The Role Of NF-κB In The Up-regulation Of GLT-1Via Activated P38MAPK During The Induction Of Brain Ischemic Tolerance Induced By CIP

Objective: Cerebral ischemic preconditioning (CIP), a previous,short-time, slight cerebral ischemia that is not lethal to neurons, can reduceischemia-reperfusion injury and induce brain ischemic tolerance. Althoughmany studies confirmed that CIP can induce brain ischemic tolerance, itsmechanism has not yet been clear. NF-κB is detected from the nucleus of Blymphocytes which is associated with the enhancer of immunoglobulin к lightchain. In most cells, NF-κB is present as a latent, inactive, IκB-bound complexin the cytoplasm. When a cell receives any of a multitude of extracellularsignals, NF-κB rapidly enters the nucleus and activates gene expression. Inaddition, some studies have found that some of IκB-independent signals, suchas p38MAPK, modifed NF-κB phosphorylation, regulated its activity, andmade it more effective in promoting gene transcription. On the one hand, ourprevious study has shown that CIP induced brain ischemic tolerance byup-regulating glial glutamate transporter-1(GLT-1) by p38MAPK pathway.On the other hand, another study has indicated that the specific inhibitor ofNF-κB, BAY11-7082, dose-dependently depressed the brain ischemictolerance induced by CIP. However, it is necessary to elucidated that whetherNF-κB participates in the up-regulating of GLT-1protein via p38MAPKpathway during brain ischemic tolerance induced by CIP. Therefore, thepresent study is aimed to observeThe role of NF-κB in the up-regulation ofGLT-1via activated p38MAPK during the induction of brain ischemictolerance induced by CIP.Method: One hundred and nity-eight adult male Wistar rats (280-320g)were used. At first, bilateral vertebral arteries were electrocauterized underchloral hydrate anesthesia (350mg/kg) administered by abdominal injection.2 day-interval, the rats were randomly divided into the following experiments.1The expression of NF-κB p50protein, NF-κB p50/p65dimer and the activityof NF-κB binding with DNA during the induction of brain ischemic toleranceinduced by CIPThe details are as follows:①s ham group(n=27): the bilateral commoncarotid arteries (BCCAs) were separated under ether anesthesia, but withoutoccluding the blood flow;②CIP group(n=27): the BCCAs were clamped for3min then reperfused with the blood flow;③i schemic insult(II) group (n=27):the BCCAs were clamped for8min then reperfused with the bloodflow;④CIP＋II group (n=27): a CIP for3min was preformed then reperfused,2day-interval, a lethal ischemic insult for8min was given then reperfusedwith the blood flow. All the groups were divided into9time points: immediatetime point (0min),30min,15min,3h,6h,1d,2d,4d and7d after thesham operation or the last operations (n=3in each time point). At thedetermined time point, the rats were sacrificed by decapitation. Western blotwas used for observing the expression of NF-κB p50protein.Co-immunoprecipitation (COIP) was used for observing the expression ofNF-κB p50/p65dimers. And electrophoretic mobility shift assay (EMSA)was used for NF-κB DNA binding activity.2The effects of SB203580, an inhibitor of p38MAPK, on the expression ofNF-κB p50protein, NF-κB p50/p65dimeric complexs and activity of NF-κBbinding with DNAThe groups were as follows:①DMSO+CIP+IIgroup (n=18);②S B203580+CIP+II group(n=18): The rats were injected with3%DMSOsolution or5nmol SB203580into the right lateral cerebral ventriclerespectively according to the design30min before CIP, the other process wasthe same as that of CIP+II group. Each group was divided into6time points:immediate time point (0min),3h,6h,1d,4d and7d after the shamoperation or the last operations (n=3in each time point). Western blot, COIPand EMSA were respectively used for observing whether SB203580has effecton the expression of NF-κB p50protein, NF-κB p50/p65dimers and on the activity of NF-κB binding with DNA.3The effects of BAY11–7082, an inhibitor of NF-κB, on the expression ofGLT-1proteinThe groups were as follows:①D MSO+CIP+II group(n=18);②B AY11–7082+CIP+II group (n=36): The rats were injected with3%DMSOsolution or BAY11–7082into the right lateral cerebral ventricle respectivelyaccording to the design30min before CIP, the other process was the same asthat of CIP+II group. According to the doses of BAY11–7082used,BAY11–7082+CIP+II