CN114107427A - Experimental method for screening FASN inhibitor in peony seed meal monomer compound based on computer simulation - Google Patents

Abstract

The invention belongs to the technical field of pharmaceutical analysis, and discloses an experimental method for screening Fatty Acid Synthase (FASN) inhibitors in peony seed meal monomer compounds based on computer simulation, which comprises the following specific operation steps: s1, molecular docking; s2, detecting cell viability; s3, western immunoblot; s4 and detecting apoptosis. According to the invention, whether four monomer compounds in the peony seed meal extract have anti-tumor activity caused by inhibition of FASN activity and expression is determined by screening with the help of a molecular docking method, and a full-enzyme dimer structure of FASN is tried to be constructed by a homologous modeling means, so that a new thought and a new method are provided for screening of FASN inhibitors, a large number of invalid experiments can be effectively avoided, the success rate of experimental operation is improved, and the experimental operation cost is reduced.

Description

Experimental method for screening FASN inhibitor in peony seed meal monomer compound based on computer simulation

Technical Field

The invention belongs to the technical field of pharmaceutical analysis, and particularly relates to an experimental method for screening a FASN inhibitor in a peony seed meal monomer compound based on computer simulation.

Background

Breast cancer is a common malignant tumor endangering female health at present, scientists are always dedicated to searching for a method for eradicating the disease, and in recent years, although great progress is made, the survival rate of breast cancer patients is greatly improved, the internal pathogenesis of the breast cancer patients is still not fully understood, the method and the effectiveness of the breast cancer prevention, treatment and recovery are still not ideal, and FASN expression and activity far higher than normal level are found in tissue samples of the breast cancer and the like in the 90 th 20 th century, so that the breast cancer patients begin to serve as potential cancer treatment targets.

Although a plurality of FASN inhibitors have been reported in recent 20 years, the FASN inhibitors which have high activity and low toxicity and can be used for drug development are still very lack, monomer compounds in peony seed meal are virtually screened by a molecular docking means, the activity of the compounds is verified by subsequent cell experiments, and the potential FASN inhibitors are searched by taking the monomer compounds as entry points and have wide prospects.

Peony is a traditional Chinese medicine, seed meal of peony contains a plurality of stilbenes compounds, through in vitro cell experiments, the peony seed meal extract is found to have an inhibiting effect on the activity of FASN in breast cancer cells for the first time, and the monomer compounds with potential FASN inhibiting activity exist in the peony seed meal, the FASN inhibiting activity of the stilbenes compounds in the peony seed meal is researched through computer simulation, due to the complexity of FASN conformation with the molecular weight of 273kDa, most of the previous domestic and foreign researches are virtual screening aiming at single structural domain of FASN, the invention constructs a human FASN (hFASN) holoenzyme structure through a homologous modeling means, performs molecular docking on the stilbenes monomer compounds in the peony seed meal and all structural domains of FASN, combines functional verification of cell experiments, and finds that Epsilon-Viniferin, Suffrutinosol A and Ampelopsin D have the ability of inhibiting FASN, is a natural FASN inhibitor, can induce cancer cells to generate apoptosis by inhibiting FASN, and lays the foundation of methodology for screening FASN inhibitors from natural product libraries by computer simulation.

Disclosure of Invention

The invention aims to solve the problems, and provides an experimental method for screening a FASN inhibitor in a peony seed meal monomer compound based on computer simulation, so as to solve the problems in the background art.

In order to achieve the purpose, the invention provides the following technical scheme: the experimental method for screening the FASN inhibitor in the peony seed meal monomer compound based on computer simulation comprises the following specific operation steps of:

s1, molecular docking;

s2, cell viability detection-MTT method;

s3, western immunoblot;

s4, detecting apoptosis;

the molecular docking in the step S1 comprises the following specific operation steps:

installing AutoDock and Openbabel software in a Linux system environment;

1) preparing a receptor pdb file, checking the receptor structure by using PyMOL software, and removing structures such as crystal water and a small molecular ligand which are possibly contained;

2) converting the ligand and receptor files into pdbqt files in batch by using an AutoDock Tool;

3) respectively putting the receptor pdbqt files into sequentially arranged folders;

4) setting related parameters such as ligand and receptor names, box size, coordinates and the like in the script to carry out automatic dock docking;

5) and extracting a log file generated by molecular docking.

The method is used for detecting whether the peony seed meal extract has an inhibition effect on Fatty Acid Synthase (FASN) and breast cancer cells by the matching treatment of the above operation steps; an operator analyzes the binding capacity of a compound Ampelopsin D and three other compounds in the peony seed meal to the KS active center of FASN through a molecular docking technology; then, obtaining structures of seven active centers of FASN through modeling, and analyzing the binding capacity of Ampelopsin D and each active center through molecular docking; various figures and data show that Ampelopsin D has strong binding capacity to multiple active centers on FASN, suggesting that it is likely to be a FASN inhibitor; proved by various cell experiments, Ampelopsin D is proved to have the capability of inhibiting FASN activity and various activities on cancer cells.

As a preferred technical scheme of the invention, the specific operation steps of the cell viability detection-MTT method in S2 are as follows:

1) when the cell density in the 96-well plate is 70-80%, adding serum-free DMEM containing different drug concentrations to treat the cells for 24h, and repeating 6 wells for each drug concentration;

2) preparing 5mg/mL MTT solution from MTT and DMSO in advance;

3) preparing a serum-free culture solution and an MTT solution (5mg/mL) into a mixed solution of 0.5mg/mL according to a ratio of 9: 1;

4) discarding the culture solution, adding PBS into each hole for cleaning, and adding 100 mu L of mixed solution into each hole;

5) culturing at 37 deg.C for 1 h;

6) discarding the mixed solution, adding and blowing 100 mu L DMSO into each hole;

7) the absorbance at 492nm was measured using a microplate reader.

As a preferred technical scheme of the invention, the specific operation steps of the Western blotting in S3 are as follows:

1) connecting the cells to a six-hole plate, adding serum-free culture solution containing different drug concentrations to treat the cells for 24 hours when the cell density is 70-80%, and repeating for 3 holes at each drug concentration;

2) washing cells with PBS, adding 120 mu L of lysis solution, and scraping the cells to a centrifuge tube;

3) performing ultrasonic treatment on ice for 4 min;

4) centrifuge at 4 ℃ and transfer the supernatant to a new centrifuge tube.

