CN112063665B

CN112063665B - Preparation method and application of Piericidin (Piericidin)

Info

Publication number: CN112063665B
Application number: CN202010826262.9A
Authority: CN
Inventors: 洪斌; 王丽; 解云英; 何维; 李星星; 孙红敏; 侍媛媛; 张秀敏; 杜郁
Original assignee: Institute of Medicinal Biotechnology of CAMS
Current assignee: Institute of Medicinal Biotechnology of CAMS
Priority date: 2020-08-17
Filing date: 2020-08-17
Publication date: 2022-05-27
Anticipated expiration: 2040-08-17
Also published as: CN112063665A

Abstract

The invention relates to a preparation method and application of Piericidin (Piericidin), wherein the preparation method comprises the following steps: (1) fermenting the CPCC 205386 strain and harvesting fermentation liquor, and centrifuging the fermentation liquor to divide the fermentation liquor into supernate and mycelium; (2) extracting the supernatant of the fermentation liquor by using a macroporous resin adsorption elution method to obtain a fermentation liquor extract, and extracting mycelia by using acetone to obtain a mycelium extract; (3) mixing the fermentation liquor extract and the mycelium extract, and purifying the active ingredients by preparative HPLC to obtain the piericidin; the Piericidin (Piericidin) is Piericidin A1, A2, A3, C1, C2 and C3. The application is the treatment of PCSK 9-related cardiovascular diseases.

Description

Preparation method and application of Piericidin (Piericidin)

Technical Field

The invention belongs to the technical field of medical biology, and particularly relates to a preparation method and application of Piericidin (Piericidin).

Background

Cardiovascular Disease (CVD) has become one of the major threats to human health worldwide. Low-density lipoprotein cholesterol (LDL-C) is an important risk factor for atherosclerotic cardiovascular disease (ASCVD) [1]. Therefore, modulating LDL-C levels is an important tool in the treatment of cardiovascular diseases. PCSK9 was first discovered and demonstrated in 2003 as a third cardiovascular disease susceptibility gene, and inhibition of PCSK9 was effective in lowering LDL-C levels [2]. The PCSK9 drug which is currently marketed only has two monoclonal antibodies, namely Alirocumab

And Evolcumab

They have good therapeutic effect and safety [3,4 ]]However, there are also limitations on the high price and inconvenient administration mode [5]. Based on the advantages of easy synthesis, low price, convenient administration mode and the like of small molecule drugs, the PCSK9 small molecule inhibitor becomes a research hotspot.

Secondary metabolism of microorganisms, especially actinomycetes, is an important source of natural products. Many small molecule compounds with pharmaceutical activity have been found in these natural products. Statistically, over 5 million natural products of microbial origin, most of which are produced by bacteria, particularly actinomycetes, have been identified since the first discovery of penicillin by fleming, an british scientist in 1928. 58% of antibiotic drugs and 32% of anticancer drugs approved in the last 35 years are natural products derived from microorganisms or semisynthetic derivatives thereof.

Compared with compound libraries and other screening libraries, the microbial crude product library has lower production cost, is rich in various natural products, and provides greater possibility for the discovery of novel active natural product medicaments with new structures. The national new drug (microorganism) screening fermentation liquor crude product library of the research institute has the fermentation liquor crude product samples of 76,000 bacterial strains at present, and the samples have wide sources, thereby providing a source for large-scale screening.

[ reference documents ]

[1]Boekholdt SM,Arsenault BJ,Mora S,et al.Association of LDL Cholesterol,non-HDL Cholesterol,and Apolipoprotein B Levels With Risk of Cardiovascular Events Among Patients Treated With Statins:A Meta-Analysis.JAMA,2012,28；307(12):1302-9.

[2]Timms K,Wagner S,Samuels M,et al.A Mutation in PCSK9 Causing Autosomal-Dominant Hypercholesterolemia in a Utah Pedigree.Human Genetics,2004,114(4):349-353.

[3]Goodman SG,Steg PG,Szarek M,et al.Sustained Low-Density Lipoprotein Cholesterol Lowering with Alirocumab in ODYSSEY OUTCOMES.J Am Coll Cardiol,2020,75(4):448-451.

