| Cerebral ischemic disease is the greatest cause of death in China. Oxidative stress induced bymitochondria dysfunction plays a key role in the pathogenesis during ischemic/reperfusioninjury. SOD2, as known as manganese superoxide dismutase, is an important antioxidantenzyme to attenuate the mitochondrial oxidative stress. The SOD2activation could reduceexperimental ischemic injury significantly. However, SOD2activation involved the cerebralischemic tolerance is unknown. As a healing art in traditional Chinese medicine,electroacupuncture (EA) administration is safe, reliable, effective and easy to operate, andits effect has been acknowledged worldwide. Our previous studies have demonstrated thatEA pretreatment elicits the neuroprotective effect against cerebral ischemic injury throughcannabinoid receptor type1receptor (CB1R) and its mediated protective signaling pathwayssuch as pro-survival pathway the signal transducer and activator of transcription3(STAT3). And some studies have also confirmed that neuroprotective effect of EA pretreatment wasrelated to the antioxidant systems. But whether EA pretreatment SOD2signal by activatingthe brain play a protective effect is not clear In the present study, we tried to investigatewhether manganese superoxide dismutase were involved in the EA pretreatment via CB1Ractivation in stroke mice and whether CB1R mediated SOD2activation via STAT3phosphorylation. Experiment1Effect of EA pretreatment on SOD2expression after cerebralischemia-reperfusion in miceObjectivesTo testify whether EA pretreatment could increase the expression of SOD2after cerebralischemia-reperfusion in C57B/6mice.Methods24male C57BL/6mice were divided into4groups randomly: sham operation group (shamgroup), a singlle electro-acupuncture pretreated group (EA group), middle cerebral arteryocclusion ischemic model group (MCAO group), EA pretreatment plus MCAO group (EA+MCAO group). In sham group, mice were injected with10%chloral hydrate (350mg/kg,i.p.) and than were induced sham surgery; in EA group, after the injection of10%chloralhydrate anesthesia intraperitonealy, mice were subjected to EA pretreatment for30minutesonly; in MCAO group, after anesthesia, mice were subjected to a transient middle cerebralartery occlusion model by a monofilament for1h, then the filament was withdrawn to letthe cerebral blood flow recover; in EA+MCAO group, mice were treated with EApretreatment, and then subject to transient MCAO model at2h after pretreatment. Theconduction of western-blot and immunofluorescence were performed at2h after reperfusionto measure the SOD2protein expression. Results1. EA pretreatment up-regulated SOD2protein expression2h after cerebralischemia/reperfusion.There is no statistic difference of SOD2protein expression between sham and EA groupwithout MCAO injury. However, EA pretreatment increased the expression of SOD2ascompared with MCAO group at2h after reperfusion (P<0.05).2. EA pretreatment increase the SOD2positive neurons2h after cerebralischemia/reperfusion.The immunohistochemical staining to investigate the location of SOD2in ischemicpenumbra; SOD2was mainly co-located with neuronal maker NeuN. Consisting with thewestern-blot result, the SOD2positive neurons in EA+MCAO groups were more than thatin MCAO group at2h after reperfusion.ConclusionEA pretreatment up-regulated the SOD2protein expression and increased SOD2positiveneuronal cells at2h after reperfusion.Experiment2SOD2up-regulation was involved in the antioxidant and neuroprotectiveeffects of EA pretreatmentObjectives1. To investigate the deficit of SOD2by administrating SOD2-siRNA could reverse theantioxidant effects of EA pretreatment.2. To determine whether SOD2-siRNA could reverse the neuroprotective effect of EApretreatment.Methods1. The verify of SOD2-siRNA interference performance24male C57B/6mice were divided into4groups: sham group (sham group), a free waterinjection group (vehicle group), SOD2-siRNA group and control siRNA group. In sham group, after intraperitoneal injection of10%chloral hydrate, mice were only carried outwith sham operation; in SOD2-siRNA group, control siRNA group, vehicle group, micewere anesthetized with10%chloral hydrate, then they were given SOD2small interferenceRNA, scramble SOD2small interference RNA control siRNA and solvent respectively byintracerebroventricular injection. The tissue was taken as ischemic penumbra area at72hafter intracerebroventricular injection for western-blot to measure the change of SOD2expression.2. To testify whether the deficiency of SOD2by SOD2-siRNA reversed the antioxidanteffect of EA pretreatment.The46male C57B/6mice were randomly divided into six groups: sham operation group(sham group), a single electro-acupuncture pretreated group (EA group), ischemic modelgroup (MCAO group), EA pretreatment group (EA+MCAO group), SOD2-siRNA group(EA+SOD2-siRNA group) and control siRNA group (EA+control siRNA group). In whichsham group, MCAO group, EA group, EA+MCAO group, the approach were performed asmentioned earlier; in EA+SOD2-siRNA group or EA+control siRNA groups, mice wereanesthetized with intraperitoneal injection of10%chloral hydrate, and then administratedwith