Rho激酶抑制剂法舒地尔通过抑制MLK3－JNK信号通路保护脑缺血再灌注大鼠海马CAl区神经元

目的探讨Rho激酶抑制剂法舒地尔对脑缺血再灌注大鼠混合性谱系激酶（mixed—lineagekinase3，MLK3）、c—Jun—N末端激酶（c-JunNH2-terminalkinase，JNK）磷酸化、胱冬酶-3表达以及海马CAl区神经元损伤的影响。方法72只Sprague—Dawley大鼠随机分为假手术组、缺血再灌注组、生理盐水对照组和法舒地尔组。四血管结扎法制作大鼠全脑缺血模型。免疫印迹分析检测MLK3和JNK磷酸化水平以及胱冬酶-3表达，采用焦油紫染色图像分析检测海马CAl区存活神经元数量。结果缺血再灌注6h时，法舒地尔组MLK3磷酸化水平（1．13±0．03）显著低于生理盐水对照组（2．08±0．01，P=0．0003），但MLK3水平无显著差异（P=0．69）；缺血再灌注3d时，法舒地尔组JNK磷酸化水平（1．27±0．02）显著低于生理盐水对照组（2．09±0．01，P=0．0002），但JNK水平无显著差异（P=0．83）；缺血再灌注6h时，法舒地尔组胱冬酶-3表达水平（1．28±0．02）显著低于生理盐水对照组（2．10±0．01，P=0．0006）；缺血再灌注5d时，法舒地尔组海马CAl区存活锥体细胞数量为（82．8±3．2）个，显著多于生理盐水对照组的（11．8±1．6）个（P〈0．05）。结论法舒地尔能显著抑制缺血再灌注诱导的MLK3、JNK磷酸化水平以及胱冬酶-3蛋白表达，进而减轻海马CAl区神经元损伤。

Rho kinase inhibitor fasudil protect neurons in hippocampal CA1 region following cerebral ischenda reperfusion through inhibiting MLK3-JNK signal transduction pathway in rats

Objective To investigate the effect of Rho kinase inhibitor fasudil on mixed lineage kinase 3 （MLK3）, c-Jun NH2-terminal kinase （JNK） phosphorylation, caspase-3 expression, and neuronal injury in hippocarnpal CA1 region follwong cerebral ischemic rep erfusion in rats. Methods A total of 72 Sprague-Dawley rats were randomly divided into sham operation, ischemia-reperfusion, normal saline, and fasudil groups. A global cerebral ischemic model was prepared by four-vessel ligation. The levels of MLK3 and JNK phosphorylation, and caspase-3 expression were detected by Western blot analysis. Cresyl violet staining was used to detect the numbers of survival neurons in hippocampal CA1 region. Results When 6 hours after ischemia-reperfusion, the level of MLK3 phosphorylation in the fasudil group （1.13 ± 0. 03） was sigaificantly lower than that in the normal saline group （2. 08 - 0. 01,P = 0. 000 3）, while the levels of MLK3 was no significant difference. When 3 hours after ischemia-reperfusion, the level of JNK phosphorylation in the fasudil group （1.27 - 0. 02） was significantly lower than that in the normal saline group （2. 09 - 0. 01, P = 0. 000 2）, while the levels of JNK was no signaificant difference. When 6 hours after ischemia-reperfusion, the expression level of caspase-3 in the fasudil group （1.28 ± 0. 02） was significantly lower than that in the normal saline group （2. 10±0. 01 ,P=0. 000 6）. When 5 days after ischemia-reperfusion, the pyramidal cells in hippocampal CA1 region almost completely disappeared in the ischemia-reperfusion group, and only a few cells left （9. 8 ± 2. 1）. The numbers of survival pyramidal cell （8. 28 ±3.2） in hippocampal CA1 region in the fasudil group was significantly more than that in the normal saline group （11.8 ± 1.6, P 〈 0. 05）. Conclusions Fasudil may significantly inhibit the ischemia-reperfusion-induced phosphorylation of MLK3 and JNK, as well as the expression of caspase-3, and thus reduce neuronal injury in hippocampal CA1 region.