[4]Murphy SA,Pedersen TR,Gaciong ZA,et al.Effect of the PCSK9 Inhibitor Evolocumab on Total Cardiovascular Events in Patients with Cardiovascular Disease:A Prespecified Analysis From the FOURIER Trial.JAMA Cardiology,2019,4(7):613-619.

[5]Kazi DS,Moran AE,Coxson PG,et al.Cost-effectiveness of PCSK9 Inhibitor Therapy in Patients With Heterozygous Familial Hypercholesterolemia or Atherosclerotic Cardiovascular Disease.JAMA.2016,16；316(7):743-53.

[6]Wang X,Chen X,Zhang X,et al.A small-molecule inhibitor of PCSK9 transcription ameliorates atherosclerosis through the modulation of FoxO1/3 and HNF1alpha.EBioMedicine,2020,52:102650.

Disclosure of Invention

The invention firstly relates to a preparation method of a group of Piericidin (Piericidin), which is characterized by comprising the following steps:

(1) fermenting I03A-00300 strain (China pharmaceutical microorganism culture Collection management center preservation number: CPCC 205386) and harvesting fermentation liquor, centrifuging the fermentation liquor at 4500rpm for 25min, and separating into supernatant and mycelium;

(2) adsorbing and enriching active components in the supernatant by using macroporous resin, extracting active components of mycelia by using acetone, and combining the two parts to obtain a fermentation crude extract;

(3) purifying the crude extract by preparative HPLC to obtain the piericidin;

the Piericidin is Piericidin A1, A2, A3, C1, C2 and C3, and the structural formula is shown as follows.

The process for fermenting the CPCC 205386 strain in the step (1) comprises the following steps:

1) inoculating the strain into an ISP2 seed culture medium, culturing at 28 deg.C and 220rpm for 48h to obtain a seed solution;

2) inoculating the strain in A1 culture medium at 5-10% of inoculation amount, culturing at 28 deg.C and 220rpm for 5d to obtain fermentation liquid.

The step (2) of adsorbing and enriching the active components in the supernatant by using macroporous resin comprises the following steps: adjusting pH of the supernatant to below 4.8, adsorbing to macroporous resin D4006 filler, eluting with deionized water, 30% acetone and 100% acetone respectively, collecting acetone eluate, and lyophilizing to obtain crude extract P1;

the step of extracting the mycelium by using acetone in the step (2) comprises the following steps:

extracting mycelium with acetone to obtain mycelium crude extract, extracting mycelium crude extract with 90% methanol and petroleum ether, retaining petroleum ether, and removing solvent to obtain mycelium extract P2;

and (3) purifying the crude extract by using preparative HPLC to obtain the piericidin, wherein the method comprises the following steps: the crude extracts P1 and P2 were combined and subjected to HPLC semipreparative column (

C18 OBD^TM Prep Column

5 μm,10mm × 250mm), 82.5% methanol-water isocratic elution for 30min, collecting six major components at 254nm absorptionThe components F1-F6 are the components of piericidin A1, A2, A3, C1, C2 and C3.

The invention also relates to the following applications of the piericidin A1, A2, A3, C1, C2 and C3:

(1) preparing an agent that inhibits expression of PCSK 9;

(2) the preparation of medicine for treating diseases related to PCSK9 gene, such as cardiac vascular diseases, preferably atherosclerosis cardiac vascular diseases.

The invention has the advantages that the constructed PCSK9 transcription inhibitor screening cell model is used for carrying out high-throughput screening on samples in a crude fermentation liquor library of a new drug (microorganism) screening center in the institute, 6 strains of positive strains with better activity and repeatability are finally obtained, a series of separation and purification and cell model detection activities are carried out on fermentation products of the positive strains I03A-00300(CPCC 205386) by taking activity as a guide, the compound Piericidin A1 and homologues thereof are finally determined to have PCSK9 expression inhibition activity, and the expression of downstream gene LDLR which can be regulated by PCSK9 and the uptake effect of LDL cholesterol are proved to be up-regulated at a cell level. The series of compounds are proved to be expected to be developed into novel antiatherosclerotic lead compounds or drug candidates.

Drawings

FIG. 1 shows a dose-effect relationship curve of a crude extract sample of a positive strain fermentation broth on a PCSK9 expression inhibitor screening model.

FIG. 2, effect of crude samples of positive strain fermentation broth on PCSK9 mRNA in HepG2 cells.

Figure 3 effect of crude positive strain fermentation broth samples on PCSK9 and LDLR protein levels in HepG2 cells.

