重组人血小板活化因子乙酰水解酶治疗脑梗塞的实验研究
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摘要
背景和目的：脑血管病是临床的常见病,其发病率高、死亡率高、致残率高,严重危害人类健康。其中,缺血性脑血管病的发病占70%,多见于60岁以上的老年患者。随着老龄化社会的到来,脑血管病带来的经济和社会负担严重影响了我国老年人的健康状况和生活质量。因此,寻找高效而安全的药物已成为我们实验研究的重点。
一旦脑缺血超过6分钟,即可引发脑组织能量的耗竭,信号传导发生异常,从而导致线粒体功能障碍,大脑的神经功能进行性的不可逆性的全面损伤。当脑缺血再恢复血流供应时,器官组织的细胞功能、代谢障碍及结构破坏不但没有得到恢复反而加重的现象,称为再灌注损伤。缺血性脑血管疾病的发病机制比较复杂,目前研究的药物主要有：兴奋性氨基酸受体拮抗剂、钙离子拮抗剂和自由基清除剂等,但其治疗急性缺血性脑卒中的效果仍不能完全肯定。第一个治疗该病的溶栓药t-PA于1996年被美国FDA批准并逐渐成为目前公认的有效方法。但溶栓治疗限于严格的时间窗和诊疗条件,仅2%-5%的卒中患者能进行该项治疗；同时溶栓治疗虽可通过血流再灌注改善脑缺血区的神经细胞功能,但对于缺血所引起的脑组织再灌注损伤束手无策。而对各种类型脑保护剂的研究表明,很多药物临床疗效欠佳,或有较严重副作用。由此可见,深入研究脑血管病的发病机制,进而开发出有效的抗缺血性脑损伤药物,已经成为当代神经病学研究迫切需要解决的课题之一
血栓形成是缺血性脑血管病的主要致病因素。血小板在血栓的发病中有十分重要的作用,因此抑制血小板活化已经成为治疗脑卒中的常规疗法。研究表明,脑缺血时,血小板功能异常亢进使血液处于高凝状态,这是造成继发性脑损伤的重要原因之一。目前临床上广泛使用抗血小板药物预防高危人群中脑卒中的发生,从而有效降低缺血性脑卒中的发生率。因此,针对血小板的粘附和聚集被认为是开发抗栓药物非常有前景的靶点。目前已有的抗血小板药物按其作用机理大体可分为四类：(1)特异性抑制二磷酸腺苷(Adenosine diphosphate, ADP)活化血小板的药物Ticlopidine和Clopidogrel,选择性抑制ADP活化的血小板聚集；(2)增高血小板内环核苷酸含量的药物,如西洛他唑和双密达莫；(3)抑制血小板花生四烯酸(Arachidonic acid, AA)代谢的药物,包括环氧酶抑制剂(阿司匹林)和血栓素A2(Thromboxane A2, TXA2)合成酶抑制剂(苯酸咪哇),通过抑制环氧酶或TXA2合成酶,阻碍AA衍变为TXA2；(4)血小板膜纤维蛋白原受体拮抗剂,如Abciximab,通过阻断纤维蛋白原与血小板受体GPⅡ b/Ⅱ1a的结合而有效地抑制血小板聚集。
血小板活化因子(platelet-activating factor, PAF)是磷脂类生物活性介质及细胞因子并日益受到重视。它在极低浓度下通过与特异性G蛋白受体结合而活化多种细胞,介导炎症和其他疾病,影响消化、心脑血管、生殖、皮肤和呼吸等多种组织系统。大量动物和临床实验表明,PAF参与了脑梗塞的发生发展病理过程,与动脉粥样硬化、缺血性脑血管病的转归密切关联。PAF是最强的诱导血小板聚集剂,一方面,PAF能进一步促进血小板桥联作用,启动血栓形成和机化,此环节不受阿司匹林的作用影响；另一方面,与继发性缺血性脑损伤有关,如免疫促炎、促血栓形成及自由基、兴奋性氨基酸释放、钙超载、即早基因表达等。因此,机体内PAF若异常升高,将导致临床脑梗塞的发生及继发性脑损害。PAF受体拮抗剂可通过抑制血小板聚集和自由基、兴奋性氨基酸释放、钙超载发挥梗塞后脑保护作用。早已证实,急性脑梗塞患者血浆和脑梗塞模型鼠脑组织中PAF含量明显高于对照组。PAF抑制剂的代表-银杏提取制剂是治疗临床脑梗塞的常用药物之一。
血管内PAF的代谢失活是由于2位乙酰基被水解,催化此反应的酶即为血浆型PAF乙酰水解酶(platelet-activating factor acetylhydrolase, PAF-AH),故该PAF-AH和PAF的动态平衡对维持PAF的正常活性起重要作用。大多数炎症性疾病PAF-AH轻度升高。另外,妊娠、高血压、血管性疾病、缺血性卒中、尿毒症、艾滋病等可出现升高现象,而升高的PAF-AH可明显减轻PAF和氧化磷脂的炎症介质效应。因而推测PAF-AH含量及活性的降低与动脉硬化性形成、脑梗塞的发生发展有关。此外,PAF-AH还可显著抑制氧化应激连锁反应,对血管内皮具有保护性作用。
人血浆型PAF-AH主要来源于巨噬细胞,其基因位于6p.12p.21.1,含12个外显子,可编码441个氨基酸,分子量为44-kDa。Tjoelker等首次成功建立人血浆型PAF-AH DNA重组体,证实所表达的重组血浆型PAF-AH (rPAF-AH)且有同等催化底物PAF的活性,可抑制PAF诱导的炎症反应。研究发现,用rPAF-AH尾静脉注射可显著减轻胰腺炎模型大鼠胰腺损伤程度,生物半衰期达7小时。而rPAF-AH更可作为预防呼吸窘迫综合征和降低败血症死亡率的有效手段。多中心、随机、双盲、安慰剂对照三期临床试验结果显示,应用rPAF-AH治疗败血症的患者对rPAF-AH耐受性好、无抗体产生。目前采用rPAF-AH治疗疾病的研究集中局限于炎症反应方面,国内则尚未见有rPAF-AH相关文献报道。
基质金属蛋白酶(matrix metalloproteases, MMPs)是一种能够分解细胞外基质(ECM)的锌依赖性蛋白水解酶家族,在人体内参与多种生理和病理过程。相对分子质量为72x103(72KD)的MMP-2和相对分子质量为92x103(92KD)的MMP-9为MMP家族的2个成员,均可降解基底膜的ECM成分,其底物包括Ⅳ型、Ⅴ型胶原酶、纤维连接蛋白、弹性蛋白和变性的基质胶原。正常情况下,MMP-2以潜伏的形式存在于脑组织内,可被膜结合蛋白酶膜型金属蛋白酶(membrane type matrixmetallo-proteinase, MT-MMp)激活。
近年来,有人发现脑缺血再灌注后出现MMPs增加,尤其是MMP-9和MMP-2活性增加与脑微血管通透性、血脑屏障(Blood brain barrier, BBB)通透性、BBB崩溃、脑水肿和炎细胞侵入明显相关。BBB破坏后,一方面可引起血液中的血浆蛋白、多种毒性物质及代谢产物进入脑内,促进并加重血管源性脑水肿；另一方面为炎性因子侵入脑组织大开方便之门。在局部脑缺血所致的出血变化时,MMP-9的表达明显增强,而MMP-2则不增强,提示两者在降解基底膜引起的神经元损伤以及局灶性脑缺血导致出血性转化早期的潜在作用。其中MMP-9(明胶酶B)激活时能降解参与血脑屏障基膜的主要成分纤粘蛋白和粘蛋白,破坏血脑屏障,引起血脑屏障的通透性升高或进一步开放,缺血后脑组织MMP-9活性增高是缺血后脑损伤的重要机制之一。近年来有研究表明,脑缺血再灌注可增强MMP的表达,予MMP抗体或MMP抑制剂可减轻脑缺血再灌注损伤,改善血管源性水肿,以及保护神经细胞免受损伤等。
