title: Fasudil hydrochloride protects neurons in rat hippocampal CA1 region through inhibiting GluR6-MLK3-JNKs signal pathway.
abstract: Fasudil hydrochloride (FH), a Rho kinase (ROCK) inhibitor, has been reported to prevent cerebral ischemia in vivo from increasing cerebral blood flow and inhibiting inflammatory responses. However, it is uncertain by what mechanism a ROCK inhibitor can directly protect neurons against ischemic damage. The present study was designed to evaluate whether FH decreased the increased phosphorylation of glutamate receptor 6 (GluR6) and its downstream in GluR6-MLK3-JNKs signal transduction pathway following global transient cerebral ischemia, as a result of protecting against neuronal apoptosis and death. Transient cerebral ischemia was induced by the Pulsinelli-Brierley four-vessel occlusion method. FH (15 mg/kg) was administered to rats by intraperitoneal injection 30 min before ischemia. The phosphorylation and protein expression of GluR6 at 6 h during reperfusion were detected using immunoprecipitation and immunoblotting analysis. The phosphorylation and protein expression of Mixed lineage kinase 3 (MLK3) at ischemia/reperfusion (I/R) 6 h and c-Jun N-terminal kinase (JNK) at I/R 3 d were detected using immunoblotting analysis, respectively. The same method was used to detect the expression of caspase-3 at I/R 6 h. Furthermore, we also use TUNEL staining and Cresyl violet staining to examine the survival neurons in rat hippocampal CA1 regions after 3 and 5 d reperfusion, respectively. Our study indicated that FH could inhibit the increased phosphorylation of GluR6 and MLK3 and the expression of caspase-3 at peaked 6 h of reperfusion and the phosphorylation of JNK (3 d) (p < 0.5). The results of TUNEL staining and Cresyl violet showed that the number of surviving pyramidal neurons in rats hippocampal CA1 subfield increased markedly in FH-treated rats compared with ischemic groups after 3 or 5 d of reperfusion following ischemia (p < 0.5). These results suggested that FH, as a ROCK inhibitor, may be partly responsible for its protective effects against such damage by taking part in GluR6-MLK3-JNKs signaling pathway which modulates ischemic damage. Taken together, this is the first study investigating Rho and ROCK as the upstream of GluR6 taking part in GluR6-MLK3-JNKs signal transduction pathway following cerebral ischemia.
authors.affiliation: Department of Neurology, The Second Affiliated Hospital of Xuzhou Medical College, Xuzhou, 221000, People's Republic of China, wxeqq@163.com.
authors.forename: Xiu-E
authors.initials: XE
authors.lastname: Wei
authors.forename: Feng-Yu
authors.initials: FY
authors.lastname: Zhang
authors.forename: Kai
authors.initials: K
authors.lastname: Wang
authors.forename: Qing-Xiu
authors.initials: QX
authors.lastname: Zhang
authors.forename: Liang-Qun
authors.initials: LQ
authors.lastname: Rong
mesh_terms: D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D056547:CA1 Region, Hippocampal / Q000166:cytology* / Q000187:drug effects; D053148:Caspase 3 / Q000378:metabolism; D019610:Cytoprotection / Q000187:drug effects*; D004789:Enzyme Activation / Q000187:drug effects; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D020930:MAP Kinase Kinase Kinases / Q000378:metabolism; D020935:MAP Kinase Signaling System / Q000187:drug effects*; D008297:Male; D009474:Neurons / Q000166:cytology* / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D010766:Phosphorylation / Q000187:drug effects; D051381:Rats; D017207:Rats, Sprague-Dawley; D018092:Kainic Acid Receptors / Q000737:chemistry / Q000378:metabolism; D015427:Reperfusion Injury / Q000378:metabolism / Q000473:pathology / Q000517:prevention & control; D012694:Serine / Q000378:metabolism; D000097767:Mitogen-Activated Protein Kinase Kinase Kinase 11; D000097806:GluK2 Kainate Receptor
chemical_list: D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D053148:Caspase 3; D048031:JNK Mitogen-Activated Protein Kinases; D020930:MAP Kinase Kinase Kinases; D018092:Kainic Acid Receptors; D012694:Serine; D000097767:Mitogen-Activated Protein Kinase Kinase Kinase 11; D000097806:GluK2 Kainate Receptor; C049347:fasudil