ATP敏感性钾通道开放剂对脑缺血／再灌注损伤的保护作用及信号转导机制研究

Protective effect of ATP sensitive potassium channel opener on cerebral ischemia/reperfusion injury and its signal transduction mechanism

目的 观察ATP敏感性钾通道（KATP）开放剂对大鼠脑缺血／再灌注（I／R）损伤后神经元凋亡的保护作用及其信号转导机制。方法 将200只雄性Wistar大鼠随机分为假手术组（A组）、缺血组（B组）、KATP开放剂治疗组（C组）及KATP开放剂和阻断剂联合治疗组（D组）。应用线栓法制备大鼠大脑中动脉闭塞（MCAO）模型，各组于缺血后2h进行再灌注。各组于再灌注6、12、24、48和72h分别取5只大鼠脑组织标本，用末端脱氧核苷酸转移酶介导的dUTP缺口末端标记法（TUNEL）检测神经元凋亡，用免疫组化法检测天冬氨酸特异性半胱氨酸蛋白酶-3（caspase-3）和caspase-9的蛋白表达；各组其余5只大鼠用逆转录-聚合酶链反应（RT-PCR）观察caspase-3和caspase-9的mRNA表达。结果 B、C、D组再灌注后各时间点凋亡神经元数以及caspase-3、caspase-9的蛋白和mRNA表达均显著高于A组（P〈0．05或P〈0．01），C组各指标均显著低于B组和D组（P〈0．05或P〈0．01），而B组与D组各指标间比较差异均无显著性（P均〉0．05）。结论 KATP开放剂能显著减少脑I／R损伤后神经元凋亡以及caspase-3、caspase-9的mRNA和蛋白表达，提示KATP开放剂可能通过抑制线粒体通路减少脑I／R损伤后的神经元凋亡。

Objective To study the protective effect of ATP sensitive potassium channel （KATP） opener against neuronal apoptosis following focal cerebral ischemia/reperfusion （I/R） and its signal transduction mechanism. Methods Two hundred male Wistar rats were randomly divided into four groups： sham operation group （A group）, I/R group （B group）, KATP opener treatment group （C group） and KATP opener and blocker treatment group （D group）. The middle cerebral artery occlusion （MCAO） by intraluminal suture method was used to reproduce cerebral ischemia, and perfusion was restored 2 hours after MCAO. Five rats in each group were used. Brain sections were made 6, 12, 24, 48 and 72 hours after I/R injury, and neuronal apoptosis was detected by terminal deoxynucleotidyl transferase -mediated deoxyuridine 5 triphosphate nick end labeling （TUNEL）. The expressions of caspase - 3 and caspase - 9 proteins were detected by immunohistochemical method. Another five rats in each group were used for assessing the expressions of caspase - 3 mRNA and caspase - 9 mRNA by reverse transcription - polymerase chain reaction （RT - PCR）. Results The number of apoptotic neurons, the expression of caspase - 3 and caspase - 9 mRNA and protein in B, C and D groups were significantly higher than A group at all time points （P〈0. 05 or P〈0. 01）. The number of apoptotic neurons, the expressions of caspase -3 and caspase - 9 mRNA and protein in C group were significantly lower than B and D groups at all time points （P〈0. 05 or P〈0. 01）. There were no differences between B and D groups at all time points （all P〉0.05）. Conclusion KATP opener can significantly mitigate neuronal apoptosis and inhibit the expressions of caspase - 3 and caspase - 9 mRNA and protein after cerebral I/R injury. This result indicates that KATP opener can decrease neuronal apoptosis by inhibiting mitochondria signaling pathway.