The Expression Of MMP9 In Cerebral Ischemia-reperfusion Damage And The Intervention Of Ginkgo Leaf

Ischemic cerebrovascular disease is the common disease on clinical. Thedisease incidence, the crippling rate and the mortality are very high. People havegenerally researched on the pathogenesis of Ischemic Cerebrovascular disease,including the free radical production, excitability toxicity, Ca2 + overload, thecell factor function and so on. There exists cerebral ischemia-reperfusion damageafter cerebral ischemia-reperfusion. The destruction of blood brain barrier(BBB) is considered the key point of cerebral ischemia-reperfusion damage. Atpresent, the approved main factor that BBB permeability increases in cerebralischemia is: (1) the damage of the oxygen free radical to the basilar membraneafter cerebral ischemia;(2) the degradation of the proteinase to the basilarmembrane constitutive protein after cerebral ischemia;(3) energy metabolismdisorder, the functional lesion of the endothelial cell and the spongiocyte thatcomposes BBB and so on. The most important reason for the damage of theintegrality of the structure or the function and permeability increase of BBB is thedamage basilar membrane. In recent years, with the research of the function ofgelatinase in the damage of cerebral ischemia, the relationship between theMMPS activeness and cerebral ischemia causes the attention. In the recentresearch it is demonstrated that the proteinase especially MMPs plays a vital rolein the damage of BBB in cerebral ischemia. MMPs is a group of zinc-dependenceproteinase which can degrades the extracellular matrix protein. Its activity isnormally controlled in the low level. It is secreted by means of proenzyme. Theactivated proenzyme can have an effect. Under the physiologies and pathologycondition, its activity obviously increases. It can have an effect of the damage andthe repair of the organization. Many factors accommodate the synthesis and thesecretion of the MMPS. Some can promote its synthesis and secretion, such asepidermis growth factor, cell factor, inflammation factor, nitrogen monoxide(NO). The extracellular matrix can play an important role in the maintenance ofthe normal cell function and the normal cell structure, by the means of theaccommodation of the cell's growth, the differentiation, the fission, the apoptosisand so on. If the extracellular matrix ingredient is abnormality, it can cause thecell damage. MMP9 is called gelatinase B which is one of the matrix metalproteinas. It is mainly synthesed and secreted by neutral granular cell,histoleucocyte, vessel endothelial cell, smooth muscle cell, horizontal cell, gittercell, macrophage and so on. MMP9 destroys ECM and the basilar membranethrough the degradation of IV, V collagen. There results in the permeabilityincrease of the blood vessel, the BBB opening and vasogenic brain edema.In recent years with the development of the diagnosis and the treatment forcerbral infarction, there improves obviously the cerebral apoplexy patient'sprognosis. The research for its etiology and the pathogenesis is still progressedvery slowly. More and more attention about the cause of disease and thepathogenesis treatment is paid. The extract of Ginkgo Biloba Leaf (GbE) is theimportant ingredient which withdraws from the precious plant gingko's leaf inour country and mainly includes the flavonoid, terpenoid. The flavonoidaccounts for 24%, the latter accounts for 6%. It has the protective function forthe ischemia, the oxygen deficit, dropsy as well as the blood brain barrier.Some research indicated that the GbE can obviously reduce the area of cerbralinfarction of the rat with cerebral ischemia, reduce the neurologic impairmentafter cerebral ischemia, reduce the apoptosis quantity of nerve cells at the edgearea of cerebral ischemia, slow down the neuron damage area of cerebralischemia, and bring the remarkable brain protective function. It is still not clearwhether it plays its role through the adjust of the expression and the secretion ofMMP9. This experiment, through the observe the expression of MMP9 incerebral ischemia-reperfusion damage and the study for the intervention ofGinkgo leaf, can furtherly reveal the protective function mechanism of GbE incerebral ischemia-reperfusion damage.Research technique: 1st, the animal model preparation and the grouping.The healthy male Wistar rats (provided by Jilin University foundation animaldepartment) weight 250-350g. They are adaptably fed. After two weeks theybecome the animal model of protocerebrum ischemia-reperfusion by making theuse of the double side arteria carotis communis sandwich method. 