P38MAPK Participates In The Up-regulation Of GLT-1during The Induction Of Brain Ischemic Tolerance By Cerebral Ischemic Preconditioning

Mitogen-activated protein kinases (MAPKs) is a family ofSerine/threonine protein kinase which exists widely in many kinds of cells.Many studies have confirmed that MAPKs pathway is an important signaltransduction system, which could transduct extracelluar signals to the cell andits nucleus, and plays an improtant role in series of cell biology reactionsincluding cell proliferation, transformation, differentiation and apoptosis.MAPKs family mainly includes four members which are ERK, JNK, p38MAPK and ERK5. p38MAPK was found by Brewster and Han in1993.Glutamate is the main excitatory neurotransmitter in the central nervoussystem. Aberrantly raised glutamate in the extracellular fluid can causeexcitatory toxic effect and damage neurons during brain ischemia. As lack ofglutamate metabolic enzymes in synaptic cleft, the regulation of itsconcentration mainly rely on the excitatory amino acid transporters (EAATs).It is generally accepted that EAAT2is very important in maintaining theglutamate level in extracellular fluid. EAAT2is also named as glial glutamatetransporter-1(GLT-1), because its protein mainly expresses in the member ofastrocytes. Therefore, modulating the function of GLT-1might be an effectivemethod to prevent excitatory toxic effect caused by excessive glutamate.Our previous study has proved that cerebral ischemic preconditioning(CIP) increased the expression of p-p38MAPK protein and GLT-1protein,and the up-regulation of p-p38MAPK protein was earlier than theup-regulation of GLT-1protein. The specific inhibitor of p38MAPK,SB203580, could inhibit the brain ischemic tolerance induced by CIP and theup-regulation of GLT-1protein. Antisense oligodeoxynucleotides (AS-ODNs)are a kind of synthetic single nucleotide fragments, which can combine with the DNA or mRNA of the target gene and then inhibit protein exprssion. Tofurther investigate the role of p38MAPK signaling pathway in theup-regulation of GLT-1during the induction of brain ischemic tolerance byCIP, the present study was undertaken to observe the effect of p38MAPKAS-ODNs on the brain ischemic tolerance and the up-regulation of GLT-1protein induced by CIP.1The activation of p-p38MAPK and expression of GLT-1protein duringthe induction of brain ischemic tolerance by CIPMethods: Forty healthy male Wistar rats (280-300g) with permanentocclusion of the bilateral vertebral arteries and recovered for2days wererandomly divided into4groups as follows:①Sham group (n=10): thebilateral vertebral arteries were separated and exposed but without occludingthe blood flow;②CIP group (n=10): the bilateral common carotid arterieswere clamped for3min, and then reperfused with the blood flow;③Ischemicinsult (II) group (n=10): the bilateral common carotid arteries were clampedfor8min, and then reperfused with the blood flow;④CIP+II group (n=10):the bilateral common carotid arteries were first clamped for3min as a CIP,and then reperfused with the blood flow. After a2days-interval, the bilateralcommon carotid arteries were clamped for8min as II. All the rats weresacrificed and separated the hippocampal CA1subfield at6h and2d after thesham operation or the last time of global brain ischemia (n=5in each timepoint), and western blot was used for analyzing the expression of p-p38MAPK and GLT-1protein.Results:The expression of p-p38MAPK protein Western blot analysis showedthat there was a certain amount of basic expression of p-p38MAPK protein inthe hippocampal CA1subfield of sham group at6h time point. Comparedwith the sham group, either CIP or II induced up-regulation in the expressionof p-p38MAPK protein(P<0.05). The integral optical density (IOD) valuereached1.25fold (P<0.05) or1.64fold (P<0.05) of the sham level in CIP andII gropus, respectively. Compared with the II group, the expression of p-p38 MAPK protein in the CIP+II group was decreased obviously (P<0.05), closeto the basic level. The changes in expression of p-p38MAPK protein in eachgroup at2d time point were similar to the results at6h time point.The expression of GLT-1MAPK protein A certain amount of GLT-1protein was observed in the hippocampal CA1subfield of sham group at6htime point. Compared with the sham group, the expression of GLT-1proteinin the CIP group was significantly increased, the IOD value was1.18fold(P<0.05) of the basal value. The expression of GLT-1protein in the II groupwas significantly down-regulated compared either with sham or CIP group,the IOD value was0.283fold (P<0.01) of the basal value and0.24fold(P<0.01) of CIP group. Compared with the II group, the expression of GLT-1protein in the CIP+II group was increased obviously (P<0.05)，close to thebasic level. The changes in expression of GLT-1protein in each group at2dtime point were corresponded to the results at6h time point.Summary:①CIP moderately up-regulated the expression of p-p38MAPK and inhibited the excessively up-regulation of p-p38MAPK inducedby ischemic insult.②CIP obviously up-regulated the expression of GLT-1protein and significantly inhibited the down-regulation of GLT-1induced byischemic insult.2The effect of p38MAPK AS-ODNs on the expression of p-p38MAPKand GLT-1protein during the induction of brain ischemic tolerance by CIPMethods: One hundred and ten healthy male Wistar rats (280-300g)with permanent occlusion of the bilateral vertebral arteries and recovered for2days were randomly divided into7groups as follows:①Sham group (n=10);②CIP group (n=10);③CIP+II group (n=10);④p38MAPK AS-ODNs+CIP group (n=30);⑤p38MAPK AS-ODNs+CIP+II group (n=30);⑥p38MAPK S-ODNs+CIP group (n=10);⑦p38MAPK S-ODNs+CIP+II group(n=10). Group④and⑤was respectively divided into three subgroups (5nmol,10nmol and15nmol subgroup) according to the dose of p38MAPKAS-ODNs (n=10in each subgroup). The dose of p38MAPK S-ODNs was15nmol. p38MAPK AS-ODNs and p38MAPK S-ODNs were dissolved in15 μL artificial cerebrospinal fluid (ACSF) and injected once a day for5consecutive days from one day before occluding the bilateral vertebral arteriesto the day after CIP. The rats for6h time point of CIP group were onlyinjected4times.All the rats were sacrificed and separated the hippocampal CA1subfieldat6h and2d after the sham operation or the last time of global brain ischemia(n=5in each time point). Western blot analysis was used for detecting theexpression of p-p38MAPK and GLT-1protein.Results: Compared with the CIP group, the expressions of p-p38MAPKand GLT-1protein in the group of p38MAPK AS-ODNs+CIP weredecreased obviously in each dose of p38MAPK AS-ODNs (5nmol,10noml,15nmol)(P<0.05) at6h time point. The inhibition showed cleardose-dependency. Specifically, the expression of p-p38MAPK and GLT-1protein in10subgroup declined obviously compared with5nmol subgroup(P<0.05) and the expression of p-p38MAPK and GLT-1protein in15nmolsubgroup was significantly down-regulated compared with10nmol subgroup(P<0.05). The expression of p-p38MAPK and GLT-1protein had nosignificant difference between p38MAPK S-ODNs+CIP group and CIPgroup (P>0.05).Compared with the CIP+II group, the expressions of p-p38MAPK andGLT-1protein in p38MAPK AS-ODNs+CIP+II group were decreasedobviously in each dose of p38MAPK AS-ODNs (5nmol,10noml,15nmol)(P<0.05) at6h time point. The inhibition showed clear dose-dependency.Specifically, the expression of p-p38MAPK and GLT-1protein in10nmol subgroup was down-regulated obviously compared with the5nmolsubgroup (P<0.05) and the expression of p-p38MAPK and