CN117531014A - Application of anti-silencing function 1A inhibitor in histone lactic modification mediated atherosclerosis - Google Patents

Abstract

The invention discloses an application of an anti-silencing function 1A inhibitor in atherosclerosis mediated by histone lactate modification. The invention firstly defines the regulating mechanism of ASF1A on histone H3K18 lactate modification, effectively prevents histone H3K18 lactate modification and EndMT and AS mediated by the histone H3K18 lactate modification from happening, provides a new prevention and treatment drug development path and drug action target point for AS diagnosis and treatment, has very important medicinal value, and inhibits or knocks out substances expressed by ASF1A to be expected to be candidate drugs for AS treatment.

Description

Application of anti-silencing function 1A inhibitor in histone lactic modification mediated atherosclerosis

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to application of a substance for inhibiting anti-silencing function 1A (anti-silencing function A, ASF 1A) activity in preparation of medicines for preventing and treating atherosclerosis-related vascular diseases.

Background

As the population ages more and more, the prevalence, morbidity and mortality of cardiovascular diseases (Cardiovascular diseases, CVDs) continue to rise, creating a significant health threat and economic burden to humans worldwide. In recent years, in the population dying from diseases in China every year, the number of CVDs accounts for 40% of the total number of deaths, and CVDs become the first cause of diseases and deaths of residents in China.

Atherosclerosis (AS) is the leading cause of CVDs, which among others have been shown to lead to the highest global mortality rates, mainly coronary artery disease (Atherosclerosis involving the arteries supplying the heart) and stroke (Atherosclerosis involving the arteries supplying the brain), making Atherosclerosis the leading cause of death worldwide.

Atherosclerosis is a typical chronic inflammatory vascular disease caused by a variety of factors. The aortic blood vessel is divided into an inner membrane, a middle membrane and an outer membrane from inside to outside, and a monolayer of cells mainly formed by endothelial cells (Endothleial cells, ECs) covers the inner wall of the blood vessel to form the inner membrane of the blood vessel, so that the exchange of oxygen and nutrient substances between blood and tissues is regulated. Thus, ECs play a key role in maintaining the barrier function of blood vessels. It has long been recognized that endothelial cell dysfunction is the initial process of AS, and is primarily characterized by upregulation of chemokines and adhesion molecules, focal penetration, recruiting circulating monocytes into the intima AS foam cells, ultimately leading to plaque formation. Activation and injury of the endothelium at the site of AS susceptibility is one of the major initiation factors for AS development, mainly in vulnerable areas of arterial blood vessels. When endothelial dysfunction occurs, vascular intimal permeability increases, causing leakage of the endothelial layer, leading to massive lipid infiltration and macrophage infiltration, thereby initiating the arteriosclerotic pathological process. Thus, maintaining the integrity of ECs is the first barrier to protect vascular function, the key to develop new strategies to prevent and treat AS.

Endothelial-mesenchymal transition (EndMT) is a special process of cell transformation of ECs under the action of a variety of stimuli such as high fat, in which ECs are transformed into mesenchymal cells under a variety of stimuli and gradually lose endothelial-specific markers, obtaining a mesenchymal phenotype, and damaging both barrier function and secretory function of ECs. Abnormal occurrence of EndMT, which frequently occurs in atherosclerosis-prone areas, has been determined to induce neointimal hyperplasia and endothelial cell dysfunction, thereby promoting endothelial dysfunction and atherosclerosis. There are studies showing that EndMT can promote AS by reducing ECs-characteristic marker expression, increasing expression of extracellular matrix components such AS fibronectin and adhesion molecules. However, the specific molecular mechanisms by which endothelial cells develop EndMT leading to AS remain unclear.

