent, a disintegrant, and a lubricant.

[0037] In certain embodiments, the dosage form of the drug comprises granules, capsules, tablets, pills or oral liquids.

[0038] The fourth technical solution provided by the present invention is the use of a short peptide in the preparation of a product for inhibiting macrophage foaming, wherein the amino acid sequence of the short peptide is FLR or LPSVL.

[0039] The fifth technical solution provided by the present invention is an activator of FPN protein, which contains one or two short peptides of FLR and LPSVL.

[0040] The sixth technical solution provided by the present invention is the use of a short peptide in the preparation of a product for preventing, alleviating and / or treating atherosclerosis, wherein the amino acid sequence of the short peptide is FLR or LPSVL.

[0041] In certain embodiments, the product is a medicine or a food.

[0042] In certain embodiments, the drug further contains a drug carrier and / or a pharmaceutical excipient.

[0043] In certain embodiments, the drug carrier comprises microcapsules, microspheres, nanoparticles and liposomes.

[0044] In certain embodiments, the pharmaceutical excipient comprises an excipient and an additive.

[0045] In certain embodiments, the pharmaceutical excipients include one or more of a stabilizer, a flavoring agent, a coloring agent, a filler, a binder, a wetting agent, a disintegrant, and a lubricant.

[0046] In certain embodiments, the dosage form of the drug comprises granules, capsules, tablets, pills or oral liquids.

[0047] The technical effects of the present invention are as follows:

[0048] The present invention relates to a method for batch screening of drugs for inhibiting macrophage foaming, which obtains drugs for inhibiting macrophage foaming through virtual screening and targeted screening. This method has extremely important guiding significance for targeted batch screening of drugs.

[0049] After virtual screening, the present invention needs to undergo two cell experiments, Nile red screening and CE / TC detection screening to verify the inhibitory effect of the drug. This effectively avoids the subjective error of traditional small-scale drug screening and saves a lot of experimental consumption. It is of great significance for screening drugs for macrophage foaming. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 The present invention is a schematic flow chart of a method for screening short peptides for inhibiting macrophage foaming.

[0051] Figure 2 Nile red staining screening results.

[0052] Figure 3 CE / TC test result diagram.

[0053] Figure 4 Oil red O staining and semi-quantitative results.

[0054] Figure 5 FPN-FLR molecular docking results.

[0055] Figure 6 FPN-LPSVL molecular docking results diagram. DETAILED DESCRIPTION

[0056] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0057] The raw materials used in the embodiment:

[0058] 1. Source of THP-1 cells: Wuhan Prosai Life Science Co., Ltd.

[0059] 2. Source of oxidized low-density lipoprotein: Guangzhou Yiyuan Biotechnology Co., Ltd.

[0060] 3. Source of short peptides: synthesized by Shanghai Sangon Biotechnology Co., Ltd.

[0061] 4. Simvastatin Source: Shanghai Yuanye Biotechnology Co., Ltd.

[0062] Example 1

[0063] See also Figure 1 , a method for screening short peptides that inhibit macrophage foaming, specifically comprising the following steps:

[0064] S1 Use Lib-dock molecular docking to screen the database in the first step

[0065] S11 Database preparation: 80 fat-soluble short peptides with 2-9 amino acid residues and GRAVY value greater than 0 were selected from black bean protein hydrolysate as the database to be screened;

[0066] S12 Preparation of lipid-soluble short peptide structures: batch downloading of peptide structures using Pymol and PyCharm;

[0067] S13 Selection of docking protein receptors: CD36, ABCA1, ABCG1, FPN, etc., key proteins that regulate macrophage foaming, select a protein for docking. This time, the FPN protein is selected, and the protein structure is downloaded from the Structural Bioinformatics Protein Database (https: / / www.pdbus.org / ). The FPN protein number is 8c03;

[0068] S14 Molecular docking: a. Receptor protein preparation: remove small molecule ligands, add hydrogen, define and edit protein docking sites. b. Ligand preparation: download drug structure, generate configuration, and minimize energy. The database to be screened and the docking results are shown in Table 1, and the specific docking parameters are shown in Table 2.

[0069] Table 1 Short peptide sequences to be screened

[0070]

[0071]

[0072] Table 2 Specific parameters of LibDock molecular docking

[0073]

[0074] S15 virtual screening: The drug structures were docked with the target proteins in batches, and the 21 drugs with the highest docking scores were selected for the next step of screening. The docking screening results are shown in Table 3.

