The Role Of HIF-1α In The Cerebral Ischemic Tolerance Induced By Electroacupuncture Pretreatment

As the aging of the population in our country is a growing problem, the incidence ofischemic stroke has been increasing, while only a small part of stroke patients receivethrombolytic therapy because of a narrow time window and side effects of thethrombolytic agent, therefore, new strategies focusing on neuroprotection are urgentlyneeded. Electroacupuncture (EA) has been shown to produce clinically beneficial effectson stroke patients, EA pretreatment can also induce tolerance against cerebral ischemia.However, the signaling mechanisms mediating the effects of EA pretreatment are unclear.Hypoxia-inducible factor1α (HIF-1α) is a transcription factor that plays a key role inregulating the neuronal apoptosis and reconstruction of microcirculation after ischemia.The neuroprotective effects of HIF1-α and its downstream target genes have been widelyconcerned. But the role of HIF-1α in the neuroprotection of EA pretreatment and theregulation mechanism are currently unclear. In this study, we explore the expression ofHIF-1α before and after cerebral ischemia/reperfusion to investigate the role of HIF-1α inthe neuroprotection induced by EA pretreatment, and to verify the crosstalk between HIF-1α and Notch pathway in cerebral ischemia/reperfusion injury. The current study is toidentify the signals that mediate the neuroprotection of EA pretreatment, and toprovide new theoretical basis for the clinical application of EA pretreatment.Experiment1The effect of EA pretreatment on the expression of HIF1-α in focalcerebral ischemic injuryObjectives1. To study the influences of cerebral ischemia/reperfusion on the expression ofHIF1-α.2. To study the effect of EA pretreatment on the expression of HIF1-α before andafter cerebral ischemia/reperfusion.Methods96Male SD rats, weighing280~320g, were divided into two groups: Control(CON)and Electroacupuncture pretreatment(EA) groups (n=48each), Animals in EA group werepretreated with EA(1mA,2/15Hz,30min/d,5d). Twenty-four hours after the lastpretreatment, a transient middle cerebral artery occlusion (MCAO) was performed inanimals of CON and EA groups (n=40each). At24h after the last EA pretreatment and2h,6h,24h,48h and72h after ischemia/reperfusion, the rats (n=8for each time point) from2groups were anesthetized and killed. Realtime PCR(RT-PCR) was performed to examinethe level of HIF-1α and HO-1mRNA in ischemic penumbra at each time points. Westernblotting was performed to examine the expression of HIF-1α protein in ischemicpenumbra at each time points. Double immunofluorescence staining was performed todetect the distribution characteristics and cellular localization of HIF-1α in ischemicpenumbra.Results1. RT-PCR results showed that there was no significant differences between EA andCON groups in the mRNA level of HIF-1α or HO-1genes before MCAO (P>0.05). The HIF-1α or HO-1mRNA in EA group was higher at2h,6h or24h after reperfusion thanCON group (P<0.05). However, the HIF-1α or HO-1mRNA in EA group was lower at48hor72h after reperfusion than that in CON group (P<0.05).2. Western blot results showed that HIF-1α level was significantly elevated at2hafter reperfusion in EA group (P <0.05, vs. before I/R) and reached the maximum at48hafter reperfusion. Furthermore, the HIF-1α level of EAgroup was significantly higher thanthat in CON group at2h,6h,24h or48h after reperfusion(P<0.05).3. Double immunofluorescent staining showed that HIF-1α immunoreactivities werecolocalized with NeuN immunoreactivities, the number of HIF-1α or NeuN positive cellsin EA group was more than that in CON group at24h after reperfusion. Furthermore, thenumber of HIF-1α/NeuN double-labelled neurons in EA group was significantly more thanthat in CON group at24h after reperfusion.ConclusionEA pretreatment significantly increased HIF-1α expression in the ischemic penumbrafollowing reperfusion, and the effect of EA pretreatment on HIF-1α expression may beneuron-specific.Experiment2The effect of HIF-1α inhibitor2ME2on the neuroprotective effect of EApretreatmentObjectives1. To study the effect of HIF-1α inhibitor2ME2on the neuroprotective effect of EApretreatment.2. To study the role of HIF1-α in the tolerance against focal cerebral ischemiainduced by EA pretreatment.Methods80Male SD rats, weighing280~320g, were divided into five groups: Sham, CON, EA,2ME2and EA+2ME2groups (n=16each), Rats in Sham group were only given the same surgical procedure without arterial occlusion. Animals in EA and EA+2ME2groups werepretreated with EA (1mA,2/15Hz,30min/d,5d). The2ME2was given by intraperitonealinjection at30min before MCAO in2ME2and EA+2ME2groups. At24h afterischemia/reperfusion, the