摘要
目的探讨线粒体分裂抑制剂（mdivi-1）在脑缺血再灌注损伤中对能量代谢的影响。方法将体外培养8d的Wistar大鼠海马神经元分为4组：正常对照组、赋形剂组、mdivi-1组、缺血再灌注损伤组，后两组利用海马神经元氧糖剥夺法建立缺血再灌注损伤模型，mdivi-1组建模前给予mdivi—1预处理40min。缺血6h再灌注20h后应用Western blotting检测各组海马神经元线粒体分裂蛋白1（Drp1）、B细胞淋巴癌／白血病-2（Bcl-2）和Bcl-2相关X蛋白（Bax）的表达水平，应用流式细胞术检测线粒体膜电位。应用酶标仪法检测三磷酸腺苷（ATP）含量及Na^＋．K^＋．ATP酶、Ca^2＋-Mg^2＋-ATP酶活性以及线粒体呼吸链酶复合体Ⅰ、Ⅱ、Ⅲ、Ⅳ活性。结果与正常对照组比较，mdivi-1组和缺血再灌注损伤组Drp1（1．001±0．276；1．985±0．301）、Bax（2．752±0．786；4．225±1．107）表达水平明显升高，Bcl-2（0．749±0．128；0．336±0．109）表达水平明显降低，线粒体膜电位降低的细胞数（72．5％；92．7％）均升高，ATP含量[（74．129±5．773）μmoL／g蛋白；（36．986±5．945）μmoL／g蛋白]及NnKiATP酶活性[（4．348±0．451）U／mg蛋白；（1．709±0．477）U／mg蛋白]、Ca^2＋-Mg^2＋-ATP酶活性[（1．955±0．287）U／mg蛋白；（1．123±0．181）U／mg蛋白]以及线粒体呼吸链酶复合体Ⅰ、Ⅱ、Ⅲ、Ⅳ活性[（15．445±1．699）nmol/（min·mg）、（17．065±1．070）nmol／（min·mg）、（32．123±1．652）nmol／（min·mg）、（2．814±0．180）nmol／（min·mg）；（6．810±1．725）nmol／（min·mg）、（9．473±0．751）nmol／（min·mg）、（23．010±1．716）nmol／（min·mg）、（1．598±0．181）nmol／（min·mg）]均明显降低，差异均有统计学意义（P〈0．05）。与缺血再灌注损伤组比较，mdivi-1组Drpl、Bax表达水平明显减少，Bcl-2表达水平明显升高，线粒体膜电位降低的细胞数减少，ATP含量及NnnATP酶、Ca^2＋-Mg^2＋-ATP酶活性以及线粒体呼吸链酶复合体Ⅰ、Ⅱ、Ⅲ、Ⅳ活性均明显升高，差异均有统计学意义（P〈0．05）。结论mdivi-1可通过抑制线粒体分裂，有效地改善能量代谢障碍，从而减轻脑缺血再灌注损伤。
Objective To investigate the effect ofmitochondrial division inhibitor （mdivi-1） on energy metabolism in hippocampus neurons of rats after ischemia reperfusion injury （IRI）. Methods The hippocampus neurons from Wistar rats were in vitro cultured for 8 d and then, divided into four groups： normal control group, vehicle group, IRI＋mdivi-1 treatment group and IRI group. IRI models in the later two groups were established by method of oxygen glucose deprivation; and pretreatment with mdivi-1 for 40 min was given to the IRI＋mdivi-1 treatment group before IRI. After ischemia for 6 h and reperfusion for 20 h of hippocampus neurons, Western blotting was employed to examine the protein expressions of dynamin-related protein 1 （Drpl）, B cell lymphomadewkmia-2 （Bcl-2） and Bcl-2 associated X protein （Bax）; mitochondrial membrane potential （△φm） was examined by flow cytometry;enzyme standard instrument was used to examine the ATP content, mitochondrial complex activity, Na^＋-K^＋-ATPase and Ca^2＋-Mg^2＋-ATPase activity. Results As compared with those in the normal control group, the expressions of Drpl （1.001±0.276 and 1.985±0.301）, Bax （2.752±0.786 and4.225±1.107） were up-regulated markedly, those of Bcl-2 （0.749 ±0.128 and 0.336 ±0.109） were significantly down-regulated, percentage of low mitochondrial membrane potential △φm cells was significantly increased （72.5% and 92.7%）, ATP content （[74.129±5.773] and [36.986±5.945] μmol/gprot）, Na^＋-K^＋-ATPase activity （[4.348±0.451] and [1.709±0.477] U/mgprot）, Ca^2＋-Mg^2＋-ATPase activity （ [1.955±0.287] and [1.123±0.181] U/mgprot） and activities ofmitochondrial complex Ⅰ, Ⅱ, Ⅲ and Ⅳ [ （15.445±1.699）, （17.065±1.070）, （32.123±1.652）, （2.814±0.180） and （6.810±1.725）, （9.473±0.751）, （23.010±1.716）, （1.598±0.181）] nmol/（min.mg） decreased in the IRI＋mdivi-1 treatment group and IRI group, with significant differences （P〈 0.05）. IRI＋mdivi-1 treatment group had markedly reduced Drpl and Bax protein expressions, significantly improved Bcl-2 protein expression, significantly reduced percentage of low mitochondrial membrane potential △φm cells, and significantly increased brain ATP content, Na^＋-K^＋-ATPase and Ca^2＋-Mg^2＋-ATPase activity, mitochondrial complex Ⅰ-Ⅳ activity as compared with IRI group （P〈0.05）. Conclusion Mdivi-1, by inhibitting mitochondrial fission, can significantly improve mitochondrial energy metabolism and ameliorate cerebral ischemia/reperfusion injury.
出处
《中华神经医学杂志》
CAS
CSCD
北大核心
2014年第6期562-567,共6页
Chinese Journal of Neuromedicine
基金
国家自然科学基金（81371448）
