CN110339344B - Application of nuclear receptor Rev-erb alpha in preparation of antiplatelet drugs - Google Patents

Abstract

The invention provides an application of a nuclear receptor Rev-erb alpha in preparing antiplatelet drugs, wherein the amino acid sequence of the nuclear receptor Rev-erb alpha is shown as SEQ ID NO. 2. The invention also provides application of the Rev-erb alpha inhibitor in preparing a medicament for inhibiting platelet activity. The present invention demonstrates the presence of the nuclear receptor Rev-erb α in human and mouse platelets. The functional deletion of Rev-erb alpha in vivo can prolong the bleeding time and inhibit the carotid artery thrombosis induced by ferric chloride. It was found that Rev-erb α, either genetically deleted or pharmacologically inactivated, inhibits ADP pathway mediated platelet aggregation. The Rev-erb α inhibitor SR8278 significantly reduces the release of α granules upon ADP-induced platelet activation. The role of the nuclear receptor Rev-erb α is exerted by inhibiting ADP pathway mediated platelet activation and granule release following activation.

Description

Application of nuclear receptor Rev-erb alpha in preparation of antiplatelet drugs

Technical Field

The invention belongs to the field of biological medicines, relates to a cardiovascular medicament, and particularly relates to a nuclear receptor for treating thrombotic diseases caused by platelet activation, in particular to application of the nuclear receptor Rev-erb alpha in preparation of antiplatelet medicaments.

Background

In recent years, with the aging process of population accelerating, social pressure increasing and natural environment continuously deteriorating, cardiovascular diseases become the first killer seriously harming the health of residents in China. The morbidity and mortality of the medicine are increased year by year and are far higher than those of tumors and other diseases. Therefore, the search and discovery of effective preventive and therapeutic strategies is urgent. Myocardial infarction is the leading cause of death of cardiovascular diseases and is concerned by basic medicine and clinicians. Research shows that the plaque rupture in coronary artery induces thrombosis as a direct pathogenic factor, and the thrombus causes blockage or stenosis of a lumen, so that myocardial ischemia, hypoxia, necrosis and apoptosis are caused. In the process of forming thrombus in coronary artery, blood platelets in the lumen are adhered, gathered and released, and simultaneously induce blood coagulation waterfall, which is the most main reason for forming thrombus. Therefore, antiplatelet therapy is one of the core strategies for the prevention and treatment of cardiovascular diseases. At present, the antiplatelet drugs are limited in types, and mainly comprise ADP receptor antagonists, platelet glycoprotein IIb/IIIa receptor antagonists, arachidonic acid metabolism inhibitors and the like. Although antiplatelet drugs are often effective in inhibiting thrombosis, their safety, especially the risk of bleeding, has been a concern for the majority of clinical medical personnel. Reviewing the existing clinical data, there are no other simple and effective alternatives except for rational selection and standardized administration of drugs to reduce the risk of bleeding. Therefore, the search for highly effective antiplatelet drugs with few side effects has become an important direction for basic and clinical research.

Nuclear Receptors (NRs) are a superfamily of transcription factors with a variety of physiological functions. Nuclear receptors, as molecular targets for a variety of FDA-approved drugs, play important roles in metabolism, differentiation, reproductive development, and homeostatic maintenance. The traditional concept is that the nuclear receptor can regulate the transcription of target genes by combining with corresponding ligands, and further plays roles in regulating growth and development, substance metabolism and the like. However, recent studies have shown that nuclear receptors, in addition to gene transcriptional regulation, also interact with cytoplasmic proteins and are involved in the pathophysiological processes of disease [8-10].