Epigenomic and epigenetic studies have provided some new insight into various diseases caused by non-resolvable inflammation, such as cancer, pulmonary fibrosis and atherosclerosis. There is growing evidence that epigenetics plays an extremely important role in cardiovascular disease, including DNA methylation, histone post-translational modifications, chromatin remodeling, non-coding RNAs, and the like. Histones are the core structure of nucleosomes and can affect chromatin accessibility in a variety of ways. The tail of histones is limited by a variety of post-translational modifications such as methylation, acetylation, phosphorylation, glycosylation, SUMO, ubiquitination, and the like. These histone modifications can significantly regulate chromatin structure and gene expression by changing chromatin microenvironment, and play a vital role in the occurrence and development of many diseases such as atherosclerosis. New studies have found that lactic acid produced by glycolysis can be used as a precursor substance to add a lactoyl group to a lysine residue of histone, and that it serves as a post-translational modification of histone to perform a gene transcription regulatory function, called histone lactonization. Such histone modifications affect gene expression by altering the physical accessibility of DNA molecules to proteins involved in DNA transcription. Furthermore, recent studies have shown that histone lactic acid is closely related to the occurrence and development of various diseases, such as tumors, neurodegenerative diseases, and cardiovascular diseases.

There is increasing evidence that aerobic glycolysis can stimulate histone lactogenesis in macrophages. The energy supply that consumes glucose and accompanies the production of lactic acid even in the presence of oxygen is known as the Warburg effect. Although the Warburg effect is described as a metabolic feature associated with tumors, it is now believed that it is a physiological phenomenon, and that the metabolic pattern of ECs is similar to that of the Warburg effect, and that aerobic glycolysis is still adopted even under aerobic conditions. Glycolysis is the primary energy metabolism of ECs in normal physiological conditions, meeting the ATP demand of 85% of ECs. Glycolysis can ensure normal metabolism and proliferation of ECs in AS-prone areas, maintain ECs steady state and vascular endothelial integrity, protect blood vessels and reduce AS development. However, some studies have shown that while glycolysis is a protective mechanism for ECs, excessive glycolysis is a aggravating endothelial dysfunction. Numerous studies have shown that atherosclerotic plaques develop well in arterial openings, bifurcations and curved sites, and that low shear stress in blood flow is closely related to the occurrence of atherosclerosis. Vascular endothelial cells exposed to turbulent blood flow in the AS-prone region are affected by laminar shear stress, which results in increased glycolysis and excessive glycolysis, resulting in abnormally elevated lactate and histone lactate formation. Excessive glycolysis can induce excessive proliferation of ECs, trigger vascular endothelial permeability, damage endothelial monolayer barrier, and further accelerate AS process, causing up-regulation of inflammatory response. It is currently believed that increased aerobic glycolysis of vascular endothelial cells in the vulnerable areas of atherosclerosis is responsible for inflammation and atherosclerotic lesions. Previous studies have shown that the Warburg effect is also involved in EndMT for pulmonary arterial hypertension. However, the effect of aerobic glycolysis on EndMT in atherosclerosis is currently unknown.

HATP300, histone acetyltransferase, is a potential histone lactated "writer" protein. P300 can catalyze the transfer of lactyl groups from lactyl-coa to histones. It is not known whether P300-dependent histone lactate requires a cofactor for precise regulation of a specific target gene. ASF1A is an evolutionarily well-preserved histone H3/H4 chaperone and is also an important regulator of gene transcription. Histone chaperone ASF1A in humans can regulate CBP-mediated acetylation of histone H3K56 in cells in vivo, and ASF1A has been reported to interact with P300 in drosophila and HeLa cells to form complexes that regulate histone modification. Previous studies have shown that ASF1A plays a key role in the development and progression of tumors, assembly of replication-coupled ribosomes in anemia, and acceleration of the development of chronic myelogenous leukemia by activating Notch signaling, but has not been reported in the cardiovascular field. Histone epigenetic modifications are closely related to the occurrence and progression of various diseases, but it is unknown whether ASF 1A-mediated epigenetic modifications are involved in the process of EndMT.

At present, the role of histone lactogenesis in atherosclerosis and its mechanism have not been studied and reported. Whether histone chaperone ASF1A and histone lactate modification are involved in EndMT and atherosclerosis has also yet to be explored.

