M型钾离子通道开放剂对脑梗死再灌注损伤的保护作用
The protective effect and mechanisms of M-type (KCNQ) potassium channel openers on acute cerebral ischemia reperfusion

摘要：目的 探讨M型钾离子通道开放剂对缺血性脑卒中的脑保护作用及其可能机制。方法 选取C57BL/6J小鼠60只，采用抽签法随机分为假手术组(10只，Sham组)、对照组 (10只，MCAO组)、治疗组(40只，RTG组)，按照给药时间的不同再将RTG组分为RTG 0h、RTG 1h、RTG 3h、RTG 6h四个亚组，每组10只。采用线栓法制作小鼠大脑中动脉缺血再灌注模型，于脑缺血2h后再灌注。RTG组给予瑞替加滨(10.5mg/kg)，假手术组和缺血再灌注组给予等量生理盐水。采用TTC染色法检测脑梗死体积；Longa评分法进行神经功能评分；苏木素-伊红(HE)染色观察海马神经元的形态变化；免疫组化法和Western blotting法测定小鼠缺血半影区半胱氨酸蛋白酶-3(Caspase-3)和细胞膜蛋白CD40L的表达水平。结果 假手术组未发现脑梗死病灶，海马神经元无明显改变，Caspase-3阳性细胞数少见，无CD40L表达。MCAO组和RTG组均可见大脑中动脉供血区梗死灶，但RTG四个治疗组梗死灶均较MCAO组明显缩小(P<0.05)，RTG 6h组较RTG 0h、RTG 1h、RTG 3h给药组小鼠脑梗死体积增大，但差异无统计学意义(P>0.05)。RTG各组较MCAO组神经功能明显改善。MCAO组海马区脑组织肿胀与坏死明显，RTG各治疗组脑水肿和神经元坏死病理损害明显较轻。RTG组Caspase-3阳性细胞数较MCAO组明显减少(P<0.05)，0、1、3h治疗组最为显著。RTG 0h、RTG 1h及RTG 3h组梗死周围区CD40L含量均较MCAO组明显下降(P<0.05)，RTG 6h下降不明显(P>0.05)。结论 M型钾离子通道开放剂瑞替加滨对缺血性脑卒中具有脑保护作用，其机制可能是降低神经细胞的兴奋性，减轻缺血半影区的炎症反应，从而抑制细胞凋亡。M型钾离子通道开放剂的脑保护作用具有时间依赖性，即超过一定的时间窗脑保护作用会减弱。
ABSTRACT: Objective To investigate the effect and possible mechanisms of M-type potassium channel openers on brain damage after middle cerebral artery occlusion (MCAO) in male mice. Methods We randomly divided 60 male mice into six groups: sham group (n=10), MCAO group (n=10) and RTG-treatment group (n=40). RTG-treatment group was further divided into RTG 0h group, RTG 1h group, RTG 3h group and RTG 6h group, with 10 in each, according to different time of drug administration. The temporary MCAO reperfusion model was constructed by the suture method, reperfusion 2h after cerebral ischemia. In RTG-treatment groups, a single dose of 10.5mg/kg RTG was injected at various designated time points (0 hour, 1 hour, 3 hour and 6 hour after reperfusion). The sham-operation group and MCAO group received the same volume of saline. Infarct size was measured by TTC staining, the neurological function was scored with Longa 5-point scale, HE staining was used to observe the morphological changes of hippocampal neurons. In the ischemic penumbra, the expression levels of caspase-3 and the membrane protein of CD40L were detected by immunohisto-chemistry and Western blotting. Results In the sham-operation group, brain tissue had no obvious change, no infarction was observed, caspase-3(+) cells were hardly found and there was no CD40L expression. However, the infarction volume and neurological outcome scores in RTG-treatment group were lower than those in MCAO group (P<0.05). HE staining showed that hippocampal neurons were obviously swollen and necrotic in MCAO group. The pathological damages such as brain edema and neuron necrosis were ameliorated significantly in RTG-treatment group. A lot of caspase-3(+) cells and CD40L