The Nuclear receptor Rev-erb α, also known as NR1D1 (Nuclear receptor family 1, group D, memberr 1), is a widely expressed protein and abundant in brain, heart, liver, kidney, skeletal muscle and other organs. It is both biological clock gene and clock-controlled gene, and its expression has obvious biological clock rhythm. Rev-erb alpha plays a role of a transcription factor, not only participates in growth and development of organisms and regulation of biological clocks, but also plays a significant role in aspects of immune inflammation, substance metabolism and the like. It has been proved that Rev-erb alpha can inhibit the generation and development of atherosclerosis by regulating the inflammatory reaction of macrophage and vascular smooth muscle cell. Interestingly, rev-erb α, while reducing myocardial remodeling following myocardial infarction in mice, aggravated the perioperative myocardial ischemia reperfusion injury. However, whether or not it has a role other than transcriptional regulation and its role in anucleated platelets is still unknown. Rev-erb α is a member of the nuclear receptor superfamily, the specific physiological ligand Heme (Heme) of which has been demonstrated. Unfortunately, however, physiological inhibitors thereof have not been discovered. According to the structural characteristics, a recent researcher designs an exogenous receptor inhibitor SR8278 and proves the effectiveness of the exogenous receptor inhibitor SR8278, which indicates that Rev-erb alpha can be used as a potential drug target, and provides a new direction for preventing and treating future diseases.

Human Rev-erb α, which consists of 614 amino acid residues, was shown to be highly expressed in platelets. The Rev-erb alpha gene is positioned on the antisense strand of human chromosome 17, and the site information is as follows: homo sapiens chromosome 17, GRCh38.P12Primary Assembly, NC _000017.11 (40092784.. 40100725, composition); the sequence of the transcript (NM-021724.5) (mRNA) is shown in SEQ ID NO. 1; the protein amino acid sequence is shown in SEQ ID NO. 2.

Disclosure of Invention

The invention aims to provide application of a nuclear receptor Rev-erb alpha in preparation of an antiplatelet medicament, and aims to solve the technical problem of poor safety of the antiplatelet medicament in the prior art.

The invention provides an application of a nuclear receptor Rev-erb alpha in preparing an antiplatelet medicament.

Furthermore, the amino acid sequence of the nuclear receptor Rev-erb alpha is shown as SEQ ID NO. 2.

The invention also provides application of the Rev-erb alpha inhibitor in preparing a medicament for inhibiting platelet activity.

Experiments prove that the nuclear receptor Rev-erb alpha is expressed in human and mouse blood platelets. Rev-erb alpha deletion can prolong bleeding time of mice and inhibit ferric chloride (FeCl) ₃ ) Induced carotid thrombosis. In vitro studies have shown that Rev-erb alpha, either genetically deleted or pharmacologically inactivated, inhibits ADP pathway-mediated platelet aggregation. Further experiments indicate that Rev-erb alpha inhibitor SR8278 significantly reduces alpha when ADP induces platelet activationAnd (4) releasing the particles. The nuclear receptor, rev-erb α, affects platelet activity primarily by affecting platelet particle release.

Compared with the prior art, the invention has the advantages of positive and obvious technical effect. The invention discovers a nuclear receptor Rev-erb alpha with potential anti-platelet activity, and the inhibition of the nuclear receptor Rev-erb alpha can effectively inhibit platelet aggregation and reduce thrombosis. The Rev-erb alpha inhibitor SR8278 can inhibit platelet aggregation induced by ADP. The role of the nuclear receptor Rev-erb α is exerted mainly by inhibiting ADP pathway mediated platelet activation and granule release after activation.

Drawings

FIG. 1 shows the expression of Rev-erb α in human and mouse platelets.

FIG. 2 is a graph showing the effect of Rev-erb α deletion on bleeding and thrombosis.

FIG. 3 is a graph showing the effect of Rev-erb α deletion on platelet aggregation function.

FIG. 4 shows the effect of the Rev-erb α inhibitor SR8278 on human platelet aggregation function.

FIG. 5 shows that the Rev-erb alpha inhibitor SR8278 reduces the level of platelet particle release.

Detailed Description

The invention will be further described in connection with specific embodiments with reference to the following drawings in order to better understand the invention.

The following are specific examples of the invention which are intended to be illustrative of the invention and not limiting.