Disclosure of Invention

In view of the above, the present application provides a pharmaceutical use of a histone chaperone ASF1A inhibitor based on inhibition of histone lactic acid H3K18la production.

Specifically, the application firstly provides application of an ASF1A inhibitor in preparing a medicine for preventing and treating cardiovascular diseases; in particular to the application in preparing the medicine for preventing and curing atherosclerosis.

Furthermore, the ASF1A inhibitor can be used as a key for influencing the lactic modification of histone H3K 18.

The aim of the invention can be achieved by the following technical scheme:

use of an ASF1A inhibitor for the preparation of a medicament for the treatment and/or prophylaxis of atherosclerosis.

The ASF1A gene sequence is as follows:

https://www.ncbi.nlm.nih.gov/nuccore/NC_000006.12report＝fasta&from ＝118894152&to＝118909171

the ASF1A amino acid sequence is as follows:

https://rest.uniprot.org/uniprotkb/Q9Y294.fasta

preferably, the ASF1A inhibitor is a substance that inhibits or knocks out ASF1A expression, and is selected from siRNA of ASF1A, a gene editing system that specifically knocks out ASF1A, or other small molecule compounds capable of specifically inhibiting ASF 1A.

As a preferred aspect of the present invention, the siRNA sequence of ASF1A is as follows:

5’→3’GAGCAGUAAUCCAAAUCUATT(SEQ ID NO.1)，

3’→5’UAGAUUUGGAUUACUGCUCTT(SEQ ID NO.2)。

use of a reagent for detecting ASF1A in the preparation of an auxiliary diagnostic kit for atherosclerosis.

The application of ASF1A as a detection target in screening atherosclerosis therapeutic drugs.

A method for screening atherosclerosis therapeutic drugs comprises detecting ASF1A content in plasma before and after administration, and evaluating therapeutic effect of atherosclerosis therapeutic candidate drugs by reducing ASF1A content.

Apoe with endothelial cell specific knockout of Asf1a ^KO Mouse (Apoe) ^KO Asf1a ^ECKO ) Is used for preparing the medicine for treating atherosclerosis.

The invention has the beneficial effects that:

the inventor discovers Apoe through experiments such as Western Blot ^KO Mouse aortic vascular tissue, mouse aortic endothelial cells (mouse aortic endothelial cells, MAECs) and human coronary endothelial cells(human coronary artery endothelial cells, HCAECs) histone lactate modification, endMT, is activated by oxidized low-density lipoprotein (ox-LDL). Under the stimulation of pathogenic factors, abnormally elevated glycolysis produces abnormally elevated lactate, which may lead to lactate modification associated with AS lesions, involving in AS formation. The invention firstly defines the regulating mechanism of ASF1A on histone H3K18 lactate modification, effectively prevents histone H3K18 lactate modification and EndMT and AS mediated by the histone H3K18 lactate modification from happening, provides a new prevention and treatment drug development path and drug action target point for AS diagnosis and treatment, has very important medicinal value, and inhibits or knocks out substances expressed by ASF1A to be expected to be candidate drugs for AS treatment.

Drawings

FIG. 1 shows the expression level of ASF1A after ox-LDL induction of HCAECs: as shown in FIG. 1, AS disease model was established using 50. Mu.g/mL ox-LDL treatment of HCAECs induced injury, cells were collected 24h later, and ASF1A protein expression levels were detected by Western Blot. (n=3;. P < 0.05)

FIG. 2 is the effect of ASF1A knockout on Pan Kla, H3K18la, H3K9la and Snail protein expression levels after ox-LDL induction of HCAECs: transfection of small interfering RNA (siRNA) into HCAECs knocked out ASF1A, further administration of 50 mug/mL ox-LDL to treat HCAECs induced damage, collection of cells after 24H, extraction of HCAECs holoprotein, and Western Blot detection of Pan Kla, H3K18la, H3K9la and Snail protein expression levels. (n=3;. P <0.01,;. P <0.001;ns,no significance)

