The Changes Of The Expression Of NF-κB P50、NF-κB P65 And PPARγ Protein In The CA1 Hippcampus During The Induction Of Brain Ischemic Tolerance Induced By Cerebral Ischemic Preconditioning

Objective: A large number of studies show that pretreatment with a slight,short-time cerebral ischemic preconditioning(CIP) which would not damagethe pyramidal neurons will reduce the ischemia-reperfusion injury andproduce brain ischemia tolerance(BIT). The protective effect of BIT isaccomplished through activating the endogenous protective mechanism of theorganism. From the theory had been put forward by Kitagawa, a famousscientist of Japan, it has been proved to be right by a quantity of literaturesthat has been studied by lots of scientists worldwide. Nuclear factor kappa B(NF-κB) is a kind of nuclear transcription factor, it is belongs to the signalchannel depended on the regulated protein hydrolase, which closely related tothe occurrence, growth, differentiation, apoptosis, infection and movement oftumor. In addition, NF-κB and IκB will become the complexus in thecytoplasmduring the quiescent condition. When some factors stimulate the cell,IκB will be phosphorylated and dissociated with NF-κB, which will activatethe NF-κB to let it take part in the transcription process. The NF-κB familycontains a large number of subtype, P50 and P65 are the most important twosubtypes, which greatly contribute to immune function coordination andanti-infection.The changes of their expression with time during the inductionof BIT induced by CIP.Peroxisome prolifer atoractivated receptors(PPAR) is a member of nucleartranscription factor superfamily, which could be activated by ligand. It playsan essential role in regulating the growth, differentiation, apoptosis andoperation of cells. PPAR family contains three kinds of subtypes: PPARα,PPARβ and PPARγ. PPARγ could produce many kinds of biological effectsafter it was activated, which protect the cerebral tissues survival fromischemic insult. On the other hand, PPARγ could mitigate a lot of clinicaldisease, such as liver cirrhosis, atherosclerosis, hypertension andhyperlipidemia. Taken all the above together, the aim of this study is toobserve the changes of the expressions of NF-κB P50, NF-κB P65 and PPARγprotein in the CA1 hippocampus during the induction of BIT induced by CIP,which will provide the theoretical basis for prevention and treatment ofcerebral ischemic disease in clinic.Methods : There are one hundred and two healthy male Wister rats(280-300g) were used for global brain ischemia by four-vessel occlusion(4VO). They were randomly divided into six groups:(1) Control group(n=3): without any treatment.(2) Sham group(n=3): exposure of bilateral vertebral arteries andbilateral common carotid arteries without blocking-up the blood flow.(3) Vertebral artery occlusion(VAO) group(n=24): occlusion of bilateralvertebral arteries permanently and exposure of bilateral common carotidarteries without blocking-up the blood flow.(4) CIP group(n=24): occlusion of bilateral vertebral arteriespermanently and occlusion of bilateral common carotid arteries for 3 minbefore reperfusion.(5) Ischemic insult(II) group(n=24): occlusion of bilateral vertebralarteries permanently and occlusion of bilateral common carotid arteries for 8min before reperfusion.(6) CIP+II group(n=24): occlusion of bilateral vertebral arteriespermanently; occlusion of bilateral carotid arteries for 3 min as cerebralischemic preconditioning, reperfusion for two days, before reperfusion again,occlusion of bilateral common carotid arteries for 8 min as ischemic insult.Except the control group and sham group, all the other groups were wasfurther divided into 8 time points : immediate time point(0min), 15 min, 30 min, 1 h, 3 h, 6 h, 2 d and 7 d after the sham operation or the last operations(n=3 in each time point).In the determined time point, the neuronal pathological evaluation ofpyramidal neurons in CA1 hippocampas was observed by thionine staining,and the expression of NF-κB P50, NF-κB P65 and PPARγ in CA1 hippocampas was observed by immunohistochemistry.Result:1 Neuropathological evaluationBy using thionine staining, pyramidal neurons in the CA1 hippocampuswere observed in good condition in control group, which arranged in orderwith 2 to 3 cell layers, the cells are intact and the nucleus is clearly visible. Incomparison with control group, no significant difference was observed inpyramidal neurons of the sham group(P>0.05). In comparison with shamgroup, no significant difference was observed in the pyramidal neurons of theVAO group(P>0.05).No neuronal damage was observed in the CA1 hippocampus at each timepoint of CIP group. In comparison with VAO group, neither cellularmorphology nor neuronal density was different at each time point of CIPgroup(P>0.05).In II group, no significant neuronal damage was observed in the CA1 hippocampus during the initial 6 h. However, some pyramidal neuronsbecome atrophy, degeneration and necrosis at 