The reagents and experimental methods used in the examples are as follows:

molecular biochemical reagents: adenosine Diphosphate (ADP), ferric chloride (FeCl 3), prostacyclin, rev-erb alpha inhibitor SR8278, protease and phosphatase inhibitors, beta-mercaptoethanol, sodium dodecyl sulfate, etc. were purchased from Sigma USA, RIPA lysate was purchased from Thermo USA, 30% methylenebisacrylamide was purchased from san-Jose, chao, triethanolamine Buffer (TBS), tween-20, phosphate Buffer (PBS), rev-erb alpha primer, etc. were purchased from Probiotics, horseradish peroxidase secondary antibody was purchased from Jackson USA, ECL chemiluminescence kit was purchased from Millipore USA, isoflurane was purchased from Hunan fibrate China, ribonucleic acid reverse transcription kit, deoxyribonucleic acid amplification kit was purchased from Takara Japan, rev-erb alpha antibody, GAPDH antibody was purchased from abcam USA, and CD62P-PE antibody was purchased from BD USA.

Example 1

Separation and purification of platelets: after informed consent and examination by ethical committee, 20ml of peripheral blood vein of healthy volunteers is taken, and purified platelets of healthy people are obtained for standby after density gradient centrifugation. In addition, blood is taken from abdominal aorta, whole blood of 8-12 weeks old wild type C57BL/6J mice is obtained, and purified platelets of the mice are obtained for standby after density gradient centrifugation. The specific purification steps are as follows: (1) Carrying out anticoagulation on the whole blood with sodium citrate according to the volume ratio of 2:1, adding 37-degree preheated physiological saline, and then mixing the mixture according to the volume ratio of 1:5000 adding prostacyclin, and centrifuging at 1100 rpm under the conditions of acceleration of 9 and deceleration of 0 for 10 minutes to obtain platelet-rich plasma; (2) Carefully pipette the upper platelet-rich plasma layer into another centrifuge tube at a volume ratio of 1: adding prostacyclin at 2500 deg.c, centrifuging at 800 rpm and acceleration of 9 and deceleration of 0 for 5min to further remove red blood cells and white blood cells from the platelet-rich plasma; (3) Taking the supernatant obtained in the second step into another centrifuge tube, and mixing the supernatant and the centrifuge tube according to the volume ratio of 1: adding prostacyclin into 5000, centrifuging for 10 minutes at 2200 r/min under the conditions of acceleration of 9 and deceleration of 4, and removing supernatant to obtain platelet; (4) Resuspending the platelet pellet with a modified benchtop solution, followed by 1: adding prostacyclin into the mixture at 5000 rpm, accelerating the speed to 9, decelerating the speed to 4, centrifuging the mixture for 10 minutes, removing supernatant, and obtaining precipitate which is purified and washed platelets. And (3) extracting protein and total RNA from the purified and washed platelets and organs respectively, then performing immunoblotting and real-time fluorescent quantitative PCR detection respectively, and verifying the existence of Rev-erb alpha from the levels of the protein and mRNA respectively.

Human and mouse platelets and organs were removed from liquid nitrogen. And adding lysate into one part of the blood, grinding, extracting corresponding tissue protein, performing immunoblotting detection, and verifying the existence of Rev-erb alpha in the blood platelet from the protein level. The other portion was added with 1ml of Trizol separately, total RNA was extracted using chloroform-isopropanol method, followed by reverse transcription into cDNA, and then subjected to real-time fluorescent quantitative PCR detection, the presence of which was confirmed from RNA level. (FIG. 1) Panel A: the heart, kidney, liver, brain and skeletal muscle of the mouse are taken as positive controls, and the platelet memory Rev-erb alpha of the mouse is verified respectively from the protein level and the mRNA level. And B, drawing: the liver, kidney, brain and skeletal muscle of human are taken as positive controls, and Rev-erb alpha in human blood platelet is confirmed from protein level and mRNA level respectively.