FIG. 4 is a flow chart of the preparation of piericidin.

FIG. 5, HPLC chromatograms of six active components F1-F6.

FIG. 6, structural formula of compound F1-F6.

Figure 7 effect of compound Piericidin a1 on HepG2 cell PCSK9 protein levels.

Figure 8, effect of compound Piericidin a1 on HepG2 cell PCSK9 protein levels (high sample concentration).

Figure 9 effect of compound Piericidin a1 on LDLR protein levels in HepG2 cells.

Figure 10, effect of compound Piericidin a1 on HepG2 cell LDLR protein levels (high sample concentration).

FIG. 11, effect of compound Piericidin A1 on the uptake of DiI-LDL by HepG2 cells.

FIG. 12, effect of compound Piericidin A1 on the uptake of DiI-LDL by HepG2 cells (high sample concentration).

Detailed Description

Treatment of fermentation broth samples

Screening sample sources: fermentation liquor sample library of national new drug (microbe) screening center;

screening fermentation liquor samples and preparing: samples of the fermentation broth sample pools were prepared according to standard procedures for strain fermentation broth samples and dissolved in 100% DMSO as solvent. After the sample is taken out from the fermentation liquid bank, the sample is diluted 5 times by 100% DMSO to be used as a sample to be screened.

High throughput screening

Taking out pGL4-PCSK9-P HepG2 cells cultured in a cell incubator (the construction process of cell strains is disclosed in an article published before the unit: reference document [6]), observing the growth state of the cells under a microscope, and carrying out PBS rinsing, trypsinization and beating on the cells with the fusion degree of about 90 percent to obtain single cells;

② mixing the cell suspension evenly, measuring the cell density, diluting the cell to 5X10⁵One/ml, inoculating in 96-well plate, adding 100 μ l per well;

③ after about 20 hours, the cells are fully attached to the wall, the original culture medium containing the serum is discarded, and the MEM culture medium without the serum is added into each hole with 200 mul;

adding 1 mul fermentation liquor sample to be tested into each hole (the DMSO concentration is 0.5%, and the DMSO concentration has no obvious influence on the cell state through the early-stage construction model detection in the laboratory), meanwhile, taking 0.5% DMSO as a solvent control, setting a blank control only added with the culture medium, and putting the blank control into a cell culture box for continuous culture.

Observing the growth state of the cells under a microscope after 24 hours, marking and recording dead or deformed cell pores, and detecting the luciferase activity of the cells after the observation is finished;

sixthly, processing the screening result

The regulation rate of the screened fermentation liquor sample on the activity of the cell luciferase is as follows:

example one screening of crude fermentation broth

1. Preliminary screening

The primary screened samples were dissolved in 100% DMSO and stored in 96-well plates, diluted 5-fold with 100% DMSO as the samples to be screened.

Screening Using 96-well plates, PCSK9 expression inhibitor screening model cells (pGL4-PCSK9-P HepG2 cells) had a density of 6X 10⁶100 mul of plate is paved in each hole per ml, 200 mul of serum-free culture medium is replaced after 24h of culture, 1 mul of sample to be tested is added in each hole, the culture is continued for 24h, the growth state of the cells is observed under a microscope, dead or deformed cell holes are marked and recorded, and the luciferase activity of the cells is detected after the observation is finished;

and (4) processing a screening result, wherein the regulation rate of the screened fermentation liquor sample on the luciferase activity of the cells is as follows:

。

in the experiment, crude fermentation liquid extracts of total 12,560 samples with fermentation liquid sample library numbers of 1073B-1110B, 1127B-1180B and 1726B-1788B are primarily screened. The firefly luciferase detection system was used with a downregulation > 50% as primary screen positivity. The screening result shows that 350 positive fermentation broth samples are obtained by primary screening, and the positive rate is 2.8%. By means of re-detecting the positive fermentation liquid, utilizing a multiple proportion dilution sample and the like to eliminate samples with accidental errors, false positives and weak activity in the experiment, 39 crude samples of the positive fermentation liquid with better activity are finally obtained, and the positive rate is 0.31%.

