| The mechanism of cerebral ischemic reperfusion ( CIR ) is a complicated process with many factors, which is main relative to the reflect of oxidation and inflammation, calcium intoication, brain edema , cellular apoptosis and so on. In 1993 Linnik announced that cellular apoptosis was concerned with the ischemic neuron death . Now more and more studies has noticed that cellular apoptosis is one of the important styles resulted in brain injury.Some studies has shown that neuronic apoptosis is under the regulation of a series of genes. Amonge these genes, cysteine - asparate protease ( caspase ) has close relationship with apoptosis. Caspases splits target protein specificly after aminosccinic acid residue. They are activated in proper order by protease cascade reaction when they are stimulated by certain signal. They play a key role in cellular apoptosis. Amonge which Caspase - 3 is the most important enzyme of effect,which play a hinge role in the process of apoptois.Ginkgo biloba Extract(EGb761) is a kind of material extracted from Gink-go biloba leaves, which can antioxidant, clear free radical, suppress the delivery of excitability amino acid, anti - inflammatory and suppress cellular apoptosis and so on. EGb761 can resist ischemic cellular apoptosis, lessen ischemic cellular damage. A large quantity of studies has shown that EGb761 can prolong the ischemic animal survival time obviously.At present, there is nearly no report about the influence of EGb761 on cas-pae -3 . In this test,we make middle cerebral artery occlusion( MCAO) modelto observe the expression of caspase - 3 in rats after cerebral ischemic repeifusion and the influnce of EGb761 on caspase -3. To further explore the pathogne-sis of cerebral ischemic repeifusion and the possible mechanism of EGb761 for cerebral protection.Materials and Methods1. Animal and groupingMale Wistar rats weighing 250 - 300g were distributed to 4 groups at random , that is sham - operation group, MCAO repeifusion group , MCAO reperfu-sion group treated with 50mg/kg lOOmg/kg EGb761.2. materialsImmunohistochemistry, first antibody is rabbit anti mouse Caspase - 3 poly-clonal antibody ( working concentration is 1:75) .3. Methods3. 1 Making rat MCAO model by using Nagasawa modified way 3. 2 Detecting the level of Caspase - 3 expression through immunohistochemistry3.3 TTC staining to determine the infarction volumeResult1. The encephalic tissue section HE staining observed in light microscope.The encephalic tissue cortical cells have normal form and structure in control ,sham - operation opposite side of ischemia groups. Neurons decrease,put in disorder,the peripheral diastema of cell widen. A lot of neurons become ischemia and necrosis, cytoplast swelling, cytoplasm staining blandly, karyoplast dissolve and structure not clear in frontal and parietal lobe cortex, infracortical medulla and basal gnglia region,and have many contract neurons in infarct peripheral region of ischemic repeifusion side.2. TTC stainingNormal tissure is red, infarction tissure is white. There is no infarction areain sham - operation group, in MCAO reperfusion group the infarction area is mainly located in frontal and parietal lobe cortex, infracortical medulla and basal ganglia region, in MCAO reperfusion group treated with 50mg/kg lOOmg/kg EGb761, its infarction volume is smaller obviously than MCAO reperfusion group3. The expression of Caspase - 3 proteinsham - operation group has a small quantity of Caspase - 3 protein expression. In MCAO reperfusion group treated with 50mg/kg, lOOmg/kg EGb761, Caspase - 3 protein is mainly located in ischemic penumbra ( IP) , and the expression enhances obviously compared with sham - operation group. The differences are statistically significant( P <0. 01) .DiscussionNow a large quantity of studies has shown that excess cellular apoptosis is the main reason of cerebral ischemic reperfusion, and play an important role in the late onset neuronic death. Apoptosis is a series of initiative cell death process in the stimulus of all kinds of death signals. About the definite mechanism of apoptosis now is unknown. Some scholar think that it is main relative to the reflect of oxidation and inflammation, calcium intoication, brain edema, free radical, excitability amino acid, inflammatory injury and so on. Now studies show that Caspases family have an important role in ischemic damage.The access of cellular apoptosis is mainly devided into cellular membrane receptor iter , mitochondrion iter and endoplasmic reticulum iter. Caspase - 3 is the key of this three iters. It is called death protease. Ni discovered that after cerebral ischemic in rats, in the neuron of cornu CA1 area, Caspase -3 mRNA peaked at 24h timepoint. And this high expression can last for 72h,then declined after 96h of ischemic. In our MCAO model,we find in rats following focal cerebral ischemic 1 h, reperfusion 6h there has a high expression of Caspase -3 , compared with sham — operation group,P <0. 01. The differences are statistically significant. This supports seriously that cerebral ischemic damage induced the expression of Caspase - 3.The main effective composition of Ginkgo biloba Extracts (EGb761) is 24% flavonoids and 6% poncitrin. EGb761 has many en°ecs,such as antioxidant, clear free radical, suppress the delivery of excitability amino acid, anti - inflammatory and suppress cellular apoptosis and so on. In our test,EGb761 SOmg/kg^ lOOmg/kg were injected intraperitoneally to rats, the result is that group CAD compared with B, its infartion volume decreased, Caspase — 3 protein reduced ( P <0. 01). This result shows that EGb761 can suppress cellular apoptosis by reducing the expression of Caspase - 3, it has good brain protective effect. Otherwise , our test also shows that the infarction volume and Caspase - 3 protein of lOOmg/kg EGb761 group is smaller than 50mg/kg EGb761 group. The difference between two groups is statistically significant ( P <0. 01 ). This manifested that the effect of EGb761 has amount -effect relationship amonge certain dose.Conclusion1. We confirmed that Caspase — 3 protein was induced by cerebral ischemic reperfusion2. EGb761 can reduce cellular apoptosis by suppress the expression of Caspase - 3 protein3. EGb761 can decrease the infarction volume4. The effect of large dose of EGb761 is superior to the one of small dose of EGb761.
|
PDF Full Text Request