Long Term Neurite Outgrowth Enhancing Effect and Neurite Regeneration Effect of an Active Substance from a Brown Alga Sargassum Macrocarpum on Rat Pheochromocytoma PC12D Cells

Abstract

We have previously purified a novel nerve growth factor (NGF)-potentiating substance, MC14 from a brown alga Sargassum macrocarpum and founded that it was capable of significantly enhancing NGF-mediated neurite outgrowth from rat pheochromocytoma PC12D cells in 48 hours. In this study, we attempted to investigate the long term effect of MC14 on neurite outgrowth enhancing activity by monitoring the neurite outgrowth activity for a period of 28 days with constant renewal of NGF and MC14 in the medium. The result showed that MC14 promoted and subsequently maintained the neurite outgrowth of PC12D cells. For the treatment of 6.3 μg/ml MC14 in the presence of 25 ng/ml NGF, the percentage of neurite-bearing cells gradually increased and maintained at about 60% during the treatment period of 28 days. There was a 3-fold enhancement compared to the effect of 25 ng/ml NGF alone. Apart from long term enhancement of neurite outgrowth, the effect of MC 14 on promoting neurite regeneration on degenerated PC12D cells was also studied in order to further investigate the capability of MC14 to promote the reconstruction of neurites of degenerating neuronal-like cells. The neurites of differentiated PC12D cells were mechanically sheared by trituration and the cells were then plated and subjected to MC14 treatment. Phase-contrast microscopic observation revealed that all of the MC 14-treated cells exhibited higher proportion of neurite regeneration compared to the untreated cells. Approximately 2.7-fold enhancement of neurite regeneration was observed for the cells treated with 3 µg/m1 MC14 in the presence of 10 ng/ml NGF for 48 hours compared with the MC 14-untreated cells at the same NGF concentration. Our findings demonstrated that MC14 may not only enhance and support the NGF-mediated neurite outgrowth of naive PC 12D cells in long term treatment but also promote neurite regeneration on the neurite-degenerated cells.