血管内皮生长因子(vascular endothelial growth factor,VEGF)是强效的有丝分裂原,可直接并选择性地作用于内皮细胞,促进新生血管生成。中枢神经系统有多种因子可促进血管新生,而许多研究均表明在血管新生过程中,VEGF是主要的因子之一。VEGF可令细胞间的接触松弛,并使稠密的细胞外基质瓦解,进而促进新的微血管形成。VEGF在血管新生中的主要作用包括：①上调细胞间和血管细胞的粘附分子的水平；②诱导内皮细胞上相应受体和配体的表达;③促进血管内皮细胞的增殖和迁移；④诱使骨桥钻合蛋白与上调的内皮细胞表面的整合素受体结合；⑤增加丝氨酸蛋白酶的活性,从而降解特定区域的细胞外基质,促进血管生成。缺氧或缺血在多数组织可以引致血管新生。而VEGF在局灶性脑缺血的血管新生中发挥不可忽视的作用。中枢神经缺血后,在梗死灶周围有一圈缺血相对较轻的区域,称半暗带(penumbra)。半暗带血供的恢复对神经细胞的存活具有举足轻重的意义。VEGF基因的启动子区含有缺氧诱导因子-1(HIF-1)反应元件。当脑部发生缺氧或低氧时,VEGF基因5’端低氧反应元件与HIF-1结合,启动环二磷酸鸟苷酸(cGMP)途径,VEGF的表达即可受到相应调节。目前公认缺氧是调节VEGF及其受体表达的主要因素之一。脑缺血后,低氧信号激活VEGF/VEGF受体系统,促使VEGF在半暗带区域高表达。随即诱导内皮细胞增殖,促进半暗带大量新生血管形成,加速侧枝循环的建立,改善相应缺血脑组织的供血供氧,减少神经元凋亡或坏死,最终减轻缺血性脑损伤。
VEGF对神经元的直接作用是神经营养作用,可促进体外培养的神经元轴突生长、增加神经突触的数目并延长其长度,提高神经元的存活率。正常情况下,脑内无VEGF表达,但当受到某种损害时如缺氧炎症肿瘤时能诱导VEGF表达已被许多体内及体外实验证实。
奥扎格雷钠为血栓烷(TX)合酶抑制剂,能阻止前列腺素H2(PGH2)生成血栓烷A2(TXA2),并促使血小板所产生的PGH2转向内皮细胞,令内皮细胞可利用PGH2合成PGI2,从而起到抑制血小板的聚集和扩张血管作用。动物试验表明,在大脑中动脉阻塞小鼠模型中,静脉给药能使其血浆TXB2水平降低,可预防性治疗脑梗塞。
金纳多(extract of ginkgo biloba leaves injection)产于德国,为银杏提取物,作为强效PAF拮抗剂,具有抑制血小板聚集,抗血栓,改善血液动力学与血液流变学,改善心脑代谢及抗炎性损伤等作用。从本世纪20年代起,银杏叶制剂就用于治疗外周及中枢血液循环障碍以及脑功能障碍等症。目前,作为银杏叶提取物的金纳多注射液已在亚洲地区广泛使用,成为治疗脑梗塞的常见药物。本研究选择安全有效的金纳多注射液和奥扎格雷钠注射液作为rPAF-AH的阳性对照,希望能对rPAF-AH的神经保护作用做出较为全面的评估。
本研究使用美国peprotech公司制作的rPAF-AH,观察不同浓度rPAF-AH对兔富含血小板血浆PAF诱导的血小板聚集率的影响和量效关系；另一方面,观察静脉注射rPAF-AH后：(1)采用线拴法制备的大脑中动脉缺血再灌注模型小鼠脑梗塞的梗塞体积、神经行为学的改变；(2)模型鼠脑MMPs、VEGF的蛋白表达；(3)模型鼠脑MMPs、VEGF的基因表达。初步探讨rPAF-AH治疗脑梗塞的有效性和可行性,评估该生物蛋白制剂的临床开发、应用前景。
材料与方法：
一采用线栓法建立小鼠大脑中动脉缺血再灌注模型,尾静脉注射rPAF-AH预处理后观测模型小鼠神经行为学和脑梗死灶大小的改变,采用Western blot研究MM P-2、MMP-9和VEGF的表达及活性；rt-PCR研究MM P-2、MMP-9和VEGF基因表达的改变,同时与奥扎格雷钠注射液、金纳多注射液及假手术对照组比较。
二于兔富含血小板血浆(PRP)中分别加入rPAF-AH,使后者达到不同的最终浓度；另设一份不加rPAF-AH的PRP做为对照。各份PRP中加入10-8mol浓度的PAF并测血小板最大聚集率。观察不同浓度rPAF-AH对兔富含血小板血浆PAF诱导的血小板聚集率的影响并和金纳多比较。
结果：
1. rPAF-AH预处理组降低脑梗塞小鼠行为障碍的神经行为学评分、脑梗死灶,与手术模型组比较,差异有显著性意义(P<0.01)
2. Western blot分析结果表明,与手术模型组比较,rPAF-AH预处理可降低脑缺血再灌注损伤所诱导的MMP-2蛋白、MMP-9蛋白的表达及其活性水平(P<0.01)；上调了VEGF蛋白的表达及其活性水平,差异有统计学意义(P<0.001)。
3. rt-PCR分析结果表明,与手术模型组比较,rPAF-AH预处理可降低脑缺血再灌注损伤所诱导的MMP-2mRNA、MMP-9mRNA的表达及其活性水平(P<0.01)；上调了VEGF mRNA的表达及其活性水平,差异有显著性意义(P<0.001)。
4.所有加入金纳多注射液或rPAF-AH的PRP,其血小板最大聚集率都比对照PRP的低,且rPAF-AH对血小板最大聚集率的影响较金纳多注射液更大,有显著性意义(P<0.01)。较高浓度的rPAF-AH,其血小板最大聚集率也较低。
结论：
1.小鼠脑缺血再灌注早期能诱导脑组织MMP-2, MMP-9的表达上调,从而导致BBB通透性增加和血管源性脑水肿的形成。rPAF-AH预处理对小鼠脑缺血再灌注损伤有一定的保护作用,可能是通过包括对MMP-2, MMP-9的表达抑制和VEGF的表达促进及稳定BBB通透性在内的多种途径来完成的。
2. rPAF-AH与金纳多注射液相比,能够降低PAF所诱导的血小板聚集,降低具有统计学意义(P<0.01)。
Background and purpose:Cerebrovascular disease are very common disease in clinic.Its high incidence, high mortality, high disability rate, have already done serious harm to human health. Among them, ischemic cerebrovascular diseases accounted for70%, which is more common in patients aged more than60years old. As the aging of population accelerates, cerebrovascular disease have been bringing more and more economic and social burden of serious impact on the elderly health status and quality of life. Therefore, searching for the high effective and safe drug has become our experimental research focus.
