| Stroke is one of the leading causes of long-term disability and the first leading causeof death in China，and the number has increased year by year. Epidemiological studieshave shown that there are1,600,000to2,000,000new cases of stroke every year, andamong7,000,000cases of this diease we are confronting now,70%of them are ischemiastroke patients. Cerebral ischemia reperfusion injury can cause more serious injury ofbrain cell after the reperfusion. Patients with ischemia stroke could have a neurologicaldeficit and the prognosis is very poor. Although recombinant tissue plasminogen activator(rtPA) has been shown to be an effective drug to treat stroke, it must be administeredwithin3.5hours of stroke onset, and only a few patients (less than3%) can benefit from this therapy. The physical, emotional, and financial toll stroke inflicts on the patients andtheir families cannot be overstated. Therefore, the development of new methods for strokeintervention is a major imperative. We have found that electroacupuncture could inducethe ischemia tolerance after the cerebral ischemia reperfusion injury, but the exactmechanism of this phenomenon still need to be investigated. It is well-known that signaltransducer and activator of transcription3(STAT3) plays an essential role in cell survivaland proliferation. Therefore, we investigated whether STAT3is involved in EApretreatment-induced neuroprotection via cannabinoid CB1receptors (CB1R) aftertransient focal cerebral ischemia in rats. This study was divided into two parts, in part onethe role of STAT3activation in EA pretreatment induced ischemia tolerance after cerebralischemia/reperfusion injury was investigated, while the mechanism between EApretreatment and the activation of STAT3was investigated in part two.Experiment1Role of STAT3activation in EA pretreatment induced ischemia tolerance aftercerebral ischemia/reperfusion injuryMethods1． The neuroprotect effects of EA pretreatment in cerebral ischemia/reperfusioninjury24male SD rats were divided into3groups: Sham, MCAO, and EA groups. In EAgroup, rats were given EA pretreatment (30minutes,1~2mA,2~15hz)2hours before theMCAO operation. Animals in MCAO and EA groups were subjected to MACO for2hours and were killed at72hours after reperfusion. Neurobehavioral evaluation andinfarct assessment were used to detect the neuroprotection effect of EA pretreatment.2． Changes of the STAT3activation after EA pretreatment20male SD rats were divided into3groups as described in1. At6and24hours afterreperfusion animals were killed, and western blot were used to detect the expression ofpSTAT3(Ser727).3． The distribution characteristics and cellular localization of pSTAT3aftercerebral ischemia/reperfusion 12male rats were divided into3groups as described in1. At6hours afterreperfusion, NeuN/pSTAT3(Ser727) double-staining immunohistochemistry wereperformed to detect the pSTAT3positive cells.Experement2Changes of phosphorylated STAT3in EA pretreatment induced ischemia tolerance1． The effect of PpYLKTK on the expression of pSTAT3(Ser727)12male rats were divided into3groups: Sham, EA+Pp and EA+PBS groups.At6hours after reperfusion, western blot was used to detect the expression ofpSTAT3(Ser727).2． The effect of STAT3activation on the neuroprotection of EA pretreatment aftercerebral ischemia/reperfusion injury32male rats were divided into4groups: Sham, MCAO, EA+PBS and EA+Pp groups.At72hours after reperfusion, neurobehavioral evaluation and infarct assessment wereused to detect the effect of PpYLKTK on the neuroprotection induced by EA pretreatment.Experiment3The effect of STAT3activation on brain cell apoptosis after cerebralischemia/reperfusion injury16male rats were divided into3groups as described in5. At24hours afterreperfusion, TUNEL and caspase-3staining were used to detect the effect of STAT3activation on brain cell apoptosis after cerebral ischemia/reperfusion injury.16male rats were divided into3groups as described in5. At24hours afterreperfusion, western blot was used to detect the effect of STAT3activation on theexpression of