辛伐他汀通过PPARγ抑制胶原诱导的血小板活化相关机制的研究

发布时间：2018-06-23 03:29

本文选题：辛伐他汀 + 血小板活化　；参考：《河北医科大学》2015年博士论文

【摘要】：目的:血栓形成是涉及临床多种疾病的重要病理生理过程,在心血管疾病领域,特别是在动脉粥样硬化、冠心病、心肌梗死等疾病的发生发展过程中,起着非常重要的作用,常可引起严重危害人类健康的临床综合征。在血栓形成过程中,一方面血小板可以通过释放多种细胞因子及其类似物促进炎症进展,同时也可通过凝血因子相继酶解激活,瀑布式放大凝血过程,促进血栓形成。因此探讨血小板活化的机制及其影响因素,一直是心血管病研究领域的重点和热点。已有研究发现,血脂升高特别是低密度脂蛋白(low-density lipoprotein,LDL)水平增高与血小板活化密切相关。羟甲基戊二酰辅酶A(3-hydroxymethyl-3-methylglutaryl coenzyme A,HMG-Co A)还原酶抑制剂(简称他汀类药物)是目前临床常用的调脂药,可通过抑制体内胆固醇合成步骤中的限速酶(HMG-Co A还原酶),在调节脂质代谢过程。多项研究证实,他汀类药物具有多种疗效显著的抗动脉粥样硬化作用,其中包括抗血小板作用。近年研究发现,他汀类药物,特别是脂溶性他汀,如辛伐他汀等,可直接影响血小板功能,抑制血小板激活,并且可独立于其降低胆固醇水平之外发挥上述作用,然而,所涉及的相关机制尚不十分明确。过氧化物酶体增值物激活受体(peroxisome proliferator-activated receptors,PPARs)属于核受体超家族,研究已经证实PPARs激活后通过调节脂代谢,脂肪酸氧化及糖代谢平衡等多种机制发挥抗炎、延缓动脉粥样硬化进展等作用。而他汀类药物可作为PPARs的配体,通过激活PPARs,参与调节多种病理生理过程。血小板虽然是无核细胞,但研究发现,在血小板胞浆中三种PPARs亚型(PPARα,PPARβ,PPARγ)均有表达,并可通过非基因调控途径影响血小板功能,然而,具体的机制尚不清楚。目前研究证实,多种诱导剂(如胶原,ADP,花生四烯酸等)可快速激活血小板。当血小板活化后,在其表面表达多种血小板膜糖蛋白(如可溶性P-选择素,即CD62P;CD40L;CD63等),这些糖蛋白大多存在于静息血小板胞浆颗粒膜上,当血小板活化后,分泌胞浆颗粒,颗粒膜与质膜融合,进而表达于血小板表面;此外,血小板活化也可使其原有跨膜糖蛋白发生构象改变(如GPⅡb/Ⅲa),从而促进血小板粘附聚集。此外血小板激活后,可引起胞浆内钙离子浓度([Ca2+]i)升高及环磷酸腺苷(cyclic adenosine monophosphate,c AMP)生成减少,这些都可以反应血小板活化状态。文献报道,血小板胞浆c AMP水平升高可通过多种机制抑制血小板功能,PI3K/Akt及血小板丝裂原活化蛋白激酶(mitogen-activated protein kinases,MAPKs)信号系统激活与c AMP水平密切相关。因此,本研究通过探讨辛伐他汀体外干预对胶原诱导的血小板聚集粘附,对血小板活化标志物表达、胞浆内钙离子浓度变化、血小板胞浆c AMP水平、所涉及的PI3K/Akt及血小板MAPKs信号系统的影响,以及血小板胞浆PPARs活化对上述影响的作用,力求从新的角度阐述他汀类药物抗血小板活化的作用机制。方法:本研究首先应用全血阻抗法探讨辛伐他汀对胶原诱导的血小板聚集的作用,并通过ELISA法检测辛伐他汀对血小板PPARs活化的影响,随后应用全血阻抗法和激光共聚焦技术探讨PPARs活化对辛伐他汀抑制血小板聚集及粘附的影响。此外,利用流式细胞术、分光光度法、ELISA法等进一步证实辛伐他汀具有抑制胶原诱导的血小板活化的作用,同时PPARγ激活在辛伐他汀抑制血小板活化中发挥重要作用。通过应用蛋白免疫印迹、免疫共沉淀技术进一步探讨辛伐他汀影响胶原诱导的血小板活化所涉及的信号转导通路,以及PPARγ激活对相关信号传导通路的影响及作用方式。本研究旨在揭示辛伐他汀对胶原诱导的血小板活化的影响,及PPARs活化的作用,为了解辛伐他汀的抗血小板作用提供新的理论依据。结果:我们的研究发现,辛伐他汀体外孵育血小板可激活PPARα及PPARγ,对PPARβ无明显活化作用。同时,随着药物浓度升高,及孵育时间延长,辛伐他汀可呈浓度和时间依赖性的抑制血小板聚集。当应用PPARα及PPARγ拮抗剂(GW6471,GW9662,终浓度均为10μmol/L)预孵育全血样本时,结果显示,虽然辛伐他汀具有PPARα及PPARγ双重激活作用,但是只有PPARγ拮抗剂(GW9662)可以逆转辛伐他汀对胶原诱导的血小板聚集的抑制作用,拮抗PPARα虽有逆转趋势,但是无统计学意义。通过采用共聚焦显微镜观察血小板粘附功能,也得到类似的结论,证实辛伐他汀可以通过PPARs激活,发挥抑制血小板粘附的作用,并且,PPARγ活化在其中起主要作用。进一步的研究发现,辛伐他汀体外孵育可呈剂量相关性抑制胶原诱导的血小板CD62p表达升高(10μmol/L辛伐他汀组49.9%±4.21,30μmol/L辛伐他汀组26.8%±2.89 v.s.阳性对照组74.6%±4.25,P0.05),以及血小板PAC-1表达升高(10μmol/L辛伐他汀组55.6%±6.01,30μmol/L辛伐他汀组35.3%±4.85 v.s.阳性对照组85.2%±4.16,P0.05)。当联合PPARs拮抗剂孵育,PPARγ拮抗剂(GW9662)可显著逆转不同浓度辛伐他汀对血小板CD62p表达的抑制作用(10μmol/L辛伐他汀联合GW9662组62.3%±3.41 v.s.10μmol/L辛伐他汀组49.9%±4.21 P0.05,30μmol/L辛伐他汀联合GW9662组37.8%±2.59 v.s.30μmol/L辛伐他汀组26.8%±2.89,P0.05),及对血小板PAC-1表达的抑制作用(10μmol/L辛伐他汀联合GW9662组74.5%±5.68 v.s.10μmol/L辛伐他汀组55.6%±6.01 P0.05,30μmol/L辛伐他汀联合GW9662组47.9%±3.85 v.s.30μmol/L辛伐他汀组35.3%±4.85,P0.05),而PPARα拮抗剂(GW6471)未观察到明显的逆转作用。类似的结果也反映在血小板胞浆钙离子浓度变化上,我们的研究发现,辛伐他汀体外孵育可呈剂量相关性抑制胶原诱导的血小板胞浆[Ca2+]i升高(3μmol/L辛伐他汀组981±35.62 nmol/L,30μmol/L辛伐他汀组654±43.54 nmol/L,50μmol/L辛伐他汀组392±30.55 nmol/L v.s.