摘要
目的：探讨丹酚酸 A（salvianolic acid A, SAA）对缺血性脑损伤大鼠的保护作用及其可能机制。方法54只成年雄性 Sprague－Daw ley 大鼠随机分为假手术组、脑缺血组和 SAA 组，每组18只。采用线栓法制作永久性大脑中动脉闭塞模型。 SAA 组在模型制作后0 h和6 h腹腔注射 SAA （3 mg/kg），其余各组注射等体积生理盐水。模型制作后24 h进行神经功能缺损评分，应用2,3,5－氯化二苯四氮唑染色检测脑梗死体积。采用 TUNEL 染色法检测细胞凋亡，免疫组化染色及蛋白质印迹法检测缺血皮质 Wnt3a、β－连环蛋白和磷酸化糖原合酶激酶3β（phospho－glycogen synthase kinase 3β, p－GSK－3β）表达。结果神经功能缺损评分显示，假手术组未见神经功能缺损（评分为0分），SAA 组神经功能缺损评分（中位数和四分位数间距）较脑缺血组显著降低[（3（2~3）分对4（3~5）分；Z =－2.679，P =0.007]。假手术组无梗死灶， SAA 组梗死体积较脑缺血组显著缩小[（79.038±10.665）mm3对（212.702±8.029）mm3；t =24.525，P 〈0.001]。假手术组仅见极少数阳性细胞，SAA组和脑缺血组 TUNEL 阳性细胞数量分别为（29.667±1.366）个/HP和（63.333±0.894）个/HP，前者显著少于后者（t =14.115，P 〈0.001）。免疫组化染色显示，假手术组、脑缺血组和 SAA 组 Wnt3a 阳性细胞数量分别为（35.500±2.572）个/HP、（18.056±3.765）个/HP和（29.000±2.376）个/HP，3组间存在显著性差异（F =115.972，P 〈0.001），而且 SAA 组显著多于脑缺血组（P 〈0.01）；假手术组、脑缺血组和 SAA 组 p－GSK－3β阳性细胞数量分别为（7.944±2.127）个/HP、（37.444±3.434）个/HP和（11.222±1.734）个/HP，3组间存在显著性差异（F =730.580，P 〈0.001），而且 SAA 组显著少于脑缺血组（ P 〈0.01）；假手术组、脑缺血组和 SAA 组β－连环蛋白阳性细胞数量分别为（26.722±26.722）个/HP、（16.556±1.854）个/HP 和（21.333±1.940）个/HP，3组间亦存在显著性差异（ F =33.385，P 〈0.01），而且 SAA 组显著多于脑缺血组（P 〈0.01）。蛋白质印迹分析显示，假手术组、脑缺血组和 SAA 组 Wnt3a 表达水平分别为1.000±0.190、0.800±0.185和1.198±0.262，3组间存在显著性差异（F =9.621，P 〈0.001），而且 SAA 组显著高于脑缺血组（P 〈0.01）；假手术组、脑缺血组和SAA 组 p－GSK－3β表达水平分别为0.650±0.150、1.290±0.250和1.190±0.250，3组间亦存在显著性差异（F =19.668，P 〈0.001），而且 SAA 组显著高于脑缺血组（P 〈0.01）；假手术组、脑缺血组和SAA 组β－连环蛋白表达水平分别为1.200±0.210、0.500±0.120和1.100±0.220，3组间存在显著性差异（F =33.385，P 〈0.001）（P 〈0.01），而且 SAA 组显著高于脑缺血组（P 〈0.01）。结论 SAA 对缺血脑损伤大鼠具有一定的保护作用，其机制可能与上调 Wnt3a 和β－连环蛋白表达以及下调 p－GSK－3β表达有关。
Objective To investigate the protective effect of salvianolic acid A （SAA） on permanent focal cerebral ischemia in rats and its possible mechanisms. Methods Fifty-four adult male Sprague-Daw ley rats w ere randomly divided into a sham operation group, a cerebral ischemia group, and a SAA group （ n =18 in each group）. A model of permanent middle cerebral artery occlusion w as induced by the intraluminal suture method.At 0 h and 6 h after modeling, the rats of the SAA groups w ere intraperitonealy injected SAA （3 mg/kg）. The other groups w ere injected equal volume of saline. At 24 h after modeling, the neurological deficit scores w ere performed. 2,3,5-Triphenyl tetrazolium chloride （TTC） staining w as used to detect cerebral infarction volume. TUNEL staining w as used to detect cel apoptosis. Both immunohistochemical staining and Western blotting w ere used to detect the expressions of Wnt3a, β-catenin, and phosphor-glycogen synthase-kinase-3β（p-GSK-3β） in the ischemic cortex. Results The neurological deficit scores show ed that no neurological deficits w ere observed in the sham operation group （score 0）. The neurological deficit score in the SAA group （median and interquartile range） w as significantly low er than that in the cerebral ischemia group （3 [2-3] vs.4 [3-5]; Z = -2.679, P =0.007）. No infarcts w ere observed in the sham operation group. The infarct volume in the SAA group w as reduced significantly compared w ith the