Example 2

Establishing a mouse bleeding model: 8-12 weeks old Rev-erb alpha function-deficient mice or wild type C57BL/6J control mice were placed in an anesthesia container, after their anesthesia stabilized, they were fixed on an operating plate, and an anesthesia mask was used to maintain anesthesia by continuous inhalation of isoflurane at a concentration of 2% (ventilation rate 1.5L/min). After the mouse breathes stably, the tail end of the mouse tail is subtracted by 3mm by using disinfected scissors, the tail of the mouse is placed in physiological saline of 37 ℃, and timing is started until no blood flows out completely, wherein the judgment standard is that no blood flows out within 1 min. The termination time is then recorded. The bleeding time of the mice is the time from the start of timing to the complete cessation of blood. Establishing a carotid artery thrombosis model: ferric chloride (FeCl) ₃ ) The animal thrombus model manufactured by damaging the common carotid artery has the advantages of simple operation method, strong controllability and high repeatability, is close to the tissue morphological characteristics of spontaneous thrombus, and is a thrombus model internationally recognized at present. Taking 7 mice each with 8-12 weeks old Rev-erb alpha deletion and same-nest WT, injecting 1% sodium pentobarbital (10 ml/Kg) into the abdominal cavity, fixing the mice on a small animal operation board after anesthesia and stable respiration, disinfecting the neck skin of the mice with alcohol, separating the common carotid artery at one side of the mice, carefully stripping connective tissues and adipose tissues around the common carotid artery, and cutting a sealing membrane with proper length to be arranged under the common carotid artery so as to prevent ferric chloride from burning tissues outside the blood vessel. Completely immersing 1X 2mm filter paper in 10% ferric chloride solution, and placing the filter paper on carotid artery (closely attached) after the filter paper is completely soaked. After 5min of action, the common carotid artery was carefully cut out and placed in 10% formaldehyde solution and fixed for 24 hours at room temperature.

Rev-erb α deleted (KO) mice on C57BL/6J background were constructed by Shanghai Square model Biotechnology, inc. Carrying out tail-shearing bleeding experiments (1200 seconds if the bleeding time is more than 1200 seconds) and carotid thrombosis experiments on SPF (specific pathogen free) KO mice 8-10 weeks old and littermate Wild Type (WT) control mice, and detecting the influence of Rev-erb alpha deletion on the platelet function of the mice. As a result, KO mice were found to have a significantly prolonged bleeding time and significantly reduced thrombosis as compared with WT mice. (FIG. 2)

A, picture A: the bleeding time of the WT mice is obviously prolonged compared with that of the KO mice, and the average bleeding time of the KO mice is 3 times that of the WT mice.

And B, drawing: after the blood vessels are burned by ferric chloride, the thrombus area of the KO mouse is obviously reduced compared with that of the WT mouse.

Example 3

Platelet aggregation experiments in mice. And (3) detecting the platelet aggregation function: (1) mouse washed platelet acquisition: 1ml syringes were prepared and about 100ul of 3.8% sodium citrate anticoagulant was drawn into each syringe (anticoagulant was added to whole blood as 1:9). Intraperitoneal injection of 1% sodium pentobarbital (10 ml/Kg), after anesthesia of the mice, fixing the mice, sterilizing the abdomen, opening the abdominal cavity, separating the abdominal aorta, puncturing the abdominal aorta of the mice with a 1ml syringe, carefully withdrawing the whole blood until resistance is felt. The syringe is pulled out, and the mixture is quickly reversed and mixed. Whole blood from mice of the same genotype was mixed and subjected to gradient centrifugation as described above to obtain mouse-washed platelets.

And (3) respectively obtaining WT mice and KO mice to wash platelets, and detecting platelet aggregation function, wherein after stimulation by low-concentration ADP (10 uM), the platelet aggregation rate of the KO mice is obviously lower than that of the WT mice, and no obvious difference exists when stimulation by high-concentration ADP (20 uM or 40 uM). (FIG. 3) Panel A: under low concentrations of ADP (10 uM) stimulation, the platelet aggregation rate in KO mice was significantly lower than that in WT mice. B. And (C) diagram: there was no significant difference in platelet aggregation rates between WT and KO mice stimulated with 20uM and 40uM concentrations.

Example 4

Human platelet aggregation assay. Platelet rich plasma preparation (PRP): blood of 20ml of healthy volunteers was extracted and subjected to density gradient centrifugation to obtain platelet-rich plasma. (3) detection of aggregation function: the water bath kettle is opened in advance, and the subsequent experiment is carried out after the water bath kettle is heated to 37 ℃. 225ul of washed platelets are placed in a collection tube, added to a rotor and placed in a collection detection hole for baseline measurement; different concentrations of the aggregation inducing agent ADP were then added separately and the platelet aggregation curves over 3 minutes were recorded. Three independent replicates were performed separately.