[0075] Table 3 Short peptide sequences obtained by molecular docking screening

[0076]

[0077] S2 Nile Red Staining Screening

[0078] S21 cell culture: Complete cell culture medium composition: RPMI-1640 + 10% fetal bovine serum + 1% penicillin / streptomycin (100x). When the cell state is poor, the concentration of fetal bovine serum can be appropriately increased. When the THP-1 cell state is good and the THP-1 cell density reaches 80-90%, subsequent experiments are carried out.

[0079] S22 cell induction: After the cells stabilized, the cells were collected at 800 r / min and the cell density was adjusted to 6 x 10 5 ~10 6 / mL uniform cell suspension, and add phorbol ester to the complete medium at a final concentration of 100ng / mL. The cell suspension was added to a 96-well plate at a volume of 100μL / well and cultured for 48h to induce THP-1 cells into macrophages. Subsequent experiments were performed after the cells attached to the wall and extended pseudopodia.

[0080] S23 drug-added culture: The foam induction medium is RPMI-1640 + 0% fetal bovine serum + 1% penicillin / streptomycin (100x). Set up a normal group, a foam cell model group, a drug treatment group, and a Yangshen group. The conditions of the normal group are a medium with 0% fetal bovine serum, the conditions of the foam cell model group are a medium with 0% fetal bovine serum + 50μg / mL ox-LDL, the conditions of the short peptide intervention group are a medium with 0% fetal bovine serum + 50μg / mL ox-LDL + 10μM drugs, and the conditions of the Yangshen group are a medium with 0% fetal bovine serum + 50μg / mL ox-LDL + 100μM simvastatin.

[0081] S24 Nile red staining: remove the cells and remove the culture medium, wash twice with PBS solution, add 4% paraformaldehyde to fix for 10-20 minutes, wash twice with PBS solution, stain with Nile red reagent for 10-20 minutes, and wash twice with PBS.

[0082] S25 numerical detection: fluorescence microplate reader Ex / Em=552 / 636nm detection value, Nile red staining screening results see Figure 2 Compared with the normal group, the lipid accumulation in the model group was about 1.45-1.65 times. The lipid accumulation in the simvastatin group was about 1.0-1.1 times. In the treatment group, the lipid accumulation in the MFVPHY group was about 1.14 times, the lipid accumulation in the FQTLFK group was about 1.11 times, the lipid accumulation in the FLR group was 0.88 times, and the lipid accumulation in the LPSVL group was 0.68 times. There was a significant difference with the model group, and the effect was obvious. Therefore, it was determined to screen four peptide sequences: MFVPHY, FQTLFK, FLR, and LPSVL.

[0083] S3 passed CE / TC screening

[0084] Several peptides with the best inhibition of foaming were selected for cell experiments to detect cell TC, FC, and CE. The inhibitory effect of short peptides was determined by CE / TC to exclude false positives caused by uneven cell plating in Nile red staining experiments. The screening results were observed by Oil Red O. THP-1 was cultured at 8x10 5 ~10 6 / mL density, 2.5mL / well was evenly plated in a 6-well plate, and the cells were collected for detection after induction of modeling and drug treatment.

[0085] S31 cell culture and induction: Cell culture conditions were the same as those for S21 and S22;

[0086] S32 cell lysis: About 5 million cells were added with 120 μL lysis buffer and lysed on ice for 20 min, centrifuged at 12000 r / min for 10 min, and the supernatant was collected for testing;

[0087] S33 TC, FC detection: Mix 180 μL of TC and FC reagent working solution with 20 μL of cell supernatant, and incubate in a 37°C water bath for 20 minutes. Detect OD value at 550 nm using a microplate reader. Perform the same operation to draw FC and TC standard curves for subsequent calculations;

[0088] S34 calculation: Calculate TC and FC according to the standard curve. CE = TC-FC. The CE / TC result is as follows: Figure 3 CE / TC is a key indicator for quantitatively describing foaming. The higher the CE / TC value, the more severe the foaming. The experimental results showed that the CE / TC value of the normal group was 0.31, the value of the model group was 0.44, and the value of the simvastatin group was 0.33. The values ​​of the MFVPHY group and the FQTLFK group were both 0.41, which was not significantly different from the model group. The values ​​of the FLR group and the LPSVL group were 0.32 and 0.33, respectively, which were significantly different from the model group. Therefore, it was determined that the peptide sequences with inhibitory effects were FLR and LPSVL.

[0089] S4 Oil Red O to observe the inhibitory effect of short peptides

[0090] S41 Oil Red O dye preparation: Mix Oil Red O powder with 100 mL of isopropanol to obtain a saturated Oil Red O solution. Mix the saturated Oil Red O solution with deionized water at a ratio of 3:2. Filter through a 0.22uM filter membrane and store at 4°C away from light for later use.

