捕食应激致大鼠海马钙/钙调素依赖性蛋白激酶途径调控紊乱

Dysfunction of Ca^(2+)/calmodulin-dependent protein kinases cascades in hippocampi of predator-stress-model rats

目的 探讨创伤后应激障碍 (PTSD)样行为异常的神经生物学机制。方法 将 16 0只Wistar大鼠随机分为捕食应激组和对照组 (每组各 80只 )。采用流式细胞仪、荧光标记术和免疫印迹法 ,检测捕食应激后 4 8h内大鼠海马细胞内游离Ca2 + 浓度与钙调素 (CaM )相对活性平均通道荧光 ,以及海马组织总CaM、Ca2 + /CaM依赖性蛋白激酶IIα (CaMKⅡα)与IV(CaMKⅣ )的表达变化。结果实验后即刻捕食应激大鼠海马细胞内游离Ca2 + 浓度明显增高 [应激组 (2 81± 70 )nmol/L ,对照组(2 0 7± 5 1)nmol/L ,P <0 0 1],12h增至顶峰 [应激组 (332± 84 )nmol/L ,对照组 (2 2 0± 5 4 )nmol/L ,P <0 0 1],2 4h仍明显增高 [应激组 (2 73± 6 7)nmol/L ,对照组 (2 0 0± 4 8)nmol/L ,P <0 0 1];实验后即刻游离CaM平均通道荧光则同步降低 (应激组 2 2± 0 5 ,对照组 3 4± 0 8,P <0 0 1;2 4h应激组2 7± 0 7,对照组 3 3± 0 8,P <0 0 5 ) ;而海马总CaM于实验后第 2 4h、CaMKⅡα和CaMKⅣ于实验后第 12h内表达明显增高 (P <0 0 1)。结论 捕食应激后早期海马细胞Ca2 + CaM及其依赖性蛋白激酶途径调控紊乱 ,在严重心理应激所致实验大鼠PTSD样行为异常中可能有意义。

Objective To explore the neurobiological basis involved in the pathogenesis of the lasting emotionality following posttraumatic stress disorder (PTSD). Methods All 160 Wistar male rats were randomly divided in two groups, in which 80 no injuriously exposed to rats were predator stress group, and the other 80 as normal controls. The hippocampus intracellular free calcium was determined with Fura-2/AM, free calmodulin (CaM) with flowcytometry, and the expressions of total CaM, Ca 2+ /CaM dependent kinase Ⅱα (CaMKⅡα) and Ⅳ (CaMKⅣ) with Western blotting. Results In comparison with controls, the intracellular free calcium level in stress rats was significantly increased immediately after cat exposure [(281±70) nmol/L vs. (207±51) nmol/L, P <0.01], and reached the peak after 12 hours [(332±84) nmol/L vs. (220±54) nmol/L, P <0.01], and it still elevated after 24 h [(273±67) nmol/L vs. (200±48) nmol/L, P <0.01]. The mean channel fluorescence of intracellular free CaM decreased synchronously (2.2±0.5 in stress rats vs. 3.4±0.8 in controls immediately after predator stress, P <0.01, and 2.7±0.7 vs. 3.3±0.8 at 24 h after cat exposure, P <0.05). The Western blotting analysis revealed that the expressions of total CaM in 24 h, CaMKⅡα and CaMKⅣ in 12 h after predator stress in stress rats were significantly elevated ( P< 0.01) respectively. Conclusion The dysfunction of Ca 2+ /CaM dependent kinases signaling cascades in hippocampi at the early stage following severe psycological stress may play an important role in the long-term neuropsychological sequelae in rats with PTSD-like behavior.