Once the brain ischemia lasts more than6minutes, can cause energy depletion, signal transduction abnormalities occur, leading to mitochondrial dysfunction, brain nerve function of irreversible comprehensive injury. If blood supply is restored after cerebral ischemia, organ and tissue cell functions, metabolic disorders and structural damage will not be restored or will even be aggravated. Such a phenomenon is called the reperfusion injury. The pathogenesis of ischemic cerebrovascular diseases is quite complex, and medications related to recent studies are:excitatory amino acid receptor antagonists, calcium antagonists and free radical scavenger. But it is still not entirely sure whether they can cure the acute ischemic stroke. FDA of America in1996approved the first treatment of the disease with thrombolytic drug t-PA, and gradually thrombolytic therapy is recognized as an effective method. But given the time window of thrombolysis and treatment conditions, only2%in5%of the patients are suitable receive this treatment; at the same time thrombolytic therapy can establish cerebral ischemia reperfusion of blood flow, but does not solve the ischemia caused by brain tissue reperfusion injury. Various types of brain protective agent researches suggest that many drugs' clinical curative effect is poor, or even have serious side effects. Therefore, the in-depth study of the pathogenesis of cerebrovascular diseases, leading to the development of effective drug against ischemic brain injury, is one of the most important research topics of contemporary neurology.
Thrombosis in ischemic cerebrovascular diseases is the main pathogenic factor. Platelet thrombosis plays a very important role, and thus inhibiting platelet activation has become the treatment of stroke in the conventional therapy. Studies show that, during cerebral ischemia and abnormal platelet function, hyperfunction of blood at high coagulation state, which is caused by secondary brain injury is one of the important reasons. The clinical use of antiplatelet drug prevention in high-risk populations of midbrain stroke, thereby effectively reduces the incidence rate of cerebral arterial thrombosis. Therefore, the platelet adhesion and aggregation is considered to be very promising targets of the development of antithrombotic drug.
The current antiplatelet drugs according to their mechanisms of action can be divided into the following four categories:(1) specific inhibition of two adenosine monophosphate (Adenosine diphosphate, ADP) activated platelet drugs:Ticlopidine and Clopidogrel, selective inhibition of ADP activation of platelet aggregation;(2) drugs of increased platelet cyclic nucleotide content, such as cilostazol and double density dipyridamole;(3) drugs of inhibition of platelet peanut acid (Arachidonic four acid, AA) metabolism including epoxy enzyme inhibitor (aspirin) and thromboxane A2(Thromboxane A2, TXA2) synthase inhibitor (benzoic acid with wow), by inhibiting the enzyme cyclooxygenase or TXA2synthase, and blocking AA development for TXA2;(4) platelet membrane fibrinogen receptor antagonists, such as Abciximab, by blocking the fibrinogen and platelet receptor GP ⅡB/Ⅱla combination and effective inhibition of platelet aggregation.
Platelet activating factor (platelet-activating factor, PAF) is a phospholipid class of biologically active mediators and cytokines and is attracting more and more attention. In the extremely low concentrations, it is bound with specific G protein receptor, which activates multiple cells, and mediates inflammation and other diseases, thus affecting digestion, cardiovascular, reproductive, skin and respiratory and other organization system. Large amounts of animal and clinical experiments show that, PAF is involved in the occurrence and development of cerebral infarction pathological processes, which usually leads to atherosclerosis and ischemic cerebrovascular disease. PAF is the strongest induced platelet aggregation agent. On one hand, PAF can further promote platelet bridging function, initiation of thrombosis and machine, and this link is not affected by the effect of aspirin effect; on the other hand, with the secondary ischemic cerebral injury, such as immune inflammatory, PAF promotes thrombosis and free radicals, the release of excitatory amino acids, calcium overload, immediate early gene expression. Therefore, the abnormal increase of PAF, will cause the clinical occurrence of cerebral infarction and secondary brain damage. PAF receptor antagonists can inhibit platelet aggregation and free radicals, the release of excitatory amino acids, calcium overload and have an infarction cerebral protective effect. It was proved that, in patients with acute cerebral infarction and rats in cerebral infarction model, the plasma content of PAF in brain tissue was significantly higher than that of the control group. PAF inhibitors (like Ginkgo extract preparations) are one of the commonly used drugs in the clinical treatment of cerebral infarction.
The enzyme2acetyl which is the plasma PAF acetylhydrolase (PAF-AH) is hydrolyzed to catalyze this reaction and this leads to intravascular PAF metabolic inactivation. The dynamic balance between PAF-AH and PAF to maintain normal activity plays an important role in PAF. PAF-AH mildly elevated in the majority of inflammatory diseases. In addition that, the elevation of PAF-AH can also be found in pregnancy, hypertension, vascular disease, ischemic stroke, uremia, AIDS. It can significantly reduce PAF and proinflammatory oxidized phospholipids medium effect. So the contents of PAF-AH and decreased activity and arteriosclerosis formation, are related to the occurrence and development of cerebral infarction. In addition, the PAF-AH also could significantly inhibit the chain reaction of oxidative stress, and have endothelial protective effects.
Human plasma PAF-AH comes mainly from macrophages, whose genes are located on the6p.12p.21.1, containing12exons, encoding441amino acids, molecular weight of44-kDa. Tjoelker's first successful establishment of human plasma PAF-AH recombinant DNA, confirmed that the expression of the recombinant plasma type PAF-AH has the same catalytic substrate PAF activity, and can inhibit PAF induced inflammatory reaction. Study found that, using recombinant human plasma PAF-AH tail vein injection can significantly reduce the pancreatitis in rat model of pancreatic injury severity, the biological half-life of7hours. Recombinant human plasma PAF-AH even can be used for the prevention of respiratory distress syndrome and as effective means to reduce sepsis mortality. A multicenter, randomized, double-blind, placebo-controlled phase in three clinical trial results shows, the application of recombinant human plasma PAF-AH in treatment of sepsis patients on recombinant human plasma PAF-AH is well tolerated, and no antibody is produced. The focus of the recombinant plasma type PAF-AH for the treatment of disease research is confined to the inflammatory response, and in China, there is no recombinant plasma PAF-AH report.
Matrix metalloproteinases (matrix metalloproteases, MMPs) is a decomposition of the extracellular matrix (ECM) zinc dependent protein hydrolase family, and they are involved in a variety of physiological and pathological processes. The MMP family of2members:the relative molecular mass of72x103(72KD) MMP-2and the relative molecular mass of92x103(92KD) MMP-9, can degrade the basement membrane of the ECM component, the substrate including type IV, type V collagenase, fibronectin, elastin and degeneration of the collagen matrix. Under normal circumstances, MMP-2is present in latent forms in the brain, can be membrane-bound proteases membrane type metalloproteinases (membrane type matrixmetallo-proteinase, MT-MMp) activated.
