目的观察浅低温联合线粒体分裂抑制剂(mitochondrial division inhibitor 1,Mdivi- 1)对大鼠海马缺血-再灌注(ischemia-reperfusion,IR)损伤后线粒体的影响。方法健康清洁级雄性SD大鼠40只,体重280~320g,采用随机数字表法分为五组:假手术组(Sham组)、全脑IR组(IR组)、浅低温组(H组)、线粒体分裂抑制剂组(M组)和浅低温联合线粒体分裂抑制剂组(HM组),每组8只。采用经食管心脏起搏诱发心跳骤停+心肺复苏方法制备大鼠全脑IR损伤模型,即缺血4 min,再灌注6h。Sham组不经历心跳骤停和心肺复苏,其余操作与IR组一致;H组和HM组于再灌注即刻体表降温,15min之内将直肠温度降至32~34℃,自制变温箱维持6h,其余组维持正常体温;M组和HM组于再灌注即刻静脉注射Mdivi-1(1.2mg/kg),其余组注射等容积二甲基亚砜(DMSO)。于再灌注6h处死大鼠取双侧海马,采用Western blot法检测线粒体发动相关蛋白-1(dynamin- related protein 1,Drp1)和细胞色素C(cytochrome Cyt-C)蛋白含量,电镜下观察海马区椎体细胞中线粒体结构形态。结果IR组、H组、M组和HM组Drp1和Cyt-C蛋白含量明显高于Sham组(P<0.05);H组、M组和HM组Drp1和Cyt-C蛋白含量明显低于IR组(P<0.05);HM组Drp1和Cyt-C蛋白含量明显低于H组和M组(P<0.05),H组和M组Cyt-C蛋白含量差异无统计学意义。Sham组线粒体呈短棒状,结构清晰完整,其他各组中可见不同程度的线粒体分裂,膜间隙肿胀,基质包含物沉积,空泡形成,嵴模糊或消失,HM组病变较轻。结论浅低温联合线粒体分裂抑制剂可以减轻大鼠全脑缺血-再灌注后线粒体损伤,联合效果优于单独应用。
英文摘要:
Objective To investigate the effect of mild hypothermia combined with mitochondrial divison inhibitor 1 in mitochondrial after cerebral ischemia-reperfusion(IR).Methods Fourty male healthy Sprague-Dawley(SD)rats,weighing 280-320 g,were randomly divided into 5 groups (n=8 each):group Sham,group IR,hypothermia group(group H),Mdivi-1 group(group M)and hypothermia+Mdivi-1 group(group HM).Animal models of global cerebral IR were established by transoesophageal cardiac pacing inducing cardiac arrest followed by cardiopulmonary resuscitation(ischemia 4 min and reperfusion 6 h).The group Sham was similarly treated to group IR except the cardiac arrest and cardiopulmonary resuscitation.In groups H and HM,the core temperature was cooled down to 32-34℃ within 15 min starting from the beginning of reperfusion,and maintained for 6 h.In the other groups,the core temperature was maintained at the normal temperature.In groups M and HM,the animals were given Mdivi-1(1.2 mg/kg)intravenously at the beginning of the reperfusion and the other groups were given the same volumn of dimethylsnlfone(DMSO).After 6 hof reperfusion, the rats were sacrificed,and bilateral hippocampi were immediately removed for determination the protein level of dynamin-related proten 1 (Drp1)and cytochrome C (Cyt-C)expression by Western blot and obsevation of the mitochondrial structure of pyramidal cell in hippocampal CA1 under electronic microscope.Results Compared with group Sham,the expression of Drp1 and Cyt-C was up-regulated in groups IR,H,M and HM (P<0.05).Compared with group IR,the expression of Drp1 and Cyt-C was down-regulated in groups H,M and HM (P<0.05).Compared with groups H and M,the expression of Drp1 and Cyt-C was down-regulated in group HM (P<0.05).There was no significant difference in the expression of Drp1 and Cyt-C between groups H and M.The mitochondria were rod-shaped with clear and sound structure in group Sham,while mitochondria showed various degree of fission,swollen structures,matrix deposit,vacuoles formation and cristae collapse in other groups.The changes of group HM were relatively slight.Conclusion Mild hypothermia combined with mitochondrial divison inhibitor 1 alleviate mitochondrial damage after global cerebral IR of rats.The combined effect is better than that of any individual application.