Study Of Protective Effect Of FMCL On Cerebral Ischemia-reperfusion Damage And Its Mechanism

Cerebrovascular disease (CVD) is a devastating clinical condition,whichseverely do harm to human healthy.The incidence and mortality from CVD havethe tendency of increasing with life span elongation.CVD is the third leadingcause of death in our country and even more is the first leading cause in someareas. There are 1.5million new CVD patients every year at present in ourcountry.75% survival patients were severely disabled which bring heavy distressand economic loss for himself,the family and the social. Ischemic cerebrovascular disease (ICD) is the central nervous system diseasecaused by the artery block temporary or permanent.It approximately hold 80% inthe cerebrovascular disorders.Although the treatment of the ICD has gained someexpectativelly progress in the animal model but was disappointed in clinicexperiment.Currently thrombolysis is the main therapy method ,but there aresome dangers and restrictions for hemorrhage and reperfussion injury.Furthermorethe occurrence of reperfussion is very frequently in ischemic brain tissue andtherefore its damage will inevitable.Cerebral ischemia-reperfusion (CIR) injury isthe damage even severely when the blood stream recanalize in the ischemic braintissue which has experenced cerebral ischemia for some time.Thereforeinvestagate the mechanism of the cerebral ischemia injury and actively developthe protective drug for the damage have very important theoretical significanceand utility value.Currently it is widely confirmed that nerve cell experienced two death style:necrosis and apoptosis after focal cerebral ischemia. Necrosis usually take up inthe region that ischemia occurrences very fast and very severely, but apoptosisoccurrences in the region of light ischemia. The damages of cerebral ischemia arecerebral infarction and reperfusion that blood stream recanalize. The region thataffected by reperfusion injury is ischemia penumbra region. The main pathologicchange is apoptosis in the area.Flavone mixture of crataegus leaves (FMCL) is the mixture of anti freeradical. It extracted from the crataegus leaves in flower season. Its main chemicalingredients are saponins, ketone, alkaloid. It is a mixture of over 30 kinds ofsaponins, mainly steroid saponins. Domestic research materials showed thatFMCL had the effect of lowering blood pressure and stimulating heart function,lowering lesterol. The injection of FMCL yixintong has applied in cardio-vascularsystem diseases for some years and has gained good social effect and economiceffect. But the effect of FMCL on cerebral ischemia has not been reported inChina at present. Our research team has done a lot of studies on FMCL.Theresearch of our research team has showed that FMCL has the effect of improvingarterial blood streaming and ameliorating the blood viscosity, anti-thromboticeffect, inhibiting platelet aggregation. But we don,t know wether FCML hasprotective effect on cerebral ischemia and how.Wether there are somerelationships between the protective effect and anti-apoptosis.Its mechanism needcontinue to be investigated.The aim of this work is to observe the protective effects of FMCL on cerebralischemia-reperfussion injury and its possible mechanism on the level of invivo,cell,molecul and gene through setting up the neurons ischemia andreperfussion in vivo or in vitro model.Thus can provide a theoretical basis forbetter, safer use and targeting application of this therapy in clinical treatment.Experiment in vivo: through setting up cerebral ischemia-reperfusion modelin rats by blocking middle cerebral artery, the effects of FMCL on the level ofSOD, MDA, NO, Ca2+, infarction area in the brain tissue and the neurologicdeficit score were detected. At the same time, samples were taken to store in thefixation liquid for HE and TUNEL immunohistochemical performance andtransmission electron microscope.The results of the experiment in vivo showed that FMCL could significantlyreduce the content of MDA and could increase the activity of SOD in brain tissue;decrease the positive cells of neurons apoptosis, and reduce the infarction area. Italso could significantly increase the neurologic deficit score and coulddownregulate the protein expression of CytC. This indicated that FMCL couldincrease the anti-oxidation activity of the enzyme, enhance the ability of removingfree radicals and reduce the free radical from ischemia-reperfusion, so that itprotected the cellular