5) The BCA method is used for determining the protein concentration and leveling;

6) adding 4 × Loading Buffer in proportion, boiling the sample at 95 ℃ for 10min, and storing the sample at-20 ℃;

electrophoresis

1) Preparing SDS-PAGE separation gel: the ddH was then formulated according to the gel formulation₂Mixing O, 30% AB, 1.5% MTris-HCL (pH 8.8), 10% AP and TEMED in a vortex manner, adding 7mL of separation gel into each gel preparation plate, adding water for supplementing, and waiting for solidification;

2) preparing SDS-PAGE concentrated gel: preparing ddH2O, 30% AB, 1M Tris-HCl (pH6.8), 10% AP and TEMED according to a gel formula, uniformly mixing by vortex, pouring water in a gel making plate, adding concentrated gel for supplementing, inserting a comb, and waiting for solidification;

3) preparing 1 XRunning Buffer: diluting 5 XRunning Buffer and ultrapure water according to the proportion of 1:4, and reversing and uniformly mixing;

4) placing the rubber plate in an electrophoresis tank, adding 1 × Running Buffer, pulling out a comb, and adding a proper amount of Marker or sample into each hole;

5) electrophoresis: performing constant-pressure 80V electrophoresis for 30min, and adjusting to 120V electrophoresis to make the strip run to the bottom of the gel;

rotary film

1) Prepare 1 × Transfer Buffer: diluting 5 times Transfer Buffer, anhydrous methanol and ultrapure water according to the proportion of 1:1:3, and reversing and mixing uniformly;

2) film shearing: soaking PVDF in methanol for activation;

3) film transfer: placing the sponge and the three layers of filter paper on a film Transfer clamp, soaking the sponge and the three layers of filter paper through a Transfer Buffer, placing the gel on the black part of the film Transfer clamp, covering the film on the gel, clamping the film Transfer clamp, inserting the film into a groove, and transferring the sample from the gel to the film fully by constant current of about 250mA for 2.5 hours;

incubating antibodies

1) Washing the membrane: diluting 10 × TBS solution to 1 × TBST solution in advance, and cleaning with 1 × TBST solution for 10min each time for 3 times after membrane transfer;

2) and (3) sealing: preparing sealing liquid in advance according to the proportion of adding 1g of skimmed milk powder into every 20ml of LTBST, putting the membrane into the sealing liquid, and shaking for 1h at 37 ℃;

3) incubating the primary antibody: diluting the primary antibody with a primary antibody diluent, and placing the membrane in a primary antibody incubation solution; incubating overnight at 4 ℃;

4) washing the membrane: washing the membrane for 3 times by TBST;

5) incubation of secondary antibody: putting the membrane into a confining liquid containing a secondary antibody, and shaking for 1h at 37 ℃;

6) washing the membrane: washing the membrane for 3 times by TBST;

development

1) Preparing ECL luminescent liquid: mixing the two solutions according to the proportion of 1: 1;

2) developing by using an exposure machine, selecting a Chemi option of an ImageLab software blot, uniformly dripping 160 mu L of luminous liquid before exposure, obtaining a strip, quantitatively analyzing the gray value of the strip by using ImageJ software, mapping by using GraphPad Prism software, and repeating each experiment for three times.

As a preferred technical scheme of the invention, the apoptosis detection in S4 comprises the following specific operation steps:

1) inoculating the cells to a six-hole plate, and adding serum-free culture solution containing different drug concentrations to treat the cells for 24 hours when the cell density is 70-80%;

2) collecting the culture solution to a centrifuge tube, digesting adherent cells for 5min by using trypsin, blowing the digested cells to a single cell state by using 1mL of the culture solution which is just sucked, merging the cells into the centrifuge tube, and centrifuging at 1100g for 4min at 4 ℃;

3) cells were washed 2 times with 0.5mL PBS, 1100g, 4min, centrifugation at 4 ℃;

4) resuspending cells in Annexin V-FITC conjugate;

5) adding Annexin V-FITC and PI in sequence;

6) flow detection: respectively selecting FITC/PE or FL1/FL2 channels;

7) and (3) fluorescent microscope detection: the cell suspension was dropped onto a glass slide and viewed with a cover slip.

As a preferred technical scheme, the experimental reagent related to the experimental method of the FASN inhibitor in the peony seed meal monomeric compound comprises a Fatty Acid Synthase (FAS) activity detection kit, a BeyoClickTMEdU-594 cell proliferation detection kit, an annexin V-FITC apoptosis detection kit, a cell cycle and apoptosis detection kit, an immunostaining blocking solution, an immunostaining washing solution, a RIPA lysate, a BCA method protein quantification kit, tetramethylethylenediamine (MED), Sodium Dodecyl Sulfate (SDS), a protein molecular weight standard Marker, a 4% immunohistochemical stationary liquid, Acrylamide (Acrylamide), dithiot-alditol (DTT), thiazole blue (MTT), a primary anti-dilution solution, bromophenol blue, palmitic acid, Tween-20, anhydrous methanol, Adapalene, Celecoxib, alecetib, Lumacaftor, Tris-HCL, potassium chloride, hydrochloric acid, ethanol, sodium chloride, skim milk powder, and the like, Fetal bovine serum, polyvinylidene fluoride membrane (PVDF membrane), developer, glycerol, Trizmabase, glycine, dimethyl sulfoxide (DMSO), Ammonium Persulfate (AP), pancreatin (without EDTA), pancreatin (with EDTA), high-sugar culture solution, PBS;

the experimental antibody comprises a FASN Rabbit mAb, a PARP Rabbit mAb, a PERK Rabbit mAb, a CHOP Rabbit mAb, a BiP Rabbit mAb, a beta-Actin Rabbit mAb, Anti-Bcl-2antibody, Anti-Bax antibody, Anti-IRE1 antibody, Anti-ATF6 antibody, Anti-DDIT3 antibody, goat Anti-Rabbit IgG-HRP and goat Anti-mouse IgG-HRP;

the experimental apparatus comprises: the device comprises an electric heating constant temperature blast drying box, an ultrasonic cell smashing instrument, an electric heating constant temperature water bath kettle, an ice maker, a metal constant temperature bath, a constant temperature oscillator, a low-speed centrifuge, a vortex mixer, an ultra-low temperature refrigerator, an electronic analysis balance, an inverted biological microscope, a pipettor, a vacuum pump, a circumference shaking table, an ultra-clean workbench, a chemiluminescence imaging analysis system, a table type refrigerated centrifuge, a multifunctional microplate reader, a flow cytometer, a pipettor, a table type pH meter, a magnetic stirrer, an intelligent upright fluorescence microscope, a carbon dioxide incubator, an ultraviolet visible spectrophotometer, an inverted fluorescence microscope and an ultrapure water integrated system.

