Study On The Role Of PI3K/Akt Pathway In The Neuroprotection Induced By Electroacupuncture Pretreatment

Background: Our country will enter an Aging Society! New data shows thatthe number of elderly people over the age of80will be nearly four times of thatis now by2050. Ischemic stroke is "the leading killer" of the middle aged andelderly people. But now there are not effective methods to prevent and cure it.Therefore it makes perfect clinical and social value to prevent and alleviate thebrain damage caused by cerebral ischemia. In recent years, it has been found anumber of different stimuli can induce a similar effect of ischemic tolerance,electroacupuncture(EA) included. EA has many advantages in clinical applicationsuch as security, convenience and simplicity, but it rests a riddle about itsmechanism. Mounting findings suggest that Notch pathway may be involved inthe pathological changes that occur in cerebral ischemia and our preliminarystudy indicates that Notch pathway perform an important part in EA pretreatment; PI3K/Akt pathway is recognized as an antiapoptotic pathway by which theneuron survive and EA also could activate it; Notch pathway and PI3K/Aktpathway form a delicate balance in kinds of cells, including neural stem cells, toregulate proliferation, differentiation and survival. Then a boldness of conceptioncomes that EA pretreatment may protect the adult from ischemia/reperfusionthrough Notch/PI3K/Akt pathway.In this study, first we investigate the role of PI3K/Akt pathway activation onthe neuroprotection induced by EA pretreatment; then detect if PI3K/Akt is thedownstream of the Notch pathway during the process of EA precondition.Part1The effect of EA pretreatment on the activity of Notch andPI3K/Akt pathway in ischemic cortex after cerebral ischemia/reperfusion1Method1.1The effect of EA pretreatment on expression of Notch1-NICD andpAkt(Ser473)18Male SD rats were divided into two groups: Con group and EA group(n=9each). Animals in EA group were pretreated with EA (1mA,2/15Hz,30min/d,5d) or only intraperitoneal injection (40mg/kg) of pentobarbital sodium.24h after the last pretreatment, all the animals both in Con group and EA groupreceived the surgeon of middle cerebral artery occlusion (MCAO) for120min.Rats were killed just before ischemia,2h or24h after the infusion(n=3at eachtime points). Western blot was performed to detect the difference of protein level of Notch pathway between the two groups at each time points in ischemic cortex,the same as PI3K/Akt pathway.1.2The distribution characteristics and cellular localization of Notch1-NICDand pAkt(Ser473) after cerebral ischemia/reperfusion18male SD rats were divided into two groups (n=9each) as described in1.1,and also received the same treatment. NICD/NeuN double-stainingimmunohistochemistry was performed to detect the Notch1-NICD positive cells;and pAkt/NeuN for the pAkt positive cells.2Result2.1The effect of EA pretreatment on the activity of Notch pathway inischemic cortex after cerebral ischemia/reperfusionNICD is regarded as the activation maker of Notch pathway. Western blotanalysis revealed that before reperfusion and2h after reperfusion, there is nostatistically significant difference in Notch1-NICD level in cortex between Congroup and EA group; Notch1-NICD level began to rise at24h after reperfusionin EA group and was significantly higher than control group(P<0.05).The similar phenomenon could be found by double immunofluorescentstaining. And Notch1-NICD was partially localized to cytoplasm and nuclei ofNeuN positive cells, which is the marker of neuron.2.2The effect of EA pretreatment on the activity of PI3K/Akt pathway inischemic cortex after cerebral ischemia/reperfusionWestern blot analysis revealed that before reperfusion, there issignificant difference in pAkt(Ser473) level in cortex between Con groupand EA group; It reached the peak at2h and remained high at24h afterreperfusion. Date revealed that at24h after reperfusion, there is no statisticallysignificant difference in pAkt(Ser473) level in cortex between Con group and EAgroup, but still higher than Con group before ischemia(P<0.05). A proportion of pAkt positive cells overlapped with NeuN positive cell, andpAkt was mostly localized to cytoplasm