CN110339344A - Nuclear receptor Rev-erb α is preparing the application in antiplatelet drug - Google Patents

Nuclear receptor Rev-erb α is preparing the application in antiplatelet drug Download PDF

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CN110339344A
CN110339344A CN201910726078.4A CN201910726078A CN110339344A CN 110339344 A CN110339344 A CN 110339344A CN 201910726078 A CN201910726078 A CN 201910726078A CN 110339344 A CN110339344 A CN 110339344A
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erb
rev
ser
nuclear receptor
platelet
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CN110339344B (en
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卜军
童仁洋
徐龙伟
袁安彩
沈薇
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Renji Hospital Shanghai Jiaotong University School of Medicine
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    • A61K38/1783—Nuclear receptors, e.g. retinoic acid receptor [RAR], RXR, nuclear orphan receptors
    • A—HUMAN NECESSITIES
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors

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Abstract

The present invention provides nuclear receptor Rev-erb α to prepare the application in antiplatelet drug, and the amino acid sequence of the nuclear receptor Rev-erb α is as shown in SEQ ID NO.2.The present invention also provides application of the Rev-erb alpha inhibitor in the drug that preparation inhibits biologically active pdgf.The invention demonstrates that there are nuclear receptor Rev-erb α in people and mouse platelets.The bleeding time can be extended in body Rev-erb α afunction, inhibit the carotid artery thrombosis of iron chloride induction.The study found that the platelet aggregation that whether genetics missing or pharmacology inactivation Rev-erb α can inhibit ADP access to mediate.The release of α particle when Rev-erb alpha inhibitor SR8278 substantially reduces the activation of ADP induced platelet.The effect of nuclear receptor Rev-erb α is the particle release after the platelet activation by inhibiting ADP access to mediate and activation to play.