72 Wistar ratare stochastically divided into 3 big groups, Namely sham operated group: 12,cerebral ischemia-reperfusion model group: 30, which according to ischemic30min, reperfusion 6h, 24h, 48h, 3 days, 5 days are divided averagely into 5groups, the GbE treatment group: 30, which according to ischemic 30minsimilarly, reperfusion 6h, 24h, 48h, 3 days, 5 days are averagely divided into 5groups. The treatment group rats will be given the medicine (7.129 mg /kg-1 ·d-1) by peritoneal injection 1 week before treatment , the sham operatedgroup rats and cerebral ischemia-reperfusion group rats will be given the samevolume of physiological saline by peritoneal injection as the treatment group rat.The rats which do not conform to the model determination standard are notincluded. 2nd, model establishment: Anaesthetize the rats with 10% chloralhydrate (400mg/Kg) by peritoneal injection. Fix the rats on the supine decubitus.Disinfecting with Iodophors in conventional. center incisal opening at cervicalpart. Separate the double side arteria carotis communis of the cerebralischemia-reperfusion model group (abbreviation model group) and GbE treatmentgroup (abbreviation treatment group). Shut the double side arteria carotiscommunis with small bulldog clamp to block the blood for about 30 minutes.Then unclamp bulldog clamp. Restore the blood stream. Suture the wound byeach layer. We do the same thing with the sham operated group as the above twogroups but only separate the double side arteria carotis communis instead ofshutting. In the course of the operation we use infra-red lamp andthermal-insulation pulvinus in order to make the rats' rectal temperaturemaintenance at 36.0℃-37.0℃. After the operation we raise the rats under thesame condition, execute the rats in various time spot to take the specimen of thebrains separately. 3rd, brain tissue protein withdrawing: Each group of rats wasrapidly beheaded separately in the corresponding time ,we put the brain into -196℃ nitrogen canister to preserve. Afterwards, taking out the brain tissue,weighing, cutting to pieces, we put the brain tissue in the PBS(PH7.0,20mmol/l)at 4℃ for 24 hours. The PBS includes the 0.5% Triton X-100. Standardize thetissue saturation to 400mg/ml, after dousing, centrifugate with 12000r/min(4℃)for 20 min, then put them in -80℃refrigerator to preserve. 4th, MMP9 densitydetermination: After the gelatinase spectral method (SDS-PAGE enzymograph),measure the expression of MMP9 of each group of brains organization atdifferent time.Findings: Dynamic change of MMP9: There are few MMP9 in the locus ofischemia after cerebral ischemia-reperfusion for 6h of the model group, theexpression of MMP9 was obviously elevated after reperfusion 24h, after 48h itreaches the peak, 3d later it has the drop, 5d later the levels are lower. On thecontrary we have not seen any expression of MMP9 of the sham operated group.There has the significance difference for the sham operated group comparingwith the model group after reperfusion for 24h and 48h. After reperfusion for 24hfor the gingko leaf treatment group, the expression of MMP9 was also obviouslyelevated, but is smaller than the model group. After reperfusion for 48h it reachesthe peak which is low comparing with the model group. After 3 days and 5 daysit drops. There has the significance difference for the gingko leaf treatment groupcomparing with the model group after reperfusion for 24h and 48h .Conclusion1st, protocerebrum cerebral ischemia-reperfusion may cause MMP-9 toexpress unusually and change dynamicly.2nd, the Extract of Ginkgo Biloba Leaf have the intervention function tothe expression of MMP-9, it can reduce the up-regulation degree of the synthesisof MMP-9.3rd, the possible mechanism that the Extract of Ginkgo Biloba Leaf havethe intervention function to the expression of MMP-9 is that it can reduce theup-regulation degree of the synthesis of MMP-9, reduce the pathology damagefrom inflammation after cerebral ischemia, reduce the cerebral edema andimprove the blood circulation of partial brain.