In recent years, it was found after cerebral ischemia there is an increase of MMPs, especially MMP-9and MMP-2activity and cerebral microvascular permeability of blood brain barrier (Blood brain, barrier, BBB) permeability, to which BBB collapse, brain edema and inflammatory cell influx were significantly related to. Once BBB is destroyed, it can cause the blood plasma protein, a variety of toxic substances and metabolites into the brain, and then promotes and exacerbates vasogenic brain edema; and at the same time, opens the floodgates for inflammatory factor to invade brain tissue. In local cerebral ischemia caused by bleeding changes, MMP-9expression was significantly increased, while MMP-2is not enhanced, and the tips of both in the degradation of basement membrane induced neuronal injury and focal cerebral ischemia caused hemorrhagic transformation in the early potential function. The MMP-9(gelatinase B) when activated can degrade the involvement of blood brain barrier,which is the main component of fiber mucins and mucin. The disruption of the blood-brain barrier, causing blood brain barrier permeability increased or further opening, and the increase of MMP-9activity in ischemia brain tissue are important mechanisms in cerebral ischemia injury. In recent years, studies have shown that, cerebral ischemia reperfusion can enhance the expression of MMP, and the MMP antibodies or MMP inhibitors may reduce cerebral ischemia reperfusion injury, ameliorate vasogenic edema, and protect neural cells against injury.
Vascular endothelial growth factor (vascular endothelial growth factor, VEGF) is an effective mitogen, and it can directly and selectively act on endothelial cells, and promote the formation of new blood vessels. Central nervous system has a plurality of angiogenic factors, including vascular endothelial growth factors which are recognized as major involving angiogenesis factors. Vascular endothelial growth factor can relax the cell-cell contact, and the collapse of specific regions of the extracellular matrix, thus forming a new micro vascular. VEGF's major roles in angiogenesis include:increasing intercellular adhesion molecules and vascular cell adhesion molecule level; inducing endothelial cell on the corresponding ligand and receptor expression; leading to the proliferation and migration of vascular endothelial cells; upregulating of endothelial cell surface expression of integrin receptors, and inducing bone bridge drilling photosynthetic proteins to bind; upregulating of serine protease activity, thereby degrading the dense extracellular matrix, promoting angiogenesis. Hypoxia or ischemia in most tissues can cause angiogenesis. And vascular endothelial growth factor in focal cerebral ischemia angiogenesis plays an important role. Ischemia after cerebral infarction, is relatively light in a circle around the area of ischemia called the penumbra (penumbra). The restorationof the blood supply of Penumbra has great significance on neuronal survival. In the VEGF gene promoter region there is hypoxia inducible factor-1(HIF-1) response element. When there is hypoxia or low oxygen, HIF1and VEGF5'end the binding of the gene hypoxia response element, through a ring two? two-ring guanosine monophosphate acid (cGMP) pathways to regulate the expression of VEGF. It is generally accepted that hypoxia is VEGF and its receptor expression in the main regulating factors. After cerebral ischemia, hypoxia as a signal for the activation of VEGF/VEGF receptor system, prompted the penumbra of high expression of VEGF, and the latter can be induced through the proliferation of endothelial cells, thus promoting the penumbra extensive neovascularization, accelerating the establishment of collateral circulation, improving the brain tissue perfusion and oxygen supply involvement, reducing neuronal apoptosis or necrosis volume, and eventually reducing ischemic brain injury.
VEGF has direct neurotrophic effects on the neurons, and can promote cultured neuronal survival and axonal growth, and increase in neurite number and length. VEGF stimulates axon growth and increases the survival of nerve cells. Under normal circumstances, there is no expression of VEGF in the brain. But when subjected to certain damage such as inflammatory tumor hypoxia, VEGF expresses and it has been used by many in vivo and in vitro experiments confirmed.
Ozagrel Sodium Injection, which is the thromboxane synthase inhibitor, can prevent prostaglandin H2(PGH2) formation of thromboxane A2(TXA2), and induce platelet derived PGH2to endothelial cells, endothelial cells to synthesize PGI2, which acts to inhibit platelet aggregation and vascular dilatation function. Animal experiments show that, intravenous injection can reduce plasma TXB2levels, and have preventive effects on mouse middle cerebral artery occlusion caused by cerebral infarction.
Extract of Ginkgo Biloba Leaves of Injection, produced in Germany, as a potent platelet activating factor (plateletactivatefactor) and PAF antagonist, can inhibit platelet aggregation, antithrombotic, improve hemodynamics and blood rheology, thus improving the treatment effect of cardiovascular and metabolic and inflammatory injury. Since the first two decades of this century, ginkgo leaf preparation has been used in the treatment of peripheral and central circulatory disorders and brain dysfunction. At present, Ginaton injection as the Ginkgo biloba extract has been widely used in Asia, and become common drugs for treatment of cerebral infarction. On the safe and effective Ginaton injection and Austria grips Gray sodium injection rPAF-AH acts as a positive controller, and a comprehensive assessment to the rPAF-AH neuroprotection is expected to be made.
This study using the American company peprotech produced by recombinant PAF-AH, to observe the effects of different concentrations of recombinant PAF-AH on rabbit platelet rich plasma PAF induced platelet aggregation rate effects and dose-effect relationship; on the other hand, intravenous injection of recombinant PAF-AH:(1) uses the line to preparation of middle cerebral artery ischemia reperfusion model in mouse models, cerebral infarction infarction volume, ultrastructural changes;(2) model rat brain MMPs, VEGF protein expression;(3) model rat brain MMPs, VEGF gene expression. It is a preliminary study of validity and feasibility of recombinant human plasma PAF-A in the treatment of cerebral infarction, the biological protein preparations in clinical development and the application prospect are also discussed.
Materials and methods:
l.An model established by suture method in mice with cerebral ischemia/reperfusion, tail vein injection of rPAF-AH after pretreatment; observation of mouse neural behavior and cerebral infarction size changes, using the Western blot on MM P-2, MMP-9and VEGF expression and activity; rt-PCR on MMP-2, MMP-9and VEGF gene expression changes, at the same time and Austria grips Gray sodium injection, Ginaton injection and sham operation control group.
2.rPAF-AH were added to two rabbit samples of platelet rich plasma (PRP), and they have different final concentration of rPAF-AH; another one without rPAF-AH PRP as the control group. Each copy of the PRP with10-8mol concentration of PAF is used to measure the maximum platelet aggregation rate. Different concentrations of recombinant PAF-AH on rabbit platelet rich plasma PAF will induce different platelet aggregation rate and a comparison of effects of ginaton will be made.
Result:
1rPAF-AH pretreated group was markedly reduced cerebral infarction mouse behavior disorders of nervous behavior score, cerebral infarction (P<0.01).
2Western blot results show, compared with the operation of model group, rPAF-AH preconditioning reduces ischemia reperfusion injury induced by MMP-2protein, the expression of MMP-9protein and activity levels (P<0.01); apparent up-regulation of VEGF expression and activity levels (P<0.001).
3rt-PCR analysis results show that, compared with the operation of model group, rPAF-AH preconditioning reduces ischemia reperfusion injury induced by MMP-2mRNA, MMP-9mRNA expression and activity level (P<0.01); VEGF mRNA markedly upregulated expression and activity levels (P<0.001).
4All join Ginaton injection or rPAF-AH PRP, the maximum platelet aggregation rate than control low PRP, and rPAF-AH on platelet aggregation rate effects of Ginaton injection more obviously (P<0.01). A high concentration of rPAF-AH, the maximum platelet aggregation rate is low.
Conclusion:
1rPAF-AH injection in mice with cerebral ischemia reperfusion early can induce brain tissue MMP-2, MMP-9upregulates the expression of BBB, resulting in increased permeability and vasogenic brain edema formation. rPAF-AH preconditioning on ischemia reperfusion brain injury in mice have a protective effect, probably by including inhibits the expression of MMP-2, MMP-9and promotes VEGF expression,so that stable BBB permeability.