Description

Nuclear receptor Rev-erb α is preparing the application in antiplatelet drug
Technical field
The invention belongs to biomedicine field, it is related to a kind of cardiovascular drugs, more particularly to a kind of for treating because blood is small Plate activates the nuclear receptor for leading to thrombotic diseases, and specifically nuclear receptor Rev-erb α is preparing answering in antiplatelet drug With.
Background technique
In recent years, as aging of population progress faster, social pressures are continuously increased and natural environment is continuous worsening etc., the heart Vascular diseases have become the number one killer that China seriously endangers residents ' health.Its morbidity and mortality increases year by year, far high In tumour and other diseases.Therefore, explore and find that effective prevention and treatment strategy is extremely urgent.Myocardial infarction is as painstaking effort Pipe disease primary cause of death is paid close attention to by preclinical medicine and clinician.Studies have shown that plaque rupture induces in coronary artery Thrombosis is its direct pathogenic factor, and the latter makes lumen that obstruction or narrow occur, in turn result in myocardial ischemia-anoxemia necrosis and Apoptosis.In coronary thrombosis forming process, blood platelet sticks, assembles and discharges in lumen, while inducing blood coagulation waterfall Cloth is the main reason of thrombosis.Therefore, Antiplatelet therapy is one of core strategy of prevention and cure of cardiovascular disease.Mesh Before, antiplatelet drug species limitation, wherein mainly short of money including adp receptor antagonist, platelet glycoprotein IIb/IIIa receptor Anti-agent and arachidonic acid metabolism inhibitor etc..Although antiplatelet drug tends to effective inhibition thrombosis, but it is pacified Quan Xing, especially bleeding risk are all the time worried for vast clinical staff.Existing clinical data is looked back, except rationally choosing It selects and the medication that standardizes reduces outside bleeding risk, have no other simple and effective alternative solutions.Therefore, it explores efficiently and pair is made Based on being had become with small antiplatelet drug and the important directions of clinical research.
Nuclear receptor (Nuclear receptors, NRs) is a kind of transcription factor superfamily with different physiological roles. Molecular target of the nuclear receptor as a variety of FDA approval drug, in terms of metabolism, differentiation, reproductive development and stable state Play important function.Traditional concept think nuclear receptor can by with corresponding ligand binding, regulate and control the transcription of target gene, into And the effects of playing regulation growth and development, metabolism.However, recent research indicate that nuclear receptor is in addition to gene transcription regulation function Outside, it can also be combined with each other with plasmosin, participate in the pathophysiological process [8-10] of disease.
Nuclear receptor Rev-erb α, also known as NR1D1 (Nuclear receptor subfamily 1, group D, It member1), is a kind of popularity expression albumen, in the internal organs rich content such as brain, heart, liver, kidney, skeletal muscle.Itself It is both clock gene and clock-controlled genes, expression has apparent biological clock rhythm.Rev-erb α is with the angle of transcription factor Color is not only involved in body growth development and adjusts with biological clock, and plays in terms of immune inflammation, metabolism etc. very important Effect.Existing research proves that Rev-erb α can pass through the inflammatory reaction of adjusting macrophage and vascular smooth muscle cells, suppression Brake the occurrence and development of pulse atherosclerosis.It is interesting that Rev-erb α is while Myocardial Remodeling after mitigating murine myocardial infarction, But heart myocardial ischemia during perioperative period reperfusion injury has been aggravated.However, its whether have the function of other than transcriptional control and its Effect in anuclear platelet is still unknown.The a member of Rev-erb α as nuclear receptor superfamily, the physiological of specificity Ligand-ferroheme (Heme) has been found.Unfortunately, the inhibitor of physiological is not yet found.There is research recently Person devises exogenous acceptor inhibitor SR8278 according to its architectural characteristic, and demonstrates its validity, illustrates Rev-erb α It can be used as potential drug target, this will provide the prevention and treatment of future disease new direction.
People Rev-erb α is made of 614 amino acid residues, through we demonstrate that it has higher expression in blood platelet. Rev-erb α gene is located on No. 17 chromosome antisense strands of people, and site information is as follows: Homo sapiens chromosome 17, GRCh38.p12Primary Assembly, NC_000017.11 (40092784..40100725, complement)；Its Transcript (NM_021724.5) (mRNA) sequence is as shown in SEQ ID NO.1；Its protein amino acid sequence such as SEQ ID NO.2 It is shown.
Summary of the invention
The purpose of the present invention is to provide nuclear receptor Rev-erb α to prepare the application in antiplatelet drug, described this Kind of nuclear receptor Rev-erb α will solve antiplatelet drug peace in the prior art preparing the application in antiplatelet drug The bad technical problem of full property.