Platelet-rich plasma was obtained from healthy volunteers and stimulated with low concentrations of ADP (5 uM), while Rev-erb α inhibitor SR8278 (concentrations 0uM, 10uM, 20uM, 30uM, respectively) and control (0.1% dmso) were administered, and as a result, it was found that as the concentration of inhibitor SR8278 increased, the platelet aggregation rate gradually decreased, indicating that pharmacological inhibition of Rev-erb α significantly inhibited the platelet aggregation function. (FIG. 4) A, B FIG: with the increase of the concentration of the inhibitor SR8278, the platelet aggregation rate is gradually reduced.

Example 5

And (3) detecting the release function of human platelet particles: CD62P is an important indicator of granule release after platelet activation. Obtaining platelet rich plasma from human, adjusting the platelet count to 6 × 10 with Platelet Poor Plasma (PPP) ⁷ Perml, different concentrations of the Rev-erb. Alpha. Inhibitor SR8278 or 0.1% dimethyl sulfoxide control were added. After incubation at room temperature for 15min, the PE-labeled CD62P antibody and the aggregation inducing agent ADP were added to the above platelets, respectively. After mixing gently, incubate at 37 ℃ in the dark for 20min at room temperature. Adding 500ul 4% paraformaldehyde fixing solution, fixing at room temperature for 10min, and performing on-machine detection by flow cytometer.

Platelet-rich plasma of healthy volunteers is obtained, low-concentration ADP (5 uM) stimulation is performed, meanwhile, rev-erb alpha inhibitor SR8278 with the concentration gradient is given, then flow detection is performed on CD62P expression quantity, the CD62P expression quantity is gradually reduced along with the increase of the inhibitor concentration, and the result shows that the release of platelet particles can be obviously inhibited by pharmacological inhibition of Rev-erb alpha. (FIG. 5) A, B, C, D: the expression level of P-selectin on the surface of the platelet gradually decreases with the increase of the concentration of the inhibitor. E, drawing: the positive expression rate of p-selectin of 10uM, 20uM and 30uM relative to 0uM is gradually reduced with increasing inhibitory concentration by taking 0uM as a control.

Sequence listing

<110> Shanghai university of traffic medical college affiliated renji hospital

Application of <120> nuclear receptor Rev-erb alpha in preparation of antiplatelet drugs

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Val Ser Pro Ser Lys Ser Thr Ser Asn Ile Thr Lys Leu Asn Gly Met

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Gln Gln Asn Ile Gln Tyr Lys Arg Cys Leu Lys Asn Glu Asn Cys Ser

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Pro Lys Arg Glu Lys Gln Arg Met Leu Ala Glu Met Gln Ser Ala Met

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Asn Leu Ala Asn Asn Gln Leu Ser Ser Gln Cys Pro Leu Glu Thr Ser

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Pro Thr Gln His Pro Thr Pro Gly Pro Met Gly Pro Ser Pro Pro Pro

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Ala Pro Val Pro Ser Pro Leu Val Gly Phe Ser Gln Phe Pro Gln Gln

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Leu Thr Pro Pro Arg Ser Pro Ser Pro Glu Pro Thr Val Glu Asp Val

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Ile Ser Gln Val Ala Arg Ala His Arg Glu Ile Phe Thr Tyr Ala His

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Gly Ser Pro Pro Ala Thr Thr Pro His Arg Trp Glu Asn Gln Gly Cys

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Glu Ala Leu Asn Gly Leu Arg Gln Ala Pro Ser Ser Tyr Pro Pro Thr

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Trp Pro Pro Gly Pro Ala His His Ser Cys His Gln Ser Asn Ser Asn

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Gly His Arg Leu Cys Pro Thr His Val Tyr Ala Ala Pro Glu Gly Lys

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Ala Pro Ala Asn Ser Pro Arg Gln Gly Asn Ser Lys Asn Val Leu Leu

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Pro Asp Leu Arg Thr Leu Asn Asn Met His Ser Glu Lys Leu Leu Ser

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Claims (1)

1. The application of the nuclear receptor Rev-erb alpha inhibitor in preparing the medicine for inhibiting the platelet activity is disclosed, wherein the amino acid sequence of the nuclear receptor Rev-erb alpha is shown as SEQ ID NO. 2.

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