Abstract:

Objective To discuss the effect of ghrelin on the differentiation of the adipose mesenchymal stem cells （ADSCs） into the neurons，and to clarify its possible mechanism. Methods The ADSCs were randomly divided into blank group， neural differentiation inducer group， and ghrelin group （given 600 μg·L^－1 Ghrelin）， U0126 group （given 40 ng·L^－1 U0126）， Ghrelin+U0126 group （given 600 μg·L^－1 Ghrelin+40 ng·L^－1 U0126），and Ghrelin receptor blocker D-Lys3-GHRP-6 （D-Lys3-GHRP-6） group （given 10^－10 g·L^－1 D-Lys3-GHRP-6）. The pathomorphology of the cells in various groups was observed under inverted microscope；the positive expression rates of neurofilament protein （NF）-200 and tubulin （Tuj-1） in the cells in various groups were detected by immunofluorescence method； the expression levels of the mitogen activated protein kinase （MAPK）/extracellular signal-regulated kinase （ERK） pathway proteins，neuron specific nuclear protein （NeuN）， Tuj-1， neuron specific enolase （NSE），NF，growth hormone secretagogne receptor（GHSR），fetal liver kinase 1（Flk1）， and CD29 proteins in the cells in various groups were detected by Western blotting method. Results On the 10th day after cell culture， the ADSCs in blank group showed the fusions of long spindle and spiral shapes； the body of the cells in neural differentiation inducer group contracted，and the long spindle like cell body was transformed into the round-like shape， the protrusion length extended， and the number of neuron-like cells was increased； in Ghrelin group， the number of body protrusions of the cells was increased and the number of round-like like cells was increased； there was a small amount of cell body contraction and circular transformation in U0126 group and D-Lys3-GHRP-6 group. The immunofluorescence assay results showed that compared with blank group， the positive expression rates of NF-200 and Tuj-1 in the cells in neural differentiation inducer group were increased （P<0.05）； compared with neural differentiation inducer group， the positive expression rates of NF-200 and Tuj-1 in the cells in Ghrelin group were increased （P<0.05）， while the positive expression rates of NF-200 and Tuj-1 in the cells in U0126 group and D-Lys3-GHRP-6 group were decreased （P<0.05）； compared with Ghrelin group， the positive expression rates of NF-200 and Tuj-1 in the cells in U0126 group， D-Lys3-GHRP-6 group， and Ghrelin+U0126 group were decreased （P<0.05）. The Western blotting results showed that compared with blank group， the expression levels of NSE， NeuN， Tuj-1， NF，and GHSR proteins，and the ratios of phosphorylated MAPK（p-MAPK）/MAPK and phosphorylated ERK（p-ERK）/ERK in the cells in neural differentiation inducer group were increased （P<0.05）， while the expression levels of Flk1 and CD29 proteins were decreased （P<0.05）； compared with neural differentiation inducer group， the expression levels of NSE， NeuN， Tuj-1， NF，and GHSR proteins， and the ratios of p-MAPK/MAPK and p-ERK/ERK in the cells in Ghrelin group were increased （P<0.05）， while the expression levels of Flk1 and CD29 proteins were decreased （P<0.05）； the expression levels of NSE， NeuN， Tuj-1， NF， and GHSR proteins， and the ratios of p-MAPK/MAPK and p-ERK/ERK in the cells in U0126 and D-Lys3-GHRP-6 groups were decreased （P<0.05），and the expression levels of Flk1 and CD29 proteins were increased （P<0.05）； compared with Ghrelin group， the expression levels of NSE， NeuN， Tuj-1， NF，and GHSR proteins， and the ratios of p-MAPK/MAPK and p-ERK/ERK in the cells in U0126 group， D-Lys3-GHRP-6 group， and Ghrelin+U0126 group were decreased （P<0.05）， while the expression levels of Flk1 and CD29 proteins were increased （P<0.05）. Conclusion Ghrelin can induce the differention of the ASCs into the neurons，and it mechanism is related to activating the MAPK/ERK pathway.

Key words: Ghrelin, Mitogen-activated protein kinase, Extracellular signal-regulated kinase, Adipose-derived mesenchymal stem cells, Neural differentiation