Effect and mechanism of fasudil on proliferation and differentiation of neural stem cells / 中国组织工程研究

ABSTRACT

BACKGROUND: The self-repairing ability of damaged brain and spinal cord is limited. It is important to search for potential therapeutics to promote the proliferation and differentiation of neural stem cells. OBJECTIVE: To investigate the effect of fasudil on the proliferation and differentiation of primary cultured neural stem cells and its potential mechanism. METHODS: Neural stem cells were obtained from the brain tissue of 15-day-old fetal rats in vitro. The expression of Nestin in the cells was detected by immunofluorescence. After treatment with fasudil at different concentrations (50, 100, 200 μmol/L) for 24, 48 and 72 hours, the proliferation rate of neural stem cells was detected by MTT, and the apoptosis rate was detected by flow cytometry. After further treatment with autophagy inhibitor, necrosis inhibitor, apoptosis inducer and ferroptosis inducer, the proliferation rates of neural stem cells were detected by MTT and the levels of malondialdehyde were detected by biochemical method. The expression of Nestin, doublecortin, microtubuleassociated protein and glial fibrillary acidic protein in neural stem cells were detected by western blot and immunofluorescence after treatment with 200 μmol/L fasudil for 10 days. RESULTS AND CONCLUSION: The positive expression of Nestin protein in primary cultured neural stem cells was observed. The proliferation rate of neural stem cells increased gradually with the increase of fasudil concentration as well as with the prolongation of action time (P < 0.05). Both apoptosis inhibitor and ferroptosis inhibitor can increase the proliferation rate of neural stem cells (P < 0.05). Fasudil increased the proliferation rate of neural stem cells treated by apoptosis inducer and ferroptosis inducer (P < 0.05). Fasudil and ferroptosis inhibitors both decreased the level of malondialdehyde in neural stem cells, while ferroptosis inducers increased the level of malondialdehyde in neural stem cells (P < 0.05). After treatment with fasudil, the expression of doublecortin and glial fibrillary acidic protein protein in neural stem cells increased, and the expression of Nestin decreased (P < 0.05). To conclude, fasudil can improve the survival of neural stem cells by inhibiting apoptosis and ferroptosis, and moreover, it can promote the proliferation and differentiation of neural stem cells into neuron-like cells and glial cells.