2 d time point. At the same time,the arrangement of pyramidal neurons becomes irregular. At 7 d time point,almost all of pyramidal neurons in the CA1 hippocampus become necrosis orinvisible. Compared with corresponding time point of VAO group, there issignificant difference of neuronal density in the CA1 hippocampus at 2 d and7 d time point(P <0.05).There is no significant neuronal damage in the CA1 hippocampus at 0min, 6 h and 2 d time point in CIP+II group. At 7 d time point, only a fewpyramidal neurons in the CA1 hippocampus become necrosis, and thearrangement of pyramidal neurons in the CA1 hippocampus was almostregular. Compared with 7 d time point of II group, the neuronal density in theCA1 hippocampus increased and the arrangement become regular clearly atthe same time point in CIP+II group(P <0.05).2 The changes of the expression of NF-κB P50 protein in the CA1 hippocampus during the induction of brain ischemic tolerance induced by CIPIn control, sham and VAO group, there is little expression of NF-κB P50 protein in karyon but some in cytoplasm of pyramidal neurons in the CA1 hippocampus. Compared with control group, the expression of NF-κB P50 protein is similar in sham group. Compared with sham group, the expressionof NF-κB P50 protein is also similar at each time point of VAO group.In CIP group, there is little expression of NF-κB P50 protein in karyonbut some in cytoplasm at 0 min time point. From 30 min time point, NF-κBP50 protein begin to express in karyon and the IOD become increasingsignificantly(P <0.05). From 1 h time point, the expression of NF-κB P50 protein increased more clearly, and the expression of NF-κB P50 proteinreached the peak at 6h time point(P <0.05). At 2 d time point, both theexpression of NF-κB P50 protein and its IOD decreased obviously, which issimilar to that of VAO group at the same time point. This level maintains to 7d time point.In II group, there is little expression of NF-κB P50 protein in karyon butsome in cytoplasm at 0 min time point. From 30 min time point, NF-κB P50 protein begin to express in karyon. Compared with VAO group, both theexpression of NF-κB P50 protein and its IOD increased significantly at 30 min,1 h, 3 h, 6 h time point, and reached the peak at 6 h time point(P <0.05). At 6h time point, there is much NF-κB P50 protein expressed both in karyon andcytoplasm. At 2 d time point, the expression of NF-κB P50 protein and its IODdecreased obviously, which is similar to that of VAO group at the same point(P>0.05). Due to many pyramidal neurons in the CA1 hippocampus necrosisat 7 d time point, the expression of NF-κB P50 protein decreased obviously,which is much lower than that of VAO group(P <0.05).In CIP+II group, there is little expression of NF-κB P50 protein in karyonbut some in cytoplasm at 0 min time point. From 30 min time point, NF-κBP50 protein begin to express in karyon. Compared with 0 min time point, theexpression of NF-κB P50 protein increased slightly, no significant change wasobserved at 30 min time point. Compared with 0 min time point, theexpression of NF-κB P50 protein increased significantly at 1 h, 3 h, 6 h timepoint(P <0.05). At 6 h time point, there is much NF-κB P50 protein expressedboth in karyon and cytoplasm. At 2 d time point, both the expression of NF-κBP50 protein and its IOD decreased obviously, which is similar to that of VAOgroup at the same time point.At 6 h time point, compared with VAO group, the expression of NF-κBP50 protein increased clearly both in CIP group and in II group(P <0.05).Compared with CIP group, the expression of NF-κB P50 protein furtherincreased in II group(P<0.05). Compared with II group, the expression ofNF-κB P50 protein significantly decreased in CIP+II group(P <0.05).At 7 d time point, compared with VAO group, no difference of NF-κBP50 protein expression was observed in CIP group(P>0.05), but significantlydecreased in II group(P<0.05). Compared with II group, the expression ofNF-κB P50 protein significantly increased in CIP+II group(P <0.05).3 The changes of the expression of NF-κB P65 protein in the CA1 hippocampus during the induction of brain ischemic tolerance induced by CIPIn control, sham and VAO group, the expression of NF-κB P65 protein isat a certain level in both karyon and cytoplasm of the pyramidal neurons.In CIP group, no significant difference of the expression of NF-κB P65 protein in the CA1 hippocampus was observed during the initial 30 min(P>0.05); from 1 h time point, the IOD become increasing significantly, and afurther increase was observed at 3 h and 6 h time point. At 3 h and 6 h timepoint, there is much NF-κB P65 protein expressed both in karyon andcytoplasm of the pyramidal neurons, and then reached the peak at 6h timepoint(P <0.05). From then on, the change trend of NF-κB P65 proteinexpression is similar to that of NF-κB P50 protein in CIP group.In II group, no significant difference of the expression of NF-κB P65 protein in the CA1 hippocampus was observed during the initial 