阳性对照组1287±50.1 nmol/L,P0.05)。同时,当联合PPARs拮抗剂孵育,结果显示,PPARγ拮抗剂(GW9662)可显著逆转不同浓度辛伐他汀对血小板胞浆[Ca2+]i升高的抑制作用(3μmol/L辛伐他汀联合GW9662组1159±38.62 nmol/L v.s.3μmol/L辛伐他汀组981±35.62 nmol/L P0.05,30μmol/L辛伐他汀联合GW9662组989±37.55 nmol/L v.s.30μmol/L辛伐他汀组654±43.54 nmol/L P0.05,50μmol/L辛伐他汀联合GW9662组555±37.2 nmol/L v.s.50μmol/L辛伐他汀组392±30.55 nmol/L,P0.05),而PPARα拮抗剂(GW6471)未观察到明显的逆转作用。通过对血小板c AMP水平及VASP-Ser157磷酸化的研究也证实,不同浓度辛伐他汀体外孵育可显著增加血小板c AMP水平(3μmol/L辛伐他汀组9.5±0.36 pmol/m L,30μmol/L辛伐他汀组22.4±0.72 pmol/m L,50μmol/L辛伐他汀组30.5±0.64 pmol/m L v.s.阳性对照组5.2±0.58pmol/m L,P0.05)。而血小板胞浆c AMP是抑制血小板活化的重要因素。同时,血小板c AMP升高可引起VASP Ser 157磷酸化,而辛伐他汀体外孵育可呈剂量依赖性促进VASP Ser 157磷酸化,这与其升高血小板c AMP水平作用一致。当联合PPARs拮抗剂孵育,结果显示,PPARγ拮抗剂(GW9662)可显著逆转不同浓度辛伐他汀对血小板c AMP的升高作用(3μmol/L辛伐他汀联合GW9662组7.1±0.54 pmol/m L v.s.3μmol/L辛伐他汀组9.5±0.36 pmol/m L P0.05,30μmol/L辛伐他汀联合GW9662组15.4±0.68 pmol/m L v.s.30μmol/L辛伐他汀组22.4±0.72 pmol/m L P0.05,50μmol/L辛伐他汀联合GW9662组21.5±0.71 pmol/m L v.s.50μmol/L辛伐他汀组30.5±0.64 pmol/m L,P0.05),而PPARα拮抗剂(GW6471)未观察到明显的逆转作用。与之一致的,当联合GW9662拮抗共同孵育,结果表面,拮抗PPARγ活性,可逆转辛伐他汀对血小板VASP Ser157磷酸化的抑制作用。通过对相关信号传导通路的研究发现,辛伐他汀体外孵育可呈剂量依赖性抑制胶原诱导的血小板胞浆Akt磷酸化,而后者可进一步调控血小板聚集、分泌等多种反应。当联合PPARγ拮抗剂GW9662与辛伐他汀共同孵育,可显著逆转后者对胶原诱导的血小板Akt磷酸化的抑制作用。此外丝裂原活化蛋白激酶(mitogen-activated protein kinases,MAPKs)信号传导通路在血小板活化中具有重要作用,血小板c AMP水平变化及Akt磷酸化与MAPKs信号传导通路(ERK1/2,p38 MAPK,JNK)活化密切相关。我们的研究发现,辛伐他汀可呈剂量依赖性抑制胶原诱导的血小板p38 MAPK及ERK1/2磷酸化,而对JNK磷酸化无明显影响。联合PPARγ拮抗剂(GW9662)与辛伐他汀共同孵育,可逆转辛伐他汀对胶原诱导的血小板p38 MAPK及ERK1/2磷酸化的抑制作用。更进一步的,通过免疫共沉淀的方法证明,辛伐他汀可呈剂量依赖性的增加胶原诱导的血小板胞浆PPARγ与p38 MAPK及ERK1/2的结合,结果表明,辛伐他汀可通过PPARγ与MAPKs(p38 MAPK,ERK1/2)信号通路产生直接相互作用,并可通过此种作用影响MAPK通路(p38MAPK及ERK1/2)磷酸化。结论：1辛伐他汀具有PPARα及PPAR双重激活作用,并可呈时间及剂量依赖性抑制胶原诱导的血小板聚集和粘附,PPARγ活化在其中发挥重要作用。2辛伐他汀主要通过PPARγ活化抑制胶原诱导的血小板活化标志物P-选择素及PAC-1表达,并抑制血小板胞浆[Ca2+]i浓度升高,同时可通过PPARγ活化促进血小板c AMP水平及VASP Ser157磷酸化。3辛伐他汀可通过PPARγ活化抑制胶原诱导的血小板Akt磷酸化,及MAPKs(p38 MAPK,ERK1/2)信号传导通路磷酸化,并且通过PPARγ与p38 MAPK及ERK1/2的直接作用发挥抑制作用,进而影响血小板功能。
[Abstract]:Objective: thrombosis is an important pathophysiological process involved in various clinical diseases. It plays a very important role in the development of cardiovascular diseases, especially in the process of atherosclerosis, coronary heart disease and myocardial infarction. It can often cause serious clinical syndromes which seriously harm human health. In the process of thrombosis, one of the most important diseases is in the process of thrombosis. Platelets can promote the progress of inflammation by releasing a variety of cytokines and their analogues, and can also be activated by successive enzymatic hydrolysis of coagulation factors, and a waterfall type enlargement of coagulation processes to promote thrombus formation. Therefore, the mechanism of platelet activation and its influencing factors have been the focus and hot spot in the research field of cardiovascular disease. It is found that the increase of blood lipid, especially