线粒体大麻素受体1在大鼠海马神经元缺氧复氧损伤中对线粒体分裂的影响  

Effect of Mitochondrial CB1 Receptor on the Mitochondrial Fission in a Rat Hippocampal Neuron Model of Hypoxia/Re-oxygenation Injury

作　　者：李振东 郝翠[2] 凌勇[3] 李瑜[1] 李玲玉[1] 王鹏[1] 王士雷[1] LI Zhen-dong1,HAO Cui2,LING Yong3,LI Yu1,LI Ling-yu1,WANG Peng1,WANG Shi-lei1(1 Department of Anesthesiology, Affiliated Hospital of Qingdao University Medical college, Qingdao, Shandong, 266003, China; 2 Institute of Cerebrovascular Disease, Affiliated Hospital of Qingdao University Medical college, Qingdao, Shandong, 266003, China; 3 Department of Pharmacy, Affiliated Hospital of Qingdao University Medical college, Qingdao, Shandong, 266003, Chin)

机构地区：[1]青岛大学附属医院麻醉科,山东青岛266003 [2]青岛大学附属医院脑血管病研究所,山东青岛266003 [3]青岛大学附属医院药房,山东青岛266003

出　　处：《现代生物医学进展》2018年第4期648-651,694,共5页Progress in Modern Biomedicine

基　　金：国家自然科学基金面上项目(81371448)

摘　　要：目的:探讨线粒体CB1受体(mitochondrial cannabinoid receptor1,mtCB1)在大鼠海马神经元缺氧复氧损伤中对线粒体分裂的影响。方法:原代培养新生的Wistar大鼠海马神经元,将培养至第8天的海马神经元采用随机数字表分为5组(n=60):正常组(N组):正常培养,不做任何处理;缺氧复氧组(H/R组):采用氧糖剥夺法构建海马神经元缺氧复氧损伤模型,缺氧6h,复氧20 h;缺氧复氧组+ACEA+AM251组(H/R+ACEA+AM251组):缺氧6 h结束后立即加入ACEA和AM251,终浓度分别为1μmol/L、10μmol/L,复氧20 h;缺氧复氧+ACEA+Hemopressin(H/R+ACEA+Hemo组):缺氧6h结束后立即加入ACEA和Hemopressin,终浓度分别为1μmol/L、10μmol/L,复氧20 h;缺氧复氧+赋形剂组(H/R+V组):同样于缺氧6h结束后立即加入二甲基亚砜(DMSO),终浓度<0.1%,复氧20 h。使用激光共聚焦显微镜检测细胞内Ca^(2+)的浓度,流式细胞仪检测细胞凋亡率,Western blot检测凋亡诱导因子(AIF)、线粒体分裂相关蛋白Drp1、Fis1,细胞凋亡相关蛋白细胞色素C(Cytc)和Rho相关的卷曲蛋白激酶1(ROCK1)的表达。结果:与N组相比,H/R组、H/R+ACEA+AM251组、H/R+ACEA+Hemo组和H/R+V组的细胞内Ca^(2+)浓度、细胞凋亡率、以及AIF、Drp1、Fis1、Cytc、ROCK1蛋白的表达水平均明显增加(P<0.05);与H/R组相比,H/R+ACEA+Hem组上述各检测指标明显降低(P<0.05),H/R+ACEA+AM251组和H/R+V组各指标比较差异无统计学意义(P>0.05)。结论:线粒体CB1受体(mtCB1受体)可能通过降低细胞内ROS的含量来减少细胞内Ca^(2+)浓度和ROCK1的表达,进而抑制线粒体分裂,并最终减轻海马神经元缺氧复氧损伤。Objective： To evaluate the effect of mitochondrial CB1 receptor（mtCB1） on mitochondrial fission in a rat hippocampal neuron model of hypoxia/re-oxygenation（H/R） injury. Methods： Primarily cultured hippocampal neurons obtained from Wistar rats were divided into 5 groups（n=60） using a random number table： normal group（N group）：were cultured in normal medium,without any administration; H/R group： the hippocampal neurons were subjected to oxygen-glucose deprivation（OGD） for 6 h followed by re-oxygenation for 20 h; H/R＋ACEA＋AM251 group： ACEA and AM251 were add with respectively final concentration 1 umol/L, 10 umol/L during the 20 h re-oxygenation; H/R＋ACEA＋ Hemopressin group： ACEA and Hemopressin were add into culture medium with respectively final concentration 1 umol/L, 10 umol/L during the re-oxygenation for 20 h; H/R ＋ Vehicle group（H/R＋V group）： DMSO was add into culture medium with final concentration 0.1 % during the 20 h re-oxygenation. Laser scanning confocal microscope was used to measure Ca^2＋concentration in cytoplasm. The apoptosis rate was tested by flow cytometry. Western blot was adopted to examine the expression of apoptosis-inducing factor（AIF）, dynamin-related protein 1（Drp1）, fission 1（Fis1）, apoptosis-related protein cytochrome c（Cytc）, and Rho-associated coiled-coil containing protein kinase（ROCK1）. Results： Compared to the N group, the apoptosis rate,Ca^2＋concentration, the expression of AIF, Drp1, Fis1, Cytc and ROCK1 were significantly increased in the other four groups（P〈0.05）;Compared to the H/R group, those detection indexes mentioned above were significantly decreased in H/R＋ ACEA＋ Hemopressin group（P〈0.05）, and there were no significant differences were observed in H/R＋ACEA＋AM251 group and H/R＋ Vehicle group（P〈0.05）;Conclusion： The reduction of ROS induced by mtCB1 can alleviate the expression of ROCK1 and Ca^2＋concentration in cytoplasm, andinhibit the mitochondrial fission, and

关 键 词：线粒体CB1受体 线粒体分裂 海马神经元 缺氧复氧损伤 

分 类 号：Q244[生物学—细胞生物学] R338[医药卫生—人体生理学]