GLT-1protein in15nmol subgroup was depressed significantly compared with the10nmol(P<0.05). The expression of p-p38MAPK and GLT-1protein had nosignificant difference between p38MAPK S-ODNs+CIP+II group and CIP+II group (P>0.05).At2d time point, the changes in the expressions of p-p38MAPK and GLT-1protein in each corresponding group were consistent with those at6htime point.Summary: p38MAPK AS-ODNs could significantly inhibited theup-regulation of p-p38MAPK and GLT-1protein in a dose-dependent mannerin rats either after CIP or during the induction of brain ischemic tolerance byCIP.3The role of p38MAPK AS-ODNs in the induction of brain ischemictolerance induced by CIPMethods: Forty-five healthy male Wistar rats (280-300g) withpermanent occlusion of the bilateral vertebral arteries and recovered for2dayswere randomly divided into7groups as follows:①Sham group (n=5);②CIPgroup (n=5);③II group (n=5);④CIP+II group (n=5);⑤ACSF+CIP+IIgroup (n=5);⑥p38MAPK AS-ODNs+CIP+II group (n=15);⑦p38MAPKS-ODNs+CIP+II group (n=5). Group⑥was divided into three subgroups(5nmol,10nmol and15nmol subgroup) according to the dose of p38MAPKAS-ODNs (n=5in each subgroup). The dose of p38MAPK S-ODNs was15nmol. All the rats were sacrificed at7d after the sham operation or the globalbrain ischemia to observe delayed neuronal death (DND) in the hippocampalCA1subfield by thionin staining.Results: Neuropathological evaluation showed that in the sham group,pyramidal neurons in the hippocampal CA1subfield were arranged in orderwith2to3layers, the outline of the neurons was intact, nucleus was full andnucleolus was clear, no neuronal damage was observed. The histological grade(HG) was0and neuronal density (ND) value was196±6.93mm-1. Comparedwith the sham group, no obvious neuronal damage was observed in thehippocampal CA1subfield in the CIP group, neither the HG nor the ND valuehad obvious difference. Significant DND was observed in the II group, almostall of the pyramidal neurons in the hippocampal CA1subfield were vanished,the HG was Ⅱ～Ⅲ grade and ND value was17.33±6.11mm-1, whichsignificantly changed compared those in sham group (P<0.01). In the CIP+IIgroup, pyramidal neurons in the hippocampal CA1subfield were arranged in order, nucleus was full and nucleolus was clear, which indicated that CIPcould obviously inhibited DND induced by ischemic insult. Compared withthe II group, the HG decreased obviously (P<0.05), the ND value was178.67±6.11mm-1, increased distinctly (P<0.05). In ACSF+CIP+II group, noobvious neuronal damage was observed, neither HG nor ND was differentfrom that of CIP+II group (P>0.05). Compared with the ACSF+CIP+IIgroup, significant DND in the hippocampal CA1subfield was observed in thep38MAPK AS-ODNs+CIP+II group. The HG was increased and ND wasdecreased. The content of DND showed a clear dose-dependency with thedose of p38MAPK AS-ODNs. In5nmol p38MAPK AS-ODNs+CIP+IIgroup, pyramidal neurons in the hippocampal CA1subfield were sporadicallydead, the HG wasⅠgrade, the ND value was123.56±11.33mm-1. In10nmolp38MAPK AS-ODNs+CIP+II group, pyramidal neurons in thehippocampal CA1subfield were flakily dead, the HG was Ⅱ grade, the NDvalue was93.11±15.76mm-1. In15nmol p38MAPK AS-ODNs+CIP+IIgroup, almost all of the pyramidal neurons in the hippocampal CA1subfieldwere dead, the HG was Ⅲ grade, the ND value was57.11±8.50mm-1. In thep38MAPK S-ODNs+CIP+II group, no obvious neuronal damage wasobserved in the hippocampal CA1subfield, compared with ACSF+CIP+IIgroup, neither ND nor HG had obvious difference (P>0.05).Summary: p38MAPK AS-ODNs significantly inhibited the induction ofbrain ischemic tolerance by CIP in a dose-dependent manner.Conclusion: p38MAPK participates in the up-regulation of GLT-1during the induction of brain ischemic tolerance by CIP.