Compared with the prior art, the invention has the following beneficial effects:

the invention determines whether four monomer compounds in the peony seed meal extract have anti-tumor activity caused by inhibition of FASN activity and expression by screening with the help of a molecular docking method, tries to construct a whole enzyme dimer structure of FASN by a homologous modeling means, although compounds with outstanding anti-tumor effect cannot be compared in the four peony seed meal monomer compounds in the research, trial and error and pavement are carried out for subsequent experiments, a new thought and a new method are provided for screening the laboratory FASN inhibitor, a large number of invalid experiments can be effectively avoided in subsequent experimental operation, the success rate of experimental operation is improved, the experimental operation cost is reduced, and the application adopts interdisciplinary combination of biological experiments and a computer, the biological experiments can ensure that operators can effectively understand the whole experimental process, and can continuously improve and discover new problems and new methods in the experiments, the computer rapidly completes the screening of the designated data through fixed data analysis and processing, the combination of the designated data and the designated data can ensure the great improvement of the working efficiency, and meanwhile, the high fidelity of the whole experiment can be ensured, and errors can be effectively avoided.

Drawings

FIG. 1 is a schematic view of the overall operation of the present invention;

FIG. 2 is a schematic diagram showing the inhibitory effect of the peony seed meal extract and peony seed oil on MDA-MB-231 cell viability and intracellular FASN activity according to the present invention;

FIG. 3 is a schematic diagram showing the apoptotic effect of the peony seed meal extract (0,5,10, 20. mu.g/ml) of the present invention on MDA-MB-231 cells;

FIG. 4 is a schematic representation of the effect of the invasion migration ability of MDA-MB-231 cells of the invention by the peony seed meal extract;

FIG. 5 is a schematic diagram of the two-dimensional and three-dimensional structures of Ampelopsin D of the present invention;

FIG. 6 is a schematic diagram of seven domains of the fatty acid synthase of origin according to the present inventors;

FIG. 7 is a schematic representation of the DH domain of a human fatty acid synthase from three sources according to the invention;

FIG. 8 is a schematic diagram of the structures of the monomer and dimer of the human fatty acid synthase of the present invention;

FIG. 9 is a schematic diagram of the active pocket of the present invention that is predicted by DoGSiteSCore to score the first three of each domain;

FIG. 10 is a schematic representation of the molecular docking results and predicted activity pockets of the present invention;

FIG. 11 is a schematic diagram showing the hydrogen bonding interaction and hydrophobic interaction of the amino acid residues of each domain with the ligand small molecule AmpelopsinD according to the present invention, shown by Ligplot;

FIG. 12 is a schematic diagram showing the effect of four peony seed meal extraction compounds of Epsilon-Viniferin, Suffruticosol A, Ampelopsin D and Suffruticosol B on the cell viability of human breast cancer cell lines (MDA-MB-231 and MCF-7);

FIG. 13 is a schematic diagram showing the change of the expression level of FASN in peony extract-induced cells according to the present invention;

FIG. 14 is a schematic diagram of the induction of apoptosis by peony extract of the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

As shown in fig. 1 to 14, the invention provides an experimental method for screening a FASN inhibitor in a peony seed meal monomer compound based on computer simulation, and the experimental method for screening the FASN inhibitor in the peony seed meal monomer compound based on computer simulation specifically comprises the following operation steps:

s1, molecular docking;

s2, cell viability detection-MTT method;

s3, western immunoblot;

s4, detecting apoptosis;

the molecular docking in the step S1 comprises the following specific operation steps:

installing AutoDock and Openbabel software in a Linux system environment;

1) preparing a receptor pdb file, checking the receptor structure by using PyMOL software, and removing structures such as crystal water and a small molecular ligand which are possibly contained;

2) converting the ligand and receptor files into pdbqt files in batch by using an AutoDock Tool;

3) respectively putting the receptor pdbqt files into sequentially arranged folders;

4) setting related parameters such as ligand and receptor names, box size, coordinates and the like in the script to carry out automatic dock docking;

5) and extracting a log file generated by molecular docking.

The peony seed meal is proved to have physiological effects of protecting the cardiovascular system, regulating blood fat, resisting inflammation and the like, is paid more and more attention, has wide development prospect of medicinal value, and has more remarkable antitumor activity as shown by early-stage experiments of crude extracts of the peony seed meal; FASN is expressed in a low amount and activity in most normal tissues, exhibits high expression and activity in various cancer cells including breast cancer cells, FASN inhibitors can induce cancer cells to produce apoptosis, and molecular docking based on compound and protein structures is a powerful tool for evaluating the binding capacity of ligand small molecules to FASN, and contributes to understanding biological processes at an atomic level.

The focus of the research is to explore whether the peony seed meal extract contains a compound monomer with an inhibition effect on FASN by means of computer-assisted screening means such as molecular docking, molecular dynamic simulation and the like, and explore the anti-cancer effect of the peony seed meal extract.

The specific operation steps of the cell viability detection-MTT method in S2 are as follows:

1) when the cell density in the 96-well plate is 70-80%, adding serum-free DMEM containing different drug concentrations to treat the cells for 24h, and repeating 6 wells for each drug concentration;

2) preparing 5mg/mL MTT solution from MTT and DMSO in advance;

3) preparing a serum-free culture solution and an MTT solution (5mg/mL) into a mixed solution of 0.5mg/mL according to a ratio of 9: 1;

4) discarding the culture solution, adding PBS into each hole for cleaning, and adding 100 mu L of mixed solution into each hole;

5) culturing at 37 deg.C for 1 h;

6) discarding the mixed solution, adding and blowing 100 mu L DMSO into each hole;

7) the absorbance at 492nm was measured using a microplate reader.

The specific operation steps of the Western blotting in S3 are as follows:

1) connecting the cells to a six-hole plate, adding serum-free culture solution containing different drug concentrations to treat the cells for 24 hours when the cell density is 70-80%, and repeating for 3 holes at each drug concentration;

2) washing cells with PBS, adding 120 mu L of lysis solution, and scraping the cells to a centrifuge tube;

3) performing ultrasonic treatment on ice for 4 min;

4) centrifuge at 4 ℃ and transfer the supernatant to a new centrifuge tube.