FIG. 3 is the effect of ASF1A knockout on EndMT-related marker expression levels after ox-LDL induction of HCAECs: as shown in FIG. 3, HCAECs were given siRNA knockdown ASF1A, and further given ox-LDL at 50. Mu.g/mL to stimulate HCAECs to induce damage, sample RNA was collected, and quantitative real-time PCR (quantitative real-time polymerase chain reaction, RT-qPCR) was performed to detect EndMT-related marker expression levels. The results are presented in heat map and bar graph form, blue (low value), red (high value). The color of each box in the heat map corresponds to the average of the corresponding 3 independent experimental data. (n=3;. P <0.05, & p <0.01, & p < 0.001)

FIG. 4 shows the formation of an endothelial cell specific knockout Asf1a for ASInfluence: as shown in fig. 4, apoe ^KO Asf1a ^WT And Apoe ^KO Asf1a ^ECKO Mice were fed normal diet (NC; laboratory mice maintained on diet, cooperative) or high fat diet (high-fat diet, HFD; atherosclerosis model diet, 21% fat and 1.25% cholesterol, cooperative) for 12 weeks, aortic blood vessels were isolated and mice were examined for aortic plaque formation with oil red O staining. (n=6 mice per group; p<0.001)

FIG. 5 is the effect of endothelial cell specific knockout Asf1a on AS formation and EndMT: as shown in fig. 5, apoe ^KO Asf1a ^WT And Apoe ^KO Asf1a ^ECKO Mice NC or HFD were fed for 12 weeks, aortic vessels were isolated, aortic root were frozen embedded for serial sections, and endothelial marker platelet-endothelial cell adhesion molecule (platelet endothelial cell adhesion molecule-1, CD 31) (green) and mesenchymal marker alpha-smooth muscle actin (alpha-Smooth MuscleActin, alpha-SMA) (red) double immunofluorescent staining of mice aortic vessel sections were examined for intimal EndMT occurrence. (n=6 mice per group)

FIG. 6 is the effect of endothelial cell specific knockout Asf1a on EndMT: as shown in fig. 6, apoe ^KO Asf1a ^WT And Apoe ^KO Asf1a ^ECKO Mice NC or HFD were fed for 12 weeks, aortic vessels were isolated, MAECs were extracted, sample RNA was extracted, and EndMT marker expression was detected by RT-qPCR. The results are presented in heat map and bar graph form, blue (low value), red (high value). The color of each box in the heat map corresponds to the average of the corresponding 10 independent experimental data. (n=10 mice per group;p)<0.01,***p<0.001)

FIG. 7 is the effect of endothelial cell specific knockout Asf1a on EndMT morphology: as shown in fig. 7, apoe ^KO Asf1a ^WT And Apoe ^KO Asf1a ^ECKO Mice were fed NC or HFD for 12 weeks, aortic vessels were isolated, MAECs were extracted, and NC or HFD was observed under electron microscopy as Apoe ^KO Asf1a ^WT And Apoe ^KO Asf1a ^ECKO Aortic endothelial cell morphology in mice. (n=6 mice per group; p<0.001)

FIG. 8 is a diagram ofEffect of endothelial cell specific knockout Asf1a on histone lactate modification: as shown in fig. 8, apoe ^KO Asf1a ^WT And Apoe ^KO Asf1a ^ECKO Mice NC or HFD were fed for 12 weeks, aortic vessels were isolated, MAECs were extracted, total cellular proteins were extracted, and Western Blot was used to detect protein expression levels of Pan Kla, H3K18la, H3K9 la. (n=6 mice per group; p<0.001；ns，no significance)

Detailed Description

The following examples will provide those skilled in the art with a thorough understanding of the present invention and are not intended to limit the present invention in any way.

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. The described embodiments are not all embodiments, but only a portion of the embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Example 1

1.1 induction of human coronary endothelial cells:

the human coronary artery endothelial cells were stimulated with ox-LDL at 50 μg/mL for 24h to induce lesions to establish an AS endothelial lesion cell model.