The present invention provides nuclear receptor Rev-erb α to prepare the application in antiplatelet drug.
Further, the amino acid sequence of the nuclear receptor Rev-erb α is as shown in SEQ ID NO.2.
The present invention also provides application of the Rev-erb alpha inhibitor in the drug that preparation inhibits biologically active pdgf.
It is demonstrated experimentally that nuclear receptor Rev-erb α has expression in people and mouse platelets.Rev-erb α missing can prolong The long mouse bleeding time inhibits iron chloride (FeCl3) induction carotid artery thrombosis.Vitro study discovery, whether gene The platelet aggregation of ADP access mediation can be inhibited by learning missing or pharmacology inactivation Rev-erb α.Further experiment shows The release of α particle when Rev-erb alpha inhibitor SR8278 substantially reduces the activation of ADP induced platelet.Nuclear receptor Rev-erb α is main Biologically active pdgf is influenced by influencing granule of platelet release.
The present invention is compared with prior art, its technical effect is that actively and apparent.Have present invention finds one kind and dives In the nuclear receptor Rev-erb α of anti-platelet activity, inhibit nuclear receptor Rev-erb α that can effectively inhibit platelet aggregation, reduces Thrombosis.The platelet aggregation that Rev-erb alpha inhibitor SR8278 can inhibit ADP to induce.The effect master of nuclear receptor Rev-erb α If the particle after platelet activation and activation by inhibiting ADP access to mediate discharges to play.
Detailed description of the invention
Fig. 1 is Rev-erb α expression in people and mouse platelets.
Fig. 2 is influence of the Rev-erb α missing to bleeding and thrombosis.
Fig. 3 is influence of the Rev-erb α missing to platelet aggregation.
Fig. 4 is influence of the Rev-erb alpha inhibitor SR8278 to human platelet aggregation function.
Fig. 5 is that Rev-erb alpha inhibitor SR8278 reduces granule of platelet emission levels.
Specific embodiment
With reference to the accompanying drawings, the present invention is further illustrated in conjunction with specific embodiments, to more fully understand this Invention.
The following is specific embodiments of the present invention, and the embodiment described is rather than to limit this hair for describing the present invention It is bright.
Reagent used in the examples and experimental method are as follows:
Molecular biochemistry reagent: adenosine diphosphate (ADP) (ADP), iron chloride (FeCl3), prostacyclin, Rev-erb α inhibit Agent SR8278, protease and inhibitors of phosphatases, beta -mercaptoethanol, lauryl sodium sulfate etc. are purchased from the U.S. Sigma, and RIPA is split It solves liquid and is purchased from the U.S. Thermo, 30% methylene diacrylamide is purchased from as assist His Majesty sea, Triethanolamine buffer (TBS), tween- 20, phosphate buffer (PBS), Rev-erb α primer etc. are purchased from raw work biology China, and horseradish peroxidase secondary antibody is purchased from The U.S. Jackson, ECL chemical luminescence reagent kit are purchased from the U.S. Millipore, and isoflurane is purchased from composite tablet China, ribose core Purchased from Takara Japan, Rev-erb Alpha antibodies, GAPDH antibody are purchased from for sour Reverse Transcriptase kit, picodna amplification kit The U.S. abcam, CD62P-PE antibody are purchased from the U.S. BD.
Embodiment 1
It separates simultaneously platelet purification: being examined through informed consent and Ethics Committee, take healthy volunteer periphery blood vains It is spare to obtain Healthy People platelet purification after density gradient centrifugation by 20ml.In addition, being obtained 8-12 weeks through abdominal aortic blood It is spare to obtain mouse platelet purification after density gradient centrifugation for week old wild type C57BL/6J Mouse whole blood.Specific purifying Step are as follows: (1) the above-mentioned whole blood anticoagulant through sodium citrate is added to the physiological saline of 37 degree of preheatings according to volume ratio 2:1, then Prostacyclin is added according to volume ratio 1:5000, through 1100 revs/min, acceleration 9, deceleration 0 is centrifuged 10 minutes, obtains Take platelet rich plasma；(2) upper layer platelet rich plasma is carefully drawn in another centrifuge tube, is added according to volume ratio 1:2500 Prostacyclin, through 800 revs/min, acceleration 9, deceleration 0 is centrifuged 5 minutes, is further removed in platelet rich plasma Red blood cell and leucocyte；(3) it takes second step supernatant into another centrifuge tube, prostacyclin is added according to volume ratio 1:5000, Through 2200 revs/min, acceleration 9, deceleration 4 is centrifuged 10 minutes, removes supernatant, and precipitating is blood platelet；(4) with changing Pellet platelets are resuspended in good tyrode, after according to volume ratio 1:5000 prostacyclin is added, through 2200 revs/min, acceleration is 9, deceleration 4 is centrifuged 10 minutes, removes supernatant, and precipitating is purifying washing blood platelet.By above-mentioned purifying washing blood platelet and Each internal organs extract albumen and total serum IgE respectively, immunoblotting and real-time fluorescence quantitative PCR detection are then carried out respectively, respectively from egg White and mRNA level in-site confirms the presence of Rev-erb α.