5) The BCA method is used for determining the protein concentration and leveling;

6) adding 4 × Loading Buffer in proportion, boiling the sample at 95 ℃ for 10min, and storing the sample at-20 ℃;

electrophoresis

1) Preparing SDS-PAGE separation gel: the ddH was then formulated according to the gel formulation₂Mixing O, 30% AB, 1.5M Tris-HCl (pH 8.8), 10% AP and TEMED in a vortex manner, adding 7mL of separation gel into each gel preparation plate, adding water for supplementing, and waiting for solidification;

2) preparing SDS-PAGE concentrated gel: preparing ddH2O, 30% AB, 1M Tris-HCl (pH6.8), 10% AP and TEMED according to a gel formula, uniformly mixing by vortex, pouring water in a gel making plate, adding concentrated gel for supplementing, inserting a comb, and waiting for solidification;

3) preparing 1 XRunningbuffer: diluting 5 XRunning Buffer and ultrapure water according to the proportion of 1:4, and reversing and uniformly mixing;

4) placing the rubber plate in an electrophoresis tank, adding 1 × Running Buffer, pulling out a comb, and adding a proper amount of Marker or sample into each hole;

5) electrophoresis: performing constant-pressure 80V electrophoresis for 30min, and adjusting to 120V electrophoresis to make the strip run to the bottom of the gel;

rotary film

1) Prepare 1 × Transfer Buffer: diluting 5 times Transfer Buffer, anhydrous methanol and ultrapure water according to the proportion of 1:1:3, and reversing and mixing uniformly;

2) film shearing: soaking PVDF in methanol for activation;

3) film transfer: placing the sponge and the three layers of filter paper on a film Transfer clamp, soaking the sponge and the three layers of filter paper through a Transfer Buffer, placing the gel on the black part of the film Transfer clamp, covering the film on the gel, clamping the film Transfer clamp, inserting the film into a groove, and transferring the sample from the gel to the film fully by constant current of about 250mA for 2.5 hours;

incubating antibodies

1) Washing the membrane: diluting 10 × TBS solution to 1 × TBST solution in advance, and cleaning with 1 × TBST solution for 10min each time for 3 times after membrane transfer;

2) and (3) sealing: preparing sealing liquid in advance according to the proportion of adding 1g of skimmed milk powder into every 20ml of LTBST, putting the membrane into the sealing liquid, and shaking for 1h at 37 ℃;

3) incubating the primary antibody: diluting the primary antibody with a primary antibody diluent, and placing the membrane in a primary antibody incubation solution; incubating overnight at 4 ℃;

4) washing the membrane: washing the membrane for 3 times by TBST;

5) incubation of secondary antibody: putting the membrane into a confining liquid containing a secondary antibody, and shaking for 1h at 37 ℃;

6) washing the membrane: washing the membrane for 3 times by TBST;

development

1) Preparing ECL luminescent liquid: mixing the two solutions according to the proportion of 1: 1;

2) developing by using an exposure machine, selecting a Chemi option of an ImageLab software blot, uniformly dripping 160 mu L of luminous liquid before exposure, obtaining a strip, quantitatively analyzing the gray value of the strip by using ImageJ software, mapping by using GraphPad Prism software, and repeating each experiment for three times.

The specific operation steps of apoptosis detection in S4 are as follows:

1) inoculating the cells to a six-hole plate, and adding serum-free culture solution containing different drug concentrations to treat the cells for 24 hours when the cell density is 70-80%;

2) collecting the culture solution to a centrifuge tube, digesting adherent cells for 5min by using trypsin, blowing the digested cells to a single cell state by using 1mL of the culture solution which is just sucked, merging the cells into the centrifuge tube, and centrifuging at 1100g for 4min at 4 ℃;

3) cells were washed 2 times with 0.5mL PBS, 1100g, 4min, centrifugation at 4 ℃;

4) resuspending cells in Annexin V-FITC conjugate;

5) adding Annexin V-FITC and PI in sequence;

6) flow detection: respectively selecting FITC/PE or FL1/FL2 channels;

7) and (3) fluorescent microscope detection: the cell suspension was dropped onto a glass slide and viewed with a cover slip.

The experimental reagent related to the experimental method of the FASN inhibitor in the peony seed meal monomeric compound comprises a Fatty Acid Synthetase (FAS) activity detection kit, a BeyoClickTMEdU-594 cell proliferation detection kit, an Annexin V-FITC apoptosis detection kit, a cell cycle and apoptosis detection kit, an immunostaining confining liquid, an immunostaining washing liquid, a RIPA lysate, a BCA protein quantification kit, Tetramethylethylenediamine (TEMED), Sodium Dodecyl Sulfate (SDS), a protein molecular weight standard Marker, a 4% immunohistochemical fixing liquid, Acrylamide (Acrylamide), dithiot-alditol (DTT), thiazole blue (MTT), a primary anti-dilution liquid, bromophenol blue, palmitic acid, Tween-20, anhydrous methanol, Adapalene, Celecoxib, alectib, Lumacaftor, Tris-HCL, potassium chloride, hydrochloric acid, ethanol, sodium chloride, skim milk powder, bovine serum, polyvinylidene fluoride (PVDF) membrane, bovine serum, Developing solution, glycerol, Trizma base, glycine, dimethyl sulfoxide (DMSO), Ammonium Persulfate (AP), pancreatin (without EDTA), pancreatin (with EDTA), high-sugar culture solution and PBS;

the experimental antibody comprises a FASN Rabbit mAb, a PARP Rabbit mAb, a PERK Rabbit mAb, a CHOP Rabbit mAb, a BiP Rabbit mAb, a beta-Actin Rabbit mAb, Anti-Bcl-2antibody, Anti-Bax antibody, Anti-IRE1 antibody, Anti-ATF6 antibody, Anti-DDIT3 antibody, goat Anti-Rabbit IgG-HRP and goat Anti-mouse IgG-HRP;

the experimental apparatus comprises: the device comprises an electric heating constant temperature blast drying box, an ultrasonic cell smashing instrument, an electric heating constant temperature water bath kettle, an ice maker, a metal constant temperature bath, a constant temperature oscillator, a low-speed centrifuge, a vortex mixer, an ultra-low temperature refrigerator, an electronic analysis balance, an inverted biological microscope, a pipettor, a vacuum pump, a circumference shaking table, an ultra-clean workbench, a chemiluminescence imaging analysis system, a table type refrigerated centrifuge, a multifunctional microplate reader, a flow cytometer, a pipettor, a table type pH meter, a magnetic stirrer, an intelligent upright fluorescence microscope, a carbon dioxide incubator, an ultraviolet visible spectrophotometer, an inverted fluorescence microscope and an ultrapure water integrated system.

Influence of crude extract of peony seed meal on breast cancer cells:

the result shows that the peony seed meal crude extract has the functions of reducing the cell activity of breast cancer cells, causing cell apoptosis, inhibiting cell invasion and transfer and reducing the activity of FASN enzyme in the cells, and shows that potential compounds in the peony seed meal can inhibit the activity of FASN and induce cancer cell apoptosis;

the peony seed meal crude extract causes the reduction of breast cancer cell activity and FASN enzyme activity:

the peony seed meal is subjected to ultrasonic oscillation extraction by using ethanol-water mixed solutions with different concentrations, and then is subjected to reduced pressure concentration to obtain different solution extracts, the cancer cell activity determination is performed on each extract, the FASN activity determination in cancer cells is performed to obtain a 50% ethanol crude extract of the peony seed meal, the 50% ethanol crude extract of the peony seed meal has the effects of remarkably reducing the cancer cell activity and remarkably inhibiting the FASN activity, the semi-inhibition concentration of the peony seed meal crude extract on the MDA-MB-231 cell activity is 5 mu g/mL, the semi-inhibition concentration of the peony seed meal crude extract on the intracellular FASN activity is 7 mu g/mL, and the semi-inhibition concentration of the peony seed oil on the MDA-MB-231 cell activity is 0.17 mu L/mL, so that the inhibition effects of the peony seed meal crude extract and the peony seed oil are relatively obvious.

