银杏内酯A、B混合物抑制永久性大脑中动脉栓塞大鼠神经细胞JNK凋亡途径

目的 研究银杏内酯A、B混合物（GKAB）对永久性大脑中动脉栓塞（pMCAO）大鼠神经元的保护作用及相关分子机制。方法 取雄性SD大鼠60只，随机分为假手术组、pMCAO模型组和GKAB处理低、中、高剂量组。除假手术组（仅分离动脉而不阻断）外，其余4组均采用阻断右侧大脑中动脉的方法制备大鼠pMCAO模型。GKAB处理低、中、高剂量组于术后10 min分别经舌下静脉注射GKAB 12.5、25、50 mg/kg，假手术组、pMCAO模型组给予中剂量组药物等容量的生理盐水。用药12 h后，采用TUNEL法检测各组大鼠脑组织神经细胞凋亡率，免疫组织化学法检测神经细胞磷酸化JNK（p-JNK）表达，蛋白质印迹法检测脑组织中p-JNK、Bcl-2、Bax、细胞色素C（Cyt C）、caspase-9、caspase-3以及cleaved caspase-9、cleaved caspase-3蛋白的表达。结果 与假手术组相比，pMCAO模型组大鼠神经细胞凋亡率和p-JNK表达水平均增加（P<0.01），凋亡相关蛋白Bax、cleaved caspase-9、cleaved caspase-3的表达均升高（P<0.01），Bcl-2、caspase-9、caspase-3的表达均降低（P<0.01）；给予GKAB处理后，与pMCAO模型组比较，GKAB处理低、中、高剂量组大鼠神经细胞凋亡率和p-JNK水平均降低（P<0.01），凋亡相关蛋白Bax、cleaved caspase-9、cleaved caspase-3的表达也呈下降趋势（P<0.01），而Bcl-2、caspase-9、caspase-3的表达呈升高趋势（P<0.01），并表现出一定的剂量依赖性。与假手术组相比，pMCAO模型组神经细胞胞质Cyt C表达升高，线粒体Cyt C表达降低（P<0.01）；与pMCAO模型组比较，GKAB低、中、高剂量组神经细胞胞质Cyt C表达逐渐降低，线粒体Cyt C表达逐渐升高（P<0.05，P<0.01）。结论 GKAB可抑制pMCAO大鼠脑神经细胞凋亡，其机制可能与其抑制JNK磷酸化、抑制JNK信号通路的激活、阻断线粒体凋亡途径有关。

Mixture of ginkgolide A and ginkgolide B inhibits JNK apoptosis pathway in neurons of rats with permanent middle cerebral artery occlusion

Objective To investigate the protective effect of ginkgolide A and ginkgolide B (GKAB) mixture on neurons of rats with permanent middle cerebral artery occlusion (pMCAO) and related molecular mechanisms. Methods Sixty male Sprague-Dawley rats were randomly divided into sham group, pMCAO permanent focal cerebral ischemia group and GKAB-treated low-, medium- and high-dose groups. In addition to the sham group (only isolated without interruption of the arteries), the rats in the remaining four groups were induced pMCAO by blocking the right middle cerebral artery. Rats in the GKAB-treated low-, medium- and high-dose groups were injected with GKAB 12.5, 25, and 50 mg/kg through sublingual vein at 10 min after pMCAO, while the sham and pMCAO groups were injected with saline of the same volume as the medium-dose group. After 12 h of treatment, the neuronal apoptosis was determined by TUNEL method, the level of phosphorylated c-Jun N-terminal kinase (p-JNK) was determined by immunohistochemistry, the expressions of p-JNK, Bcl-2, Bax, cytochrome C (Cyt C), caspase-9, caspase-3, cleaved caspase-9, and cleaved caspase-3 in brain tissues were detected by Western blotting. Results Compared with the sham group, the apoptosis rate and p-JNK expression of neurons in the pMCAO group were significantly increased (P<0.01), and the expressions of apoptosis-related proteins Bax, cleaved caspase-9 and cleaved caspase-3 in brain tissues were significantly increased (P<0.01), while the expressions of Bcl-2, caspase-9 and caspase-3 in brain tissues were significantly decreased (P<0.01). Compared with the pMCAO group, the apoptosis rate and p-JNK expression of neurons in GKAB-treated low-, medium- and high-dose groups were significantly decreased (P<0.01), the expressions of Bax, cleaved caspase-9 and cleaved caspase-3 protein were significantly decreased (P<0.01), and the expressions of Bcl-2, caspase-9 and caspase-3 were significantly increased (P<0.01) in a dose-dependent manner. Compared with the sham group, the expression of Cyt C in cytoplasm in the pMCAO group was significantly increased, and the expression of mitochondrial Cyt C was significantly decreased (P<0.01). Compared with the pMCAO group, the expressions of Cyt C in cytoplasm in the GKAB-treated low-, medium- and high-dose groups were significantly decreased in a dose-dependent manner, and the expressions of mitochondrial Cyt C were significantly increased (P<0.05, P<0.01). Conclusion GKAB can inhibit neuronal apoptosis after pMCAO in rats, and its mechanism may be related to the inhibition of JNK phosphorylation and JNK signaling pathway and the block of mitochondrial apoptosis pathway.