| Objectives:1.To observe the effect of electroacupuncture on the neurological functional recovery in SD rat after focal cerebral ischemia/reperfusion;2.To probe the impact of electroacupuncture on the infarct volume in SD rat after focal cerebral ischemia/reperfusion;3.To investigate the effect of electroacupuncture on the expression of CD34 microvessels and CD34~+VEGFR2~+vessels in SD rat ischemic cerebral cortex after focal cerebral ischemia/reperfusion;4.To explore the influence of electroacupuncture on the protein expression of AKT,p-AKT,mTOR,p-mTOR,P70S6K,p-P70S6K in SD rat ischemic cerebral cortex after focal cerebral ischemia/reperfusion;5.To probe the effect of electroacupuncture on the expression of MMP-9,VEGF,HIF-1αprotein and mRNA in SD rat ischemic cerebral cortex after focal cerebral ischemia/reperfusion;6.To explore the effect of electroacupuncture and RAPA on the activation of AKT/mTOR/P70S6K signal pathway,topreliminary determine the meachanism of electroacupuncture promoting angiogenesis in rat ischemic cerebral cortex.Methods:A total of 180 healthy male adult Sprague-Dawley(SD)rats were randomly assigned tosham operation group(sham),model group(I/R),model+electroacupuncture group(I/RE),RAPA plus electroacupuncture group(I/RER).The model of focal cerebral ischemia/reperfusion was prepared by the thread embolism method.All groups were divided into 12 h,1 d,2 d,3 d and 7 d sub-groups after reperfusion 2 h.Electroacupuncture was given at"Baihui"(GV 20)and"Siguan"on the left side(Hegu LI 4/Taichong LR 3)acupoints for 30 min every turn,per day for once.Neurological functional recovery was tested by 18 score of neurological function.TTC staining was used to detect the infarction area of each group at 7 d after reperfusion.CD34~+VEGFR2~+vessels were quantified by doublelabeling immunofluorescenceat(IF)at 7 d after reperfusion.Immunohistochemical(IHC)method was used to detect the expression of CD34~+microvessels.Western Blot was applied to detect the protein expression of AKT/mTOR/P70S6K and HIF-1/VEGF signal pathway in SD rat ischemic cerebral cortex.The mRNA expression of HIF-1/VEGF pathway were measured by qRT-PCR in SD rat ischemic cerebral cortex.qRT-PCR and IHC were applied to test the expression of MMP-9 mRNA and positive cells respectively.Results:1.The neurological functional scores at each time point in each groupAs time went on,the neurological functional score was gradually increased.However,there was no significant difference after reperfusion 12h,1 d and 2 d.Compared with I/R group,electroacupuncture significantly improved the recovery of neurological function at 3 d and 7 d after reperfusion(P<0.01).The neurological functional score in I/RE was higher compared with I/R group at 3 d and 7 d after reperfusion(P<0.05).2.The impact of electroacupuncture on the infarct volume in each group at 7 d after reperfusionCompared with I/R group,electroacupuncture significantly reduced infarct volume at 7 d after reperfusion(P<0.01).The infarct volume in I/RER was higher compared with I/R group at 7d after reperfusion(P<0.01).3.The effect of electroacupuncture on the expression of CD34~+microvessel in rat ischemic cerebral cortex in each group at 3 d and 7 d after reperfusionCompared with Sham group,The number of CD34~+microvessel in I/R was higher(P<0.05).Comparedwith I/R group,electroacupuncture significantly promoted the number of CD34~+microvessel at 3 d and 7 d after reperfusion(P<0.01).The number of CD34~+microvessels were decreased in I/RER compared with I/R group(P<0.05).4.The effect of electroacupuncture on the expression of CD34~+VEGFR2~+vessels in rat ischemic cerebral cortex in each group at 7d reperfusionCompared with I/R group,electroacupuncture significantly promoted the number of CD34~+VEGFR2~+vessels at 7 d after reperfusion(P<0.01).The number of CD34~+VEGFR2~+vessels was decreased in I/RER compared with I/R group(P<0.01).5.The influence of electroacupuncture on the AKT/mTOR/P70S6K signal pathway in rat ischemic cerebral cortex after focal cerebral ischemia/reperfusionThe expression of p-AKT was changed insignificantly at 12 h after reperfusion in I/R group compared with Sham group,while it was increased significantly at 1 d,2 d,3 d after reperfusion in I/R group(P<0.05)and peakedat2dafterreperfusion;ComparedwithI/Rgroup,electroacupuncture significantly enhanced the expression of p-AKT at each time in each group(P<0.001).The the expression of p-AKT was decreased in I/RER compared with I/RE group(P<0.01).Comparedwith S group,The expression of p-mTOR and p-P70S6K were improved significantly at 2d,3 d,7 d after reperfusion(P<0.05)and peaked at 3 d;Compared with I/R group,electroacupuncture significantly enhanced the expression of p-mTOR and p-P70S6K at each time point in each group(P<0.001).The expression of p-mTOR and p-P70S6K were reduced in I/RER compared with I/RE group(P<0.01).6.The impact of electroacupuncture on the expression of MMP-9mRNA and MMP-9 positive cellsCompared with Sham group,The expression of MMP-9 mRNA was significantly increased at each time point in I/R group(P<0.001)and peaked at 3 d after reperfusion;Compared with I/R group,electroacupuncture cut down the expression of MMP-9 mRNA at 1 d(P<0.05)and remarkably promoted the expression at 3 d and 7 d(P<0.01).The expression of MMP-9 mRNA was decreased in I/RER at each time point in each group(P<0.01).There were little MMP-9 positive cells in Sham group.Compared with Sham group,The expression of MMP-9positive cells were enhanced significantly at each time in I/R group(P<0.001)and peaked at 7 d after reperfusion;Compared with I/R group,electroacupuncture cut down the expression of MMP-9 positive cells at 1d(P<0.05)and remarkably promoted the expression at 3 d and 7 d(P<0.01).The expression of MMP-9 positive cells were decreased significantly in I/RER at each time point in each group(P<0.01).7.The influence of electroacupuncture on the VEGF/HIF-1 signal pathway inratischemiccerebralcortex afterfocalcerebral ischemia/reperfusionThe expression of HIF-1α、VEGF mRNA and protein were increased in I/R group compared with Sham group(P<0.01);Compared with I/R group,electroacupuncture enhanced the expression of HIF-1α、VEGF mRNA and protein(P<0.001).The expression of HIF-1α、VEGF mRNA and protein were decreased in I/RER(P<0.01).Conclusions1.Electroacupuncture can promote the recovery of neurological function;2.Electroacupuncture can activate AKT/mTOR/P70S6K and VEGF/HIF-1 signal pathway;3.Electroacupuncture can increase the density of CD34~+microvessel and CD34~+VEGFR2~+vessels in rat ischemic cerebral cortex and decrease infarct volume.4.Electroacupuncture can improve angiogenesis mediated MMP-9and reduce infarct volume and promote recovery of neurological functionvia the activation of AKT/mTOR/P70S6K signal pathway so as to play a important role in against the injury of cerebral ischemia. |
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