Neurprotective Effects And Mechanisms Of Fasudil Mesylate As A Selective Rho Kinase Inhibitor On Ischemic Brain Damage

Aim To investigate the neuroprotective effect of fasudil mesylate (FM) and the underlyingmechanisms.Methods 1.The relaxation effect of FM was studied using cerebralvasospasm (CVS) in vivo and isolated aortic rings in vitro.2.FM was investigated for itsneuroprotective potential in rats with ischemia following middle cerebral artery occlusion(MCAO) and reperfusion.Rats were randomly assigned to sham,vehicle or FM groups.Infarct volume,neurological deficit score,morphology and molecular biological markerswere assessed.FM (10 mg·kg^-1) or NS was administered at 48h,24h and 2h before MCAO.Immunohistochemistry and western blot were used for detection of inducible nitric oxidesynthase (iNOS) and endothelium nitric oxide synthase (eNOS).3.The model of oxygenand glucose deprivation (OGD) insult in rat cortical and hippocampal brain slices was usedto investigate the neuroprotective effect of FM in vitro.FM (10,100μM) wereco-incubated with the slices for 30 min prior to and during OGD insult respectively.Brainslices viability was evaluated by using the 2,3,5-triphenyl tetrazolium chloride (TTC)method;The fluorescence of propidium iodide (PI) staining was used for quantification ofcellular survival,and lactate dehydrogenase (LDH) activity in incubation medium wasassessed to evaluate the degree of injury.Brain slices were collected at the end of theexperiment and stored at -70℃for analysis of iNOS and superoxide dismutase (SOD).4.The effect of FM on hydrogen peroxide (H₂O₂)induced neurotoxicity in PC12 cells wasinvestigated.PC12 cells were damaged by 300μM H₂O₂ for 12 h.Thiazoyl bluetetrazolium bromide (MTT) method and LDH release were detected to determine cellactivity.Hoechst 33258 staining and Acridine orange (AO)/Ethidium bromide (EB)double-staining were used to observe morphology changes of the cells. 2',7'-dichlorodihydrofluorescin diacetate (DHCF-DA) staining and nitroblue tetrazolium(NBT) method were used for quantification of intracellular ROS in PC12 cells.The qPCRand western blot method were used to test the mRNA and protein expression of Bax andbcl-2.Resultsâ… (1) FM (0.35,1.2,3.5 mg·kg^-1) decreased cerebrovascular resistance(CVR) and increased cerebral blood flow (CBF) dose-dependently,and the relaxationeffects of FM on cerebral vessels were much stronger than on peripheral vessels.(2) FMshowed dose-dependent relaxation of isolated aortic rings contracted by pretreatment withMethoxamine (Met) or KCL.The relaxation IC₅₀ of FM to the rabbit and rat aortic ringscontracted by KCL are 37.15μtM and 0.74μM respectively,and the relaxation IC₅₀ of FMand Prasozin (Pra) to the rabbit aortic rings contracted by Met are 27.54μM and 0.01μMrespectively.In addition,the relaxation action of FM had no obvious difference inendothelium-intact and endothelium-removal groups.(3) The Met dose-effect relationshipcurve was significantly shifted to the right by 0.3μM and 3μM of FM,and E_max wasdecreased.â…¡Gross anatomy showed that cerebral infarct size was significantly smaller inthe FM-treated than in the non FM-treated ischemic rats.In the brain regions vulnerableto ischemia of ischemic rats,FM was also found to significantly restore the enzyme proteinexpression level of endothelial nitric oxide synthase (eNOS),which was decreased inischemia.However,it remarkably reduced the protein synthesis of inducible nitric oxidesynthase (iNOS) that was induced by ischemia and reperfusion.â…¢In rat brain slices treatedwith OGD/REO injury,FM increased the neuronal cell viability by 40 % for cortex and by61% for hippocampus respectively.Finally,in the presence of OGD and FM,superoxidedismutase (SOD) activity was increased by 50 % for cortex and by 58 % for hippocampus,compared to OGD only group.â…£Pretreatment with FM prior to H₂O₂ exposuresignificantly elevated cell viability,reduced H₂O₂-induced cell apoptosis,and decreasedintracellular accumulation of reactive oxygen species (ROS).Furthermore,FM alsoreversed the upregulation of Bax/Bcl-2 ratio,the downstream cascade following ROS.Conclusions These results suggest that:1) The relaxation effects of FM on cerebral vessels were much stronger than on peripheral vessels in vivo,and the action was in anendothelium-independent manner;2) FM not only provided neuroprotective effects onneurological deficit and cerebral infarct size after MCAO/Reperfusion,but also amelioratedcell apoptosis induced by oxidative stress insult on PC12 cells directly.The mechanismsmay be involved in the increasing CBF,ameliorating vascular endothelium function,alleviation of intracellular ROS and reactive nitrogen species (RNS) generation,anddepressing of Bcl-2 family-related apoptotic signaling pathway.