To explore the changes in the histone chaperone ASF1A in AS, applicant purchased human coronary endothelial cells from scientific research laboratory and cultured them in ECM medium containing 10% fetal bovine serum, and passaged after reaching 80% -90% confluency. HCAECs were stimulated with 50 μg/mL ox-LDL for 24h to establish an AS model, while the control group was an equivalent PBS group; and extracting total cell proteins, and detecting the expression level of the ASF1A protein by using a Western Blot method. As shown in FIG. 1, the ASF1A protein expression level of HCAECs was significantly increased after ox-LDL stimulation induced injury (FIG. 1). The experimental results suggest that: endothelial cells ASF1A were upregulated during AS.

Example 2

2.1 transfection of Small interfering RNA (siRNA) into human coronary endothelial cells

(1) HCAECs were seeded into petri dishes for transfection with the following transfection system:

A：ECM 125μL+siRNA6.25μL

B：ECM 125μL+lipo30003μL

(2) Mixing the solution A and solution B, gently mixing with a pipette, and standing at room temperature for 5min. Mixing the two liquids, gently mixing with a pipette, standing at room temperature for 15-20min, and immediately transfecting.

(3) The cells were replaced with fresh, pre-warmed complete medium prior to transfection, the mixture from step (2) was added to the wells and the plates gently shaken to distribute the complexes evenly.

(4) Cells were cultured by static culture at 37℃for 4-6h and then changed to fresh pre-warmed complete medium.

In the invention, siRNA is designed according to ASF1A gene sequence, and the sequence of the siRNA is:

5’→3’GAGCAGUAAUCCAAAUCUATT，

3’→5’UAGAUUUGGAUUACUGCUCTT。

2.2 administration of HCAECs ox-LDL stimulation

HCAECs were stimulated with 50 μg/mL ox-LDL for 24h to induce lesions.

2.3 RT-qPCR

(1) The treated samples were collected and total RNA was extracted as described in Trizol kit. Wearing a mask to avoid RNase so as to prevent RNA degradation, and using a RNase gun head and DEPC water;

(2) Cells in six well plates were washed with pre-chilled PBS and 1ml of ltrizol was added to each well;

(3) After 10s, the cell lysate was transferred to an EP tube and allowed to stand on ice for 10min for lysis;

(4) Adding 200 mu L chloroform into each tube, mixing, and placing on ice for cracking for 10min;

(5) Centrifuging at 12000rpm at 4deg.C for 15min;

(6) Carefully sucking the supernatant to a new EP tube, adding equal volume of isopropanol, mixing the mixture upside down, and placing the mixture on ice for 10min;

(7) Centrifuging at 12000rpm at 4deg.C for 15min, and removing supernatant;

(8) The residual liquid was blotted down on paper to see white feathery precipitate, 75% ethanol diluted with DEPC water was added, and mixed up upside down.

(9) Centrifuging at 12000rpm at 4deg.C for 15min, and discarding supernatant ethanol;

(10) Residual ethanol is sucked off, and the mixture is placed at a vent hole of a clean bench of a cell room for 5-10min for air drying.

(11) Add 20. Mu.L DEPC water to dissolve RNA, and NanoDrop to measure RNA concentration, and place in-80℃refrigerator for use.

(12) UsingII 1st Strand cDNA Synthesis Kit reverse transcription was carried out in a total reaction volume of 20. Mu.L, and the specific composition was as follows:

RNase free ddH ₂ O To 20μL

Total RNA 1μg

ⅡBuffer plus 4μL

(13) After mixing evenly, reverse transcription is carried out by a PCR instrument:

25℃ 5min

42℃ 30min

85℃ 5min

(14) After the reverse transcription, the cDNA was added to 80. Mu. LDEPC water at a ratio of 1:4, diluting in proportion, and storing at-20 ℃ for standby.