SOD2-siRNA or control siRNA respectively by intracerebroventricular injection. At72h after administration, mice were conducted to MCAO ischemic injury. At24h afterischemia-reperfusion, tissue from ischemic penumbra area was taken to detect the content ofROS, MDA,8-OH-dG and nitrotyrosine by Elisa. The evaluation of super oxidants wasperformed by DHE satining also.3. Whether the supplementation of SOD2-siRNA reversed the neuroprotective effect ofEA pretreatment.The36male C57BL/6mice were divided into six groups: sham operation group (shamgroup), a single electro-acupuncture group (EA group), ischemic/reperfusion model group(MCAO group), EA pretreatment plus MCAO group (EA+MCAO group), SOD2-siRNAgroup (EA+SOD2-siRNA group) and control siRNA group (EA+control siRNA group).The paradigm was according as described above. At24h after ischemia-reperfusion,neurobehavioral score (Longa score) was examined and then TTC staining was conducted to measure the infarct volume and TUNEL staining were performed to examine the level ofapoptosis cells.Results1. SOD2-siRNA can significantly reduce SOD2expression in brainTo determine the involvement of SOD2in the neuroprotective effect induced by EApretreatment, we need to drive the inactivation of SOD2. Thus, we used the SOD2-siRNA todeficiency the expression of SOD2. By western-blot analysis, we showed SOD2-siRNAreduced effectively the expression of SOD2(P<0.05).2. SOD2-siRNA reversed EA pretreatment induced antioxidant effectsThe oxidative stress was measured by oxidative productions ROS, MDA,8-OHdG andnitrotyrosine at24h after reperfusion. EA pretreatment significantly reduced the generationof ROS, MDA,8-OHdG and nitrotyrosine compared with those productions in MCAOgroup (P<0.05). The siRNA microinjection72h before the onset of each EA pretreatmentreversed the antioxidant effect of EA pretreatment, the contents of ROS, MDA,8-OHdGand nitrotyrosine in EA+SOD2-siRNA group were similar as those in MCAO group.Expectably, there were no statistical differences was detected among EA+MCAO and EA+control siRNA group. DHE staining was also used to detect the crucial role of SOD2againstthe generation of super oxidants after ischemia/reperfusion. The result of staining showed areduction in the EA+MCAO group as compared with MCAO group. Not surprisingly, thelevel of DHE oxidation in EA+SOD2-siRNA group appeared to increase as compared withEA+MCAO group. Exposed to control siRNA of EA pretreated animals caused no changein DHE oxidation compared with EA+MCAO group.3. SOD2-siRNA reversed the neuroprotective effect of EA pretreatmentAs expected, the deficiency of SOD2abolished the reduction in infarct volume under thecondition of EA pretreatment as compared with EA+MCAO group (P<0.05). Additionally,SOD2siRNA reversed the improvement in neurological outcome induced by EApretreatment in EA+SOD2-siRNA group vs the EA+MCAO group (P<0.05). The EA+control siRNA showed no statistically significant differences in neurological scores andinfarct volume with those in EA+MCAO group. Cellular apoptosis was determined by TUNEL staining. The number of TUNEL positive cells in MCAO group was increased ascompared to sham group. EA pretreatment reduced the number of apoptotic cells ascompared with the MCAO group (P<0.05). The SOD2knockdown by siRNA increased thenumber of TUNEL positive cells in EA+SOD2-siRNA groups as compared withEA+MCAO group (P<0.05).ConclusionEA pretreatment reduced oxidative stress, inhibited cellular apoptosis and affordedneuroprotective effect against cerebral ischemia-reperfusion damage through theup-regulation of SOD2expression in rats.Experiment3EA pretreatment up-regulated SOD2via cannabinoid CB1R-mediatedSTAT3phosphorylationObjectives1. To determine the effect of CB1receptor antagonist AM251, SR141716on EApretreatment regulated SOD2protein expression;2. To investigate the effect of CB1receptor agonist ACEA, WIN55,212-2on theexpression of SOD2.3. To determine the effect of EA pretreatment on the phosphorylation level (Y705) ofSTAT3after cerebral ischemic/reperfusion in mice;4. To determine the effect of CB1receptor antagonist AM251, SR141716on EApretreatment regulated STAT3phosphorylation;5. To investigate the effect of CB1receptor agonist ACEA, WIN55,212-2on thephosphorylation of STAT3.Methods1. Testify whether CB1receptor antagonist AM251, SR141716reversed up-regulatedSOD2protein expression induced by EA pretreatment.The30male C57B/6mice were divided into five groups: ischemic model group (MCAO group), EA pretreatment group (EA+MCAO group), AM251group (EA+AM251group),SR141716group (EA+SR141716group), vehicle group (EA+vehicle group). The protocolof MCAO group, EA+MCAO group was mentioned earlier. The agents CB1R antagonistAM251and SR141716were dissolved in DMSO and Tween-80respectively, followed bydilution in saline. In EA+AM251and EA+SR141716groups, the does of1mg/kg waschosen for both two antagonist based on detailed data. The administration was performed at30min after EA pretreatment and then mice were subjected to ischemic injury. At2h afterreperfusion, tissue from ischemic penumbra area was collected for the assessment of SOD2protein expression by western-blot.2. To