芒柄花素磺酸钠调控线粒体凋亡通路改善脑缺血再灌注损伤的研究

Sodium formononetin-3′-sulphonate alleviates cerebral ischemia-reperfusion injury via regulating mitochondrial apoptosis pathway

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.