2rPAF-AH compared with Ginaton injection, can significantly reduce PAF induced platelet aggregation.
一旦脑缺血超过6分钟,即可引发脑组织能量的耗竭,信号传导发生异常,从而导致线粒体功能障碍,大脑的神经功能进行性的不可逆性的全面损伤。当脑缺血再恢复血流供应时,器官组织的细胞功能、代谢障碍及结构破坏不但没有得到恢复反而加重的现象,称为再灌注损伤。缺血性脑血管疾病的发病机制比较复杂,目前研究的药物主要有：兴奋性氨基酸受体拮抗剂、钙离子拮抗剂和自由基清除剂等,但其治疗急性缺血性脑卒中的效果仍不能完全肯定。第一个治疗该病的溶栓药t-PA于1996年被美国FDA批准并逐渐成为目前公认的有效方法。但溶栓治疗限于严格的时间窗和诊疗条件,仅2%-5%的卒中患者能进行该项治疗；同时溶栓治疗虽可通过血流再灌注改善脑缺血区的神经细胞功能,但对于缺血所引起的脑组织再灌注损伤束手无策。而对各种类型脑保护剂的研究表明,很多药物临床疗效欠佳,或有较严重副作用。由此可见,深入研究脑血管病的发病机制,进而开发出有效的抗缺血性脑损伤药物,已经成为当代神经病学研究迫切需要解决的课题之一
血栓形成是缺血性脑血管病的主要致病因素。血小板在血栓的发病中有十分重要的作用,因此抑制血小板活化已经成为治疗脑卒中的常规疗法。研究表明,脑缺血时,血小板功能异常亢进使血液处于高凝状态,这是造成继发性脑损伤的重要原因之一。目前临床上广泛使用抗血小板药物预防高危人群中脑卒中的发生,从而有效降低缺血性脑卒中的发生率。因此,针对血小板的粘附和聚集被认为是开发抗栓药物非常有前景的靶点。目前已有的抗血小板药物按其作用机理大体可分为四类：(1)特异性抑制二磷酸腺苷(Adenosine diphosphate, ADP)活化血小板的药物Ticlopidine和Clopidogrel,选择性抑制ADP活化的血小板聚集；(2)增高血小板内环核苷酸含量的药物,如西洛他唑和双密达莫；(3)抑制血小板花生四烯酸(Arachidonic acid, AA)代谢的药物,包括环氧酶抑制剂(阿司匹林)和血栓素A2(Thromboxane A2, TXA2)合成酶抑制剂(苯酸咪哇),通过抑制环氧酶或TXA2合成酶,阻碍AA衍变为TXA2；(4)血小板膜纤维蛋白原受体拮抗剂,如Abciximab,通过阻断纤维蛋白原与血小板受体GPⅡ b/Ⅱ1a的结合而有效地抑制血小板聚集。
血小板活化因子(platelet-activating factor, PAF)是磷脂类生物活性介质及细胞因子并日益受到重视。它在极低浓度下通过与特异性G蛋白受体结合而活化多种细胞,介导炎症和其他疾病,影响消化、心脑血管、生殖、皮肤和呼吸等多种组织系统。大量动物和临床实验表明,PAF参与了脑梗塞的发生发展病理过程,与动脉粥样硬化、缺血性脑血管病的转归密切关联。PAF是最强的诱导血小板聚集剂,一方面,PAF能进一步促进血小板桥联作用,启动血栓形成和机化,此环节不受阿司匹林的作用影响；另一方面,与继发性缺血性脑损伤有关,如免疫促炎、促血栓形成及自由基、兴奋性氨基酸释放、钙超载、即早基因表达等。因此,机体内PAF若异常升高,将导致临床脑梗塞的发生及继发性脑损害。PAF受体拮抗剂可通过抑制血小板聚集和自由基、兴奋性氨基酸释放、钙超载发挥梗塞后脑保护作用。早已证实,急性脑梗塞患者血浆和脑梗塞模型鼠脑组织中PAF含量明显高于对照组。PAF抑制剂的代表-银杏提取制剂是治疗临床脑梗塞的常用药物之一。
血管内PAF的代谢失活是由于2位乙酰基被水解,催化此反应的酶即为血浆型PAF乙酰水解酶(platelet-activating factor acetylhydrolase, PAF-AH),故该PAF-AH和PAF的动态平衡对维持PAF的正常活性起重要作用。大多数炎症性疾病PAF-AH轻度升高。另外,妊娠、高血压、血管性疾病、缺血性卒中、尿毒症、艾滋病等可出现升高现象,而升高的PAF-AH可明显减轻PAF和氧化磷脂的炎症介质效应。因而推测PAF-AH含量及活性的降低与动脉硬化性形成、脑梗塞的发生发展有关。此外,PAF-AH还可显著抑制氧化应激连锁反应,对血管内皮具有保护性作用。
人血浆型PAF-AH主要来源于巨噬细胞,其基因位于6p.12p.21.1,含12个外显子,可编码441个氨基酸,分子量为44-kDa。Tjoelker等首次成功建立人血浆型PAF-AH DNA重组体,证实所表达的重组血浆型PAF-AH (rPAF-AH)且有同等催化底物PAF的活性,可抑制PAF诱导的炎症反应。研究发现,用rPAF-AH尾静脉注射可显著减轻胰腺炎模型大鼠胰腺损伤程度,生物半衰期达7小时。而rPAF-AH更可作为预防呼吸窘迫综合征和降低败血症死亡率的有效手段。多中心、随机、双盲、安慰剂对照三期临床试验结果显示,应用rPAF-AH治疗败血症的患者对rPAF-AH耐受性好、无抗体产生。目前采用rPAF-AH治疗疾病的研究集中局限于炎症反应方面,国内则尚未见有rPAF-AH相关文献报道。
基质金属蛋白酶(matrix metalloproteases, MMPs)是一种能够分解细胞外基质(ECM)的锌依赖性蛋白水解酶家族,在人体内参与多种生理和病理过程。相对分子质量为72x103(72KD)的MMP-2和相对分子质量为92x103(92KD)的MMP-9为MMP家族的2个成员,均可降解基底膜的ECM成分,其底物包括Ⅳ型、Ⅴ型胶原酶、纤维连接蛋白、弹性蛋白和变性的基质胶原。正常情况下,MMP-2以潜伏的形式存在于脑组织内,可被膜结合蛋白酶膜型金属蛋白酶(membrane type matrixmetallo-proteinase, MT-MMp)激活。
近年来,有人发现脑缺血再灌注后出现MMPs增加,尤其是MMP-9和MMP-2活性增加与脑微血管通透性、血脑屏障(Blood brain barrier, BBB)通透性、BBB崩溃、脑水肿和炎细胞侵入明显相关。BBB破坏后,一方面可引起血液中的血浆蛋白、多种毒性物质及代谢产物进入脑内,促进并加重血管源性脑水肿；另一方面为炎性因子侵入脑组织大开方便之门。在局部脑缺血所致的出血变化时,MMP-9的表达明显增强,而MMP-2则不增强,提示两者在降解基底膜引起的神经元损伤以及局灶性脑缺血导致出血性转化早期的潜在作用。其中MMP-9(明胶酶B)激活时能降解参与血脑屏障基膜的主要成分纤粘蛋白和粘蛋白,破坏血脑屏障,引起血脑屏障的通透性升高或进一步开放,缺血后脑组织MMP-9活性增高是缺血后脑损伤的重要机制之一。近年来有研究表明,脑缺血再灌注可增强MMP的表达,予MMP抗体或MMP抑制剂可减轻脑缺血再灌注损伤,改善血管源性水肿,以及保护神经细胞免受损伤等。