determine the effect of CB1receptor agonist ACEA and WIN55,212-2on the SOD2expression.The30male C57B/6mice were divided into five groups: ischemic model group (MCAOgroup), EA pretreatment group (EA+MCAO group), ACEA group (MCAO+ACEA group),WIN55,212-2group (MCAO+WIN group), vehicle group (MCAO+vehicle group). InMCAO group, EA+MCAO group, the paradigm was according the protocol previously. InMCAO+ACEA group, MCAO+WIN group, and MCAO+vehicle group: mice were givenACEA (2.5mg/kg), the WIN55,212-2(1.5mg/kg) and solvent by intraperitoneal injection30min before MCAO operation. At2h after reperfusion, tissue from ischemic penumbraarea was collected for the detection of SOD2protein expression by western-blot.3. To determine the effect of EA pretreatment on the phosphorylation levels of STAT3aftercerebral ischemic/reperfusion in mice.The24male C57BL/6mice were divided into4groups: sham operation group (shamgroup), a single electro-acupuncture pretreatment group (EA group), ischemic model group(MCAO group), EA pretreatment group (EA+MCAO group). The paradigm was performedaccording which described above. At2h after reperfusion, western-blot was taken toexamine the STAT3phosphorylation level at Y705in each group.4. To determine the effect of CB1receptor antagonist AM251, SR141716on EApretreatment regulated STAT3activation.The30male C57B/6mice were divided into five groups: ischemic model group (MCAO group), EA pretreatment group (EA+MCAO group), AM251group (EA+AM251group),SR141716group (EA+SR141716group), vehicle group (EA+vehicle group). The protocolof five groups was performed as which mentioned above. At2h after reperfusion, tissuefrom ischemic penumbra area was collected for the detection of STAT3phosphorylationlevels by western-blot.5. To determine the effect of CB1receptor agonist ACEA and WIN55,212-2on the SOD2expression.The30male C57B/6mice were divided into five groups: ischemic model group (MCAOgroup), EA pretreatment group (EA+MCAO group), ACEA group (MCAO+ACEA group),WIN55,212-2group (MCAO+WIN group), vehicle group (MCAO+vehicle group). Theparadigm was according the protocol previously. At2h after reperfusion, tissue fromischemic penumbra area was collected for the detection of STAT3phosphorylation level bywestern-blot.Results1. CB1receptor antagonist AM251, SR141716can reverse the up-regulated expression ofSOD2induced by EA pretreatment.The expressions of SOD2were decreased in EA+AM251treated group and inEA+SR141716group compared with that in EA+MCAO group (P<0.05). However, noeffect on SOD2expression was found in the EA+vehicle treated group.2. CB1receptor agonist ACEA, WIN55,212-2can up-regulate the content of SOD2.The administration of ACEA and WIN55,212-2mimicked the up-regulation of SOD2inMCAO+ACEA treated group and in MCAO+WIN group compared with MCAO group(P<0.05). No significant change was detected in MCAO+vehicle treated group as comparedwith MCAO group.3. EA pretreatment promoted STAT3phosphorylation level.There is no statistic difference of STAT3activation between sham and EA group with outMCAO injury. EA increased the phosphorylation of STAT3at Y705as compared withMCAO group at2h after reperfusion (P<0.05). However, no significant change wasdetected of total expression of STAT3among all groups. 4. CB1receptor antagonist AM251, SR141716can reverse the up-regulated STAT3phosphorylation induced by EA pretreatment.The STAT3phosphorylation level were decreased in EA+AM251treated group and inEA+SR141716group compared with that in EA+MCAO group (P<0.05). However, noeffect on STAT3phosphorylation was found in the EA+vehicle treated group.5. CB1receptor agonist ACEA, WIN55,212-2can up-regulate the phosphorylation ofSTAT3.The administration of ACEA and WIN55,212-2mimicked the up-regulation of STAT3phosphorylation level in MCAO+ACEA treated group and in MCAO+WIN groupcompared with MCAO group (P<0.05). No significant change was detected inMCAO+vehicle treated group as compared with MCAO group.ConclusionEA pretreatment up-regulated SOD2expression through CB1receptor-mediated STAT3phosphorylation in cerebral ischemic mice.SummeryIn current study, ischemic stroke injury was induced by a transient middle cerebralartery occlusion for60min in C57BL/6mice at2h after EA pretreatment. EA pretreatmentup-regulated the SOD2protein expression and increased SOD2positive neuronal cells at2hafter reperfusion. EA pretreatment attenuated the generation of oxidative injury productsand also inhibited the cellular apoptosis to induce a neuroprotective effect against ischemicdamage. However these beneficial effects of EA pretreatment were reversed by knockdownof SOD2. Furthermore, we testified the mechanism of SOD2activation mediated by EApretreatment. Our studies confirmed that cannabinoid CB1receptor mediated SOD2up-regulation by EA pretreatment attenuates ischemic oxidative damage via STAT3phosphorylation in stroke mice, which may represent a new mechanism of EApretreatment-induced neuroprotection against cerebral ischemia in mice. This investigationsupports a potential of the clinical application of activating SOD2to defend ischemic strokedamage. |
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