apoptosis protein.Experiment4The regulation of the STAT3phosphorylation in EA pretreatment32male rats were divided into8groups: MCAO, EA, EA+AM251and EA+vehiclegroups are used to detect the effect of AM251on the STAT3activation;MCAO, EA,EA+CB1-siRNA and EA+control-siRNA groups are used to detect the effect ofCB1-siRNA on the STAT3activation. At6hours after reperfusion, western blot was used to detect the expression of pSTAT3(Ser727).24male rats were divided into6groups: MCAO, MCAO+WIN, MCAO+vehiclegroups are used to detect the effect of WIN55,212-2on the STAT3activation; MCAO,MCAO+ACEA, MCAO+vehicle groups are used to detect the effect of ACEA on theSTAT3activation. At6hours after reperfusion, western blot was used to detect theexpression of pSTAT3(Ser727).Results1． The neuroprotection of EA pretreatment on cerebral ischemia/reperfusioninjury and the changes of the expression of pSTAT3(Ser727) after EApretreatmentInfarct volume in the EA group was significantly reduced compared with the MCAOgroup72hours after reperfusion (P<0.001), and pretreatment with EA improved theneurological scores (P<0.01).The semi-quantitative analysis of the Western blots indicated that at6hours afterreperfusion, the content of pSTAT3(Ser727) in the peri-ischemic penumbra of the EAgroup was significantly higher compared with the Sham and MCAO groups. In addition,compared with Sham group the content of pSTAT3(Ser727) in the peri-ischemic penumbraof the MCAO and EA group was significantly increased24hours after reperfusion, but nodifference was found in the content of pSTAT3(Ser727) between EA and MCAO groups24hours after reperfusion.Double immunofluorescence staining revealed that pSTAT3(Ser727)-positive cellsco-localized with NeuN positive neurons.2． The effect of STAT3activation on the neuroprotection of EA pretreatmentIncreased phosphorylation of pSTAT3(Ser727) was blocked significantly by theinjection of PpYLKTK (Pp)30minutes before focal cerebral ischemia in the EA+Ppgroup compared with the EA+PBS group (P<0.001).EA pretreatment significantly decreased infarct volume at72hours after reperfusionat72hours after reperfusion when compared with the MCAO and EA+Pp groups. EApretreatment increased the neurological scores at72hours after reperfusion compared with the MCAO and EA+Pp groups and there were no statistical differences in infarct volumeand neurological scores between MCAO group and EA+Pp group.3. The effect of STAT3activation on the apoptosis of brain cells after cerebralischemia reperfusion injuryIn the EA group, the number of TUNEL and caspase-3positive cells in theperi-ischemic penumbra was significantly decreased compared with the MCAO andEA+Pp groups.At24hours after reperfusion, the ratio of Bax to Bcl-2in the peri-ischemic penumbraof rats in the MCAO group was higher than that in the Sham group. Compared with ratsonly subjected to MCAO, EA pretreatment markedly downregulated the ratio of Bax toBcl-2. Interestingly, PpYLKTK administration before ischemia clearly suppressed theincrease in Bcl-2protein contents by EA pretreatment.4. The mechanism of the regulation between EA pretreatment and thephosphorylation of STAT3At6hours after reperfusion, the expression of pSTAT3(Ser727) in the EA+AM251group was significantly decreased compared with the EA and EA+vehicle groups, but nosignificant difference was found compared with the MCAO group. The expression ofpSTAT3(Ser727) in the EA+CB1R siRNA group was significantly decreased comparedwith the EA and EA+control siRNA groupsThe expression of pSTAT3(Ser727) in the MCAO+WIN55,212-2group increasedcompared with the MCAO and MCAO+vehicle groups. The expression ofpSTAT3(Ser727) in the MCAO+ACEA group was increased compared with the MCAOand MCAO+vehicle groups.ConclusionActivation of STAT3is involved in neuroprotection by electroacupuncturepretreatment via cannabinoid CB1receptors in rats. This study tell more about themechanism of the neuroprotection from EA pretreatment, and it wil be valuable to theclinical study on EA pretreatment. |
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