low density lipoprotein (low-density lipoprotein, LDL), is closely related to platelet activation. Hydroxymethylglutamyl two acyl coenzyme A (3-hydroxymethyl-3-methylglutaryl coenzyme A, HMG-Co A) reductase inhibitor (for short, statins) is a commonly used lipid regulating agent, which can be used to inhibit the body bile. The rate limiting enzyme (HMG-Co A reductase) in the step of sterol synthesis regulates the process of lipid metabolism. A number of studies have confirmed that statins have a variety of significant anti atherosclerotic effects, including antiplatelet effects. Recent studies have found that statins, especially fat soluble statins, such as simvastatin, can directly affect blood. The function of the platelets, which inhibits platelet activation, and can be independent of its cholesterol lowering levels, but the related mechanisms are not very clear. The peroxisome activation receptor (peroxisome proliferator-activated receptors, PPARs) belongs to the nuclear receptor superfamily, and the study has confirmed the activation of PPARs. By regulating lipid metabolism, fatty acid oxidation and glucose metabolism balance and other mechanisms to play anti-inflammatory and slow the progression of atherosclerosis, statins can be used as the ligand for PPARs and regulate a variety of pathophysiological processes by activating PPARs. Although platelets are nuclear free cells, three kinds of PPARs in the cytoplasm of platelets have been found. Subtypes (PPAR alpha, PPAR beta, PPAR gamma) are expressed and can affect platelet function through non gene regulatory pathways. However, specific mechanisms are not yet clear. A variety of inducers, such as collagen, ADP, arachidic acid, etc., can quickly activate platelets, and many platelet membrane glycoproteins are expressed on the surface of the blood cells after the activation of the platelets. Soluble P- selectin, that is, CD62P, CD40L, CD63, etc., most of these glycoproteins exist on the resting platelet cytoplasmic granular membrane. When platelets are activated, they secrete cytoplasm particles, granular membranes and plasmalemma, and then express on the surface of the platelets. In addition, platelet activation can also make the original transmembrane glycoproteins conformational changes (such as GP II b/ III a). In addition to platelet activation, platelet activation can cause an increase in intracellular calcium concentration ([Ca2+]i) and a decrease in the formation of cyclic adenosine monophosphate (C AMP), which can reflect the state of platelet activation. It is reported that the elevated level of C AMP in the cytoplasm of platelets can inhibit platelet work through a variety of mechanisms. Yes, the activation of PI3K/Akt and platelet mitogen activated protein kinase (mitogen-activated protein kinases, MAPKs) signal system is closely related to