血管内皮生长因子(vascular endothelial growth factor,VEGF)是强效的有丝分裂原,可直接并选择性地作用于内皮细胞,促进新生血管生成。中枢神经系统有多种因子可促进血管新生,而许多研究均表明在血管新生过程中,VEGF是主要的因子之一。VEGF可令细胞间的接触松弛,并使稠密的细胞外基质瓦解,进而促进新的微血管形成。VEGF在血管新生中的主要作用包括：①上调细胞间和血管细胞的粘附分子的水平；②诱导内皮细胞上相应受体和配体的表达;③促进血管内皮细胞的增殖和迁移；④诱使骨桥钻合蛋白与上调的内皮细胞表面的整合素受体结合；⑤增加丝氨酸蛋白酶的活性,从而降解特定区域的细胞外基质,促进血管生成。缺氧或缺血在多数组织可以引致血管新生。而VEGF在局灶性脑缺血的血管新生中发挥不可忽视的作用。中枢神经缺血后,在梗死灶周围有一圈缺血相对较轻的区域,称半暗带(penumbra)。半暗带血供的恢复对神经细胞的存活具有举足轻重的意义。VEGF基因的启动子区含有缺氧诱导因子-1(HIF-1)反应元件。当脑部发生缺氧或低氧时,VEGF基因5’端低氧反应元件与HIF-1结合,启动环二磷酸鸟苷酸(cGMP)途径,VEGF的表达即可受到相应调节。目前公认缺氧是调节VEGF及其受体表达的主要因素之一。脑缺血后,低氧信号激活VEGF/VEGF受体系统,促使VEGF在半暗带区域高表达。随即诱导内皮细胞增殖,促进半暗带大量新生血管形成,加速侧枝循环的建立,改善相应缺血脑组织的供血供氧,减少神经元凋亡或坏死,最终减轻缺血性脑损伤。
VEGF对神经元的直接作用是神经营养作用,可促进体外培养的神经元轴突生长、增加神经突触的数目并延长其长度,提高神经元的存活率。正常情况下,脑内无VEGF表达,但当受到某种损害时如缺氧炎症肿瘤时能诱导VEGF表达已被许多体内及体外实验证实。
奥扎格雷钠为血栓烷(TX)合酶抑制剂,能阻止前列腺素H2(PGH2)生成血栓烷A2(TXA2),并促使血小板所产生的PGH2转向内皮细胞,令内皮细胞可利用PGH2合成PGI2,从而起到抑制血小板的聚集和扩张血管作用。动物试验表明,在大脑中动脉阻塞小鼠模型中,静脉给药能使其血浆TXB2水平降低,可预防性治疗脑梗塞。
金纳多(extract of ginkgo biloba leaves injection)产于德国,为银杏提取物,作为强效PAF拮抗剂,具有抑制血小板聚集,抗血栓,改善血液动力学与血液流变学,改善心脑代谢及抗炎性损伤等作用。从本世纪20年代起,银杏叶制剂就用于治疗外周及中枢血液循环障碍以及脑功能障碍等症。目前,作为银杏叶提取物的金纳多注射液已在亚洲地区广泛使用,成为治疗脑梗塞的常见药物。本研究选择安全有效的金纳多注射液和奥扎格雷钠注射液作为rPAF-AH的阳性对照,希望能对rPAF-AH的神经保护作用做出较为全面的评估。
本研究使用美国peprotech公司制作的rPAF-AH,观察不同浓度rPAF-AH对兔富含血小板血浆PAF诱导的血小板聚集率的影响和量效关系；另一方面,观察静脉注射rPAF-AH后：(1)采用线拴法制备的大脑中动脉缺血再灌注模型小鼠脑梗塞的梗塞体积、神经行为学的改变；(2)模型鼠脑MMPs、VEGF的蛋白表达；(3)模型鼠脑MMPs、VEGF的基因表达。初步探讨rPAF-AH治疗脑梗塞的有效性和可行性,评估该生物蛋白制剂的临床开发、应用前景。
材料与方法：
一采用线栓法建立小鼠大脑中动脉缺血再灌注模型,尾静脉注射rPAF-AH预处理后观测模型小鼠神经行为学和脑梗死灶大小的改变,采用Western blot研究MM P-2、MMP-9和VEGF的表达及活性；rt-PCR研究MM P-2、MMP-9和VEGF基因表达的改变,同时与奥扎格雷钠注射液、金纳多注射液及假手术对照组比较。
二于兔富含血小板血浆(PRP)中分别加入rPAF-AH,使后者达到不同的最终浓度；另设一份不加rPAF-AH的PRP做为对照。各份PRP中加入10-8mol浓度的PAF并测血小板最大聚集率。观察不同浓度rPAF-AH对兔富含血小板血浆PAF诱导的血小板聚集率的影响并和金纳多比较。
结果：
1. rPAF-AH预处理组降低脑梗塞小鼠行为障碍的神经行为学评分、脑梗死灶,与手术模型组比较,差异有显著性意义(P<0.01)
2. Western blot分析结果表明,与手术模型组比较,rPAF-AH预处理可降低脑缺血再灌注损伤所诱导的MMP-2蛋白、MMP-9蛋白的表达及其活性水平(P<0.01)；上调了VEGF蛋白的表达及其活性水平,差异有统计学意义(P<0.001)。
3. rt-PCR分析结果表明,与手术模型组比较,rPAF-AH预处理可降低脑缺血再灌注损伤所诱导的MMP-2mRNA、MMP-9mRNA的表达及其活性水平(P<0.01)；上调了VEGF mRNA的表达及其活性水平,差异有显著性意义(P<0.001)。
4.所有加入金纳多注射液或rPAF-AH的PRP,其血小板最大聚集率都比对照PRP的低,且rPAF-AH对血小板最大聚集率的影响较金纳多注射液更大,有显著性意义(P<0.01)。较高浓度的rPAF-AH,其血小板最大聚集率也较低。
结论：
1.小鼠脑缺血再灌注早期能诱导脑组织MMP-2, MMP-9的表达上调,从而导致BBB通透性增加和血管源性脑水肿的形成。rPAF-AH预处理对小鼠脑缺血再灌注损伤有一定的保护作用,可能是通过包括对MMP-2, MMP-9的表达抑制和VEGF的表达促进及稳定BBB通透性在内的多种途径来完成的。
2. rPAF-AH与金纳多注射液相比,能够降低PAF所诱导的血小板聚集,降低具有统计学意义(P<0.01)。
Background and purpose:Cerebrovascular disease are very common disease in clinic.Its high incidence, high mortality, high disability rate, have already done serious harm to human health. Among them, ischemic cerebrovascular diseases accounted for70%, which is more common in patients aged more than60years old. As the aging of population accelerates, cerebrovascular disease have been bringing more and more economic and social burden of serious impact on the elderly health status and quality of life. Therefore, searching for the high effective and safe drug has become our experimental research focus.
