目的 研究芒柄花素磺酸钠（sodium formononetin-3ʹ-sulphonate，Sul-F）调控线粒体凋亡通路改善脑缺血再灌注损伤的作用机制。方法 建立大鼠脑缺血再灌注损伤模型，随机分为假手术组、模型组、依达拉奉（3 mg/kg）组和Sul-F高、低剂量（80、40 mg/kg）组，分别于再灌注0、12 h尾iv药物干预，假手术组和模型组给予等体积的生理盐水。再灌注24 h后，Zea Longa评分法评估神经功能；2,3,5-氯化三苯基四氮唑（2,3,5-triphenyltetrazolium chloride，TTC）染色测定脑梗死体积；苏木素-伊红（hematoxylin-eosin，HE）染色观察脑缺血半暗带病理变化；原位末端标记（TdT-mediated dUTP nick end labeling，TUNEL）染色检测脑缺血半暗带细胞凋亡情况；JC-1探针流式细胞术检测脑缺血半暗带线粒体膜电位水平；透射电镜观察脑组织超微结构；ELISA法检测脑缺血半暗带丙二醛（malondialdehyde，MDA）水平及超氧化物歧化酶（superoxide dismutase，SOD）、谷胱甘肽过氧化物酶（glutathione peroxidase，GSH-Px）活性；qRT-PCR和Western blotting分别检测脑缺血半暗带B淋巴细胞瘤-2（B-cell lymphoma-2，Bcl-2）、Bcl-2相关X蛋白（Bcl-2 associated X protein，Bax）和半胱氨酸天冬氨酸蛋白酶-3（cystein-asparate protease-3，Caspase-3）mRNA和蛋白表达。结果 与模型组比较，Sul-F高剂量组大鼠神经功能评分显著降低（P＜0.01），脑梗死体积显著减小（P＜0.05），脑缺血半暗带病理损伤减轻，细胞凋亡显著减少（P＜0.01），线粒体膜电位的去极化显著逆转（P＜0.05），线粒体嵴更加清晰、自噬现象更少见，MDA含量显著降低（P＜0.01），SOD和GSH-Px活力显著升高（P＜0.05、0.01）；Bcl-2表达显著增加（P＜0.01），Bax和Caspase-3表达显著减低（P＜0.01）。结论 Sul-F通过调控线粒体凋亡相关基因Bcl-2/Bax平衡，改善线粒体氧化应激水平，减轻脑缺血再灌注损伤。

Objective To explore the effect and mechanism of sodium formononetin-3ʹ-sulphonate (Sul-F) on cerebral ischemia-reperfusion (I/R) injury. Methods The rat model of cerebral I/R injury was established, rats were randomly divided into sham group, model group, edaravone (3 mg/kg) group and high-, low-dose (80, 40 mg/kg) Sul-F groups. Drug intervention was administered through the caudal vein at 0, 12 h of reperfusion, respectively. Sham group and model group were given the equal volume of saline simultaneously. After 24 h of reperfusion, Zea Longa score was used to assess the neural function; The volume of cerebral infarction was measured by 2,3,5-triphenyltetrazolium chloride (TTC) staining; Hematoxylin-eosin (HE) staining was applied to observe the pathological changes of ischemic penumbra; TdT-mediated dUTP nick end labeling (TUNEL) was used to detect apoptosis in ischemic penumbra; JC-1 probe was applied to detect the mitochondrial membrane potential in ischemic penumbra by flow cytometry; Transmission electron microscopy was used to observe ultrastructure of brain tissue; The level of malondialdehyde (MDA) and activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH Px) were detected by ELISA; qRT-PCR and Western blotting were applied to measure the mRNA and protein expressions of B-cell lymphoma-2 (Bcl-2), Bcl-2 associated X protein (Bax) and cystein-asparate protease-3 (Caspase-3) in ischemic penumbra. Results Compared with model group, neurological function score of rats was significantly reduced in Sul-F high-dose group (P < 0.01), cerebral infarction volume was decreased (P < 0.05), pathological damage to the ischemic penumbra was reduced, apoptosis was decreased (P < 0.01), depolarization of mitochondrial membrane potential was reversed (P < 0.05), mitochondrial cristae was clearer and autophagy phenomenon was less, level of MDA was decreased (P < 0.01), activities of SOD and GSH-Px were increased (P < 0.05, 0.01), Bcl-2 expression was increased (P < 0.01), expressions of Bax and Caspase-3 were decreased (P < 0.01). Conclusion Sul-F can alleviate mitochondrial function and alleviate cerebral I/R injury by regulating the balance of mitochondrial apoptosis related genes Bcl-2/Bax.

R285.5

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