The peony seed meal crude extract can obviously induce MDA-MB-231 breast cancer cell apoptosis: the apoptosis effect of the peony seed meal on MDA-MB-231 cells is determined by flow cytometry and through FITC and PI double staining, as shown in figure 3, 98.5% of the control group live cells are obtained, and only 61.8% of the live cells are remained at 20 mu g/mL, which indicates that the peony seed meal crude extract has strong capacity of inducing cancer cell apoptosis.

The peony seed meal crude extract can obviously reduce the migration of MDA-MB-231 breast cancer cells: the high activity of FASN is beneficial to the invasion and metastasis of cancer cells, and through a cell scratch experiment, the influence of peony seed meal crude extract on the invasion and metastasis capacity of breast cancer cells is researched, and as shown in FIG. 4, the obvious invasion and metastasis phenomena of tumor cells after 24 hours of a control group exist; after 10 mu g/mL of peony seed meal crude extract is added, the invasion and metastasis characteristics of tumor cells are obviously inhibited.

Molecular docking of four peony seed meal monomer compounds: the binding sites of polyphenol compounds from natural sources reported previously are mostly KS structural domains, so that 4 compounds contained in a peony seed meal extract are selected firstly, and a small molecular ligand is downloaded from NCBI; performing molecular docking on a KS structure through an AutoDock; semi-flexible molecular docking was performed by Lamark Genetic Algorithm (LGA) in AutoDock 4.2 software; the PyMOL software was used to remove water from the receptor and other small molecules, and the docking centers were set (-13.313, 51.009, 33.26) by examination of the literature, which gave the results that Ampelopsin D was the lowest binding energy score as shown in table 1, so we chose Ampelopsin D for the next experiment.

TABLE 1 binding energies of four compounds in peony seed meal to the FASN-KS domain

Constructing the structure of hFASN holoenzyme based on homologous modeling: although Ampelopsin D has been used to comprehensively interface all domains of FASN, a complete holoenzyme structure file of FASN holoenzyme is currently lacking in the literature, and table 2 shows crystal structures of analyzed human fatty acid synthase domains that can be found in Protein structure databases (PDB), including ER (4W9N), KR (5C37), MAT (3HHD), KS (3HHD), TE (3 t), jm (2CG5), and lacking in the DH domain.

TABLE 2 currently available human FASN domains for PDB

Meanwhile, the crystal structure of the swine FASN holoenzyme has been analyzed by Maier, Leibundgut and Ban et al in 2008, and the similarity of human and swine FASN protein sequences is 78.64% by Blast measurement; therefore, it was attempted to construct a human fatty acid synthase holoenzyme by homologous modeling using the crystal structure of porcine FASN (2VZ9) and the amino acid sequence of human FASN as templates by SWISS-MODEL, and isolate the DH domain; the RMSD values of the KS-MAT domain obtained by homology modeling and the crystal structure (3HHD) of the porcine FASN and KS-MAT domain from the PDB database were calculated to be 0.319nm and 1.359nm by using PyMOL software, so that we can consider that the DH domain obtained by SWISSMODEL homology modeling using the porcine FASN holoenzyme (2VZ9) as a template has high accuracy.

The amino acid sequence of the crystal structure of the ACP domain (2CG5) was examined by PyMOL, and the amino acid position that plays a key role for activity, serine Ser 2156, was mutated to alanine Ala; taking a 2CG5 crystal structure as a template, and constructing the crystal structure of the ACP structural domain without mutation by using the corrected sequence in a SWISS-MODEL homologous modeling mode; the RMSD value of the crystal structure of the ACP structural domain obtained by homologous modeling and the crystal structure of the pro-ACP structural domain (2CG5) is 0.269 nm.

After inspection, the KR and ER domain files downloaded from PDB database are partially deleted of amino acid residues or atoms, and the residues or atoms can be automatically supplemented through SPDBV software or manually supplemented one by one through PyMOL software, so that the crystal structures of all domains of human FASN are obtained, as shown in FIG. 6; and comparing the position of the protein with that of human FASN obtained by pig FASN homologous modeling, a more real human FASN structure file can be spliced, as shown in FIG. 8(C), and the human FASN predicted protein structure is updated in the alphaFold protein structure database at 7/1/2021, as shown in FIG. 8 (B).

The DH domain of the SWISSMODEL homology modeling in the experiment is compared with the DH domain predicted by the alphaFold, the RMSD is 0.358nm, the structural similarity is high, as shown in figure 7, and the similarity of the FASN monomer structure predicted by the alphaFold is also high compared with the FASN monomer structure constructed in the experiment, as shown in figure 8(A, B).

Prediction of the potential binding pocket for each domain of hfsn: after obtaining seven domains of FASN, they are pretreated for water removal, small molecule removal, etc., and potential binding pockets of the crystal structure of each domain of fatty acid synthase are predicted by using the DoGSitisCorer on-line tool, and then ranked according to their size, surface area, and pharmaceutical acceptable score, etc. To determine the three-dimensional search space for binding of the receptor protein of the molecular docking to the ligand and the manner of binding, table 3 and fig. 9 list and show the information and location of the predicted active pocket scored three times.

ACP Domain

DH Domain

KS Domain

MAT Domain

ER Domain

KR Domain

TE Domain

TABLE 3 active pocket scoring the top three for each domain predicted by DoGSitisCorer

Carrying out molecular docking on proteins and ligands in the PDB file again by using Ampelopsin D for molecular docking of each domain of hFASN so as to test the accuracy of docking; since there is no crystal structure of the ligand-binding human fatty acid synthase KS domain in PDB, alignment accuracy was verified using the crystal structure of bacterial fatty acid synthase KS domain binding to thiolactamycin (PDB ID:2VB 8). Extraction of nisin and re-docking of the crystal structure, overlap of the re-generated structure with the original crystal structure (2VB8) gives a Root Mean Square Deviation (RMSD) of

The same applies to the MAT, DH domain. The RMSD values for each domain after docking ranged from 0.04 to 0.492, indicating that the docking protocol of AutoDock is highly accurate (table 4), and that molecular docking of the selected compound with FASN can be predicted using the same docking protocol.

