| Methods102 SPF adult male SD rats,randomly divided into 6 groups :Control group(group C,n = 12),ischemia-reperfusion group(group I/R,n = 42),low dose HCQ pretreatment group(group HCQ250,n = 12),medium dose HCQ pretreatment group(group HCQ500,n = 12),high dose HCQ pretreatment group(group HCQ1000,n =12)and extracellular regulated protein kinase 1/2(ERK1/2)inhibitor U0126 pretreatment group(group U0126,n = 12).Filamentous occlusion of the middle cerebral artery(MCAO)was used to establish rat ischemic stroke model.Group C was the same as group I/R,but not occluded the middle cerebral artery;group I/R was reperfused after MCAO for 2 h;group HCQ250,group HCQ500,group HCQ1000 and group U0126 were injected with 8 μL 250 μM,500 μM,1000 μM and 1000 μM HCQ by lateral ventricle(LV)at 24 h,48 h and 72 h,respectively,before MCAO,and U0126 group was injected with 8 μL of 1000 μM U0126 after 12 h each injection of HCQ.In the I/R group,brain was taken from the head of the rats after perfusion of MCAO for 2 h and then reperfusion at 0 h(T1),3h(T2),6h(T3),12h(T4)and 24h(T5),Detect changes in the expression of ERK1/2 and p-ERK1/2 of the Ischemic penumbra.The brains were decapitated at 6 h after reperfusion.Western blot was used to detect expression of ERK1/2,p-ERK1/2,anti-apoptotic factor Bcl-2,proapoptotic factor cleaved caspase-3,phosphorylated CREB CREB(p-CREB)and Phosphorylated Akt(p-Akt)in ischemic penumbra;Evaluation of neurological deficits after 24 and 72 h of reperfusion using a standard neurological deficit score scale,followed by brains were decapitated,then cerebral infarct volume was measured using2% TTC staining.Results(1)Compared with T1,the expression of p-ERK1/2 was up-regulated in I/R group T2(P=0.001),and the expression of p-ERK1/2 was up-regulated in ischemic penumbra T3~5(P<0.001);When T2 was compared,the expression of p-ERK1/2 in the ischemic penumbra was up-regulated in I/R group T3(P=0.025).(2)Compared with control group,neurological deficit score was reduced,and cerebral infarct volume was magnified in I/R group,HCQ250 group,HCQ500 group,HCQ1000 group and U0126 group(P < 0.05);Compared with I/R group,neurological deficit scores increased,and the volume of cerebral infarction decreased in HCQ250 group,HCQ500 group and the HCQ1000 group(P < 0.05);Compared with the HCQ1000 group,the neurological deficit score of the U0126 group decreased,and the volume of the cerebral infarction increased in U group(P < 0.05).(3)Compared with group C,the expression of p-ERK1/2 and cleaved caspase-3in I/R group,HCQ250 group,HCQ500 group,HCQ1000 group and U0126 group increased,and the expression of Bcl-2 decreased(P < 0.05);Compared with the I/R group,the expression of p-ERK1/2,Bcl-2,p-CREB and p-Akt was increased and cleaved caspase-3 expression was decreased in HCQ1000 group(P < 0.05).Compared with HCQ1000 group,The expression of p-ERK1/ 2,Bcl-2,p-CREB and p-Akt decreased,and the expression of cleaved caspase-3 increased in U0126 group(P <0.05).(4)Compared with group C,the expression of p-CREB and p-Akt in I/R group,HCQ250 group,HCQ500 group,HCQ1000 group and U0126 group increased,and the expression of Bcl-2 decreased(P < 0.05);Compared with I/R group,the expression of p-CREB and p-Akt increased in HCQ1000 group(P < 0.05).Compared with HCQ1000 group,the expression of p-CREB and p-Akt in U0126 group decreased(P < 0.05).Conclusion(1)The expression of p-ERK1/2 increased with the increase of reperfusion time after cerebral ischemia-reperfusion in rats;(2)Pretreatment with hydroxylated chloroquine decreased the cerebral infarction volume and increased neurological deficit scores after ischemia,while U0126 could antagonize the neuroprotective effect of hydroxylated chloroquine;(3)Pretreatment with hydroxylated chloroquine reduced neuronal apoptosis in ischemic penumbra after ischemia,while U0126 could antagonize the neuroprotective effect of hydroxylated chloroquine;(4)The neuroprotective effect of hydroxylated chloroquine may be related to CREB/Akt signaling pathway;... |