It is removed from liquid nitrogen the blood platelet and each organ of people and mouse.A part is separately added into lysate and is ground, Corresponding histone is extracted, immune-blotting method is carried out, confirms that there are Rev-erb α in blood platelet from protein level.Another portion It is separately added into 1ml Trizol, extracts total serum IgE using chloroform-isopropanol method, and then reverse transcription is cDNA, is then carried out Real-time fluorescence quantitative PCR detection, confirms its presence from rna level.(Fig. 1) A figure: take mouse heart, kidney, liver, brain and Skeletal muscle is positive control, confirms mouse platelets memory Rev-erb α from protein level and mRNA level in-site respectively.B figure: people is taken Liver, kidney, brain and skeletal muscle be positive control, confirm that human blood platelets memory exists from protein level and mRNA level in-site respectively Rev-erbα。
Embodiment 2
The foundation of mouse Hemorrhage Model: 8-12 weeks week old Rev-erb α afunction mouse or wild type C57BL/6J control Mouse be placed in anesthesia container in, after its anesthesia stablize after be fixed on surgical plate, anaesthetic mask continue inhaled concentration for 2% it is different Fluothane carries out maintenance anesthesia (Ventilation Rate 1.5L/min).After its steady breathing, rat-tail end is subtracted with the scissors by disinfection 3mm is held, mouse tail is placed in 37 degree of physiological saline, and start timing, until flowing out entirely without blood, judgment criteria is It is flowed out again without blood in 1min.Then the end of record time.The mouse bleeding time be start timing to blood stop completely when Between.The foundation of carotid thrombosis model: iron chloride (FeCl3) damage arteria carotis communis production animal thrombosis model, operating method Simply, controllability is high with repeatability by force, and is current internationally recognized thrombus mould close to the tissue morphology feature of spontaneous thrombus Type.8-12 weeks week old Rev-erb α missing and brood WT mouse each 7 are taken, are injected intraperitoneally 1% yellow Jackets (10ml/Kg), After its anesthesia and breathing steadily, mouse is fixed on small animal surgical plate, disinfects mouse skin of neck in alcohol, is separated small Mouse side arteria carotis communis, it is careful to remove arteria carotis communis surrounding connective tissue and adipose tissue, the sealed membrane pad of clip moderate length Under arteria carotis communis, to prevent iron chloride calcination extravascular tissue.1*2mm size filter paper is taken to be completely submerged in 10% ferric chloride solution In, after its filter paper is impregnated with completely, its smooth be placed on arteria carotis and (be closely affixed).After acting on 5min, carefully cut This section of arteria carotis communis is taken to be placed in 10% formalin, room temperature fixes 24 hours.
Rev-erb α missing (KO) mouse of C57BL/6J background is constructed by Shanghai south model organism Science and Technology Ltd.. SPF grades of KO mouse of 8-10 weeks week old and its brood wild type (WT) control mice cut tail bleeding experiment (if the bleeding time is big In 1200 seconds, then it is denoted as 1200 seconds) and carotid artery thrombosis experiment, detection Rev-erb α missing is to mouse platelets function It influences.As a result, it has been found that the KO mouse bleeding time is obviously prolonged, thrombosis is significantly reduced compared with WT mouse.(Fig. 2)
A figure: the bleeding time of WT mouse is obviously prolonged compared with KO mouse, and KO mouse bleeding time that be averaged is 3 times of WT mouse.
B figure: after iron chloride calcination blood vessel, KO mouse thrombus area is significantly reduced compared with WT mouse.
Embodiment 3
Mouse platelets focusing experiment.Platelet aggregation detection: (1) mouse washing blood platelet obtains: preparing 1ml note Emitter is several, and 3.8% sodium citrate anticoagulant of sucking about 100ul in every syringe, (anti-coagulants and whole blood are according to 1:9 It is added).1% yellow Jackets (10ml/Kg) is injected intraperitoneally, after mouse anesthesia, fixed mouse sterilizes abdomen, opens abdominal cavity, Abdominal aorta is separated, mouse abdominal aorta is pierced into 1ml syringe, carefully extracts whole blood, until feeling there is resistance.Extract injection Device is mixed by inversion rapidly.The whole blood of genotype same mouse is mixed, carries out gradient centrifugation according to the method described above, is obtained Mouse washs blood platelet.
WT mouse is obtained respectively and KO mouse washs blood platelet, platelet aggregation is detected, with low concentration ADP (10uM) After stimulation, KO mouse platelets aggregation rate is substantially less than WT mouse platelets aggregation rate, and middle and high concentration ADP stimulation (20uM or 40uM), then no significant difference.(Fig. 3) A figure: under low concentration ADP (10uM) stimulation, KO mouse platelets aggregation rate is obviously low In WT mouse.B, C schemes: at 20uM and the stimulation of 40uM concentration, WT mouse and KO mouse platelets aggregation rate are without significant difference.