TABLE 4 RMSD values for domain re-docking

Semi-flexible molecular docking was performed by Lamark Genetic Algorithm (LGA) in AutoDock 4.2 software, the PyMo l software was used to remove water and other small molecules from the receptor, and KS, MAT, KR, ER, DH, TE docking centers (-13.313, 51.009, 33.26), (17.518, 1.325, 67.106), (-0.429, 3) were set by AutoDock, respectively2.887, 8.521), (-34.5, -10.95, 27.643), (13.42, 127.505, 49.567), (0.525, 61.725, 43.075) are sized

The grid covering the active site amino acids and surrounding surface portions, rigid receptors and flexible ligands were selected, the number of genetic algorithm conformation search outputs was set to 100, the AutoDock program was run to obtain results (table 5), the docking results were obtained from at least 5 repeated docks and visualized using PyMol software, the conformationally best docking results were selected to be aligned with the active pocket predicted by the dougitescore, and their binding patterns to amino acid residues were analyzed by LigPlot plot (fig. 11)

TABLE 5 binding energy (kcal/mol) of Ampelopsin D to each domain of FASN

The molecular docking results were overlapped with the three active pockets before the scoring of each domain predicted by the DoGSitsCorer to obtain Ampelopsin D bound to the most highly scored active pocket of DH, ER, KR, KS, TE domain, respectively, and the MAT domain bound to the second most highly scored active pocket (FIG. 10), and from Table 6 it was found that Ampelopsin D could directly form hydrogen bonds with Ser2308, which is the active amino acid of TE, and bound between the four alpha helices of the active pocket, Ampelopsin D did not interact with the active amino acid of KS, but bound to the active amino acid Cys161, which may exert inhibitory effects, Ampelopsin D could form hydrophobic interactions with Gly1678, which is the active amino acid of ER, while Ampelopsin D bound to MAT and DH, respectively, which are far from Ser, His878, which is presumed to be relatively weak.

TABLE 6 Hydrogen-and hydrophobic interactions of the amino acid residues of each domain shown by Ligplot with the ligand small molecule Ampelopsin D

Influence of four peony seed meal monomer compounds on breast cancer cells

Four peony seed meal monomer compounds reduce cell viability of two breast cancer cells four peony seed meal compounds, namely Epsilon-Viniferin, Suffruticosol A, Ampelosol D and Suffruticosol B, and the cell viability inhibition of the MDAMB-231 and MCF-7 breast cancer cells is determined by setting 11 drug concentrations from 0 mu M to 100 mu M. As shown in Table 7, the inhibition of cell viability of two cell lines was reduced sequentially by four peony seed meal compounds, namely Epsilon-Viniferin, Suffruticosol A, Ampelopsin D and Suffruticosol B, wherein the IC50 values for MDA-MB-231 cell line were 27.0. mu.M, 35.6. mu.M, 48.6. mu.M and 50.7. mu.M, respectively, and the IC50 values for MCF-7 cell line were 32.1. mu.M, 47.2. mu.M, 65.6. mu.M and 98.33. mu.M, respectively.

TABLE 7 IC50 values (μ M) for peony extract to induce changes in cell viability

Three peony seed meal monomer compounds inhibit the expression level of FASN of two breast cancer cells: whether three compounds, namely Epsilon-Viniferin, Suffrutinosol A and Ampelopsin D, have inhibitory effects on the expression level of FASN under the concentrations of 0 mu M, 25 mu M, 50 mu M and 70 mu M respectively is detected through Western immunoblotting, and the results are shown in FIG. 13, wherein the expression levels of the two breast cancer cell lines FASN of Epsilon-Viniferin, Suffrutinosol A and Ampelopsin D have inhibitory effects to different degrees and are in concentration gradient dependence;

it should be noted that: in FIG. 13, after different concentrations of Epsilon-Viniferin, Suffrutinosol A and Ampelopsin D treated MDA-MB-231 and MCF-7 cell lines for 24h, the expression level of FASN in the cells was detected by immunoblotting.

In the research, the apoptosis conditions of Epsilon-Viniferin, Suffruticosol A and Ampelopsin D with different concentrations of 0,25,50 and 75 mu M after the cells are treated for 24 hours are detected. The results are shown in FIG. 14, the cleavage condition of the apoptosis protein PARP is gradually obvious along with the increase of the drug concentration of Epsilon-Viniferin, Suffruticosol A and Ampelopsin D, and simultaneously the cleavage condition of the anti-apoptosis protein Bcl-2 is also obviously reduced, wherein the cleavage condition of PARP and the reduction of Bcl-2 are extremely obvious when two breast cancer cell strains are administrated at the concentration of 75 μ M;

of note in fig. 14 are: expression levels of the intracellular apoptotic protein PARP and the anti-apoptotic protein Bcl-2 were examined using Western immunoblotting 24h after different concentrations of Epsilon-Viniferin, Suffruticosol A, AmpelopsinD treated MDA-MB-231 and MCF-7 cell lines.

To sum up:

discussion in vitro cell experiments on precancerous breast cancer cells the results of experiments on anticancer activity of peony seed meal crude body indicate that monomeric compound components with antitumor efficacy may be present therein. The research hopes to screen and determine whether four monomer compounds in the peony seed meal extract have anti-tumor activity caused by inhibition of FASN activity and expression by means of methods such as molecular docking, molecular dynamic simulation and the like.

This study attempted to construct the holoenzyme dimer structure of FASN by a homology modeling approach, similar to the FASN structure based on deep learning predictions updated by AlphaFold in 7 months 2021. Trial and error and road paving are carried out for subsequent experiments, but a new idea is provided for screening the FASN inhibitor in the laboratory.

In the research, compounds with outstanding anti-tumor effect cannot be compared among four peony seed meal monomer compounds. The reasons for this may be:

1) the components in the crude extract of the peony seed meal are complex, only four monomer compound components in the peony seed meal components are obtained in the experiment, the scope and range for screening are small, and in addition, the purity of the obtained four compounds cannot be determined, so that the result reliability of in vitro cell experiments cannot be guaranteed.

2) In the experiment, only a single compound is used for treating the breast cancer cells, and the possible combined action effect among a plurality of compounds is not considered.

3) Since FASN itself is a multienzyme complex containing seven catalytically active domains, and has been mostly targeted at virtual screening of single domains, the present study was aimed at docking all domains of FASN, and fully understanding the binding properties of the screened compounds at all domains of FASN. However, the lack of comparability between multiple different domains makes it difficult to rationally analyze the results.