(15) By usingqPCR SYBR Green Master Mix the target gene is detected relatively quantitatively, and the PCR reaction system is as follows:

(16) Grouping, calculating a system (finally adding cDNA);

(17) Sealing the membrane, centrifuging the 384-hole plate, and then placing the membrane into a fluorescent quantitative PCR instrument;

(18) The reaction was performed on a Bio-Rad 480 type I quantitative PCR instrument.

The primer sequences were as follows:

siASF1A Forward:GAGCAGUAAUCCAAAUCUATT

Reverse:UAGAUUUGGAUUACUGCUCTT

to further confirm the effect of histone chaperone ASF1A on the lactate formation of HCAECs, applicant cultured HCAECs, and after transfection of small interfering RNAs of ASF1A into cells for 48H at the logarithmic growth stage, further administration of ox-LDL stimulation of 50 μg/mL was performed, and after 24H cells were collected, and protein expression levels of Pan Kla, H3K18la, H3K9la and EndMT classical transcription factors Snail were detected by Western Blot. As a result, as shown in FIG. 2, it was found that the lack of ASF1A significantly inhibited the ox-LDL induced increase in H3K18la and Snail protein levels and the EndMT process (FIG. 2).

To further clarify the role of ASF1A in EndMT and AS, applicants cultured HCAECs, transfected small interfering RNAs of ASF1A into cells during the logarithmic growth phase, and given an ox-LDL stimulus of 50 μg/mL, cells were collected 24h later and assayed for EndMT marker expression levels using RT-qPCR. During EndMT, the expression levels of the endothelial markers platelet-endothelial cell adhesion molecule (platelet endothelial cell adhesion molecule-1, cd 31), vascular endothelial cadherin (vascular endothelial cadherin, VE-cadherin), endothelial nitric oxide synthase (endothelial nitric oxide synthases, eNOS) and occluding (Occludin) are reduced, and the expression levels of VSMCs or mesenchymal stem cell-like markers such as N-cadherin (N-cadherin), calmodulin (Calponin), alpha-smooth actin (alpha-Smooth Muscle Actin, alpha-SMA), fibronectin (fibonectin 1), type I Collagen a (Collagen 1A) and Vimentin (Vimentin) are increased in endothelial cells. As a result, as shown in FIG. 3, the absence of ASF1A significantly inhibited the decrease in the expression level of the endothelial cell-specific marker induced by ox-LDL, while inhibiting the increase in the expression level of the mesenchymal marker induced by ox-LDL (FIG. 3). The above results indicate that ASF1A deficiency significantly inhibited ox-LDL induced EndMT processes.

Example 3

3.1 construction of Apoe with endothelial cell Asf1 a-specific knockout ^KO Mouse (Apoe) ^KO Asf1a ^ECKO )

There are 6 transcripts of the Asf1a gene, and exon 2 of Asf1a-201 (ENSMUST 00000020004.8) is recommended as the gene knockout region, depending on the structure of the Asf1a gene. The region contains a coding sequence of 116 bp. Knocking out this region will result in disruption of gene function. In this project, we used CRISPR-Cas9 technology to engineer the Asf1a gene. The brief procedure is as follows: the CRISPR-Cas9 system and Donor were microinjected into fertilized eggs of C57BL/6JGpt mice. Positive F0 mice were obtained after fertilized egg transplantation and genotype was confirmed by PCR and targeted amplicon sequencing. Positive F0 mice were mated with C57BL/6JGpt mice to obtain stable F1 mice strains, and the desired mutant alleles were confirmed by PCR and targeted amplicon sequencing. Thereby obtaining Asf1a ^flox/flox And (3) a mouse. The gene constructs a flox structure by inserting loxp sequences at two sides of exon No. 2. In the cre expression state, cre is nuclease, the cre enters the nucleus to recognize the flox structure, so that the cre is cyclized to cut off the sequence between two loxp, thereby achieving the purposes of knocking exon and frame shift mutation, and leading the target gene Asf1a to be incapable of normally transcribing and translating. After mating with Cre recombinase expressing mice, flox mice will be knocked out, resulting in the loss of function of the gene of interest Asf1a in specific tissues and cell types. Asf1a ^flox/flox Mice and endothelial cell specific Cdh5-cre mice were purchased from Jiangsu Jiuyaokang Biotech Co. C57BL/6J and Apoe ^KO Mice were also purchased from Jiangsu Jiuyaokang biotechnology Co., ltd and kept in constant temperature SPF-grade animal houses. By the method of ApoE ^KO Asf1a floxed (Asf 1a ^fl/fl ) The mice were hybridized with Cre recombinase system (Cdh 5-Cre) mice driven by vascular endothelial fibronectin (Cdh 5) promoter to obtain mice (Apoe) with specific deletion of Asf1a in endothelial cells ^KO Asf1a ^ECKO ). By co-nest Apoe ^KO Asf1a ^WT Mouse (Apoe) ^KO Asf1a ^fl/fl ) As a control, a normal diet (normal chow, NC; experimental mouseSustaining feed feeding, synergistic) or high-fat diet feeding (HFD; atherosclerosis model feed feeding, 21% fat and 1.25% cholesterol, synergistic organisms) were fed separately for 12 weeks, and an AS mouse model was established, simulating AS disease progression.

