Fasudil hydrochloride differentiates bone marrow mesenchymal stem cells into neurons via notch signaling

BACKGROUND： Notch signaling regulates bone marrow mesenchymal stem cell （MSC） proliferation, differentiation, and apoptosis, Notch signaling and Rho kinase signaling exhibit a crosstalk phenomenon with JAK/STAT, and both participate in the neuronal dendritic spine development. Inhibition of RhoA/Rho kinase signaling may regulate MSC differentiation into neuronal-like cells. OBJECTIVE： To investigate the effect of Notch1 signaling on the differentiation of rat MSCs into neurons induced by fasudil hydrochloride （C14H17N3O2S-HCI）, a Rho kinase inhibitor, through a siRNA approach. DESIGN, TIME AND SETTING： An in vitro cytological experiment was performed in the Cell Laboratory of Henan Academy of Medical and Pharmaceutical Sciences between December 2007 and May 2009. MATERIALS： MSCs were obtained from Wistar rat femoral bone, fasudil hydrochloride was provided by -Tianjin Chase Sun Pharmaceutical Co., Ltd. Rn-notchl-siRNa, negative control siRNA （Cy3 label） and Rn-MAPK1 control siRNA were provided by QIAGEN, Coloqne, German. METHODS： The cultured MSCs were divided into non-transfected, transfected group （transfected with Rn-Notchl-siRNA）, positive control （transfected with Rn-MAPK-1 control siRNA）, and negative control （transfected with negative control siRNA） groups. Fasudil hydrochloride was applied to induce MSCs to differentiate into neurons. MAIN OUTCOME MEASURES： The fluorescence expression by the transfected MSCs was observed under an inverted fluorescence microscope; the expression of Notch1 mRNA, Hesl mRNA, and MAPK1 mRNA in MSCs was detected by reverse transcription polymerase chain reaction; the expression of Notch1 protein, nestin, neurofilament M, and glial fibrillary acidic protein was detected by immunocytochemistry. The viability of MSCs was detected by tetrazolium bromide assay. RESULTS： MSC fluorescence increased following a 72-hour siRNA transfection, with transfection efficiencies of up to （0.91 ± 0.04）; the Notch1 mRNA and Hesl mRNA expressed by transfected MSCs was significantly decreased （P 〈 0.05） compared with non-transfected cells. Fasudil hydrochloride induced MSCs to differentiate into neurons with greater efficiency in the transfected group （P 〈 0.05）. CONCLUSION： Fasudil hydrochloride induces rat MSCs to differentiate into neurons; inhibition of Notch1 signaling and Hesl expression may jointly promote the differentiation of MSCs into neurons.

Rho kinase:A new target for treatment of cerebral ischemia/reperfusion injury

Rho kinase inhibitor fasudil hydrochloride has been shown to reduce cerebral vasospasm, to inhibit inflammation and apoptosis and to promote the recovery of neurological function. However, the effect of fasudil hydrochloride on claudin-5 protein expression has not been reported after cerebral ischemia/reperfusion. Therefore, this study sought to explore the effects of fasudil hydrochloride on blood-brain barrier permeability, growth-associated protein-43 and claudin-5 protein expression, and to further understand the neuroprotective effect of fasudil hydrochloride. A focal cerebral ischemia/reperfusion model was established using the intraluminal suture technique. Fasudil hydrochloride （15 mg/kg） was intraperitoneally injected once a day. Neurological deficit was evaluated using Longa＇s method. Changes in permeability of blood-brain barrier were measured using Evans blue. Changes in RhoA, growth-associated protein-43 and claudin-5 protein expression were detected using immunohistochemistry and western blotting. Results revealed that fasudil hydrochloride noticeably contributed to the recovery of neurological function, improved the function of blood-brain barrier, inhibited RhoA protein expression, and upregulated growth-associated protein-43 and claudin-5 protein expression following cerebral ischemia/reperfusion. Results indicated that Rho kinase exhibits a certain effect on neurovascular damage following cerebral ischemia/reperfusion. Intervention targeted Rho kinase might be a new therapeutic target in the treatment of cerebral ischemia/reperfusion.