The experimental reagent related to the experimental method of the FASN inhibitor in the peony seed meal monomeric compound comprises a Fatty Acid Synthetase (FAS) activity detection kit, a BeyoClickTMEdU-594 cell proliferation detection kit, an Annexin V-FITC apoptosis detection kit, a cell cycle and apoptosis detection kit, an immunostaining confining liquid, an immunostaining washing liquid, a RIPA lysate, a BCA protein quantification kit, Tetramethylethylenediamine (TEMED), Sodium Dodecyl Sulfate (SDS), a protein molecular weight standard Marker, a 4% immunohistochemical fixing liquid, Acrylamide (Acrylamide), dithiot-alditol (DTT), thiazole blue (MTT), a primary anti-dilution liquid, bromophenol blue, palmitic acid, Tween-20, anhydrous methanol, Adapalene, Celecoxib, alectib, Lumacaftor, Tris-HCL, potassium chloride, hydrochloric acid, ethanol, sodium chloride, skim milk powder, bovine serum, polyvinylidene fluoride (PVDF) membrane, bovine serum, Developing solution, glycerol, Trizmabase, glycine, dimethyl sulfoxide (DMSO), Ammonium Persulfate (AP), pancreatin (without EDTA), pancreatin (with EDTA), high-sugar culture solution and PBS;

the experimental antibody comprises a FASN Rabbit mAb, a PARP Rabbit mAb, a PERK Rabbit mAb, a CHOP Rabbit mAb, a BiP Rabbit mAb, a beta-Actin Rabbit mAb, Anti-Bcl-2antibody, Anti-Bax antibody, Anti-IRE1 antibody, Anti-ATF6 antibody, Anti-DDIT3 antibody, goat Anti-Rabbit IgG-HRP and goat Anti-mouse IgG-HRP;

the experimental apparatus comprises: the device comprises an electric heating constant temperature blast drying box, an ultrasonic cell smashing instrument, an electric heating constant temperature water bath kettle, an ice maker, a metal constant temperature bath, a constant temperature oscillator, a low-speed centrifuge, a vortex mixer, an ultra-low temperature refrigerator, an electronic analysis balance, an inverted biological microscope, a pipettor, a vacuum pump, a circumference shaking table, an ultra-clean workbench, a chemiluminescence imaging analysis system, a table type refrigerated centrifuge, a multifunctional microplate reader, a flow cytometer, a pipettor, a table type pH meter, a magnetic stirrer, an intelligent upright fluorescence microscope, a carbon dioxide incubator, an ultraviolet visible spectrophotometer, an inverted fluorescence microscope and an ultrapure water integrated system.

The specific preparation method of the experimental reagent comprises the following steps:

1) complete culture medium

The reagent preparation is operated in a sterile environment, and is stored in a refrigerator at 4 ℃ after being inverted and mixed uniformly.

2)PBS

3.2L of ddH were added₂0, adjusting the pH to 7.4, diluting to 4L and sterilizing.

Reagent related to immunoblotting

1) SDS-PAGE separating gel (10%)

2) SDS-PAGE concentrated gel (5%)

3)1.5M Tris-HCl,pH 8.8

Adjusting pH to 8.8 with hydrochloric acid, diluting to 500mL, and storing at 4 deg.C. 4)1.0M Tris-HCl, pH6.8

Adjusting pH to 6.8 with hydrochloric acid, diluting to 500mL, and storing at 4 deg.C.

5)10％SDS

Dissolving in water bath at 50 deg.C, and storing at room temperature.

6) 10% ammonium persulfate

After vortex mixing, the mixture can be separately loaded into a 2mL centrifuge tube and stored at-20 ℃ for later use.

7) 5X electrophoresis buffer

When in use, the product is diluted 5 times with ultrapure water.

8)5 Xtransmembrane buffer

9)1 Xtransmembrane buffer

10)10×TBS

Adding an appropriate amount of ultrapure water, after the ultrapure water is completely dissolved, adjusting the pH to 7.5 by using concentrated hydrochloric acid, and fixing the volume to 2L.

11)TBST

12)4 XLoading buffer

Mixing, and storing at-20 deg.C.

It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

Claims (5)

1. The experimental method for screening the FASN inhibitor in the peony seed meal monomer compound based on computer simulation is characterized by comprising the following steps: the experimental method for simulating and screening the FASN inhibitor in the peony seed meal monomer compound by the computer comprises the following specific operation steps:

s1, molecular docking;

s2, cell viability detection-MTT method;

s3, western immunoblot;

s4, detecting apoptosis;

the molecular docking in the step S1 comprises the following specific operation steps:

installing AutoDock and Openbabel software in a Linux system environment;

1) preparing a receptor pdb file, checking the receptor structure by using PyMOL software, and removing structures such as crystal water and a small molecular ligand which are possibly contained;

2) converting the ligand and receptor files into pdbqt files in batch by using an AutoDock Tool;

3) respectively putting the receptor pdbqt files into sequentially arranged folders;

4) setting related parameters such as ligand and receptor names, box size, coordinates and the like in the script to carry out automatic dock docking;

5) and extracting a log file generated by molecular docking.

2. The experimental method for screening FASN inhibitor in peony seed meal monomer compounds based on computer simulation according to claim 1, wherein: the specific operation steps of the cell viability detection-MTT method in S2 are as follows:

1) when the cell density in the 96-well plate is 70-80%, adding serum-free DMEM containing different drug concentrations to treat the cells for 24h, and repeating 6 wells for each drug concentration;

2) preparing 5mg/mL MTT solution from MTT and DMSO in advance;

3) preparing a serum-free culture solution and an MTT solution (5mg/mL) into a mixed solution of 0.5mg/mL according to a ratio of 9: 1;

4) discarding the culture solution, adding PBS into each hole for cleaning, and adding 100 mu L of mixed solution into each hole;

5) culturing at 37 deg.C for 1 h;

6) discarding the mixed solution, adding and blowing 100 mu L DMSO into each hole;

7) the absorbance at 492nm was measured using a microplate reader.

3. The experimental method for screening FASN inhibitor in peony seed meal monomer compounds based on computer simulation according to claim 1, wherein: the specific operation steps of the Western blotting in S3 are as follows:

1) connecting the cells to a six-hole plate, adding serum-free culture solution containing different drug concentrations to treat the cells for 24 hours when the cell density is 70-80%, and repeating for 3 holes at each drug concentration;

2) washing cells with PBS, adding 120 mu L of lysis solution, and scraping the cells to a centrifuge tube;

3) performing ultrasonic treatment on ice for 4 min;

4) centrifuge at 4 ℃ and transfer the supernatant to a new centrifuge tube.