3.2 general oil Red staining

The area of the aortic vessel plaque in the whole body of mice was examined by general oil red O staining in a 12 week established AS mouse model fed with high fat for 3.1.

A general oil red O staining step: the aortic blood vessels of the mice were removed, perivascular adipose tissue was removed with forceps, the blood vessels were fixed with 4% paraformaldehyde for 10min, and then rinsed with PBS for 10min. The dissected blood vessel is taken out after being dyed for 1h by using oil red O dyeing working solution, rinsed 3 times by using 60% isopropanol, fat plaque in the lumen is orange or bright red, other parts are nearly colorless, and finally rinsed 2 times by using distilled water. The stained general tissue was spread and fixed on a glass slide, and photographed under good light conditions.

The results of the assay are shown in FIG. 4, comparing the Apoe fed with NC or HFD ^KO Asf1a ^ECKO And Apoe ^KO Asf1a ^WT Mice, oil red O staining showed, relative to Apoe ^KO Asf1a ^ECKO Mice, apoe ^KO Asf1a ^WT The lipid burden in the aorta of mice was significantly reduced. Quantitative data statistics of mice aortic vascular oil red O staining showed a reduction in plaque area following endothelial Asf1a knockdown in mice (fig. 4).

3.3 vascular immunofluorescence

Furthermore, to further clarify the role of Asf1a in endothelial dysfunction and AS, applicant isolated NC or HFD fed Apoe ^KO Asf1a ^WT And Apoe ^KO Asf1a ^ECKO Aortic blood vessels of mice were stained with CD31 (green) and α -SMA (red) double immunofluorescence and nuclei with diamidino-2-phenylindole (4', 6-diamidino-2-phenylindole, DAPI) (blue) for aortic blood vessel frozen sections.

Applicant first isolated the proximal portion of the ascending aorta of the different groups of mice and then embedded OCT frozen section embedding agent. PBS was placed in a refrigerator pre-chilled at 4deg.C. The aorta was snap frozen at-80℃and cut into 8 μm frozen sections. The frozen sections were baked at 55℃for 15min, then fixed in 4% paraformaldehyde for 20min, followed by instilling 0.1% Triton X-10020min to rupture the membranes. Subsequently, the samples were blocked with 10% BSA for 1 hour and incubated overnight at 4 ℃ with the specific primary antibody. The next day, samples were washed with PBS and then incubated with secondary antibodies for 1h at 37 ℃. Finally, DAPI was used at 1: the nuclei were stained for 10min at a ratio of 1000. The image was obtained by observation and photographing using a confocal laser scanning microscope.

The results are shown in FIG. 5 for HFD fed Apoe ^KO Asf1a ^WT The endothelial cell marker CD31 was significantly down-regulated in the vascular intima and plaque of mice, which was found in Apoe ^KO Asf1a ^ECKO Is reversed in mice; the opposite phenomenon was seen with the mesenchymal transition cell marker α -SMA, suggesting that Asf1a may be involved in the occurrence of endothelial dysfunction, endMT (fig. 5).

