Department of Integrative Medicine, Zhongshan Hospital, Laboratory of Neurology, Institute of Integrative Medicine, Fudan University, Shanghai 200032, China.
Guangzhou University of Traditional Chinese Medicine, Guangzhou 510006, China.
J Ethnopharmacol. 2014;151(1):307-16. doi: 10.1016/j.jep.2013.10.042. Epub 2013 Nov 1.
Xiao-Xu-Ming decoction (XXMD) has been used to treat stroke and other neurological diseases for more than 1000 years. The purpose of this study was to investigate the effects of XXMD on mitochondrial damage and apoptosis after cerebral ischemia and reperfusion.
Male Sprague-Dawley rats were randomly divided into 3 groups: sham, cerebral ischemia and reperfusion (I/R), and cerebral ischemia and reperfusion plus XXMD (60 g/kg/day) (XXMD60). Focal cerebral ischemia and reperfusion models were induced by middle cerebral artery occlusion. Cerebral ischemic injury was evaluated by hematoxylin and eosin staining. Ultrastructural features of mitochondria in the penumbra of the ischemic cortex were analyzed by transmission electron microscopy. Apoptosis was evaluated by terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate nick end labeling (TUNEL) staining and cleaved caspase 3 immunohistochemistry. Proteins in the mitochondrial p53 pathway were detected by western blot and immunofluorescence.
The results showed that XXMD treatment markedly attenuated ischemic changes, preserved mitochondrial integrity, and significantly reduced apoptosis. In addition, we found that XXMD treatment reduced p53 and Bax levels and increased Bcl-2 levels in mitochondrial fractions. XXMD significantly blocked the release of cytochrome c and Smac/Diablo from mitochondria, and inhibited activation of caspase 9 and caspase 3 in cytoplasmic fractions. Increased expression of c-IAP1 was observed in the XXMD60 group.
The findings demonstrated that XXMD protected mitochondria from ischemic injury and inhibited apoptosis. The mitochondrial p53 pathway could be partially involved in the protective effects.
消眩止晕汤（XXMD）已被用于治疗中风和其他神经系统疾病超过 1000 年。本研究旨在探讨 XXMD 对脑缺血再灌注后线粒体损伤和细胞凋亡的影响。
雄性 Sprague-Dawley 大鼠随机分为 3 组：假手术组、脑缺血再灌注组（I/R 组）和脑缺血再灌注加 XXMD（60 g/kg/天）组（XXMD60 组）。采用大脑中动脉闭塞法制备局灶性脑缺血再灌注模型。苏木精-伊红染色评估脑缺血损伤。透射电镜观察缺血皮质半暗带中线粒体的超微结构特征。末端脱氧核苷酸转移酶介导的脱氧尿嘧啶三磷酸缺口末端标记法（TUNEL）染色和 cleaved caspase 3 免疫组化法评估细胞凋亡。Western blot 和免疫荧光法检测线粒体 p53 通路相关蛋白。
结果表明，XXMD 治疗显著减轻了缺血性变化，保护了线粒体的完整性，并显著减少了细胞凋亡。此外，我们发现 XXMD 治疗降低了线粒体部分的 p53 和 Bax 水平，增加了 Bcl-2 水平。XXMD 显著抑制了线粒体细胞色素 c 和 Smac/ Diablo 的释放，并抑制了细胞质部分 caspase 9 和 caspase 3 的激活。XXMD60 组中 c-IAP1 的表达增加。
这些发现表明，XXMD 可保护线粒体免受缺血性损伤并抑制细胞凋亡。线粒体 p53 通路可能部分参与了保护作用。