Once the brain ischemia lasts more than6minutes, can cause energy depletion, signal transduction abnormalities occur, leading to mitochondrial dysfunction, brain nerve function of irreversible comprehensive injury. If blood supply is restored after cerebral ischemia, organ and tissue cell functions, metabolic disorders and structural damage will not be restored or will even be aggravated. Such a phenomenon is called the reperfusion injury. The pathogenesis of ischemic cerebrovascular diseases is quite complex, and medications related to recent studies are:excitatory amino acid receptor antagonists, calcium antagonists and free radical scavenger. But it is still not entirely sure whether they can cure the acute ischemic stroke. FDA of America in1996approved the first treatment of the disease with thrombolytic drug t-PA, and gradually thrombolytic therapy is recognized as an effective method. But given the time window of thrombolysis and treatment conditions, only2%in5%of the patients are suitable receive this treatment; at the same time thrombolytic therapy can establish cerebral ischemia reperfusion of blood flow, but does not solve the ischemia caused by brain tissue reperfusion injury. Various types of brain protective agent researches suggest that many drugs' clinical curative effect is poor, or even have serious side effects. Therefore, the in-depth study of the pathogenesis of cerebrovascular diseases, leading to the development of effective drug against ischemic brain injury, is one of the most important research topics of contemporary neurology.
Thrombosis in ischemic cerebrovascular diseases is the main pathogenic factor. Platelet thrombosis plays a very important role, and thus inhibiting platelet activation has become the treatment of stroke in the conventional therapy. Studies show that, during cerebral ischemia and abnormal platelet function, hyperfunction of blood at high coagulation state, which is caused by secondary brain injury is one of the important reasons. The clinical use of antiplatelet drug prevention in high-risk populations of midbrain stroke, thereby effectively reduces the incidence rate of cerebral arterial thrombosis. Therefore, the platelet adhesion and aggregation is considered to be very promising targets of the development of antithrombotic drug.
The current antiplatelet drugs according to their mechanisms of action can be divided into the following four categories:(1) specific inhibition of two adenosine monophosphate (Adenosine diphosphate, ADP) activated platelet drugs:Ticlopidine and Clopidogrel, selective inhibition of ADP activation of platelet aggregation;(2) drugs of increased platelet cyclic nucleotide content, such as cilostazol and double density dipyridamole;(3) drugs of inhibition of platelet peanut acid (Arachidonic four acid, AA) metabolism including epoxy enzyme inhibitor (aspirin) and thromboxane A2(Thromboxane A2, TXA2) synthase inhibitor (benzoic acid with wow), by inhibiting the enzyme cyclooxygenase or TXA2synthase, and blocking AA development for TXA2;(4) platelet membrane fibrinogen receptor antagonists, such as Abciximab, by blocking the fibrinogen and platelet receptor GP ⅡB/Ⅱla combination and effective inhibition of platelet aggregation.
Platelet activating factor (platelet-activating factor, PAF) is a phospholipid class of biologically active mediators and cytokines and is attracting more and more attention. In the extremely low concentrations, it is bound with specific G protein receptor, which activates multiple cells, and mediates inflammation and other diseases, thus affecting digestion, cardiovascular, reproductive, skin and respiratory and other organization system. Large amounts of animal and clinical experiments show that, PAF is involved in the occurrence and development of cerebral infarction pathological processes, which usually leads to atherosclerosis and ischemic cerebrovascular disease. PAF is the strongest induced platelet aggregation agent. On one hand, PAF can further promote platelet bridging function, initiation of thrombosis and machine, and this link is not affected by the effect of aspirin effect; on the other hand, with the secondary ischemic cerebral injury, such as immune inflammatory, PAF promotes thrombosis and free radicals, the release of excitatory amino acids, calcium overload, immediate early gene expression. Therefore, the abnormal increase of PAF, will cause the clinical occurrence of cerebral infarction and secondary brain damage. PAF receptor antagonists can inhibit platelet aggregation and free radicals, the release of excitatory amino acids, calcium overload and have an infarction cerebral protective effect. It was proved that, in patients with acute cerebral infarction and rats in cerebral infarction model, the plasma content of PAF in brain tissue was significantly higher than that of the control group. PAF inhibitors (like Ginkgo extract preparations) are one of the commonly used drugs in the clinical treatment of cerebral infarction.
The enzyme2acetyl which is the plasma PAF acetylhydrolase (PAF-AH) is hydrolyzed to catalyze this reaction and this leads to intravascular PAF metabolic inactivation. The dynamic balance between PAF-AH and PAF to maintain normal activity plays an important role in PAF. PAF-AH mildly elevated in the majority of inflammatory diseases. In addition that, the elevation of PAF-AH can also be found in pregnancy, hypertension, vascular disease, ischemic stroke, uremia, AIDS. It can significantly reduce PAF and proinflammatory oxidized phospholipids medium effect. So the contents of PAF-AH and decreased activity and arteriosclerosis formation, are related to the occurrence and development of cerebral infarction. In addition, the PAF-AH also could significantly inhibit the chain reaction of oxidative stress, and have endothelial protective effects.
Human plasma PAF-AH comes mainly from macrophages, whose genes are located on the6p.12p.21.1, containing12exons, encoding441amino acids, molecular weight of44-kDa. Tjoelker's first successful establishment of human plasma PAF-AH recombinant DNA, confirmed that the expression of the recombinant plasma type PAF-AH has the same catalytic substrate PAF activity, and can inhibit PAF induced inflammatory reaction. Study found that, using recombinant human plasma PAF-AH tail vein injection can significantly reduce the pancreatitis in rat model of pancreatic injury severity, the biological half-life of7hours. Recombinant human plasma PAF-AH even can be used for the prevention of respiratory distress syndrome and as effective means to reduce sepsis mortality. A multicenter, randomized, double-blind, placebo-controlled phase in three clinical trial results shows, the application of recombinant human plasma PAF-AH in treatment of sepsis patients on recombinant human plasma PAF-AH is well tolerated, and no antibody is produced. The focus of the recombinant plasma type PAF-AH for the treatment of disease research is confined to the inflammatory response, and in China, there is no recombinant plasma PAF-AH report.
Matrix metalloproteinases (matrix metalloproteases, MMPs) is a decomposition of the extracellular matrix (ECM) zinc dependent protein hydrolase family, and they are involved in a variety of physiological and pathological processes. The MMP family of2members:the relative molecular mass of72x103(72KD) MMP-2and the relative molecular mass of92x103(92KD) MMP-9, can degrade the basement membrane of the ECM component, the substrate including type IV, type V collagenase, fibronectin, elastin and degeneration of the collagen matrix. Under normal circumstances, MMP-2is present in latent forms in the brain, can be membrane-bound proteases membrane type metalloproteinases (membrane type matrixmetallo-proteinase, MT-MMp) activated.
In recent years, it was found after cerebral ischemia there is an increase of MMPs, especially MMP-9and MMP-2activity and cerebral microvascular permeability of blood brain barrier (Blood brain, barrier, BBB) permeability, to which BBB collapse, brain edema and inflammatory cell influx were significantly related to. Once BBB is destroyed, it can cause the blood plasma protein, a variety of toxic substances and metabolites into the brain, and then promotes and exacerbates vasogenic brain edema; and at the same time, opens the floodgates for inflammatory factor to invade brain tissue. In local cerebral ischemia caused by bleeding changes, MMP-9expression was significantly increased, while MMP-2is not enhanced, and the tips of both in the degradation of basement membrane induced neuronal injury and focal cerebral ischemia caused hemorrhagic transformation in the early potential function. The MMP-9(gelatinase B) when activated can degrade the involvement of blood brain barrier,which is the main component of fiber mucins and mucin. The disruption of the blood-brain barrier, causing blood brain barrier permeability increased or further opening, and the increase of MMP-9activity in ischemia brain tissue are important mechanisms in cerebral ischemia injury. In recent years, studies have shown that, cerebral ischemia reperfusion can enhance the expression of MMP, and the MMP antibodies or MMP inhibitors may reduce cerebral ischemia reperfusion injury, ameliorate vasogenic edema, and protect neural cells against injury.
