The Role Of P38MAPK-PPARγ/NF-κB Signaling Pathway In The Induction Of Brain Ischemic Tolerance Induced By Cerebral Ischemic Preconditioning

Objective: Brain ischemia is one of the most important diseases thataffect human life. Many studies have confirmed that pre-treatment withsublethal ischemia to neurons can improve the neuronal tolerance to ischemiathat follows the sublethal ischemia and is usually lethal to neurons. The pre-administrated sublethal ischemia is called cerebral ischemic precon-ditio ning(CIP). Mitogen activated protein kinases (MAPKs) signaling pathway is aserine/threonine protein kinase. Many experiments have confirmed that theMAPKs signaling pathway is involved in the process of cerebral ischemia, andmaybe participate in the induction of brain ischemic tolerance. p38MAPK is asubtype of MAPKs family, which plays an important role in brain ischemictolerance induced by CIP. Activation of p38MAPK signaling pathwayphosphorylates the downstream signaling molecules and-then plays animportant role in various life activities of cells. Both PPARγ, one subtype ofPeroxisome proliferator-activated receptor (PPAR), and nuclear transcriptionfactor-kappa B (NF-κB) belong to the downstream signaling molecules of p38MAPK. PPARγ is one of the transcription factors that is activated by ligandsand involved in many physiological and pathophysiological processes, such aslipid body different-tiation and metabolism, energy conversion, and theinflammatory reaction. In the quiescent condition, NF-κB combines with IκBand forms a complex that lies in the cytoplasm. When cells are subjected tohypoxia, the phosphorylated IκB dissociates from the complex and makesNF-κB activating, and the activated NF-κB is involved in gene transcription.Therefore, we speculate that CIP possibly through the p38MAPK-PPARγ/NF-κB signaling pathway induces brain ischemic tolerance. Our previousstudies have showed that each of SB203580(p38MAPK specific inhibitor), GW9662(PPARγ specific antagonist), and BAY11-7082(NF-κB specificantagonist) could inhibit the brain ischemic tolerance induced by CIP andsuggested the role of the p38MAPK-PPARγ/NF-κB signaling pathway in theinduction of induces brain ischemic tolerance by CIP.Antisense Oligodeoxy-Nucleotides (AS-ODNs) can be combined withtarget gene according to the principle of complementary base pairing, so as toachieve the purpose of closing specific target gene and protein expression. Inorder to further prove the role of p38MAPK-PPARγ/NF-κB signal pathwayin brain ischemic tolerance induced by CIP, we observed the effects of p38MAPK AS-ODNs on brain ischemic tolerance induced by CIP and theexpressions of p38MAPK-PPARγ/NF-κB signal pathway proteins in thisexperiment.Method:Part1The changes of p-p38MAPK, PPARγ, NF-κB p50proteinexpression during the brain ischemic tolerance induced by CIP.Forty healthy male Wistar rats (280-300g) were used. At first, thebilateral vertebral arteries were occluded permanently. After recovery for twodays, the rats were divided into four groups randomly:①s ham group(n=10):the rats were subjected to exposed the bilateral common carotid arteries only,but not occluded the blood flow;②C IP group (n=10):the bilateral commoncarotid arteries were occluded for3min, and then reperfused with the bloodflow;③i schemic insult (II) group (n=10): the bilateral common carotidarteries were occluded for8min, then reperfused with the blood flow;④CIP+II group (n=10): the bilateral common carotid arteries were occluded for3min and then reperfused as CIP. After a interval for two days, the bilateralcommon carotid arteries were occluded for8min and then reperfused. Eachgroup was divided into6h and2d (n=5in each time point) subgroup. At thedetermined time point, the rats were decapitated and the hippocampal CA1region was separated for observation. Western blot analysis was used for theobservation of the expression of p-p38MAPK, PPARγ and NF-κB P50protein in hippocampal CA1subregion. Part2p38MAPK AS-ODNs inhibit