of neuon.Part2The effect of inhibition activation of PI3K/Akt pathwayon the tolerance against focal cerebral ischemia induced byEA pretreatment1Method1.1Confirmation the method of intraventricular injection2SD rats intraventricularly infused with methylene blue (MB) and weredecapitated15min later. Take out the brain and observe the distribution of bluearea.1.2The effect of LY294002on blocking PI3K/Akt pathway after EApretreatment in vivo9male SD rats were randomized into3groups (n=3each): Sham group, Congroup and LY294002group. Both EA group and LY294002group receivedtransient MCAO surgeon; LY294002group received intracerebroventricular(i.c.v) injection of10mM LY2940021h before ischemia. At2h after reperfusion,all the rats were decapitated. The level of pAkt(Ser473) expression in ischemiccortex was examined by Western blot.1.3The effect of PI3K/Akt inhibitor LY294002on the tolerance against focalcerebral ischemia induced by EA pretreatment40male SD rats were randomized into5groups (n=8each): Sham group,Con group, EA group, LY294002group and Vehicle group. Vehicle groupreceived the same treatment as LY294002group in1, except i.c.v injection of DMSO to substitute LY294002. The Neurological deficit scores (NDS) wereassessed at24h after reperfusion. Then all the animals were decapitated, thecerebral infarct volumes were measured with2%2,3,5-triphenytetrazoliumchloride (TTC) staining.2Result2.1The method of LY294002injection is rightWestern blot analysis of LY294002and Con groups revealed that pAkt levelin LY294002group at2h after reperfusion was significantly lower than that inCon group (P<0.05). The injection of LY294002i.c.v (1.5mm lateral,0.8mmposterior,3.8mm deep)1h before I/R could successfully block the activation ofPI3K/Akt pathway.2.2Intraventricular injections with the PI3K/Akt inhibitor (LY294002)attenuated the neuroprotective effect of EA pretreatment.EA pretreatment significantly increased neurological function scores andreduced infarct volumes compared with Con group (P<0.05).Neurological function scores in LY294002group were lower than those inMCAO group (P<0.05), but infarct volumes is bigger (P<0.05). There is nostatistically significant difference between Vehicle group and EA group in scoresand infarct volumes.Part3The effect of inhibiting Notch pathway on the PI3K/Aktpathway when focal cerebral ischemia after EA pretreatment 1Method1.1The effect of DAPT on block of Notch pathway after EA pretreatment12male SD rats, weighting280-320g, were randomized into4groups (n=3each): Sham, EA, EVM and EDM groups. Rats in EA, EVM and EDM groupswere subjected to cerebral ischemia/infusion at24hours after the last EApretreatment, animals in EVM and EDM groups were Intraventricularly infusedwith DMSO and DAPT respectively3h before MCAO. The brains wereharvested at24h after reperfusion. Notch1-NICD expression in ischemic cortexwas examined by Western blot.1.2The effect of DAPT on influencing PI3K/Akt pathway after EApretrearmentWith the same protein harvested from the rats in1.1, the level of Aktphosphorylation in ischemic cortex were examined by Western blot.2Result2.1DAPT could effectively inhibit the activation of Notch pathway.The level of NICD in cortex in EDM group is evidently lower than that inother group (P<0.05), it demonstrates that DAPT could successfully block theNotch pathway.2.2DAPT down regulate PI3K/Akt signaling pathwayThere is a marked decrease of pAkt expression in EDM group comparedwith EVM group and EA group (P<0.05), but still higher than that in Sham group(P<0.05).3Conclusions3.1EA could activate PI3K/Akt pathway in adult rats’ brain, as well as cerebralischemia/reperfusion; what’s more, EA pretreatment up-regulate it in ischemiacortex.3.2EA pretreatment could induce ischemia tolerance against I/R, which will be weakened by blocking PI3K/Akt pathway. Perhaps EA pretreatment protect thebrain from I/R injury through PI3K/Akt pathway.3.3During the process of EA pretreatment, Notch and PI3K/Akt have a goodcompanionship in the neuron of ischemia cortex; PI3K/Akt pathway will beinhibited by blocking Notch; in this case, PI3K/Akt pathway is likely to be thedownstream of Notch pathway. So, it is possible that Notch/PI3K/Akt mediatesthe neuroprotection of EA pretreatment.