5) The BCA method is used for determining the protein concentration and leveling;

6) adding 4 × Loading Buffer in proportion, boiling the sample at 95 ℃ for 10min, and storing the sample at-20 ℃;

electrophoresis

1) Preparing SDS-PAGE separation gel: the ddH was then formulated according to the gel formulation₂Mixing O, 30% AB, 1.5M Tris-HCl (pH 8.8), 10% AP and TEMED in a vortex manner, adding 7mL of separation gel into each gel preparation plate, adding water for supplementing, and waiting for solidification;

2) preparing SDS-PAGE concentrated gel: preparing ddH2O, 30% AB, 1M Tris-HCl (pH6.8), 10% AP and TEMED according to a gel formula, uniformly mixing by vortex, pouring water in a gel making plate, adding concentrated gel for supplementing, inserting a comb, and waiting for solidification;

3) preparing 1 XRunning Buffer: diluting 5 XRunning Buffer and ultrapure water according to the proportion of 1:4, and reversing and uniformly mixing;

4) placing the rubber plate in an electrophoresis tank, adding 1 × Running Buffer, pulling out a comb, and adding a proper amount of Marker or sample into each hole;

5) electrophoresis: performing constant-pressure 80V electrophoresis for 30min, and adjusting to 120V electrophoresis to make the strip run to the bottom of the gel;

rotary film

1) Prepare 1 × Transfer Buffer: diluting 5 times Transfer Buffer, anhydrous methanol and ultrapure water according to the proportion of 1:1:3, and reversing and mixing uniformly;

2) film shearing: soaking PVDF in methanol for activation;

3) film transfer: placing the sponge and the three layers of filter paper on a film Transfer clamp, soaking the sponge and the three layers of filter paper through a Transfer Buffer, placing the gel on the black part of the film Transfer clamp, covering the film on the gel, clamping the film Transfer clamp, inserting the film into a groove, and transferring the sample from the gel to the film fully by constant current of about 250mA for 2.5 hours;

incubating antibodies

1) Washing the membrane: diluting 10 × TBS solution to 1 × TBST solution in advance, and cleaning with 1 × TBST solution for 10min each time for 3 times after membrane transfer;

2) and (3) sealing: preparing a confining liquid in advance according to the proportion of adding 1g of skimmed milk powder into every 20mL of TBST, putting the membrane into the confining liquid, and shaking for 1h at 37 ℃;

3) incubating the primary antibody: diluting the primary antibody with a primary antibody diluent, and placing the membrane in a primary antibody incubation solution; incubating overnight at 4 ℃;

4) washing the membrane: washing the membrane for 3 times by TBST;

5) incubation of secondary antibody: putting the membrane into a confining liquid containing a secondary antibody, and shaking for 1h at 37 ℃;

6) washing the membrane: washing the membrane for 3 times by TBST;

development

1) Preparing ECL luminescent liquid: mixing the two solutions according to the proportion of 1: 1;

2) developing by using an exposure machine, selecting a Chemi option of an ImageLab software blot, uniformly dripping 160 mu L of luminous liquid before exposure, obtaining a strip, quantitatively analyzing the gray value of the strip by using ImageJ software, mapping by using GraphPad Prism software, and repeating each experiment for three times.

4. The experimental method for screening FASN inhibitor in peony seed meal monomer compounds based on computer simulation according to claim 1, wherein: the specific operation steps of apoptosis detection in S4 are as follows:

1) inoculating the cells to a six-hole plate, and adding serum-free culture solution containing different drug concentrations to treat the cells for 24 hours when the cell density is 70-80%;

2) collecting the culture solution to a centrifuge tube, digesting adherent cells for 5min by using trypsin, blowing the digested cells to a single cell state by using 1mL of the culture solution which is just sucked, merging the cells into the centrifuge tube, and centrifuging at 1100g for 4min at 4 ℃;

3) cells were washed 2 times with 0.5mL PBS, 1100g, 4min, centrifugation at 4 ℃;

4) resuspending cells in Annexin V-FITC conjugate;

5) adding Annexin V-FITC and PI in sequence;

6) flow detection: respectively selecting FITC/PE or FL1/FL2 channels;

7) and (3) fluorescent microscope detection: the cell suspension was dropped onto a glass slide and viewed with a cover slip.

5. The experimental method for screening FASN inhibitor in peony seed meal monomer compounds based on computer simulation according to claim 1, wherein: the experimental reagent related to the experimental method of the FASN inhibitor in the peony seed meal monomeric compound comprises a Fatty Acid Synthase (FAS) activity detection kit, a BeyoClickTMEdU-594 cell proliferation detection kit, an Annexin V-FITC apoptosis detection kit, a cell cycle and apoptosis detection kit, an immunostaining confining liquid, an immunostaining washing liquid, a RIPA lysate, a BCA protein quantification kit, Tetramethylethylenediamine (TEMED), Sodium Dodecyl Sulfate (SDS), a protein molecular weight standard Marker, a 4% immunohistochemical fixing liquid, Acrylamide (Acrylamide), dithiot-alditol (DTT), thiazole blue (MTT), a primary anti-diluent, bromophenol blue, palmitic acid, Tween-20, anhydrous methanol, Adapalene, Celecoxib, aletinib, Lumacaftor, Tris-HCL, potassium chloride, hydrochloric acid, ethanol, sodium chloride, skim milk powder, fetal calf serum, polyvinylidene fluoride (PVDF membrane), Developing solution, glycerol, Trizma base, glycine, dimethyl sulfoxide (DMSO), Ammonium Persulfate (AP), pancreatin (without EDTA), pancreatin (with EDTA), high-sugar culture solution and PBS;

the experimental antibody comprises a FASN Rabbit mAb, a PARP Rabbit mAb, a PERK Rabbit mAb, a CHOP Rabbit mAb, a BiP Rabbit mAb, a beta-Actin Rabbit mAb, Anti-Bcl-2antibody, Anti-Bax antibody, Anti-IRE1 antibody, Anti-ATF6 antibody, Anti-DDIT3 antibody, goat Anti-Rabbit IgG-HRP and goat Anti-mouse IgG-HRP;

the experimental apparatus comprises: the device comprises an electric heating constant temperature blast drying box, an ultrasonic cell smashing instrument, an electric heating constant temperature water bath kettle, an ice maker, a metal constant temperature bath, a constant temperature oscillator, a low-speed centrifuge, a vortex mixer, an ultra-low temperature refrigerator, an electronic analysis balance, an inverted biological microscope, a pipettor, a vacuum pump, a circumference shaking table, an ultra-clean workbench, a chemiluminescence imaging analysis system, a table type refrigerated centrifuge, a multifunctional microplate reader, a flow cytometer, a pipettor, a table type pH meter, a magnetic stirrer, an intelligent upright fluorescence microscope, a carbon dioxide incubator, an ultraviolet visible spectrophotometer, an inverted fluorescence microscope and an ultrapure water integrated system.

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