Embodiment 4
Human platelet aggregation experiment.Platelet rich plasma is prepared (PRP): being extracted healthy volunteer's blood 20ml, is carried out close Gradient centrifugation is spent, platelet rich plasma is obtained.(3) aggregation capability detect: open water-bath in advance, to its be heated to 37 degree it is laggard Row subsequent experimental.It takes 225ul washing blood platelet to be placed in aggregation pipe, after rotor is added, is put into aggregation detection hole and measures baseline； It is then respectively adding the aggregation inducing agent ADP of various concentration, and records the curve of platelet aggregation in 3 minutes.Three are carried out respectively Secondary independent repetition is tested.
Healthy volunteer's platelet rich plasma, low concentration ADP (5uM) stimulation are obtained, while giving Rev-erb alpha inhibitor SR8278 (concentration is respectively 0uM, 10uM, 20uM, 30uM) and control (0.1%DMSO), as a result, it has been found that with inhibitor SR8278 concentration increases, and platelet aggregation rate gradually decreases, and illustrates that pharmacology inhibits Rev-erb α that can significantly inhibit blood platelet Aggregation capability.(Fig. 4) A, B figure: with the increase of inhibitor SR8278 concentration, platelet aggregation rate also gradually declines.
Embodiment 5
The detection of human blood platelets particle release function: CD62P is the important indicator that particle discharges after reflecting platelet activation.It presses People's platelet rich plasma is obtained according to the above method, adjusts platelet counts to 6 × 10 with platelet poor plasma (PPP)7/ ml, point Not Jia Ru various concentration Rev-erb alpha inhibitor SR8278 or 0.1% dimethyl sulphoxide control.After being incubated at room temperature 15min, to The CD62P antibody and aggregation inducing agent ADP of PE label are separately added into above-mentioned blood platelet.After mixing gently, 37 degree are protected from light room temperature It is incubated for 20min.4% paraformaldehyde fixer of 500ul is added, then the fixed 10min of room temperature carries out machine examination on flow cytometer It surveys.
Healthy volunteer's platelet rich plasma, low concentration ADP (5uM) stimulation are obtained, while giving above-mentioned concentration gradient Then Rev-erb alpha inhibitor SR8278 carries out flow cytometer detection CD62P expression quantity, discovery increases with inhibitor concentration, CD62P Expression quantity gradually decreases, and illustrates the release that pharmacology inhibits Rev-erb α that can obviously inhibit the granule of platelet.(Fig. 5) A B C D Figure: with the increase of inhibitor concentration, platelet surface palatelet-selectin expression quantity is gradually decreased.E figure: being that 0uM is with inhibitor Control, the p- selectin positive expression rate of 10uM, 20uM, 30uM relative to 0uM, as inhibition concentration increases, positive rate is gradually It reduces.
Sequence table
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Gly His Arg Leu Cys Pro Thr His Val Tyr Ala Ala Pro Glu Gly Lys
385 390 395 400
Ala Pro Ala Asn Ser Pro Arg Gln Gly Asn Ser Lys Asn Val Leu Leu
405 410 415
Ala Cys Pro Met Asn Met Tyr Pro His Gly Arg Ser Gly Arg Thr Val
420 425 430
Gln Glu Ile Trp Glu Asp Phe Ser Met Ser Phe Thr Pro Ala Val Arg
435 440 445
Glu Val Val Glu Phe Ala Lys His Ile Pro Gly Phe Arg Asp Leu Ser
450 455 460
Gln His Asp Gln Val Thr Leu Leu Lys Ala Gly Thr Phe Glu Val Leu
465 470 475 480
Met Val Arg Phe Ala Ser Leu Phe Asn Val Lys Asp Gln Thr Val Met
485 490 495
Phe Leu Ser Arg Thr Thr Tyr Ser Leu Gln Glu Leu Gly Ala Met Gly
500 505 510
Met Gly Asp Leu Leu Ser Ala Met Phe Asp Phe Ser Glu Lys Leu Asn
515 520 525
Ser Leu Ala Leu Thr Glu Glu Glu Leu Gly Leu Phe Thr Ala Val Val
530 535 540
Leu Val Ser Ala Asp Arg Ser Gly Met Glu Asn Ser Ala Ser Val Glu
545 550 555 560
Gln Leu Gln Glu Thr Leu Leu Arg Ala Leu Arg Ala Leu Val Leu Lys
565 570 575
Asn Arg Pro Leu Glu Thr Ser Arg Phe Thr Lys Leu Leu Leu Lys Leu
580 585 590
Pro Asp Leu Arg Thr Leu Asn Asn Met His Ser Glu Lys Leu Leu Ser
595 600 605
Phe Arg Val Asp Ala Gln
610

Claims (3)

1. nuclear receptor Rev-erb α is preparing the application in antiplatelet drug.
2. application according to claim 1, it is characterised in that: the amino acid sequence of the nuclear receptor Rev-erb α is such as Shown in SEQ ID NO.2.
Application of the 3.Rev-erb alpha inhibitor in the drug that preparation inhibits biologically active pdgf.
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1602953A (en) * 2003-09-29 2005-04-06 中国人民解放军军事医学科学院野战输血研究所 Thrombogenesis inhibiting medicine
CN101273128A (en) * 2005-11-04 2008-09-24 学校法人自治医科大学 Platelet aggregation inhibitor composition
US20140363423A1 (en) * 2011-06-14 2014-12-11 Tur-Fu Huang Peptide compounds for inhibition of platelet aggregation

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1602953A (en) * 2003-09-29 2005-04-06 中国人民解放军军事医学科学院野战输血研究所 Thrombogenesis inhibiting medicine
CN101273128A (en) * 2005-11-04 2008-09-24 学校法人自治医科大学 Platelet aggregation inhibitor composition
US20140363423A1 (en) * 2011-06-14 2014-12-11 Tur-Fu Huang Peptide compounds for inhibition of platelet aggregation

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