Dickkopf-1在脑出血大鼠神经元凋亡中的作用及机制研究

摘要

目的：探讨Dickkopf‐1（DKK‐1）在脑出血大鼠神经元凋亡中的作用及机制。方法采用随机数字法将40只成年雄性SD大鼠分成对照组（n＝13）、假手术组（n＝13）及脑出血组（n＝14）。通过颅内注射自体外周血制作脑出血模型。采用平衡木行走实验和肌力测验进行脑出血模型评估，并利用Real‐time PCR和Western blot检测大鼠脑组织中Bcl‐2、Bax、p‐Akt、Akt及DKK‐1蛋白和mRNA表达水平。分离培养原代大鼠皮质神经元，分为对照组、空载体组、DKK‐1过表达组、BTBD10（Akt磷酸化激活剂）过表达组，检测细胞中DKK‐1表达水平和Akt磷酸化水平，利用流式细胞术检测各组细胞凋亡。结果脑出血组大鼠平衡木行走得分高于对照组和假手术组（P＜0.05），而肌力测验评分低于对照组和假手术组（P＜0.05）。与对照组和假手术组比较，大鼠脑出血部位脑组织Bcl‐2表达降低（P＜0.05），而Bax表达则升高（P＜0.05），p‐Akt表达水平下降（P＜0.05），DKK‐1表达水平上升（P＜0.05）。DKK‐1过表达组神经元凋亡率及 p‐Akt表达水平均高于空载体组和对照组（均P＜0.05）。Akt磷酸化激活剂BTBD10过表达组p‐Akt水平、Bcl‐2蛋白表达高于对照组和空载体组，而Bax蛋白表达则低于对照组和空载体组。B T BD10过表达组神经元存活率高于对照组和空载体组（均 P＜0.05），且BTBD10和DKK‐1共转染组大鼠神经元存活率高于DKK‐1转染组（P＜0.05）。结论 DKK‐1可能通过抑制Akt的磷酸化促进出血性脑卒中大鼠神经元的凋亡。%Objective To investigate the role and mechanism of Dickkopf‐1 (DKK‐1 ) in neuronal apoptosis of rat with intracerebral hemorrhage (ICH) .Methods Adult male SD rats (n= 40) were randomly divided into the control group (n= 13) ,the sham group (n= 13) and the ICH group (n= 14) .The ICH rat model was established by intracerebral injection of peripheral blood .This model was assessed by beam‐walking assay and muscle strength testing .Furthermore ,expressions of Bcl‐2 ,Bax ,p‐Akt ,Akt and DKK‐1 protein and the mRNA were analyzed by Real‐time PCR and Western blot .Primary rat cortical neurons were divided into groups as follows :(1) the control ,empty vector and DKK‐1 overexpression group;(2) the control ,empty vector and BTBD10 overexpression group .The expressions of DKK‐1 and phosphorylated Akt were analyzed . The apoptosis was determined by flow cytometry .Results The score of beam walking assay in the ICH group was significantly increased ,and the muscle testing score was significantly lower than that in the control and sham groups (all P<0.05) .Compared with the control and sham groups ,the expressions of Bcl‐2 and p‐Akt in brain tissues of rats with cerebral hemorrhage decreased ,whereas the expressions of Bax and DKK‐1 increased after ICH treatment (all P<0.05) .In primary rat cortical neurons ,the apoptosis rate and p‐Akt expression level in the DKK‐1 overexpression group were significantly higher than those in the empty vector and control groups (P<0.05) .Furthermore ,the p‐Akt level in the BTBD10 (an Akt phosphorylation activator) overexpression group was significantly higher than that of the empty vector and control groups (all P<0.05) .Bcl‐2 protein level in the BTBD10 overexpression group was significantly higher ,while Bax protein expression was significantly lower than those in the empty vector and control groups (all P< 0.05) .Additionally ,the neuronal survival rate in the BTBD10 overexpression group was significantly higher than that in the empty vector and control groups (all P<0.05) and the neuronal survival rate in the BTBD10 and DKK‐1 co‐transfected group was significantly higher than that in the DKK‐1 transfected group (P< 0.05) .Conclusions DKK‐1 overexpression might promote neuronal apoptosis in rats with hemorrhagic stroke through the inhibition of Akt phosphorylation .