group was further divided into1.25nmol and2.5nmolsubgroups (n=18in each subgroup). Each group (subgroup) was divided into6time points: immediate time point (0min),3h,6h,1d,4d and7d after thesham operation or the last operations (n=3in each time point). Western blotwas used for observing the expression of GLT-1protein.Results:1The expression of NF-κB p50protein during the induction of brain ischemictolerance induced by CIP and the effect of SB203580on itThe expression of NF-κB p50proein in CA1hippocampal subfield wasobserved by western blot during the induction of brain ischemic toleranceinduced by CIPIn the sham group, a little NF-κB p50protein was observed at all timepoints. Compared with the immediate time point, no significant difference wasobserved at each time point (P>0.05).After CIP for3min, the expression of NF-κB p50protein was observed atthe immediate time point. The expression of the protein was slightlyup-regulated at15min and30min time points, but no significant differencewas observed compared with the immediate time point (P>0.05). Comparedwith the immediate time point, its expression significantly increased at3h and6h time points (P <0.05), no significant difference was observed at the othertime points (P>0.05).In the ischemic insult group, the expression of NF-κB p50wasobservedat the immediate time point after cerebral ischemic for8minutes. Compared with the immediate time point, the expression of the protein wasup-regulated slightly but significantly at15min time point (P<0.05), andreached its peak at30min time point (P <0.01). In spite of a descent of theexpression at3h,6h,1d and2d time points, it was still higher than that ofthe immediate time point (P＜0.05). Compared with the immediate time point,there was significant difference at neither4d nor7d time point (P>0.05).At all time points (0min,3h,6h,1d,4d and7d) of DMSO+CIP+IIgroup, the expression of NF-κB p50protein were high. Compared with thecorresponding time point of DMSO+CIP+II group, the expression of theprotein significantly down-regulated at each time point in5nmol SB203580+CIP+II group (P <0.05).These results implicated that5nmol SB203580could depress the expression of NF-κB p50protein effectively during brainischemic tolerance induced. The result of5nmol SB203580on the expressionof NF-κB p50protein was the same as that of2.5nmol SB203580dose groupduring the induction of brain ischemic tolerance induced by CIP.2The expression of NF-κB p50/p65dimer during the induction of brainischemic tolerance induced by CIP and the effect of SB203580on itThe expression of NF-κB p50/p65dimer in CA1hippocampal subfieldhas been detected by Co-immunoprecipitation (COIP) method during theinduction of brain ischemic tolerance induced by CIP.In the sham group, the expression of NF-κB p50/p65dimer was observedat0min,15min,30min,3h and6h time points. Compared with theimmediate time point (0min), a little up-regulation was observed at15min,30min,3h and6h time points (P>0.05). The expression of the NF-κBp50/p65dimer decreased after1d of the sham operation.After CIP for3min, the expression of NF-κB p50/p65dimer wasobserved at immediate time point. The expression of NF-κB p50/p65dimerwas significantly up-regulated at15min time point compared with theimmediate time point (P＜0.01). Despite a descent was observed, theexpression of NF-κB p50/p65dimer at30min and3h time point was stillhigher than that of the immediate time point (P＜0.05). Its expression maintained at a certain level at6h,1d and2d time point, and there was nosignificant difference between each of these time points and the immediatetime point (P>0.05). Compared with the immediate time point, the expressionof the dimer significantly decreased at4d and7d time point (P＜0.05).In the ischemic insult group, the expression of NF-κB p50/p65dimer wasobserved at the immediate time point after cerebral ischemic for8minutes.The expression of NF-κB p50/p65dimer was obviously up-regulated at15min,30min and3h time point compared with that of the immediate timepoint (P＜0.05), and the peak is at30min time point. Compared with theimmediate time point, there was no significant difference at other time points(P>0.05).In the CIP+II group, the expression of NF-κB p50/p65dimer wasobserved at the immediate time point. Compared with the immediate timepoint, the expression of the dimer was obviously