The Effect Of Cerebral Ischemic Preconditioning On PPARγ Signal Pathway And The Expresion Of GLT-1

Objective: Peroxisome prolifer atoractivated receptor (PPAR) is aligand-activated transcription factor including three types of alpha, beta, andgamma subtype. PPARγ is involved in the regulation of many physiologicaland pathophysiological processes such as, lipid metabolism, glucosemetabolism, fat cell differentiation, energy balance and the inflammatoryreaction. Recent studies have found that activation of PPARγ could protect thebrain against ischemic injury. At present, ischemic cerebrovascular disease isone of the most common diseases in nervous system. Severe cerebral ischemiacould lead to cell and tissue irreversibility injury. Animal experimentsconfirmed that a short and mild previous cerebral ischemia had a protectiveeffect on neuron to survival subsequent severe ischemia which is usuallylethal to the neurons. The short and mild previous cerebral ischemia is knownas cerebral ischemic preconditioning (CIP). A set of studies confirmed theprotective effect of CIP, after Kitagawa first proposed it in the year of1990.Our previous studies observed that CIP could up-regulate the expression ofglial glutamate transporter-1(GLT-1) and thus elevate the tolerance to brainischemia. However, CIP up-regulated the expression of GLT-1whether viaPPAR signaling pathway or not has not been reported until now. This studyinvestigated the expression of PPARγ and GLT-1protein in CA1hippocampusin rat. The aim of the present study is to provide a theoretical basis of theprevention and therapy to ischemic disease for future.Methods: Cerebral ischemia rats were processed using four-vesselocclusion method (4VO).240male Wister rats in weight of280-320g wererandomly divided into the following two parts:1The expression of PPARγ protein in CA1hippocampus during theinduction of brain ischemic tolerance induced by CIPRats were first occluded the bilateral vertebral arteries permanently, after2-day interal, which wererandomly divided into four groups as follows (n=45):①sham group:exposeing the bilateral carotid arteries without blocking-up the blood flow;②CIP group: occlusion of bilateral common carotid arteries for3min, thenreperfusion;③Ischemic insult (Ⅱ) group: occlusion of bilateral commoncarotid arteries for8min, then reperfusion;④CIP+Ⅱ group: occlusion ofbilateral common carotid arteries for3min as ischemic preconditioning, thenreperfusion for48h, once again occlusion of bilateral common carotid arteriesfor8min as ischemic insult, and then reperfusion. Each group was dividedinto9time points according to the reperfusion time after the last ischemia orsham operation:0min,15min,30min,3h,6h,1d,2d,4d and7d (n=5ineach time point).At the determined time, rats were decapitated and the expression of PPARγ inCA1hippocampus was observed using western blot analysis.2The Effect of GW9662, a specific inhibitor of PPARγ, on theexpression of GLT-1protein in CA1hippocampusThe groups were divided as follows:①3%DMSO+CIP+Ⅱ group (n=30);②GW9662+CIP+Ⅱ group (n=30).3%DMSO or5nmol GW9662dissolved in3%DMSO were injected in the lateral ventricles,30minutes later, bilateralcommon carotid arteries were occluded for3min as CIP, then reperfusion for2days, once again the bilateral common carotid arteries were occluded for8min as ischemic insult, and then reperfusion again. Each group was dividedinto6time points according to the reperfusion time after the last ischemia:0min,3h,6h,1d,4d and7d. The expression of GLT-1protein in CA1hippocampus was observed using western blot analysis (n=5in each timepoint).Result:1The expression of PPARγ protein in CA1hippocampus during theinduction of brain ischemic tolerance induced by CIP.Western blot analysis showed that no significant difference in theexpressions of PPARγ protein in CA1hippocampus of sham rats was observed among each time points (P>0.05).In the CIP group, compared with the0min time point, the expression ofPPARγ protein was significantly increased at30min time point (P<0.05), andreached its peak at3h time point, the peak value of integral optical density(IOD) was about2.5fold of the0min point. From the6h time point, theexpression of PPARγ protein recovered to the levels of0min time point.In the ischemic insult group, compared with0min time point, theexpression of PPARγ protein was significantly increased at15min time pointafter cerebral ischemic insult for8min (P<0.05). The expression of PPARγprotein further increased at30min,3h, and6h time points, and the peakvalue of IOD was about10fold of the0min time point (P<0.01). Theexpression of PPARγ protein slightly fell at1d,2d and4d time points,which still was much higher than the0min time point (P<0.05). At7dtime point, the expression of PPARγ protein was still significantly higher thanthe0min time point (P<0.05). The results showed that the expression ofPPARγ protein increased significantly after ischemic insult for8min.In CIP+Ⅱ group, compared with0min time point, the expression ofPPARγ protein was significantly increased at6h time point (P<0.05), and thepeak value of IOD was about1.35fold of the0min time point. The expressionof PPARγ were significantly decreased from2d to7d (P<0.05). Nosignificant difference was observed at other time points (P>0.05)2The Effect of GW9662, a specific inhibitor of PPARγ, on theexpression of GLT-1protein in CA1hippocampusIn DMSO+CIP+Ⅱ group, the expressions of GLT-1protein were higherat each time point (including0min,3h,6h,1d,4d and7d time point).Compared with the corresponding time point in DMSO+CIP+Ⅱ group, theexpression of GLT-1protein in GW9662+CIP+Ⅱ group was significantlydecreased at each time point after5nmol GW9662was previouslyadministrated (P<0.05).Summary:1CIP caused moderate up-regulation of PPARγ protein. Ischemic insult lead to PPARγ protein increased enduringly and intensively. CIP for3min2days before the ischemic insult significantly inhibited the enduring andintensive increasing of PPARγ protein induced normally by ischemic insult for8min.2The PPARγ specific inhibitor GW9662inhibited the up-regulation ofGLT-1protein during the induction of brain ischemic tolerance induced byCIP.Conclusion: Cerebral ischemic preconditioning regulated the expressionof GLT-1protein via PPARγ signaling pathway during the induction of brainischemic tolerance.