Investigation Of The Relationship Between Cathepsin B-mediated Apoptosis And P-mek After Cerebral Ischemia-reperfusion In Rat

Background and Objective:Ischemia-reperfusion injury in neurons of penumbra is the main cause of braindamage after cerebral ischemia. The rescue of these neurons in the ischemiapenumbra which mainly died of apoptosis is the key of ischemia cerebrovasculardisease’ s treatment. Studies have found that Cathepsin B and MEK/ERK signalingpathway are involved in the process of neurons’ s apoptosis after ischemia-reperfusioninjury. There is few research covering the relationship of the two factors in apoptosis.In this experiment, ischemia-reperfusion injury model in rat was established. Electionmicroscope was used to observe the activation of lysosomes in the penumbra.Detecting the expression of Cathepsin B and p-MEK with western blot. Clarifying theinteraction between Cathepsin B and MEK/ERK signaling pathway in neurons,whichcan provides a new theory foundation for the mechanism of apoptosis afterischemia-reperfusion injury, also have a certain significance for the treatment ofischemia cerebrovascular disease.Methods:69male Sprague-Dawley Rats (260-300g,10-12weeks old, clean lever animal)were randomly divided into sham-operated control group(Sham, n=7),ischemia-reperfusion group30min (IRI-30min, n=8), ischemia-reperfusion group2h(IRI-2h, n=8), ischemia-reperfusion group6h (IRI-6h, n=8), ischemia-reperfusiongroup12h (IRI-12h, n=13), U0126intervention group30min (U0126-30min, n=5),U0126intervention group2h (U0126-2h, n=5), U0126intervention group6h(U0126-6h, n=5), U0126intervention group12h (U0126-12h, n=10). Transientmiddle cerebral artery occlusion was performed as Longa described. The interventiongroup was injected with U0126through the lateral ventricle puncture (5ug,5mlDMSO). The rats of other groups were injected with the same volume of DMSO (10ml/L) at the same time points. We use Longa’s of5levels grading evaluationneural function defect. Applying TTC staining to detect cerebral infarction volume.Election microscope was used to observe the morphology of lysosomes in thepenumbra. Detecting the expression of Cathepsin B and p-MEK in correspondingtime point and Caspase-3in12h with western blot.Results1.The success rate of model:86.42%.The relative infarct volume of model group12h is12.31±1.02%.2.sham-operated group was not seen brain infarcts,whereas model group andU0126intervention group were observed white infarction in cortical ischemia side.Inaddition,the relative infarct volume (p<0.05) of the u0126intervention group wassignificantly decreased compared with the model group,and neurological deficitsscore(p<0.05) was obviously ameliorated.3.The ischemic side of brain cortex preoptic area was observed activation oflysosome,increased number and enlarged volume of lysosome,and increased numberof secondary lysosome with electron microscope.And observed apoptosis of neuron inthe late reperfusion (12h).4.In rat ischemic side of brain cortex preoptic area,expression of Cathepsin Bprotein were observed in sham-operated group with Western blotting analysis,and,theexpression of Cathepsin B protein of model group were increased at30min,2h and6hafter cerebral ischemia/reperfusion,and reached the peak at6h,deceased at12h,buthigher than sham-operated group (p<0.01).Whereas the expression of Cathepsin Bprotein of the intervetion group was reached peak at2h afterischemia/reperfusion,decreased from2h;the expression of Cathepsin B protein at2h,6h and12h waere decreased ccomparad with model group(p<0.05)；the expressionof Cathepsin B protein of30min has no obvious difference with modelgroup(p<0.05).5. In rat ischemic side of brain cortex preoptic area,expression of p-mek wereobserved in sham-operated group with Western blotting analysis,the expression of p-mek of the intervetion group was reached peak at2h afterischemia/reperfusion,decreased from6h,but higher than sham-operated group(p<0.05);the expression of p-mek at2h,6h and12h waere decreased ccomparad withmodel group(p<0.05).6.In rat ischemic side of brain cortex preoptic area,Caspase3protein at12h afterischemia/reperfusion was lowest expressed in shan-operated group,most expressed inmodel group,and U0126intervention group was placed in the middle.Conclusions1. p-mek affect the expression of Cathepsin B protein after cerebralischemia-reperfusion in Rat.2.U0126may inhibit the MEK/ERK signal transduction pathway of apoptosisand reduce cell apoposis,and decrease cerebral infarct volnmu after cerebralischemia-reperfusion in Rat.