up-regulated at15min,30min,3h and6h time points (P <0.01). Despite a descent was observed, theexpression of the dimer at1d and2d time point was still higher than that ofthe immediate time point (P<0.05). There was no significant difference at4dor7d time point compared with that of the immediate time point (P>0.05).At all time points (0min,3h,6h,1d,4d and7d) of DMSO+CIP+IIgroup, the expression of NF-κB p50/p65dimer were high. Compared with thecorresponding time point of the DMSO+CIP+II group, the expression of thedimer significantly down-regulated at each time point in5nmol SB203580+CIP+II group (P <0.01). These results indicated that5nmol SB203580couldblock the up-regulation of NF-κB p50/p65dimer effectively during brainischemic tolerance induced by CIP.3The DNA binding activity of NF-κB during the induction of brain ischemictolerance induced by CIP and the effect of SB203580on itThe DNA binding activity of NF-κB in CA1hippocampal subfield wasdetected by EMSA.In the CIP group, NF-κB activity was observed at the immediate timepoint. NF-κB activity at15min time point was significantly enhanced and reached a peak (P＜0.01). Although a descent of NF-κB activity at30min and3h time point was observed, it was still higher than that of the immediate timepoint (P＜0.05). Its binding activity maintained at a certain level at6h,1dand2d time point, and there was no significant difference between each ofthese time points and the immediate time point (P>0.05). Compared with theimmediate time point, the binding activity significantly decreased at4d and7d time point (P <0.05).In the ischemic insult group, NF-κB binding activity had a basal level atimmediate time point after cerebral ischemic for8minutes. NF-κB activitysignificantly enhanced at15min,30min and3h time points compared withthe immediate time point (P＜0.05), and the peak was at30min time point.Compared with the immediate time point, there was no significant differenceat other time points (P>0.05).In the CIP+II group, NF-κB activity had a basal level at the immediatetime point. Compared with the immediate time point, the level of the activityinitially increased at15min time point, and further increased at30min and3h time points, then reached its peak at6h time point. In spite of a descent ofthe activity at1d and2d time points, it was still higher than that of theimmediate time point (P＜0.05). Compared with the immediate time point, nosignificant difference was observed at4d and7d time point (P>0.05).At all time points (0min,3h,6h,1d,4d and7d) of DMSO+CIP+IIgroup, the levels of NF-κB binding activity were high. Compared with thecorresponding time point of DMSO+CIP+II group, the level of the activitysignificantly decreased at each time point in5nmol SB203580+CIP+II group(P <0.01). These findings implicated that5nmol SB203580couldsignificantly depress the activity of NF-κB during the induction of brainischemic tolerance induced by CIP.4The effects of BAY11–7082, an inhibitor of NF-κB, on the expression ofGLT-1protein during the induction of brain ischemic tolerance induced byCIPThe effect of the specific inhibitor of NF-κB, BAY11–7082, on the expression of GLT-1protein was observed by western blot during theinduction of brain ischemic tolerance induced by CIP.At all time points (0min,3h,6h,1d,4d and7d) of DMSO+CIP+IIgroup, the expression of GLT-1protein were high. The level of GLT-1expression at each time point was significantly decreased by either1.25nmolor2.5nmol BAY11–7082injected30min before CIP (P <0.05). The resultsconfirmed that BAY11–7082, the specific inhibitor of NF-κB, couldsignificantly down-regulate the expression of GLT-1protein.Summary:1During the induction of brain ischemic tolerance induced by CIP, NF-κBwas significantly activeted, which up-regulating the expression of NF-κB p50protein, appeared forming active dimers, nuclear translocation from cytoplasmand increasing the DNA binding activity.2SB203580, the specific inhibitor of p38MAPK, down-regulated both theexpression of NF-κB p50protein and NF-κB p50/p65dimers, and suppressedthe DNA binding activity of NF-κB.3BAY11–7082, the specific inhibitor of NF-κB, blocked the up-regulating ofGLT-1protein induced by cerebral ischemic preconditioning.Conclusion: NF-κB plays a role in the up-regulation of GLT-1viaactivated p38MAPK during the induction of brain ischemic tolerance inducedby CIP.