[0091] The steps of culturing and inducing S42 cells were basically the same as those of S102, except that the cells were resuspended and diluted to a density of 5x10 in complete medium containing 100 ng / mL PMA. 5 / mL, and plated in a 24-well plate at a volume of 1mL / well. After 48h of induction, the cells were observed to adhere to the wall and have pseudopodia, which proved that the induction was successful.

[0092] The drug addition and culture steps of S43 are the same as those of S23.

[0093] S44 Oil Red O staining: After cell treatment, remove the culture medium, wash with PBS 1-2 times, and fix with 4% paraformaldehyde for 20 minutes. Wash with PBS 1-2 times, and stain with Oil Red O solution in the dark for 20 minutes. Remove the staining solution, wash with PBS 1-2 times, and take photos under the microscope after the background is clean. Figure 4 ). Add 400 μL / well isopropanol to wash out Oil Red O, and detect the absorbance at 540 nm to obtain semi-quantitative results. The absorbance of the normal group was 0.44, the model group was 0.57, and the simvastatin group was 0.48. The MFVPHY group and the FQTLFK group were 0.54 and 0.51, respectively, which were not significantly different from the model group. The FLR group and the LPSVL group were 0.49 and 0.48, respectively, which were significantly different from the model group. Therefore, the FLR and LPSVL peptides have a good inhibitory effect, which is consistent with the screening results.

[0094] Example 2 Effects of FLR and LPSVL on FPN, a key protein regulating macrophage foaming

[0095] The specific steps are as follows:

[0096] Preparation of ligands: The short peptides FLR and LPSVL obtained in Example 1 were selected as ligands, and linear peptides were generated using PyMol software and exported as .pdb files. The configurations were then imported into Discovery Studio 2019 Client software to generate configurations and perform energy minimization.

[0097] Preparation of receptor protein: Retrieve the FPN protein number in the PDB (https: / / www.rcsb.org / ) online database, select the appropriate protein crystal structure and download its PDB format file, edit the receptor protein using Discovery Studio 2019 Client software, remove water molecules, remove residues, and add hydrogen to the protein.

[0098] Molecular docking: In the Discovery Studio 2019 Client software, all the configurations generated by the short peptide FLR and LPSVL were selected as ligand molecules. The docking site was selected in the defined receptor protein cavity, site1 was selected as the docking site, and radius = 9 was set. Then Lib-Dock molecular docking was performed. The optimal binding conformation was selected to analyze the interaction, and the results were visualized to generate a protein-ligand interaction diagram.

[0099] The results are as follows Figure 5 , 6 As shown, the benzene ring of the peptide FLR has hydrophobic interactions with proline 189 Pro and arginine 40 Arg of the FPN protein, and the isopropyl group of FLR has hydrophobic interactions with isoleucine 186 Ile and phenylalanine 324 Phe of the FPN protein. The imino group of FLR has electrostatic interactions with aspartic acid 325 Asp. The imino group has hydrogen bonds with methionine 346 Met of the receptor protein, and asparagine 185 Asn has hydrogen bonds with the amino group of the short peptide and the oxygen atom on the peptide bond. The arginine side chain of FLR has carbon-hydrogen bonds with threonine 320 Thr. The C-terminal isobutyl group of the peptide LPSVL has hydrophobic interactions with tryptophan 470 Trp of the protein FPN, the isopropyl group has hydrophobic interactions with tyrosine 318 Tyr, and the two methyl groups of the N-terminal isobutyl group have hydrophobic interactions with tyrosine 64 Tyr and phenylalanine 508 Phe. There is an electrostatic interaction between the N-terminal amino group and the aspartic acid Asp at No. 325. There are hydrogen bond interactions between the N-terminal amino group and the pyrroline ring of proline and cysteine ​​Cys at No. 326, the oxygen atom on the peptide bond formed by P and S in the peptide segment LPSVL and isoleucine Ile at No. 327, arginine Arg at No. 466, and the amino group of serine in the peptide segment LPSVL and histidine His at No. 507. There are carbon-hydrogen bond interactions between the oxygen atom on the peptide bond formed by V and L in the peptide segment LPSVL and glycine Gly at No. 323, and the hydroxyl group of serine in the peptide segment LPSVL and valine Val at No. 321. This shows that the short peptides FLR and LPSVL can form a suitable spatial complementarity with FPN, with strong interaction, and play a role in inhibiting macrophage foaming.

[0100] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. Use of a short peptide in the preparation of a product for inhibiting macrophage foaming, characterized in that: The amino acid sequence of the short peptide is FLR or LPSVL.