15 min(P>0.05). From 30 min time point, its IOD increased obviously, and a furtherincrease was observed at 1 h, 3 h and 6 h time point. At 1 h, 3 h and 6 h timepoint, there is much NF-κB P65 protein expressed both in karyon andcytoplasm of the pyramidal neurons, and reached the peak at 6 h time point(P<0.05). At 2 d time point, the expression of NF-κB P65 protein decreasedclearly, especially in karyon. Compared with VAO group, the expression isstill in high level at 2 d time point. From then on, the change trend of NF-κBP65 protein expression is similar to that of NF-κB P50 protein in II group.In CIP+II group, no significant difference of the expression of NF-κBP65 protein in the CA1 hippocampus was observed during the initial 15 min(P>0.05). From 30 min time point, its IOD increased obviously, and a furtherincrease was observed at 1 h, 3 h and 6 h time point. At 1 h, 3 h and 6 h timepoint, there is much NF-κB P65 protein expressed both in karyon andcytoplasm of the pyramidal neurons, and reached the peak at 6 h time point(P<0.05). From then on, the change trend of NF-κB P65 protein expression issimilar to that of NF-κB P50 protein in CIP+II group.At 6 h time point, compared with VAO group, the expression of NF-κBP65 protein increased clearly both in CIP group and in II group(P <0.05).Compared with CIP group, the expression of NF-κB P65 protein furtherincreased in II group(P<0.05). Compared with II group, the expression ofNF-κB P65 protein significantly decreased in CIP+II group(P <0.05).At 7 d time point, compared with VAO group, no difference of NF-κBP65 protein expression was observed in CIP group(P>0.05), but significantlydecreased in II group(P<0.05). Compared with II group, the expression ofNF-κB P65 protein significantly increased in CIP+II group(P <0.05).4 The changes of the expression of PPARγ protein in the CA1 hippocampus during the induction of brain ischemic tolerance induced by CIPIn control, sham and VAO group, the expression of PPARγ protein is at acertain level in both karyon and cytoplasm of the pyramidal neurons.In CIP group, no significant difference of the expression of PPARγprotein in the CA1 hippocampus was observed during the initial 15 min(P>0.05). From 30 min time point, the IOD become increasing significantly,and a further increase was observed at 1 h and 3 h time point. At 1 h and 3 htime point, there is much PPARγ protein expressed both in karyon andcytoplasm of the pyramidal neurons, and then reached the peak at 3 h timepoint(P <0.05). At 6 h, 2 d and 7 d time point, both the expression of PPARγprotein and its IOD decreased obviously, which is similar to that of VAOgroup at the same time point.In II group, no significant difference of the expression of PPARγ proteinin the CA1 hippocampus was observed during the initial 15 min(P>0.05).From 30 min time point, its IOD increased obviously, and a further increasewas observed at 1 h, 3 h and 6 h time point. At 1 h, 3 h and 6 h time point,there is much PPARγ protein expressed both in karyon and cytoplasm of thepyramidal neurons, and reached the peak at 6h time point(P<0.05). Due tomany pyramidal neurons in the CA1 hippocampus necrosis at 2 d and 7 d timepoint, the expression of PPARγ protein decreased obviously, which is muchlower than that of VAO group(P <0.05).In CIP+II group, no significant difference of the expression of PPARγprotein in the CA1 hippocampus was observed during the initial 1 h(P>0.05).From 3 h time point, its IOD increased obviously, and a further increase wasobserved at 6 h time point. At 6 h time point, there is much PPARγ proteinexpressed both in karyon and cytoplasm of the pyramidal neurons, andreached the peak at 6 h time point(P <0.05). From then on, the change trendof PPARγ protein expression is similar to that of NF-κB P50 protein in CIP+IIgroup.At 6 h time point, compared with VAO group, no significant difference ofthe expression of PPARγ protein in the CA1 hippocampus was observed inCIP group(P>0.05), but significantly increased in II group(P<0.05).Compared with CIP group, the expression of PPARγ protein significantlyincreased in II group(P<0.05). Compared with II group, the expression ofPPARγ protein significantly decreased in CIP+II group(P <0.05).At 7 d time point, compared with VAO group, no difference of PPARγprotein expression was observed in CIP group(P>0.05), but significantlydecreased in II group(P<0.05). Compared with II group, the expression ofPPARγ protein significantly increased in CIP+II group(P <0.05).Conclusions:1 Cerebral ischemic preconditioning increased the expression of NF-κBP50, NF-κB P65 and PPARγ protein moderately, and the expression in karyonis the main.2 Ischemic insult upregulated the expression of NF-κB P50, NF-κB P65 and PPARγ protein extremely, and the expression in karyon is also the main.3 Cerebral ischemic preconditioning could effectively prevent theexcessive up-regulation of the expression of NF-κB P50, NF-κB P65 andPPARγ protein which is normally induced by ischemic insult.