the level of C AMP. Therefore, this study is to explore the platelet aggregation adhesion, expression of platelet activation markers, intracellular calcium concentration changes and blood concentration in vitro by the intervention of simvastatin in vitro. The effect of PI3K/Akt and platelet MAPKs signal system on the level of C AMP, the effect of platelet cytoplasm PPARs activation on the effect of platelets on the above effects, and to elaborate the mechanism of anti platelet activation by statins from a new point of view. The effect of simvastatin on platelet PPARs activation was detected by ELISA method. The effect of PPARs activation on the inhibition of platelet aggregation and adherence by simvastatin was investigated by total blood impedance and laser confocal technique. In addition, the flow cytometry, spectrophotometry, ELISA method, etc. were used to further confirm the simvastatin. It has the effect of inhibiting the activation of collagen induced platelet activation, and PPAR gamma activation plays an important role in simvastatin inhibition of platelet activation. By using protein immunoblotting, immunoblotting is used to further explore the signal transduction pathways involved in the effect of simvastatin on the activation of collagen induced platelets, and the correlation of PPAR gamma activation. This study aims to reveal the effect of simvastatin on the activation of collagen induced platelets and the role of PPARs activation to provide a new theoretical basis for understanding the antiplatelet action of simvastatin. Results: our study found that the platelets of simvastatin can activate PPAR A and PPAR gamma in vitro. There was no obvious activation of PPAR beta. At the same time, with the increase of drug concentration and prolonged incubation time, simvastatin could inhibit platelet aggregation in a concentration and time dependent manner. When using PPAR alpha and PPAR gamma antagonists (GW6471, GW9662, the final concentration of 10 u mol/L) to preincubate whole blood samples, the results showed that although simvastatin had PPAR alpha and PPA R gamma double activation, but only PPAR gamma antagonist (GW9662) can reverse the inhibitory effect of simvastatin on collagen induced platelet aggregation. Although antagonistic PPAR alpha has a reversal trend, there is no statistical significance. A similar conclusion is obtained by using confocal microscopy to observe platelet adhesion function. PPARs activation plays an important role in inhibiting platelet adhesion, and PPAR gamma activation plays a major role. Further studies have found that in vitro incubation of simvastatin can show a dose-dependent inhibition of collagen induced platelet CD62p expression (10 mu mol/L simvastatin group, 49.9% + 4.21,30 mol/L simvastatin group 26.8% + 2.89 v.s. positive control Group 74.6% + 4.25, P0.05), and