| Backgroud:Ischemic stroke has the characteristics of high rates of morbidity, mortality and disability. Therefore, to seek the effective treatment and restore the neural function have generated serious challenge in the neural medical research. The discovery of neural stem cells (NSCs) brings new hope for treatment of central nervous system injury or degenerative diseases. The brain has the ability of self-repair after the cerebral injury, such as the NSCs are activated, then they can proliferate, migrate, differentiate and integrate to improve neurological function. However, these abilities are poor. Therefore, to explore some methods of drugs and rehabilitation for enhancing NSCs proliferation, migration, differentiation and integration under the pathological condition in neurogenetic areas, in order to promote nerve functional recovery, will have the wide prospect in clinical application.Resveratrol (Resveratrol, Res) is a natural phytoalexin that is found in plants such as mulberries, polygonum cuspidatum, semen cassiae, grapes and peanuts, which has anti-oxidant, anti-inflammatory, anti-cancer, anti-aging and lipid-lowering pharmacological properties. In addition, it has been reported that resveratrol has a neuroprotective effects on cerebral ischemia/reperfusion injury in vivo, but the concrete mechanism of nerve regeneration is not clear.Objective:To investigate the effect and its mechanism of resveratrol pretreatment on proliferation of rat cortical NSCs after oxygen-glucose deprivation/reoxygenation (OGD/R) in vitro.Methods:Isolation and purification of NSCs in neonatal Sprague-Dawley (SD) rats by suspended cultivation. The 2-4 generation NSCs of adherent culture were cultured under oxygen and glucose deprivation for 150 min, and returned to normal oxygen for 24 h. (1) To study the concentration effects of resveratrol on NSCs proliferation. Four groups were studied:normal group (Norm), control group (Ctrl), vehicle group (Veh), resveratrol pretreatment group (low concentration with 1 μmol/L Res, middle concentration with 5 μmol/L Res, and high concentration with 20 μmol/L Res). (2) To investigate the mechanism of resveratrol on NSCs proliferation. Five groups were studied:normal group (Norm), control group (Ctrl),5 μmol/L resveratrol pretreatment group (Res 5), cyclopamine pretreatment group (Cyc),5 μmol/L resveratrol +cyclopamine pretreatment group (Res 5+Cyc). Cell viability was observed by CCK-8 assay. Flow cytometry cell cycle and BrdU assay measured cell proliferation. Immunofluorescence was used to identify NSCs and nuclear translocation of Gli-1. The expressions of Patched-1 (Ptc-1), Smoothened (Smo), Gli-1 mRNAs and proteins were detected with RT-PCR and Western blotting, respectively.Results:Cells both in suspended and adherent cultivation were high expression of neuroepithelial stem cell protein (nestin). Resveratrol pretreatment could significantly enhance NSCs viability and proliferation in a concentration-dependent manner after OGD/R injury. At the same time, Gli-1 entered the nucleus, the expressions of mRNAs and proteins were upregulated, which Gli-1 was in the nucleus, Ptc-1 and Smo were in the cytoplasm, and the best effective concentration of resveratrol was 5 μmol/L (P<0.05), which was inhibited by cyclopamine (P< 0.05), a Smo receptor inhibitor of Sonic hedgehog (Shh) signaling pathway.Conclusion:Shh signaling mediates resveratrol to increase NSCs viability and proliferation after OGD/R injury in vitro. |
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