Uncoupling Protein 2 Play A Role In Hyperglycemia Aggravated Cerebral Ischemia-reperfusion Injury Mediated By The Mitochondria

Objective Diabetes hyperglycemia aggravated cerebral ischemiareperfusion injury,and promoted production of mitochondrial reactive oxygen species(ROS) after ischemia. Uncoupling protein 2(UCP2) can stabilized the mitochondrial membrane potential and reduced the generation of ROS.But at present, mechanism of action and regulatory pathway of UCP2 in hyperglycemia aggravated cerebral ischemia injury are not clear.This study using UCP2 knockout mice simulated cerebral ischemia and hyperglycemia, to explore if UCP2 knockout can aggravated neuronal injury and its molecular pathways.Methods Male UCP2 knockout mice(KO-UCP2) and wild-type C57BL/6j mice were randomly divided into wild-type mice with normoglycemic focal cerebral I/R group(CNgroup), KO-UCP2 mice with normoglycemic focal cerebral I/R group(KNgroup) and KO-UCP2 mice with diabetic hyperglycemic I/R group(KHgroup).A typeⅠ diabetic rat model was induced by streptozotocin(STZ) injection and the focal cerebral I/R model was induced by 1 hour of middle cerebral artery occlusion(MCAO) followed by6 h,24h,72 h of reperfusion.Histopathology,immunohistochemistry,immunofluorescence,TTCstaining,TUNEL,RT-PC Rand western blot were applied to detect changes of the morphology and the number of neurons, apoptosis and the expression of Cyt C,Cleaved Caspase-9, Caspase-3 from each group.Results(1)At 24 h of I/R,the brain function scores of KN group were higher than CN group( P <0.05),and KH group were obviously higher than KN groupIV(P <0.05).(2)TTC staining results shown no significant infarction in sham group.At 24 h of I/R,infarct size of KN group was significantly larger than the CN group,and KH group was larger than the KN group(P <0.05).(3)Brain edema and partial shrinkage of neurons were observed in infarction area at 6 hour I/R after 1h of ischemia and worsed damage, structural failure, disorder arrangement at 24 hour of I/R.At 72 h,necrotic nerve cell shedding and appeared Inflammatory cells and a large number of glial cells in infarction area.Compared with CN,KN group, even worse cerebral edema was detected in KH group. Around the infarction area,penumbra boundary is unclear and cerebral edema were observed at 6h of I/R. Obviously cerebral edema,increased swelling neurons and cleared penumbra boundary were observed in peri-infarction area at 24 h of I/R and nearly recover to normal at 7h of I/R.Compared with CN,KN group, edema and cell injury situation in KH group was more serious, and still visible part of the nerve cell edema, telangiectasia congestion, Cell gap widened at 72 h of I/R in KH group.(4)TUNEL staining results shown apoptotic index in KN group was higher than the CN group in peri-infarction area,and KH group higher than the KN at 24 h of I/R( P <0.05).(5)Immunohistochemical results of Cyt C and Caspase-3. Positive expression of Cyt C, Caspase-3, began to appear at 6h, peaked at 24 h and decreased at 72h(P< 0.05).At 6h of I/R,positive expression of Cyt C, Caspase-3 in KN group was higher than CN group, Cyt C positive cells in KH group was significantly higher than that of KN group(P <0.05).At 24 h, expression of Cyt C, Caspase-3 in KN group was higher than CN group, and KH group was significantly higher than that of KN group(P <0.05).At 72 h. expression of Cyt C, Caspase-3 in KN group was higher than CN group, Caspase-3 positive cells in KH group was significantly higher than that of KN group(P <0.05).(6)Immunofluorescence staining shown Cleaved Caspase-9 positive cells and double immunofluorescence positive cells in KN group was significantly higher than the CN group, KH group more than KN group at6,24,72 h after reperfusion(P <0.05).(7)RT-PCRresults shown expression of Cyt C, Caspase-9, Caspase-3m RNA began to increase at 6h of I/R, KH group was significantly higher than KN group(P <0.05).Peaked at 24 h, KN group higher than the CN group, and KH group was significantly higher than that of KN group(P <0.05).When to 72 h, expression of Cyt C, Caspase-9m RNA in KN group was higher than the CN group, expression of Caspase-3m RNA in KH group was significantly higher than KN group(P <0.05).(8)Western Blot results shown Cyt C expression when reperfusion 6h, KN group was significantly higher than the CN group(P <0.05).At 24 h, Cyt C expression in KN group was higher than CN group, KH group was significantly higher than that of KN group(P <0.05).a KH group was more than KN group at 72 h of I/R.(P <0.05).Conclusion(1)Diabetic hyperglycemia aggravated cerebral ischemiareperfusion focal cerebral ischemia-reperfusion injury, which will increased the infarction area,nerve function deficit,and the number of cerebral cortex cell apoptosis around the infarction will increase.(2)knockout UCP2 aggravated focal cerebral ischemia-reperfusion injury that infarct area expanded, brain edema and neuronal shrinkage increases.(3)Cooperated with the knocking out of UCP2 gene, hyperglycemia can further aggravated the brain damage. The mechanism might be that hyperglycemia further upregulates the expression of Cyt C,Cleaved Caspase-9 and caspase-3,mitochondrial apoptotic pathways activated degree increase, which induce the increasing of apoptosis and oxidative damage.