increased expression of platelet PAC-1 (10 mu mol/L simvastatin group 55.6% + 6.01,30 mol/L simvastatin group 35.3% + 4.85 v.s. positive control group 85.2% + 4.16, P0.05). When combined with PPARs antagonist incubation, PPAR gamma antagonist (GW9662) could significantly reverse the inhibitory effect of different concentration of Simvastatin on platelet CD62p expression (10). Mol/L simvastatin combined with GW9662 group 62.3% + 3.41 v.s.10 mu mol/L simvastatin group 49.9% + 4.21 P0.05,30 micron simvastatin combined with GW9662 group 37.8% + 2.59 v.s.30 mu mol/L simvastatin group 26.8% + 2.89, P0.05), and the inhibitory effect on platelet PAC-1 expression (10 micron mol/L sympletin combined with 74.5% + 5.68. The 55.6% + 6.01 P0.05,30 mu mol/L simvastatin combined with GW9662 group 47.9% + 3.85 v.s.30 mol/L simvastatin group 35.3% + 4.85, P0.05), while PPAR a antagonist (GW6471) did not observe the obvious reversal effect. Similar results were also reflected in the change of platelet cytoplasmic calcium concentration. Our study found that simvastatin can be incubated in vitro. Dose-dependent inhibition of collagen induced platelet cytoplasmic [Ca2+]i increased (3 mu mol/L simvastatin group 981 + 35.62 nmol/L, 30 micron simvastatin group 654 + 43.54 nmol/L, 50 u mol/L simvastatin group 392 + 30.55 nmol/L v.s. positive control group 1287 + 50.1 nmol/L, P0.05). At the same time, when incubated with PPARs antagonist, the results showed that PPAR gamma antagonist (GW9662) the inhibitory effect of different concentrations of simvastatin on the elevation of platelet cytoplasmic [Ca2+]i (3 micron simvastatin combined with GW9662, 1159 + 38.62 nmol/L v.s.3 mu mol/L simvastatin group, 981 + 35.62 nmol/L P0.05,30 micron mol/L simvastatin combined with GW9662 group 989 + 37.55 nmol/L 654 + 43.54 simvastatin group) P0.05,50 mu mol/L simvastatin combined with GW9662 group 555 + 37.2 nmol/L v.s.50 mug mol/L simvastatin group 392 + 30.55 nmol/L, P0.05), but PPAR alpha antagonist (GW6471) did not observe the obvious reversal effect. The level of C AMP (3 mu mol/L simvastatin group was 9.5 + 0.36 pmol/m L, 30 micron simvastatin group 22.4 + 0.72 pmol/m L, 50 micron simvastatin group 30.5 + 0.64 pmol/m L v.s. positive control group 5.2), and platelet cytoplasm was a major factor in inhibiting platelet activation. 157 phosphorylation, while simvastatin incubated in vitro can be dose-dependent to promote VASP Ser 157 phosphorylation, which is consistent with the increase of platelet C AMP level. When combined with PPARs antagonists, the results showed that PPAR gamma antagonist (GW9662) could significantly reverse the effect of different concentrations of simvastatin on the increase of platelet C AMP (3 mu mol/L simvastatin combined. Group GW9662 7.1 + 0.54 pmol/m L v.s.3 mol/L simvastatin group 9.5 + 0.36 pmol/m L P0.05,30 