2. Rescreening of crude extract of positive fermentation broth

In order to further verify whether the fermentation product of the strain corresponding to the obtained positive fermentation broth sample has repeatability on the PCSK9 expression inhibition activity, the positive strain corresponding to the primary screening positive sample is obtained from the China pharmaceutical microbiological culture Collection center, and the positive strain is cultured and fermented again. The positive strains are subjected to repeated fermentation culture by using fermentation culture media A1 and A2 according to the original recording fermentation conditions, and all components of thalli and culture solution are extracted by using the same method as the original recording to obtain similar detection samples. And then, a PCSK9 expression inhibitor screening model is used for detecting the activity of the strain, and the results of the primary screening activity are compared to confirm whether the activity of the positive strain can be repeated and whether the activity is changed. The effect of A1 and A2 fermentation media on the production of active substances by the strains was also compared. And (3) displaying a detection result: the 31 primary screening positive strains participate in secondary screening, and 22 strains in total show PCSK9 expression inhibition activity in A1 or A2 fermentation culture medium; meanwhile, the positive rate of most positive strains in A1 is slightly higher than that of A2, and an A1 fermentation culture medium is adopted subsequently.

A1 culture medium:

glucose 0.5% (w/v); 1% (w/v) of malt extract; 1% (w/v) of cottonseed cake meal; 2% (w/v) soluble starch; 0.5% (w/v) of yeast extract; K2HPO40.05% (w/v); CaCo30.3% (w/v); NaCl 0.1% (w/v); adding water to 1000 ml; pH 7.2 ± 0.2;

sterilizing at 115 deg.C under high pressure for 20 min.

A2 culture medium:

glucose 0.5% (w/v); peptone 0.5% (w/v); soybean flour 1% (w/v); starch 2% (w/v); 0.5% (w/v) of yeast extract; beef extract 0.5% (w/v); 0.4% (w/v) of corn steep liquor; CaCo30.4% (w/v); CoCl20.002% (w/v); adding water to 1000 ml; the pH value is 7.2 plus or minus 0.2;

sterilizing at 115 deg.C under high pressure for 20 min.

6 positive strains with better activity and repeatable property are obtained by re-screening, and are respectively as follows: I03A-09264, I03A-09569, I03A-09790, I03A-09776, I03A-09770 and I03A-00300.

The medium conditions were further optimized. The final strains I03A-09264, I03A-09790, I03A-09776, I03A-09770 and I03A-00300 adopt MS culture medium, and I03A-09569 adopts ISP2 culture medium.

3. Quantitative effect relationship analysis of positive strain fermentation liquor sample

And 6 screened positive strain fermentation liquor samples are subjected to dose-effect analysis. Starting from 1/500 dilution concentration, a series of dilution times of samples were obtained by 1/10 gradient dilution (highest concentration was 1/500 dilution, lowest concentration was 1/5x 10)^-9Dilution).

The activity of the PCSK9 expression inhibitor is detected by using a PCSK9 expression inhibitor screening model, and the result is shown in figure 1: the fermentation liquor of the 6 strains has better activity of down-regulating the expression of PCSK9, wherein the fermentation liquor of the strain with the best activity is I03A-00300.

Example two, verification of Activity of Positive Strain fermentation broth samples

1. Effect on the level of transcription of PCSK9 protein

After 1/1000 diluted concentration of crude extract of positive fermentation broth is added into HepG2 cells for 24 hours, total RNA of the cells is extracted and is reversely transcribed into cDNA, and real-time PCR experiment is carried out.

The results show that 6 positive strain fermentation broth samples have influence on mRNA of PCSK9 in HepG2, and as shown in FIG. 2, crude fermentation broth samples of positive strains I03A-09264, I03A-09569, I03A-09790, I03A-09776, I03A-09770 and I03A-00300 can all significantly reduce the mRNA level of PCSK 9.

2. Effect on PCSK9 protein levels

Adding 1/1000 diluted concentration of crude extract sample cells of the positive fermentation liquid into HepG2, extracting total cell protein after 24 hours, and performing a western blot experiment. The experimental results are shown in fig. 3: the fermentation liquor samples of the 6 positive strains can obviously inhibit the expression level of the PCSK9 protein.

EXAMPLE III preparation and validation of active ingredients

The results of the first and second examples show that the fermentation broth sample of the strain I03A-00300 can significantly inhibit the PCSK9 mRNA level; the fermentation liquor sample of the strain I03A-00300 can obviously inhibit the protein level of PCSK 9. Therefore, the strain I03A-00300 was isolated and purified.

