| 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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