Vascular endothelial growth factor (vascular endothelial growth factor, VEGF) is an effective mitogen, and it can directly and selectively act on endothelial cells, and promote the formation of new blood vessels. Central nervous system has a plurality of angiogenic factors, including vascular endothelial growth factors which are recognized as major involving angiogenesis factors. Vascular endothelial growth factor can relax the cell-cell contact, and the collapse of specific regions of the extracellular matrix, thus forming a new micro vascular. VEGF's major roles in angiogenesis include:increasing intercellular adhesion molecules and vascular cell adhesion molecule level; inducing endothelial cell on the corresponding ligand and receptor expression; leading to the proliferation and migration of vascular endothelial cells; upregulating of endothelial cell surface expression of integrin receptors, and inducing bone bridge drilling photosynthetic proteins to bind; upregulating of serine protease activity, thereby degrading the dense extracellular matrix, promoting angiogenesis. Hypoxia or ischemia in most tissues can cause angiogenesis. And vascular endothelial growth factor in focal cerebral ischemia angiogenesis plays an important role. Ischemia after cerebral infarction, is relatively light in a circle around the area of ischemia called the penumbra (penumbra). The restorationof the blood supply of Penumbra has great significance on neuronal survival. In the VEGF gene promoter region there is hypoxia inducible factor-1(HIF-1) response element. When there is hypoxia or low oxygen, HIF1and VEGF5'end the binding of the gene hypoxia response element, through a ring two? two-ring guanosine monophosphate acid (cGMP) pathways to regulate the expression of VEGF. It is generally accepted that hypoxia is VEGF and its receptor expression in the main regulating factors. After cerebral ischemia, hypoxia as a signal for the activation of VEGF/VEGF receptor system, prompted the penumbra of high expression of VEGF, and the latter can be induced through the proliferation of endothelial cells, thus promoting the penumbra extensive neovascularization, accelerating the establishment of collateral circulation, improving the brain tissue perfusion and oxygen supply involvement, reducing neuronal apoptosis or necrosis volume, and eventually reducing ischemic brain injury.
VEGF has direct neurotrophic effects on the neurons, and can promote cultured neuronal survival and axonal growth, and increase in neurite number and length. VEGF stimulates axon growth and increases the survival of nerve cells. Under normal circumstances, there is no expression of VEGF in the brain. But when subjected to certain damage such as inflammatory tumor hypoxia, VEGF expresses and it has been used by many in vivo and in vitro experiments confirmed.
Ozagrel Sodium Injection, which is the thromboxane synthase inhibitor, can prevent prostaglandin H2(PGH2) formation of thromboxane A2(TXA2), and induce platelet derived PGH2to endothelial cells, endothelial cells to synthesize PGI2, which acts to inhibit platelet aggregation and vascular dilatation function. Animal experiments show that, intravenous injection can reduce plasma TXB2levels, and have preventive effects on mouse middle cerebral artery occlusion caused by cerebral infarction.
Extract of Ginkgo Biloba Leaves of Injection, produced in Germany, as a potent platelet activating factor (plateletactivatefactor) and PAF antagonist, can inhibit platelet aggregation, antithrombotic, improve hemodynamics and blood rheology, thus improving the treatment effect of cardiovascular and metabolic and inflammatory injury. Since the first two decades of this century, ginkgo leaf preparation has been used in the treatment of peripheral and central circulatory disorders and brain dysfunction. At present, Ginaton injection as the Ginkgo biloba extract has been widely used in Asia, and become common drugs for treatment of cerebral infarction. On the safe and effective Ginaton injection and Austria grips Gray sodium injection rPAF-AH acts as a positive controller, and a comprehensive assessment to the rPAF-AH neuroprotection is expected to be made.
This study using the American company peprotech produced by recombinant PAF-AH, to observe the effects of different concentrations of recombinant PAF-AH on rabbit platelet rich plasma PAF induced platelet aggregation rate effects and dose-effect relationship; on the other hand, intravenous injection of recombinant PAF-AH:(1) uses the line to preparation of middle cerebral artery ischemia reperfusion model in mouse models, cerebral infarction infarction volume, ultrastructural changes;(2) model rat brain MMPs, VEGF protein expression;(3) model rat brain MMPs, VEGF gene expression. It is a preliminary study of validity and feasibility of recombinant human plasma PAF-A in the treatment of cerebral infarction, the biological protein preparations in clinical development and the application prospect are also discussed.
Materials and methods:
l.An model established by suture method in mice with cerebral ischemia/reperfusion, tail vein injection of rPAF-AH after pretreatment; observation of mouse neural behavior and cerebral infarction size changes, using the Western blot on MM P-2, MMP-9and VEGF expression and activity; rt-PCR on MMP-2, MMP-9and VEGF gene expression changes, at the same time and Austria grips Gray sodium injection, Ginaton injection and sham operation control group.
2.rPAF-AH were added to two rabbit samples of platelet rich plasma (PRP), and they have different final concentration of rPAF-AH; another one without rPAF-AH PRP as the control group. Each copy of the PRP with10-8mol concentration of PAF is used to measure the maximum platelet aggregation rate. Different concentrations of recombinant PAF-AH on rabbit platelet rich plasma PAF will induce different platelet aggregation rate and a comparison of effects of ginaton will be made.
Result:
1rPAF-AH pretreated group was markedly reduced cerebral infarction mouse behavior disorders of nervous behavior score, cerebral infarction (P<0.01).
2Western blot results show, compared with the operation of model group, rPAF-AH preconditioning reduces ischemia reperfusion injury induced by MMP-2protein, the expression of MMP-9protein and activity levels (P<0.01); apparent up-regulation of VEGF expression and activity levels (P<0.001).
3rt-PCR analysis results show that, compared with the operation of model group, rPAF-AH preconditioning reduces ischemia reperfusion injury induced by MMP-2mRNA, MMP-9mRNA expression and activity level (P<0.01); VEGF mRNA markedly upregulated expression and activity levels (P<0.001).
4All join Ginaton injection or rPAF-AH PRP, the maximum platelet aggregation rate than control low PRP, and rPAF-AH on platelet aggregation rate effects of Ginaton injection more obviously (P<0.01). A high concentration of rPAF-AH, the maximum platelet aggregation rate is low.
Conclusion:
1rPAF-AH injection in mice with cerebral ischemia reperfusion early can induce brain tissue MMP-2, MMP-9upregulates the expression of BBB, resulting in increased permeability and vasogenic brain edema formation. rPAF-AH preconditioning on ischemia reperfusion brain injury in mice have a protective effect, probably by including inhibits the expression of MMP-2, MMP-9and promotes VEGF expression,so that stable BBB permeability.
2rPAF-AH compared with Ginaton injection, can significantly reduce PAF induced platelet aggregation.
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