u mol/L simvastatin combined GW9662 group 15.4 + 0.68 pmol/m L 22.4 + 0.72 .05), and PPAR alpha antagonist (GW6471) did not observe a significant reversal effect. When combined with GW9662 antagonism, the antagonism of PPAR gamma activity could reverse the inhibitory effect of simvastatin on the phosphorylation of VASP Ser157 in platelets. Through the study of the related signal transduction pathway, the incubation of simvastatin in vitro can be used as an agent. The amount dependent inhibition of collagen induced phosphorylation of platelet cytoplasm Akt, and the latter can further regulate platelet aggregation, secretion and other reactions. When the combined PPAR gamma antagonist GW9662 is incubated with simvastatin, the latter can significantly reverse the inhibitory effect of the latter on collagen induced platelet Akt phosphorylation. In addition, mitogen activated protein kinase (mitog En-activated protein kinases, MAPKs) signaling pathway plays an important role in platelet activation. The changes in platelet C AMP level and Akt phosphorylation are closely related to the activation of MAPKs signal transduction pathway (ERK1/2, p38 MAPK, JNK). Our study found that simvastatin can be dose-dependent inhibition of collagen induced platelets 1/2 phosphorylation has no significant effect on the phosphorylation of JNK. The co incubation of the combined PPAR gamma antagonist (GW9662) with simvastatin can reverse the inhibitory effect of simvastatin on collagen induced platelet p38 MAPK and ERK1/2 phosphorylation. Further, it is demonstrated by immunoprecipitation that simvastatin can increase collagen induced by a dose-dependent manner. The combination of platelet cytoplasm PPAR gamma and p38 MAPK and ERK1/2 shows that simvastatin can interact directly with the PPAR gamma and MAPKs (p38 MAPK, ERK1/2) signaling pathways and can affect the MAPK pathway (p38MAPK and ERK1/2) phosphorylation through this action. Conclusion: 1 simvastatin has a double activation effect and can be present. Inter and dose-dependent inhibition of collagen induced platelet aggregation and adhesion, PPAR gamma activation plays an important role in.2 simvastatin mainly through PPAR gamma activation to inhibit collagen induced platelet activation marker P- selectin and PAC-1 expression, and inhibit the increase of platelet cytoplasm [Ca2+]i concentration, and can promote blood small by PPAR gamma activation. The level of plate C AMP and VASP Ser157 phosphorylation.3 simvastatin can inhibit collagen induced platelet Akt phosphorylation and MAPKs (p38 MAPK, ERK1/2) signaling pathway phosphorylation through PPAR gamma activation, and inhibit the function of platelets by inhibiting the direct action of PPAR gamma.
【学位授予单位】：河北医科大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R96

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