电针对暂时性脑缺血大鼠线粒体功能损伤的保护作用

Protective Effect of Electroacupuncture on Mitochondrial Function in Rats with Temporal Cerebral Ischemia

目的:对电针抗氧化应激参与缺血再灌注脑功能损伤的保护机制作进一步探讨。方法:采用大脑中动脉栓塞法(middle cerebral artery occlusion,MCAO)建立大鼠脑缺血再灌注模型。Sprague-Dawley(SD)雄性大鼠分成4组:模型组(MCAO)、模型+电针组(MCAO+EA)、假手术组(Sham)和假手术+电针组(Sham+EA)。电针穴位取“水沟”和“百会”。以比色法测定线粒体细胞色素C氧化酶的活性,以凝胶电泳法检测线粒体DNA的损伤。结果:MCAO组线粒体细胞色素C氧化酶活性比Sham组显著性降低;MCAO+EA组给予电针处理后,酶活性虽然仍低于Sham组,但比MCAO组明显升高,而电针对Sham组没有明显的影响。凝胶电泳显示MCAO组核心区形成DNA梯形条带,半影区存在一定程度的DNA链断裂性损伤;电针可以减轻半影区DNA的损伤程度,而对核心区DNA损伤无明显改善。结论:缺血早期给予电针治疗对缺血再灌注脑损伤具有良好的保护作用,其可能通过减轻半影区线粒体DNA的氧化性损伤,调整线粒体呼吸链关键酶功能,缓解了脑细胞的氧化应激,从而保护脑梗塞区部分受损细胞的功能。

Objective： To explore the protective effect of electroacupuncture （EA） on the activity of cerebral cytochrome C oxidase and mitochondrial DNA （MtDNA） in cerebral ischemia-reperfusion （CUR） rats. Methods： Forty-eight male SD rats were evenly randomized into sham-operation, sham-operation＋EA, model, and model＋EA groups with 12 cases in each group. CI/R model was established by middle cerebral artery occlusion （MCAO） under anesthesia （7% chloral hydrate, 1 mL/200 g）. EA （1-4 mA, 5/20 Hz, dense-sparse waves） was applied to ＂Shuigou＇（GV 26） and ＂Baihui＇（GV 20） for 30 min first, suspen- ded for 15 min and then 30 min again. The rats in each group were sacrificed for taking out the right hemisphere of the brain and the cerebral mitochondria were isolated by differential centrifugation. Mitochondria suspension （ 1 mg/mL protein） were frozen and thawed three times under temperature of -20 ℃. The activity of complex IV （cytochrome C oxidase） was estimated by spectrophotometry and figured out according to formula： enzyme activity μmol/（min · mg mitochondria protein）] = K · cytochrome C concentration/ protein concentration [K is the ratio between nature Logarithm of OD values and time （min） of six time points at the wavelength of 550 nm）]. MtDNA was isolated and purified according to DNA isolation kit. The concentration of MtDNA was determined by spectrophotometry. DNA damage levels were measured by agarose gel electrophoresis. Results： Compared with sham-operation and sham-operation ＋ EA groups, the activity of cytochrome C oxidase was significantly lower in model group （P〈0.01）, while compared with model group, the activity of this enzyme in model＋ EA group was obviously higher （P〈0.01）,indicating an increase of the activity of cytochrome C oxidase after EA. There was no significant difference between sham and sham＋ EA in the activity of this oxidase. MtDNA in the core of the brain focus and penumbra region were both damaged seriously. DNA ladder was found in the core. DNA damage extent in the penumbra region reduced after EA treatment, but not in the core region. Conclusion： EA can reduce the injury of mitochondrial function, which may result from its effect in alleviating the oxidative stress of the brain cells in MOAO rats.