the expression of PPARγ and NF-κBp50protein after CIP.Sixty healthy male Wistar rats (280-300g) were used. At first, thebilateral vertebral arteries were occluded permanently. After a recovery fortwo days, the rats were divided into four groups randomly:①sham group(n=10);②CIP group (n=10);③p38MAPK AS-ODNs+CIP group (n=30);④p38MAPK S-ODNs+CIP group (n=10); According to the dose of p38MAPK AS-ODNs used, group③was further divided into3subgroups (5noml,10noml and15noml). In group④, the dose of p38MAPK S-ODNswas15noml. Each group/subgroup was divided into two time points,6h and2d (n=5in each time point). p38MAPK AS-ODNs and p38MAPK S-ODNswere dissolved in15μl artificial cerebrospinal fluid (ACSF) and injected oncea day for5consecutive days from first day before the occlusion of the bilateralvertebral arteries to the day after CIP. The rats for6h time point of CIP groupwere only injected4times. At the determined time point, the rats weredecapitated and the hippocampal CA1region was separated for observation.Western blot analysis was used for the observation of the expression of p-p38MAPK, PPARγ and NF-κB P50protein in hippocampal CA1subregion.Part3p38MAPK AS-ODNs inhibit the upregulation of PPARγ andNF-κB p50protein expression induced during the brain ischemic toleranceinduced by CIP.Fifty healthy male Wistar rats (280-300g) we used. At first, the bilateralvertebral arteries were occluded permanently. After a recovery for two days,the rats were divided into three groups randomly:①C IP+II group (n=10);②p38MAPK AS-ODNs+CIP+II group (n=30);③t he p38MAPK S-ODNs+CIP+II group (n=10). According to the dose of AS-ODNs p38MAPK used,group②was further divided into3subgroups (5noml,10noml and15noml).In group③, the p38MAPK S-ODNs dose was15noml. Each group/subgroupwas divided into two time points,6h and2d (n=5in each time point). p38MAPK AS-ODNs and p38MAPK S-ODNs were dissolved in15μl ACSF andinjected once a day for5consecutive days from first day before the occlusion of the bilateral vertebral arteries to the day after CIP. At the determined timepoint, the rats were decapitated and the hippocampal CA1region wasseparated for observation. Western blot analysis was used for the observationof the expression of PPARγ and NF-κB P50protein in hippocampal CA1subregion.Part4The effect of p38MAPK AS-ODNs on brain ischemic toleranceinduced by CIP.Forty-five healthy male Wistar rats (280-300g) we used. At first, thebilateral vertebral arteries was occluded permanently. After a recovery for twodays, they were divided into seven groups randomly:①sham group (n=5);②CIP group (n=5);③group II (n=5);④CIP+II group (n=5);⑤ACSF+CIP+II group (n=5);⑥p38MAPK AS-ODNs+CIP+II group (n=15);⑦p38MAPK S-ODNs+CIP+II group (n=5). According to the design, p38MAPKAS-ODNs and p38MAPK S-ODNs were dissolved in15μl ACSF andinjected once a day for5consecutive days from first day before the occlusionof the bilateral vertebral arteries to the day after CIP. According to the dose ofp38MAPK AS-ODNs used, the group⑥was divided into3subgroups (5noml,10noml and15noml). In group⑦the dose of p38MAPK S-ODNswas15noml. All animals were decapitated7days after sham operation thelast global brain ischemia/reperfusion, and the hippocampal CA1region wasseparated to observe delayed neuronal death of the pyramidal neurons throughthionine staining.Results:Part1The changes of p-p38MAPK, PPARγ, NF-κB p50proteinexpression during the brain ischemic tolerance induced by CIP.Western blot analysis showed that, at6h time point, a basal levelexpression of p-p38MAPK, PPARγ and NF-κB p50protein were observed inthe rat hippocampal CA1region of sham group. Compared with the shamgroup, each of the expression of p-p38MAPK, PPARγ and NF-κB p50proteinwas up-regulated in the CIP group, and reached1.25times,1.28times and1.36times of its own basic value, respectively (P <0.05). After II, Each of the expression of p-p38MAPK, PPARγ and NF-κB p50protein showed moresignificant up-regulation in the II group, and