摘要
第一部分Rho激酶抑制剂(盐酸法舒地尔)在缺血性脑损伤中的实验研究
目的探讨Rho激酶抑制剂(盐酸法舒地尔)对谷氨酸受体6(Glutamate receptor6, GluR6)的磷酸化、混合性的谱系激酶(Mixed lineage kinase3, MLK3)及c-Jun氨基末端激酶(c-Jun N-terminal kinase, JNK)的磷酸化水平及蛋白表达、半胱氨酸天冬氨酸蛋白酶3(Caspase-3)的蛋白表达及海马CA1区神经元凋亡及损伤的影响。
方法采用四血管阻塞(4-vessel-occlusion,4-VO)法制作大鼠全脑缺血损伤模型,将体重220-300g清洁级SD大鼠随机分成假手术组(Sham group)、缺血再灌注组(Ischemia/Reperfusion group)、生理盐水组(Physiological saline group)、盐酸法舒地尔组(Fasudil hydrochloride group)。盐酸法舒地尔组予缺血前30分钟腹腔注射(15mg.kg-1)进行干预,生理盐水组同给药组,腹腔注射等量的生理盐水。采用免疫共沉淀和免疫印迹的方法检测盐酸法舒地尔对缺血再灌注6小时GluR6的磷酸化及蛋白表达水平的影响；采用免疫印迹的方法分别检测盐酸法舒地尔对缺血再灌注6小时MLK3、缺血再灌注3天时JNK磷酸化、蛋白表达水平及缺血再灌注6小时Caspase-3蛋白表达的影响；采用TUNEL染色方法检测缺血再灌注3天盐酸法舒地尔对大鼠海马CA1区神经元凋亡的影响；采用焦油紫染色方法检测缺血再灌注5天盐酸法舒地尔对大鼠海马CA1区神经元损伤的影响。
结果
1.采用免疫共沉淀及免疫印迹的方法检测GluR6丝氨酸的磷酸化,结果显示：与假手术组相比,缺血再灌注6小时时GluR6丝氨酸的磷酸化水平显著增高(P<0.05),而同一时间点的GluR6的蛋白表达水平无明显变化(P>0.05)。与缺血再灌注组及生理盐水组相比,盐酸法舒地尔组可明显抑制GluR6丝氨酸的磷酸化高峰,差异有统计学意义(P<0.05)。
2.采用免疫印迹的方法检测MLK3、JNK的磷酸化、蛋白表达及caspase-3的蛋白表达水平,结果显示：与假手术组相比,缺血再灌注组MLK3、JNK的磷酸化水平及caspase-3的蛋白表达水平分别在缺血再灌注后6h、3d及6h不同时间点显著增高(P<0.05),而同一时间点的MLK3、JNK的蛋白表达水平无明显变化(P>0.05)。与缺血再灌注组及生理盐水组比较,盐酸法舒地尔组MLK3、JNK的磷酸化及caspase-3蛋白表达水平显著降低(P<0.05)。
3.采用TUNEL的方法检测海马CA1区神经元的凋亡情况,结果显示：与假手术组大鼠海马CA1区每1mm长度范围内TUNEL阳性细胞数(8.60±2.19)相比,缺血再灌注3天时TUNEL阳性细胞数为(41.80±3.03),两组比较有统计学意义(P<0.05)。与缺血再灌注组及生理盐水组(41.80±3.03,40.20±2.77)比较,盐酸法舒地尔组的凋亡细胞数(19.80±2.86)显著降低,有统计学意义(P<0.05)
4.采用焦油紫染色的方法观察缺血再灌注诱导的大鼠海马CA1区迟发性神经元损伤情况,结果显示：假手术组海马CA1区锥体细胞排列密集(193.3+2.9),缺血再灌组海马CA1区锥体细胞几乎完全消失,仅残留少数细胞(9.8±2.1);生理盐水组与其相似(11.8±1.6),盐酸法舒地尔组海马CA1区锥体细胞存活明显增多(82.8±3.2),与缺血再灌组比较细胞损伤明显减少,有统计学意义。
结论盐酸法舒地尔能显著抑制脑缺血/再灌注诱导的GluR6磷酸化高峰,进而抑制MLK3、JNK的磷酸化水平及caspase-3的蛋白表达,显著减少海马CA1区神经元的凋亡及损伤。
第二部分盐酸法舒地尔治疗急性缺血性脑卒中临床疗效的Meta分析
目的系统评价盐酸法舒地尔治疗急性缺血性脑卒中的近期疗效和安全性。
方法计算机检索Cochrane Library、PubMed、西文生物医学数据库、中国生物医学文献数据库、中国期刊全文数据库、中文科技期刊全文数据库,同时辅助其他检索,纳入盐酸法舒地尔治疗急性缺血性脑卒中的随机对照试验,检索时间从各数据库建库开始至2011年12月31日。2名评价者独立评价纳入研究的质量并提取资料,用RevMan5.0软件进行统计分析。
结果共纳入57篇文献,合计5945患者。Meta分析结果显示：①盐酸法舒地尔组治疗急性缺血性脑卒中的神经功能缺损临床疗效明显优于对照组[OR=3.38,95%CI(2.95,3.88),P<0.00001];②与对照组相比,盐酸法舒地尔在治疗过程中产生头痛、头晕、颜面潮红、低血压等不良反应发生率明显增高[RR=2.67,95%CI(2.06,3.46),P<0.00001]。
结论盐酸法舒地尔治疗急性缺血性脑卒中可短期内改善患者神经功能缺损,疗效显著,值得在临床中推广。
Part I:The experimental research about Rho kinase inhibitor (fasudil hydrochloride) in ischemic brain damage
Objective In this study,we investigate the effect of Rho kinase inhibitor fasudil hydrochloride on the phosphorylation and protein expression of Glutamate receptor6, Mixed lineage kinase3and c-Jun N-terminal kinase, and the effect on the expression of caspase-3, hippocampus CA1region neuronal apoptosis and death induced by cerebral ischemia followed by reperfusion.