1. The expanded fermentation of the strain I03A-00300, the separation and purification of active components and the structural identification.

(1) The expanded culture adopts two-stage fermentation, and the fermentation process comprises the following steps:

the slant culture of the strain I03A-00300 was inoculated into the seed medium ISP2 (100ml medium/500 ml Erlenmeyer flask) and cultured at 28 ℃ and 220rpm for 48 hours to obtain a seed culture.

Then inoculating the strain in A1 culture medium (1L culture medium/5L conical flask) with the inoculation amount of 5% -10%, culturing at 28 deg.C and 220rpm for 5d to obtain fermentation liquid sample.

After centrifugation, freeze-drying and dissolution in 100% DMSO, a fermentation liquid sample obtained by secondary fermentation of the strain I03A-00300 is verified to have good PCSK9 expression inhibition activity and can be repeated in a PCSK9 gene expression inhibitor model.

2. Separation, purification and structural identification of active compounds

Conditions optimization of active components in macroporous resin D4006 enriched fermentation supernatant

Adjusting the pH of the fermentation liquor, dynamically adsorbing the fermentation liquor by macroporous adsorption resin D4006, eluting the fermentation liquor by water, 30% acetone and 100% acetone, and detecting the activity of each component by utilizing a PCSK9 expression inhibitor model. The results show that the adsorption capacity of the macroporous resin on the active ingredients is enhanced along with the increase of acidity or alkalinity, wherein the macroporous resin can completely adsorb the active ingredients under the acidic condition of pH 4.80, so that the fact that the ingredients with PCSK9 gene expression inhibition activity in the fermentation liquor of the positive strain I03A-00300 possibly have acid-base amphipathy can be inferred, and therefore, the fermentation pH is adjusted to be below 4.80 in order to fully adsorb the active ingredients.

The preparation steps of the sample for structure identification are as follows:

the strain I03A-00300 is subjected to expanded fermentation by adopting secondary fermentation, and after centrifugation, the strain is divided into two parts, namely supernatant and thalli.

Adjusting pH of the supernatant to below 4.8 with HCl, adsorbing with D4006 macroporous adsorbent resin, and eluting with deionized water, 30% acetone, and 100% acetone respectively (mixing acetone eluate fractions, and lyophilizing to obtain P1 crude extract);

extracting thallus part with acetone, centrifuging, concentrating to remove solvent, re-dissolving in 90% methanol, extracting with petroleum ether, retaining petroleum ether part, centrifuging, concentrating to remove solvent to obtain mycelium extract P2; the experimental procedure is shown in fig. 4:

combine fractions P1 and P2, redissolve in 80% methanol and purify fractions by semi-preparative HPLC (

C18 OBD^TM Prep Column

5 μm,10mm × 250mm, 82.5% methanol-water isocratic elution for 30 min). The semi-preparative HPLC chromatogram is shown in FIG. 5, and has 6 absorption peaks under 254nm absorption, and the maximum ultraviolet absorption wavelengths are the same, and are 238nm and 268 nm. Collecting according to peak, vacuum concentrating and drying to obtain components F1-F6, dissolving the obtained components F1-F6 in DMSO to obtain a sample to be detected, and using the sample to be detected for activity evaluation. The results show that 6 samples can obviously inhibit the expression level of the PCSK9 protein, wherein the compound F4 has the most obvious effect.

Compound F4 was analyzed by high resolution mass spectrometry (HRESI-MS) and Nuclear Magnetic Resonance (NMR) and compared to literature data (Table 1) to confirm it as piericidin A1(piericidin A1). The remaining compounds had similar uv absorption characteristics to the F4 component, indicating that they were piericidin a1 analogs, and further confirmed by high resolution mass spectrometry (table 2) that F1 was piericidin C1, F2 was piericidin C2, F3 was piericidin C3, F5 was piericidin a2, and F6 was piericidin A3, the structural formula being shown in fig. 6.