membrane, relieve the morphology change of the cells,reduced neurons apoptosis percent and ameliorated brain function. At the sametime it also provide the clue that neurons apoptosis through two pathway: the Fasreceptor pathway and the mitochondrion pathway, and FMCL can affect themitochondrion pathway. On basis of that we will continue the study on the level ofcells next step.Experiments in vitro: simulating the hypoxia model on cultured PC12 cells,oberseve the cell activity, SOD and LDH activity, MDA content in medium, theapoptosis rate, mitochondrial membrane pertential (△ Ψ m), intracellular calciumconcentration, DNA ladder, the protein expression of Bcl-2, Bax, caspase-3P20,caspase-9 and the expression of Bcl-2, Bax, caspase-3 mRNA by MTT,flowcytometer, laser-confocal-microscopy system, agarose gel electrophoresis,western blot and RT-PCR.The results of the experiment in vitro showed that FMCL could increase thesurvival rate of PC12 cells, decrease apoptosis rate, stablize the mitochondrialmembrane potential, reduce the intracellular calcium concentration, increase theprotein expression of Bcl-2 and decrease the protein expression of Bax, caspase-9,caspase-3P20 and increase the expression of Bcl-2 mRNA, decrease theexpression of Bax, caspase-3 mRNA. The result showed that FMCL couldincrease SOD activity, increase the anti-oxidation activity of the enzyme, enhancethe ability of removing free radicals and reduce the free radical from ischemiareperfusion, keep the integrity of the membrane, inhibit the opening ofmitochondrial transition pores, thus reducing the release of apoptosis-inducingprotein. And then inhibit the activation of caspase-3, thus it inhibit the apoptosisprocess by stopping apoptosis cascade. At the same time it can regulate thecorrelative apoptosis protein expression of BcL-2 and Bax.increase the ratio ofBcl-2/Bax and then help inhibit apoptosis. While doing all that, it help keep thecalcium balance otherwise the calcium overload can induce apoptosis in theneurons.The mechanism of cerebral ischemia reperfusion is the increasion of freeradical, calcium overloading and ATP synthesizing obstruction in themitochondrion. These three factors may work separately or interact with eachother to cause cellular structure, function and metabolism destruct, and eventualcell death. The most important factor is free radical increase. It can cause lipid,protein, nucleic acid over oxidated, cellular membrane structure destructed, thebackbone of nucleic acid fragmentated, protein degradated, hyaluronic aciddepolymerized, cells collapsed, mitochondrion degenerated, the cell occurunreversible alternation and then dead. On the other hand the destruction of cellmembrane and mitochondrion membrane which result in calcium flowing intocells, mitochondrion loss its respiratory function, ATP synthesizing obstructed,pump function failure and then result in intracellular calcium overload. Thereforeto reduce intracellular calcium, protect mitochondrial function, removing freeradical is very important.Based on the above experiments in vivo and in vitro, it has been proved thatFMCL increased the ability of removing free radicals, decreased intracellularcalcium overload, and protected mitochondrion through modulating proteinexpression of Bcl-2/Bax, stablized △ Ψm, downregulated caspase-3, caspase-9,and inhibited the activation of caspase-3 which in turn inhibited the apoptosticcascade process and then protected the neurons from hypoxia damage. If patientssuffer from cerebral ischmia FMCL can be applied when thrombolysis therapywas used, it can relieve the injury of blood vessel recanalization after cerebralischemia or thrombolysis.Some new ideas in this study are:1. As anti-myocardium ishemia medicine FMCL has been applied for manyyears in clinic. It has not been reported in any literature about the effect of FMCLon cerebral ishemia. The study is the first case to investagate the protective effectof FMCL on cerebral ishemia and its mechanism2. The study will provide a new way for the application of FMCL in clinic.3. The study of the effect of FMCL on intracellular calcium overload, △ Ψmand the expression of caspase-9, caspase-3 P20 protein and Bcl-2 /Bax, caspase-3mRNA have not been reported up to now.4. Establishment of cerebral ischemia-reperfusion model together with themodel of PC12 cells apoptosis induced by H2O2 and the application of the aboveindex tests will provide a basis for new methods in research and development ofanti-cerebral ishemia medicines.