Methods Global cerebral ischemia was induced by four-vessel occlusion. The SD rats weighed220-300g were randomly divided into four groups:the sham group, the ischemia/reperfusion group, the fasudil hydrochloride group and the physiological saline group.Fasudil hydrochloride were injected intraperitoneally30minutes before ischemia. And the physiological saline group were dealed with the intraperitoneal injection of the same volume of saline. The phosphorylation and protein expression of GluR6at6hours during reperfusion were detected using immunoprecipitation and immunoblotting analysis. The phosphorylation and protein expression of MLK3at ischemia/reperfusion6hours and JNK at ischemia/reperfusion3days were detected using immunoblotting analysis,respectively. The same method was used to detect the expression of caspase-3at ischemia/reperfusion6hours. Furthermore, we also use TUNEL staining and cresyl violet staining to examine the survival neurons in rat hippocampal CA1regions after3days and5days reperfusion, respectively.
Results1. Immunoprecipitation and immunoblotting analysis were used to analyze the phosphorylation of GluR6in serine site. The results showed that the GluR6serine phosphorylation level increased significantly at6h of reperfusion comparing with the sham group (P<0.05). Fasudil hydrochloride group could inhibit the increased phosphorylation of GluR6at6h of reperfusion comparing with the ischemia/reperfusion group and saline group, respectively (P<0.05).
2. Immunoblotting analysis was used to detect the phosphorylation and protein expression of MLK3, JNK, as well as the protein expression of caspase-3. The results demonstrated that the phosphorylation of MLK3,JNK and the protein expression of caspase-3elevated remarkably at ischemia/reperfusion group comparing with the sham group (P<0.05). However, the protein expression level of MLK3and JNK was hardly alterated at the same time point (P>0.05). Fasudil hydrochloride group could suppress the phosphorylation of MLK3(6h) and JNK(3d), as well as the expression of caspase-3at6h after reperfusion comparing with ischemia/reperfusion group and saline group,respectively(P<0.05).
3. TUNEL staining was used to examine the apoptosis of the neurons in3days after reperfusion in CA1region of hippocampus. The results indicated that significant numbers of TUNEL positive cells (41.80+3.03) were observed3days after ischemia/reperfusion. The numbers of viable neurons per1mm length of CA1pyramidal cells were quantitatively analyzed. Fasudil hydrochloride markedly decreased the neuronal loss comparing with the ischemia/reperfusion group (19.80±2.86)(P<0.05).
4. Cresyl violet staining was used to examine the survival neurons after5days reperfusion. The results showed that transient brain ischemia followed5days of reperfusion induced severe cell death, while normal CA1pyramidal cells in sham-operated groups showed round and pale stained nuclei. However,Fasudil hydrochloride obviously limited the neuronal injury.
Conclusion Fasudil hydrochloride can inhibit ischemia/reperfusion induced phosphorylation of GluR6in serine site significantly and then reduce the phosphorylation of MLK3, JNK and the protein expression of caspase-3eminently. Fasudil hydrochloride can reduce the apoptosis and injury, and perform a protective effect on neuron in CA1region of hippocampus following cerebral ischemia/reperfusion.
Part II:The Meta analysis of clinical efficacy of fasudil hydrochloride on acute ischemic stroke
Objective:To evaluate the curative effect and safety of fasudil hydrochloride injection in the treatment of acute ischemic stroke.
Method:We searched relevant randomized controlled trials (RCT) from Cochrane Library, PubMed, CBM, CNKI using Computer retrieval. And in the meanwhile, other retrieval methods were adopted to gain the information what we need. The search about fasudil hydrochloride injection in the treatment of acute ischemic stroke covered from the establishment of database to31, Dec,2011. Trials data were reviewed and extracted independently by2reviewers. The data were analyzed by RevMan5.0software.
Results:57RCT, which involved5945patients, were inclued. The results of Meta analysis showed that compared with control groups, fasudil hydrochloride had a significant benefit in improve the neural function defect [OR=3.38,95%CI (2.95,3.88), P<0.00001]. On the other hand, fasudil hydrochloride increased the occurrence of headache, giddiness,facial flushing and hypotension etc.[RR=2.67,95%CI(2.06,3.46), P<0.00001].
Conclusions:Fasudil hydrochloride could improve neurological function defect significantly in short time. And it is worthy of clinical application.
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1. Shibuya M, Suzuki Y, Sugita K, et al. Effect of AT877 on cerebral vasospasm after aneurysmal subarachnoid hemorrhage:Results of a prospective placebo-controlled double-blind trial [J]. J Neurosurgery,1992,76(4):571-577.
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