胡黄连苷II抑制大鼠脑缺血再灌注损伤后线粒体细胞色素C的表达及意义研究

Effect of picroside II on mitochondria cytochrome C expression and its significance in rats after ischemia/reperfusion

目的探讨胡黄连苷II抑制大鼠脑缺血再灌注损伤后线粒体细胞色素C（CytC）的表达及意义。方法Wistar大鼠96只按随机数字表法分为假手术组、模型组、胡黄连苷II组、CytC特异性抑制剂环孢素（CsA）组、CsA＋胡黄连苷II组、CytC选择性激动剂苍术苷（Atr）组、Atr＋胡黄连苷II组、DMSO组。每组12只。采用改良Longa法制备缺血2h再灌注24h模型，并按分组给予相应干预。各组大鼠在再灌注24h后行改良神经功能缺损评分（mNSS），采用ELISA法检测脑组织活性氧（ROS）含量。采用HE染色观察神经细胞形态，采用电镜观察线粒体超微结构，采用TUNEL法检测神经细胞凋亡情况，采用免疫组化染色和Western blotting检测CytC表达水平。结果模型组大鼠与假手术组比较，mNSS评分升高，ROS含量增高，皮质区变性细胞数增多，线粒体结构破坏严重，神经细胞凋亡数和CytC蛋白表达增多，差异均有统计学意义（P〈0．05）。胡黄连苷II组大鼠与模型组比较，mNSS评分下降，ROS含量降低，皮质区变性细胞数减少，线粒体损伤程度减轻，神经细胞凋亡数和CytC蛋白表达降低，差异均有统计学意义（P〈0．05）。Atr＋胡黄连苷II组与Atr组比较，mNSS评分下降，ROS含量降低，变性细胞数及神经细胞凋亡数减少，线粒体损伤程度减轻，CytC蛋白表达降低，差异均有统计学意义（P〈0．05）。结论胡黄连苷II对局灶性脑缺血再灌注损伤可产生神经保护作用，机制可能与清除ROS、下调CytC蛋白表达和保护细胞线粒体结构有关。

Objective To investigate the effect of picroside II on mitochondria cytochrome C （CytC） expression and its significance in rats after ischemia/reperfusion. Methods Ninety-six Wistar rats were randomly divided into sham-operated group, model group, picroside II group, Cyclosporin A （CsA, specific antgonist of CytC） group, CsA＋picroside II group, atractyloside （Atr, selective agonist of CytC） group, Atr＋picroside II group and DMSO group （n=12）; the middle cerebral artery oeclusion/reperfusion models referring to Longa＇s method with medications were adopted, which were established by inserting a monofilament suture into the internal carotid artery for 2 h and then reperfusion for 24 h. After 24 h of ischemia/reperfusion, modified neurological severity scale （mNSS） scores were observed, contents of reactive oxygen species （ROS） in brain tissues were measured by enzyme-linked immunosorbent assay （ELISA）, morphology of brain tissues was observed by hematoxylin-eosin staining, ultrastructures of mitochondria were observed by transmission electron microscopy, apoptotic cells were counted by terminal deoxynucleotidyl transferase dUTP nick end labeling assay （TUNEL）, and CytC expression was determined by immunohistochemical assay and Western blotting. Results As compared with the sham-operated group, the model group had significantly increased mNSS scores, ROS contents, number ofapoptotic cells and CytC expression （P〈0.05）, and the mitochondria structure was seriously destroyed. The picroside II group had obviously decreased mNSS scores, ROS contents, number of apoptotic cells and CytC expression, and the morphology of brain tissue was improved and the mitochondria damage was reduced as compared with the model group, with significant differences （P〈0.05）. The Atr＋picroside I/group had significantly decreased mNSS scores, ROS contents, number of apoptotic cells and CytC expression （P〈0.05）, and the mitochondria damage in the Atr＋pieroside II group was reduced as compared with that in the Atr group with significant difference （P〈0.05）. Conclusion The mechanism of picroside II protecting against focal cerebral ischemia reperfusion might attribute to decrease of ROS contents, protection of mitochondria structure and down-regnlation of CytC expression in middle cerebral artery occlusion/reperfusion rats.