reached1.64times,1.74timesand2.02times of its own basic value, respectively (P<0.01). Compared with IIgroup, each of the expression of p-p38MAPK, PPARγ and NF-κB p50proteinwas significantly decreased in the CIP+II group (P <0.05), indicated that CIPin advance could obviously inhibit the excessive upregulation of p-p38MAPK,PPARγ and NF-κB p50protein expression after II. At2d time point, thechange trends in the expression p-p38MAPK, PPARγ and NF-κB p50proteinin each group were consistent with those of6h time point. The data indicatedthat CIP could moderately induce upregulation of p-p38MAPK, PPARγ andNF-κB P50protein expression, and inhibit the excessive upregulation of p-p38MAPK, PPARγ and NF-κB p50protein expression after II.Part2p38MAPK AS-ODNs inhibit the expression of PPARγ and NF-κBp50protein after CIP.At6h time point, a basal level expression of PPARγ and NF-κB p50protein were observed in the rat hippocampal CA1region of sham group.Compared with the sham group, each of the expression of PPARγ and NF-κBp50protein was up-regulated in the CIP group (P<0.05). In the p38MAPKAS-ODNs+CIP group, both the expression of PPARγ and NF-κB p50proteinwere significantly reduced in a dose-dependent manner. Compared with theCIP group, neither the expression of PPARγ nor NF-κB p50protein changedsignificantly in the p38MAPK S-ODNs+CIP group (P>0.05). At2d timepoint, the change trends of PPARγ protein and NF-κB p50protein expressionin each group were consistent with those of6h time point. The data indicatedthat p38MAPK AS-ODNs dose-dependently inhibited the upregulation ofPPARγ and NF-κB P50protein expression induced by CIP.Part3p38MAPK AS-ODNs inhibit the upregulation of PPARγ andNF-κB p50protein expression induced during the brain ischemic toleranceinduced by CIP.At6h time point, compared with CIP+II group, both the expression ofPPARγ and NF-κB p50protein were significantly reduced in the p38MAPK AS-ODNs+CIP+II group, and the inhibition showed a significantdose-dependency with the dose of p38MAPK AS-ODNs. Compared with theCIP+II group, neither the expression of PPARγ nor NF-κB p50proteinchanged significantly in the p38MAPK S-ODNs+CIP+II group (P>0.05).At2d time point, the change trends of PPARγ protein and NF-κB p50proteinexpression in each group were consis-tent with those of6h time point. Thedata indicated that p38MAPK AS-ODNs dose-dependently inhibited theupregulation of PPARγ and NF-κB P50protein expression induced during theinduction of brain ischemic tolerance by CIP.Part4The effect of p38MAPK AS-ODNs on brain ischemic toleranceinduced by CIP.In the sham group, neuropathological evaluation showed that thepyramidal 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 delayed neuronal death (DND) wasobserved in the II group, almost all of the pyramidal neurons in thehippocampal CA1subfield were vanished, the HG was Ⅱ～Ⅲ grade and NDvalue was17.33±6.11mm-1, which significantly changed compared withthose in sham group (P＜0.01). In the CIP+II group, pyramidal neurons inthe hippocampal CA1subfield were arranged in order, which indicated thatCIP could obviously inhibited DND induced by ischemic insult. Comparedwith the II group, the ND value was178.67±6.11mm-1, increased distinctly (P＜0.05). In ACSF+CIP+II group, no obvious neuronal damage wasobserved, neither HG nor ND was different from that of CIP+II group(P>0.05). Compared with the ACSF+CIP+II group, significant DND in thehippocampal CA1subfield was observed in the p38MAPK AS-ODNs+CIP+II group. The HG was increased and ND was decreased. The content of DND showed a clear dose-dependency with the dose of p38MAPK AS-ODNs.In the p38MAPK S-ODNs+CIP+II group, no obvious neuronal damagewas observed in the hippocampal CA1subfield, compared with ACSF+CIP+II group, neither ND nor HG had obvious difference (P>0.05). The dataindicated that p38MAPK AS-ODNs dose-dependently blocked the brainischemic tolerance induced by CIP.Conclusion：p38MAPK-PPARγ/NF-κB signaling pathway involved inbrain ischemic tolerance induced by CIP.