TABLE 1 of Compound F4¹³C NRM data (150MHz, DMSO-d)₆)

。

TABLE 2 high resolution Mass Spectrometry results for six Compounds F1-F6

。

EXAMPLE four Piericidin A1 Activity assay

1. Dose-effect relationship of Piericidin A1 on PCSK9 expression inhibitor screening model

The result of detection in a PCSK9 expression inhibitor screening model shows that the highest final concentration of the positive compound is 0.12mM, 1/10 gradient dilution is carried out, and the lowest detection concentration is 0.12 nM. The results showed that Piericidin a1 inhibited PCSK9 promoter activity (i.e. expression at the level of PCSK9 gene transcription) in a dose-dependent manner, with an IC50 of 3.0nM calculated using software prism version 5.0.

2. Piericidin A1 cytotoxicity assessment

The effect of Piericidin A1 on the survival and growth of HepG2 cells at different concentrations was examined using the MTT method. The results show that at concentrations between 0 and 40. mu.g/ml (0 and 100. mu.M), cell viability was more than 90% of the control group, with little effect on cell growth survival.

3. Effect of Piericidin A1 on PCSK9 expression in HepG2 cells

Three concentration gradients of 5nM, 10nM, 20nM, etc. are set to act on HepG2 cell respectively, and after 24h total cell protein is extracted for western blot experiment. The results show that the compound Piericidin a1 can significantly inhibit the expression level of PCSK9 protein in HepG2 cells (fig. 7).

The concentration of the sample is increased, four concentration gradients of 50nM, 100nM, 500nM and 1 μ M are adopted, and after 24 hours of incubation in HepG2 cells, the total cell protein is extracted for western blot experiment. The results show that the compound Piericidin a1 can significantly inhibit the expression level of PCSK9 protein in HepG2 cells (fig. 8).

4. Effect of Piericidin A1 on LDLR expression in HepG2 cells

Three concentration gradients of 5nM, 10nM, 20nM and the like are set to act on HepG2 cells respectively, and total cell protein is extracted after 24h for western blot experiment. The results show that the compound Piericidin a1 can significantly increase the expression level of LDLR protein in HepG2 cells (fig. 9).

The concentration of the sample is increased, four concentration gradients of 50nM, 100nM, 500nM and 1 μ M are adopted, and after incubation in HepG2 cells for 24h, the total cell protein is extracted for western blot experiment. The results show that the compound Piericidin a1 can significantly inhibit LDLR protein expression levels in HepG2 cells (fig. 10).

4. Effect of Piericidin A1 on LDLR-mediated uptake of LDL-C by HepG2 cells

We used DiI-fluorescently labeled LDL (DiI-LDL) and tested the differences in the uptake of DiI-LDL by HepG2 cells using flow cytometry. Three concentration gradients of 5nM, 10nM and 20nM were also set, and after 24h incubation in HepG2 cells, continued incubation for 4h with DiI-LDL addition followed by flow cytometry detection as shown in FIG. 11. Compared with a blank control cell, the cell fluorescence value of the DiI-LDL control group is obviously increased (B vs. A), and the different concentrations of Piericidin A1 can increase the DiI-LDL uptake effect of HepG2 cells to different degrees, wherein 10nM Piericidin A1 can increase the DiI-LDL uptake capacity of hepatocyte HepG2 by 15.1% (D vs. B).

The sample concentration was increased by four concentration gradients of 50nM, 100nM, 500nM and 1. mu.M, and after 24h incubation in HepG2 cells, the incubation was continued for 4h with the addition of DiI-LDL, followed by detection by flow cytometry, as shown in FIG. 12. Compared with the blank control cells, the cell fluorescence value of the DiI-LDL control group is remarkably increased (B vs. A), and the different concentrations of Piericidin A1 can increase the DiI-LDL uptake effect of HepG2 cells in a dose-dependent manner, wherein 50nM of Piericidin A1 can increase the DiI-LDL uptake capacity of hepatocyte HepG2 to 1.7 times (C vs. B), and 100nM of Piericidin A1 can increase the DiI-LDL uptake capacity of hepatocyte HepG2 to 2 times (D vs. B).

The results show that the compound Piericidin A1 can not only inhibit the expression of PCSK9 in HepG2 cells, but also promote LDLR-mediated uptake function of LDL-C by hepatocytes.

Finally, it should be noted that the above embodiments are only used to help those skilled in the art understand the essence of the present invention, and are not intended to limit the protection scope of the present invention.

Claims

1. The following uses of piericidin A1:

preparing a medicament for treating diseases related to the PCSK9 gene, wherein the diseases are atherosclerotic cardiovascular diseases;

the structural formula of the piericidin A1 is shown as follows:

。