The Experimental Research And Clinical Efficacy Meta Analysis Of Rho Kinase Inhibitor (Fasudil Hydrochloride) On Ischemic Brain Damage

Part I:The experimental research about Rho kinase inhibitor (fasudil hydrochloride) in ischemic brain damageObjective 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 strokeObjective: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.