栝楼桂枝颗粒对脑缺血再灌注损伤大鼠脑组织Ca^(2+)/CaMKⅡ/CREB信号通路的影响

Effects of Gualou Guizhi Granules on Ca^(2+)/CaMKⅡ/CREB Signaling Pathway in Brain Tissue of Rats with Cerebral Ischemia Reperfusion Injury

目的观察栝楼桂枝颗粒对脑缺血再灌注损伤大鼠脑组织Ca^(2+)/Ca MKⅡ/CREB信号通路的影响,探讨其作用机制。方法采用线栓法致大脑中动脉栓塞建立大鼠脑缺血再灌注损伤模型。SD大鼠随机分为假手术组、模型组和中药组,中药组给予栝楼桂枝颗粒药液灌胃,连续7 d。采用改良的神经功能缺损评分法进行大鼠神经功能评分,测定大鼠脑梗死面积,采用透射电镜观察大鼠海马神经元超微结构,RT-PCR检测大鼠脑组织钙活化调蛋白依赖性蛋白激酶Ⅱ(Ca MKⅡ)、c AMP反应元件蛋白(CREB)的基因表达,Western blot检测大鼠缺血侧脑组织p-CREB、p-CaMKⅡ、Ca MKⅡ、CREB和钙调蛋白(Ca M)的蛋白表达。结果与假手术组比较,模型组大鼠神经功能缺损和脑梗死面积评分显著升高,海马神经元超微结构改变明显,细胞高度水肿、细胞核和细胞质收缩,p-CREB蛋白表达降低,Ca M蛋白表达升高;与模型组比较,中药组大鼠神经功能评分和脑梗死面积显著降低,缺血区神经元超微结构改善,细胞损伤减少,p-CREB和p-CaMKⅡ蛋白表达升高,Ca M蛋白表达降低。结论栝楼桂枝颗粒能改善模型大鼠神经功能,抑制神经细胞凋亡,其作用机制可能与促进CREB、Ca MKⅡ的磷酸化,抑制Ca M的表达有关。

Objective To investigate the effects of Gualou Guizhi Granules on Ca2＋/CaMKⅡ/CREB signaling pathway in brain tissue of rats with cerebral ischemia reperfusion injury; To explore its possible mechanism of action. Methods A rat model of cerebral ischemia reperfusion injury was established by middle cerebral artery occlusion. SD rats were divided into sham-operation group, model group and TCM group. TCM group was given Gualou Guizhi Granules decoction for gavage for 7 d. The modified neural function defect scoring was used to score the nerve functions of rats and the area of cerebral infarction was determined. The ultrastructure of hippocampal neurons in rats was observed by transmission electron microscopy, and RT-PCR method was used to detect gene expressions of CaMKⅡ and CREB in the brain tissue. Western blot was used to detect the protein expressions of p-CREB, p-CaMKⅡ, CaMKⅡ, CREB and CaM in the ischemic brain tissue of rats. Results Compared with the sham-operation group, the nerve function defect and cerebral infarction area score of the model group increased significantly. Electron microscopy showed that the ultrastructural changes of hippocampal neurons were obvious, the cells were highly edematous, the nucleus and cytoplasm contracted, the protein expression of p-CREB decreased, and the protein expression of CaM increased. Compared with the model group, the nerve function score and the area of cerebral infarction of TCM group decreased significantly, ultrastructural neurons in ischemic area was improved, and cell damage was reduced, the protein expressions of p-CREB and CaMKⅡ increased, and the the protein expressions of CaM decreased. Conclusion Gualou Guizhi Granules can improve the neural functions of model rats and inhibit the apoptosis of nerve cells. Its mechanism of action may be related to the phosphorylation of CREB and CaMKⅡ and the inhibition of the expression of CaM.