2. An activator of FPN protein, characterized in that Contains one or two short peptides from FLR and LPSVL.

3. Use of a short peptide in the preparation of a drug for preventing, alleviating and / or treating atherosclerosis, characterized in that: The amino acid sequence of the short peptide is FLR or LPSVL.

4. The use according to claim 3, characterized in that: The medicine also contains a drug carrier and / or a pharmaceutical excipient.

5. A method for screening short peptides that inhibit macrophage foaming, characterized in that: The steps include: S1, Molecular docking screening: The short peptides in the black bean-derived fat-soluble short peptide database were docked with key proteins in macrophages by Lib-Dock molecular docking using Discovery Studio software, and scored to obtain the initial screening short peptides. S2, Nile red staining screening: The short peptides screened in step S1 are mixed with foam cells, and the lipid content of the foam cells is detected by Nile red to obtain the short peptides screened again; S3, CE / TC quantitative screening: The rescreened short peptides in step S2 are subjected to CE / TC detection to obtain short peptides that inhibit macrophage foaming.

6. The method according to claim 5, characterized in that Step S1 specifically includes the following steps: S11 database preparation: 80 to 200 fat-soluble short peptides with 2-9 amino acid residues and GRAVY value greater than 0 were selected from black bean protein hydrolysate as the database to be screened; S12 Preparation of lipid-soluble short peptide structure: Use Pymol to create the peptide structure of the database in step S11; S13 Docking protein receptor selection: FPN, a key protein that regulates macrophage foaming, was selected for docking; S14 Molecular docking: a. Receptor protein preparation: remove small molecule ligands, add hydrogen, define and edit the protein docking sites in step S13; b. Ligand preparation: import the peptide structure in step S12, generate configuration, and perform energy minimization; S15 virtual screening: perform batch docking calculations on the ligands and receptors in step S14, and select 20 to 50 short peptides with higher docking result scores as the initial screening short peptides.

7. The method according to claim 5, characterized in that Step S2 specifically The following steps are involved: S21 cell culture: THP-1 cells were inoculated with complete medium and cultured until the cell density reached 80-90%; S22 cell induction: 600-800 r / min. Collect the cells cultured in step S21 and adjust the cell density to 6x10 with complete medium. 5 ~10 6 / mL uniform cell suspension, and add phorbol ester to the complete culture medium with a final concentration of 100ng / mL, and culture the cell suspension for 24h to 72h to induce macrophages, until the cells adhere to the wall and extend pseudopodia; S23 drug addition culture: adding the short peptide initially screened in step S1 to the cells cultured in step S22 and co-culturing; S24 Nile red staining: remove the cells and remove the culture medium, wash twice with PBS solution, add 4% paraformaldehyde to fix for 10-20 minutes, wash twice with PBS solution, add Nile red reagent to stain for 10-20 minutes, and wash twice with PBS; S25 numerical detection: The fluorescence microplate reader Ex / Em=552 / 636nm detects the degree of lipid accumulation in the numerical response cells to obtain the multiple screening short peptides.

8. The method according to claim 5, characterized in that Step S3 specifically includes the following steps: S31 Cell culture and induction: Inoculate foam cells with complete culture medium and culture until the cell density reaches 80-90%; collect cells cultured in step S21 at 600-800 r / min and adjust the cell density to 6x10 5 ~10 6 / mL uniform cell suspension, and add phorbol ester to the complete culture medium with a final concentration of 100ng / mL, and culture the cell suspension for 24h to 72h to induce macrophages, until the cells adhere to the wall and extend pseudopodia; S32 cell lysis: About 5 million cells were added with 100-150 μL lysis buffer and lysed on ice for 20-30 min, 10,000-12,000 r / min, centrifuged for 5-10 min, and the supernatant was collected for testing; S33 TC, FC detection: 180 μL of TC and FC reagent working solutions were mixed with 20 μL of cell supernatant from step S32, and incubated in a 37°C water bath for 20 minutes. Detect the OD value at 550 nm using an ELISA reader; S34 calculation: using the OD value of step S33 to calculate TC, FC, and CE according to the standard curve, a short peptide that inhibits macrophage foaming is obtained.

9. Use of the method according to any one of claims 5 to 8 in preparing a product for inhibiting macrophage foaming, characterized in that: The product contains one or two short peptides of FLR and LPSVL.

10. Use of the method according to any one of claims 5 to 8 in the preparation of a drug for preventing, alleviating and / or treating atherosclerosis, characterized in that: The drug contains one or two short peptides of FLR and LPSVL.

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