Example 4

To confirm whether Asf1a affects the occurrence of endothelial dysfunction, applicant extracted NC or HFD fed Apoe ^KO Asf1a ^WT And Apoe ^KO Asf1a ^ECKO Aortic endothelial cells from mice were assayed for expression levels of EndMT-related markers using RT-qPCR analysis. The results are shown in FIG. 6, where endothelial cell specific knockout of Asf1a significantly inhibited the EndMT process. These further demonstrate the key role of endothelial cells Asf1a in histone lactate and the EndMT phenotype (fig. 6).

To determine the effect of endothelial cell specific Asf1a knockout at the cellular level, applicants extracted and cultured Apoe from NC-fed or HFD-fed ^KO Asf1a ^WT And Apoe ^KO Asf1a ^ECKO MAECs in mice. The results are shown in FIG. 7, which shows the results with Apoe under high fat feeding ^KO Asf1a ^ECKO From Apoe compared with mice ^KO Asf1a ^WT The cell morphology of the ECs of the mice was significantly altered, not oval, but long fusiform, with a change in the classical cell morphology phenotype of EndMT (fig. 7).

To verify that endothelial cell specific Asf1a knockout was lactating to histoneInfluence, applicants extracted and cultured Apoe from NC-fed or HFD-fed ^KO Asf1a ^WT And Apoe ^KO Asf1a ^ECKO MAECs in mice. Protein expression levels of Pan Kla, H3K18la and H3K9la were detected by Western Blot. The results are shown in FIG. 8, associated with Apoe ^KO Asf1a ^WT From Apoe compared with mice ^KO Asf1a ^ECKO The Pan Kla and H3K18la protein expression levels of MAECs of the ECs of mice were significantly increased (fig. 8).

The experimental results suggest that: asf1a is involved in histone H3K18 lactate mediated EndMT, thereby promoting the transition of mouse endothelial cells to the EndMT phenotype.

The experimental results fully prove that the modification of the lactate of the H3K18 dependent on the ASF1A participates in the progress of AS. The ASF1A inhibitor can prevent the generation of H3K18la, and can down regulate the lactic modification of histone H3K18, thus effectively inhibiting the generation and development of AS. Therefore, the ASF1A can be considered AS a new important target for clinically treating AS, and has potential clinical application value in prevention and treatment of AS.

The foregoing detailed description of the invention has been provided for the purpose of illustrating the general principles and features of the invention, and is not meant to limit the scope of the invention to the particular embodiments disclosed. The present invention is not limited to the above examples, and changes can be made without departing from the spirit of the invention, and all equivalent changes or modifications to the invention according to the gist of the present invention are intended to be within the scope of the present invention.

Claims (7)

1. Use of an asf1a inhibitor for the preparation of a medicament for the prevention and/or treatment of atherosclerosis.
2. 2. The use according to claim 1, wherein the ASF1A inhibitor is a substance that inhibits or knocks out ASF1A expression, selected from the group consisting of siRNA for ASF1A, gene editing system for specific knockdown ASF1A, or other small molecule compound capable of specifically inhibiting ASF 1A.
3. 3. The use of claim 2, wherein the ASF1A inhibitor knocks down a small interfering RNA expressed by ASF1A, having the sequence shown in SEQ ID No.1 and SEQ ID No. 2.
4. Use of an asf1a inhibitor for the preparation of a reagent for down-regulating histone lactate modification in vitro.
5. Use of asf1a as a therapeutic target in the screening of a medicament for the prevention and/or treatment of atherosclerosis.
6. 6. Use of a reagent for detecting ASF1A in the preparation of an auxiliary diagnostic kit for atherosclerosis.
7. 7. A method for screening atherosclerosis therapeutic drugs is characterized in that the ASF1A content before and after administration is detected, and the curative effect of atherosclerosis therapeutic candidate drugs is evaluated by the reduction degree of the ASF1A content.