RESUMO
This study is designed to investigate the function of the miR-145 in the protection of neural stem cells (NSCs) through targeting mitogen-activated protein kinase (MAPK) pathway in the treatment of cerebral ischemic stroke rat. In our study, rat NSCs were selected and cultured in complete medium. The light microscopy was used to observe the morphology of NSCs at different times. The quantitative reverse transcriptase real-time polymerase chain reaction (qRT-PCR) was used to detect the miR-145 and other related mRNAs of the MARK pathway. The Western blotting was used to detect the activation of MAPK pathway and neuronal specific markers. The Immunofluorescence was used to detect the expression of the neuron-specific enolase. And the cell viability was detected by Cell Counting Kit (CCK)-8 assay. The flow cytometry was used to test the cell cycle and apoptosis. The ischemic stroke rat models were established and neural stem cell transplantation was performed. The neurological function score, balance beam experiment, and cortical Nissl staining were used to evaluate the postoperative neurological function in rats. The expression of miR-145, extracellular signal-regulated kinase (ERK), and p38 mRNA in rat NSCs increased in a time-dependent manner. Compared with the Blank group, the over-expression of miR-145 promoted the expression of related mRNA and protein of the MAPK pathway in NSCs, while the decreased expression of miR-145 suppressed the MAPK Pathways. Compared with the Blank group, over-expression of miR-145 in NSCs promoted the up-regulation of Cyclin D1, Nestin, neuron-specific enolase (NSE), and Glial fibrillary acidic protein (GFAP) proteins, enhanced the activity of NSCs, and promoted cell proliferation and differentiation, while inhibited the cell apoptosis and the Cleaved-caspase 3 expression. After treatment of NSCs in the SB203580 group, the Nestin, NSE, and GFAP were decreased; cell viability, proliferation and differentiation were inhibited, while Cleaved-caspase 3 protein and cell apoptosis rate increased. The results of animal experiments showed that compared with the Blank group, the walking ability and neurological impairment recovered rapidly in the rats after transplantation of NSCs with over-expression of miR-145, and more neurons were generated in the cortex. After the transplantation of SB203580-treated NSCs, the walking ability and neurological impairment of the rats were slower and the cortical neurons were less. We conclude that miR-145 protects the function of neuronal stem cells through targeting MAPK pathway in the treatment of cerebral ischemic stroke rat.
