| Objective:The present study is performed to investigate the infarct volume and the changes about neuronal apoptosis and the expression of the protein and mRNA of cathepsin D and Caspase-9 in different time point in rat after ischemia-reperfusion, in addition to, we deal with the rat model with PepstatinA and investigate the changes of the above-mentioned indicatrix, in order that explore the possible molecular pathological mechanism of cerebral ischemia-reperfusion injury, so as to provide theoretical basis for the prevention and treatment of cerebral infarction.Methods:145 Male Sprague-Dawley rats weighing 250±20g were randomly divided into four groups: normal group (n=10), sham-operated control group (n=15), cerebral ischemia model group (model group,n=60), PepsttinA-treated group (treated group, n=60). Temporary middle cerebral artery occlusion (MCAO) model was applied, rats received intraperitoneal injections of 0.2ml/10g of PepstatinA injection for the treated group Immediately and same volume physiological saline was used for model group per 12 h after reperfusion. Neurological behavior evaluation were detected by Longa's scoring method. The pathologic changes were done by hematoxylin and eosin (HE) staining and TTC staining. The neuronal apoptosis was detected by the method of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL). The expression of cathepsin D and Caspase-9 protein and mRNA were measured with methods of immunohistochemistry and reverse transcriptttionpolymer- ase chain reaction (RT-PCR) respectively at 6,12,24 and 48h after reperfusion in cerebral cortex of rats.Results:(1)Rats in model group showed notable necrotic change and neuron loss by the HE staining and PepstatinA could lessen that damage at 48 hours after reperfusion.TTC staining showed: normal group and the sham-operated rats were no infarction, the infarction of model group were obviously, the infarct volume of the rats treated with PepstatinA were less than the model group (P<0.05).(2)The TUNEL positive cells were hardly found in the rats of the normal and the sham-operated rats. More positive cells could be found in peri-infarction at 6h after reperfusion and the apoptosis cell counting reached peak at 48h after reperfusion, PepstatinA treatment could decrease the neuronal apoptosis after reperfusion(P<0.05).(3) A very low level of cathepsin D mRNA was detected in the cerebral cortex of sham and normal rats. The expression level of cathepsin D mRNA increased significantly at 6h after reperfusion, peaked at 24h, then declined 48h after reperfusion. The cathepsin D protein had same change of expression level as that of its mRNA. PepstatinA treatment could decrease thecathepsin D mRNA and protein expression in parietal lobe cortex beginning at 6 h after reperfusion (P<0.05).(4) A very low level of Caspase-9 mRNA was detected in the cerebral cortex of sham and normal rats. The expression level of Caspase-9mRNA increased significantly at 6 h after reperfusion, peaked at 24 h, then declined at 48h. The Caspase-9 protein had same change of expression level as that of its mRNA. PepstatinA treatment could decrease the Caspase-9mRNA and protein expression level in parietal lobe cortex beginning at 6 h after reperfusion(P<0.05).Conclusions: (1)The CathepsinD mRNA and protein expression increased significantly after Cerebral ischemia-reperfusion, it may be involved in neuronal apoptosis.(2)After CathepsinD inhibitor PepstatinA intervention,Caspase-9 expression more obvious decreased, CathepsinD may be involved in mitochondrial pathway apoptosis through the endogenous rat brian neurons after ischemia-reperfusion. (3)PepstatinA can inhibit the expression of protein and mRNA of CathepsinD and Caspase-9, decreased the number of apoptosis of nerve cells,reduce infarct volume and brain water content, and reduce cerebral ischemia injury, therefore it pay a role in brain protection. |
PDF Full Text Request