rats from5groups (n=8) were killed. TUNEL staining and theexpression of Bcl2and Bax were tested for neuronal apoptosis. At72h afterischemia/reperfusion, neurobehavioral evaluation and MRI were performed. Then the ratsfrom5groups (n=8) were killed, infarct assessment was performed with TTC staining.Results1. MRI results showed that there was no infarct volume in the Sham group inT1-weighted and T2-weighted images. T1-weighted images could not show infarct volumeclearly. At72h after reperfusion, the extent of brain edema in EA group was significantlyless than that in CON group in T2-weighted images (P<0.05).2. The neurological function was assessed with the Garcia scores. At72h afterreperfusion, EA pretreatment significantly improved the neurological function scorescompared with CON group (P<0.05). However, the neurological function scores inEA+2ME2group were significantly lower than those in EA group (P<0.05). At72h afterreperfusion, brain infarct volumes in EA pretreatment group were smaller compared withCON group (P<0.05). Interestingly, the infarction volumes in EA+2ME2group weresignificantly larger than those in EA group (P<0.05). There was no difference between theCON and2ME2groups.3. TUNEL staining showed that there was no TUNEL positive cell in the brainsections of sham group at24h after reperfusion. However, there were more TUNELpositive cells in the ischemic penumbra in CON and EA+2ME2groups than those in EAgroup at24h after reperfusion(P<0.05). There was no difference between the CON and2ME2groups (P>0.05).4. Western blot results showed that EA pretreatment markedly up-regulated theBcl-2level (P<0.05, vs CON) in the ischemic penumbra at24h after reperfusion, while thelevels of Bcl-2proteins in the ischemic penumbra in the EA+2ME2group were lower thanthose in EA group (P<0.05). There was no difference between the CON and2ME2groups (P>0.05). Meanwhile, the up-regulation of Bax in the ischemic penumbra was markedlyreduced by EA pretreatment (P<0.05, vs CON). However,2ME2can not affect theexpression of Bax protein.ConclusionThe neuroprotective effect induced by EA pretreatment can be alleviated by2ME2intervention. EA pretreatment significantly reduced the number of apoptotic neurons,up-regulated the anti-apoptotic protein of Bcl-2and down-regulated the pro-apoptoticprotein of Bax in the ischemic penumbra, which may be mediated by HIF-1α.Experiment3The interaction between HIF-1α and Notch pathway in focal cerebralischemic injuryObjectives1. To study the corelation between the activation of HIF-1α and Notch pathway aftercerebral ischemia/reperfusion.2. To study whether the neuroprotection of EA pretreatment induced byup-regulation of HIF-1α is associated with Notch pathway.Methods32Male SD rats, weighing280~320g, were divided into four groups: CON, EA,EA+MW167and EA+2ME2groups (n=8each), Animals in EA, EA+MW167andEA+2ME2groups were pretreated with EA(1mA,2/15Hz,30min/d,5d). MW167wasgiven by intraventricular injection at30min before MCAO in EA+MW167group, the2ME2was given by intraperitoneal injection at30min before MCAO in EA+2ME2group.At24h after ischemia/reperfusion, the rats from4groups were anesthetized and killed.Double immunofluorescence staining was performed to detect the distributioncharacteristics and cellular localization of HIF-1α and NICD in the ischemic penumbra.Western blotting was performed to examine the level of HIF-1α and NICD in the ischemicpenumbra. Results1. Double immunofluorescent staining showed that HIF-1α and Notch1NICD wereco-expressed in the ischemic penumbra. The number of HIF-1α or NICD positive cells inEA group was more than that in CON group in the ischemic penumbra at24h afterreperfusion(P<0.05). Furthermore, the number of HIF-1α/NICD double-labeled cells inEA group was more than that in CON group in the ischemic penumbra at24h afterreperfusion (P<0.05).2. Western blot results showed that the expression of HIF-1α and NICD protein inEA group in ischemic penumbra was significantly higher than that in CON group at24hafter reperfusion (P<0.05). However, MW167which can inhibit the activation of Notchpathway decreased the expression of HIF-1α and NICD (P<0.05, EA+MW167vs EA).Furthermore,2ME2can only inhibit the expression of HIF-1α (P<0.05, EA+2ME2vs EA),but had no effect on the expression of NICD (P>0.05, EA+2ME2vs EA).ConclusionBoth of HIF-1α and Notch pathway were activated after cerebralischemia/reperfusion. Inhibition of Notch signal suppressed the expression of HIF-1α inischemic penumbra. EA pretreatment affords strong protection against transient cerebralischemic injury, and increases protein expression and activation of HIF-1α in rats, thesebeneficial effects of EA pretreatment may be mediated by activation of Notch pathway.