PI3K/Akt信号通路在头部浅低温减轻大鼠全脑缺血再灌注损伤中的作用

Role of PI3K/Akt signaling pathway in mild head hypothermia-induced reduction of global cerebral ischemia-reperfusion injury in rats

目的 评价磷脂酰肌醇3-激酶-丝氨酸-苏氨酸蛋白激酶（PI3K/Akt）信号通路在头部浅低温减轻大鼠全脑缺血再灌注损伤中的作用.方法 健康雄性SD大鼠60只,体重250～ 280 g,采用随机数字表法分为5组（n=12）：假手术组（S组）、脑缺血再灌注组（I/R组）、浅低温组（H组）、浅低温＋溶剂对照组（DM组）和浅低温＋PI3K抑制剂LY294002组（LY组）.除S组外,其余各组采用改良Pulsinelli四血管阻断法建立全脑缺血再灌注损伤模型,H组采用鼻咽腔降温法降低海马温度至33℃时夹闭双侧颈总动脉15 min复灌,期间维持海马温度32.5～ 33.5℃1h,DM组和LY组于左侧脑室缓慢注射DMSO 5μl或LY294002 5μl,20 min后处理同H组.于再灌注8h时,每组随机取6只大鼠,采用免疫组化法测定海马CA1区磷酸化FoxO3a（pFoxO3a）、Bcl-2及Bax蛋白表达水平,计算Bcl/Bax比值;余6只大鼠,采用Western blot法检测海马磷酸化Akt（p-Akt）表达水平.结果 与S组比较,I/R组、H组和DM组p-Akt、pFoxO3a、Bax蛋白表达上调,H组和DM组Bcl-2蛋白表达下调、Bcl-2/Bax比值升高,I/R组和LY组Bcl-2/Bax比值降低（P＜0.05）;与I/R组比较,H组和DM组p-Akt、pFoxO3a、Bcl-2蛋白表达上调,Bax蛋白表达下调,Bcl-2/Bax比值升高（P＜0.05）;与H组和DM组比较,LY组p-Akt、pFoxO3a和Bcl-2蛋白表达下调,Bax蛋白表达上调,Bcl-2/Bax比值降低（P＜0.05）.结论 PI3K/Akt信号通路参与了头部浅低温减轻大鼠全脑缺血再灌注损伤的过程.

Objective To evaluate the role of phosphatidylinositol 3-kinase/protein kinase B （PI3K/Akt） signaling pathway in mild head hypothermia-induced reduction of global cerebral ischemiareperfusion （I/R） injury in rats.Methods Sixty male Sprague-Dawley rats,weighing 250-280 g,were randomly divided into 5 groups （n=12 each） using a random number table：sham operation group （group S）,group I/R,mild hypothermia ＋ I/R group （group H）,mild hypothermia ＋ I/R ＋ solvent control group （group DM）,and mild hypothermia ＋ I/R ＋ PI3K inhibitor LY294002 group （group LY）.Global cerebral I/R was induced by modified four-vessel occlusion method described by Pulsinelli.In H group,when the hippocampal temperature was decreased to 33℃ using nasopharyngeal cooling,the bilateral common carotid arteries were occluded for 15 min followed by reperfusion,and hippocampal hypothermia was maintained at 32.5-33.5 ℃ for 1 h.In DM and LY groups,DMSO and LY294002 5 μl were injected into the left ventricle,respectively,and 20 min later the other procedures were similar to those previously described in group H.At 8 h of reperfusion,6 rats were sacrificed,and hippocampal specimens were obtained to detect the expression of pFoxO3a,Bcl-2 and Bax （by immuno-histochemistry）.The expression of phosphor-Akt （p-Akt） was determined by Western blot.Results Compared with group S,the expression of p-Akt,pFoxO3a and Bcl-2 was significantly up-regulated in I/R,H and DM groups,the expression of Bcl-2 was down-regulated,and the ratio of Bcl-2/Bax was increased in H and DM groups,and the ratio of Bcl-2/Bax was decreased in I/R and LY groups.Compared with group I/R,the expression of p-Akt,pFoxO3a and Bcl-2 was significantly up-regulated,the expression of Bcl-2 was down-regulated,and the ratio of Bcl-2/ Bax was increased in H and DM groups.Compared with H and DM groups,the expression of p-Akt,pFoxO3a and Bcl-2 was down-regulated,the expression of Bax was up-regulated,and the ratio of Bcl-2/ Bax was decreased in group LY.Conclusion PI3K/Akt signaling pathway is involved in reduction of global cerebral I/R injury by mild head hypothermia in rats.