cerebral ischemia group （79.038 ±10.665 mm 3 vs.212.702 ±8.029 mm 3; t = 24.525, P 〈 0.001）. Very few positive cels w ere observed in the sham operation group. The numbers of TUNEL -positive cels in the SAA group and the cerebral ischemia group w ere 29.667 ±1.366/HP and 63.333 ±0.894/HP, respectively. The former w as significantly less than the latter （ t = 14.115, P 〈 0.001）. Immunohistochemical staining show ed that the number of Wnt3a positive cels in the sham operation group, the cerebral ischemia group, and the SAA group w ere 35.500 ±2.572/HP, 18.056 ±3.765/HP, and 29.000 ±2.376/HP, respectively. There w ere significant differences among the 3 groups （ F = 115.972, P 〈 0.001）, and those in the SAA group w ere significantly more than the cerebral ischemia group （ P 〈 0.01）. The numbers of p-GSK-3βpositive cels in the sham operation group, the model group, and the SAA group w ere 7.944 ±2.127/HP, 37.444 ±3.434/HP, and 11.222 ±1.734/HP, respectively. There w ere significant differences among the three groups （F =730.580, P 〈 0.001）, and those in the SAA group w ere significantly less than the cerebral ischemia group （ P 〈 0.01）. The numbers of β-catenin positive cels in the sham operation group, the cerebral ischemia group, and the SAA group w ere 26.722 ±26.722/HP, 16.556 ±1.854/HP, and 21.333 ± 1.940/HP, respectively. There w ere also significant differences among the 3 groups （ F 〈 33.385, P 〈0.01）, and those in the SAA group w ere significantly more than the cerebral ischemia group （ P 〈 0.01）. Western blot analysis show ed that Wnt3a expression levels in the sham operated group, the cerebral ischemia group, and the SAA group w ere 1.000 ±0.190, 0.800 ±0.185, and 1.198 ±0.262, respectively. There w ere significant differences among 3 groups （ F = 9.621, P 〈 0.001）, and those in the SAA group w ere significantly higher than the cerebral ischemia group （ P 〈 0.01）. The p-GSK-3βexpression levels in the sham operation group, the cerebral ischemia group, and the SAA group w ere 0.650 ±0.150, 1.290 ± 0.250, and 1.190 ±0.250, respectively. There w ere also significant differences among the 3 groups （ F =19.668, P 〈 0.001）, and those in the SAA group w ere significantly higher than the cerebral ischemia group （P 〈0.01）. The β-catenin expression levels in the sham operation group, the cerebral ischemia group, and the SAA group w ere 1.200 ±0.210, 0.500 ±0.120, and 1.100 ±0.220, respectively. There w ere significant differences among the 3 groups （ F = 33.385, P 〈 0.001）, and those in the SAA group w ere significantly higher than the cerebral ischemia group （ P 〈 0.01）. Conclusions SAA has certain protective effect on permanent cerebral ischemia injury in rats. Its mechanism may be associated w ith the up -regulation of the expression of Wnt3a and β-catenin and the dow n-regulation of the expression of p-GSK-3β.
出处
《国际脑血管病杂志》
2016年第2期168-173,共6页
International Journal of Cerebrovascular Diseases
基金
贵州省卫生计生委科学技术基金项目（gzwjkj2014-2-157）
珠海市重点学科项目（珠卫2013070）
