瑞替加滨对缺血性脑卒中模型大鼠的保护作用及机制研究

Protective effect of retigabine on ischemic stroke and its mechanism in model rats

目的探讨M型钾离子通道开放剂—瑞替加滨(RTG)对缺血性脑卒中模型大鼠的保护作用及可能机制,为临床治疗脑卒中提供新的思路及科学参考。方法选取SD大鼠75只,随机分为假手术组(15只,Sham组),大脑中动脉闭塞(MCAO)模型组(15只,MCAO组),RTG干预组(45只)。RTG干预组大鼠均经尾静脉给予RTG10 mg/kg,按给药不同时间点再分为RTG 0 h亚组;RTG 1 h亚组;RTG 3 h亚组,每组15只。Sham组和MCAO组不做特殊处理,仅给予等量生理盐水。采用Longa 5分制评分法比较神经功能缺损评分,2,3,5-氯化三苯四唑(TTC)染色测量脑梗死体积,免疫组化检测星形胶质纤维酸性蛋白(GFAP)阳性表达细胞(即星形胶质细胞)数和细胞凋亡蛋白酶-3(Caspase-3)阳性表达细胞数。结果 Sham组无神经功能缺损症状,MCAO组大鼠均表现出一定程度的神经功能缺损症状,如:蜷缩、不能站稳、旋转和倾倒、不能行走等。与MCAO组相比,RTG干预组神经功能缺损症状评分明显降低(P均<0.05),但RTG不同作用时间各亚组间差异无统计学意义(P>0.05)。TTC染色图像显示:Sham组未见梗死灶,MCAO组脑组织可见不同程度的梗死灶,RTG 0 h、1 h、3 h干预组梗死灶体积[(14.06±1.68)%、(14.08±1.60)%、(14.10±2.14)%]较MCAO组明显降低[(28.50±1.02)%,P均<0.05],但RTG不同作用时间各亚组间差异无统计学意义(P>0.05)。免疫组化检测Sham组可见少量星形胶质细胞及Caspase-3阳性细胞;RTG干预组的星形胶质细胞数及Caspase-3阳性细胞数均较MCAO组明显减少(P均<0.05),但RTG不同作用时间各亚组间差异无统计学意义(P>0.05)。结论 RTG对大鼠缺血性脑卒中具有保护作用,其作用机制可能与RTG促进钾离子外流抑制神经元的兴奋性、减轻脑梗死灶周围区炎症,抑制Caspase-3的表达而减少神经元的凋亡有关,但其是否具时间依赖性,有待进一步研究。

Objective To investigate the protective effect of retigabine （ RTG）, a M type potassium channel openers, on ischemic stroke and its potential mechanism in model rats to provide a new ideas and scientific reference for the clinical treatment of stroke. Methods Seventy - five SD rats were randomly divided into sham operation group （ sham group, n = 15 ）, middle cerebral artery occlusion （MCAO） model group （ MCAO group, n = 15 ） and RTG intervention group （ n = 45 ）. In RTG intervention group, rats were given RTG （ 10 mg/kg） via rat tail vein and was re-divided into RTG 0 h group, RTG 1 h group and RTG 3 h group （n = 15, each） according to different administration time. No special treatment was given except for the same amount of normal saline in sham group and MCAO group. Longa Five Mark Scoring method was used to compare the neural function deficit scores. 2,3,5-Triphenyhetrazolium chloride （TTC）staining method was used to measure cerebral infarction volume. Immunohistochemistry was used to detect the number of glial fibrillary acidic protein （GFAP）positive-expression cells （astrocytes） and the number of apoptotic protease-3 （Caspase-3） positive-expression cells. Results There was no symptoms of neurological function deficit in sham group. All the rats in MCAO group showed neurological function deficit symptoms of a certain degree such as curling up, unable to stand firmly, rotating and toppling, unable to walk, etc. The neurological function deficit symptoms score in RTG intervention group significantly decreased compared with MCAO group （ all P 〈 0.05 ）, but there was no statistical difference among three subgroups （ P 〉 0.05 ）. TTC staining image displayed that no infarction focus was found in sham group ;infarction foci of different degree infarction were found in MCAO group ; compared with MCAO group [ （28.50 ± 1.02） % ], infarction loci volumes in subgroups RTG 0 h [ （ 14.06 ± 1.68） % ], RTG 1 h [ （ 14.08 ± 1.60） % ] and RTG 3 h [ （ 14.10 ± 2.14） % ] decreased significantly（ all P 〈 0.05 ） , but there was no statistical difference among the subgroups （ P 〉 0.05 ）. Immunohistochemistry showed a small quantity of astroeytes and Caspase-3 positive-expression cells; the number of astrocytes and the number of Caspase-3 positive-expression cells in RTG intervention group were all significantly less than those in MCAO group（ all P 〈 O. 05 ） , but there was no statistical difference among the subgroups （P 〉 0.05 ）. Conclusion RTG has a protective effect on ischemic stroke in rats, and its mechanism may be related to promoting potassium efflux, inhibiting neuronal excitability, decreasing inflammation of surrounding area of cerebral infarction focus and decreasing neuronal apoptosis by inhibiting Caspase-3 expression, but it should be studied further that whether it has time dependence.