Effects Of Inosine On Neuronal Protection And Neurite Outgrowth Of Rat Pheochromocytoma (PC12) Cells

Center Nervous System (CNS) injuries often lead to calamitous results, mainly due to secondary cell death, adverse environment and lack of neurotrophic factors which can benefit regeneration of injured nervous fibers. Therefore, to explore the effective measures to treat the CNS injury is a long term effort of neuroscientists. Inosine, one of the catabolic products of purine nucleotide, has long been clinically used to supplementally treat a variety of diseases due to its involvement in energy synthesis. Recently, it has been reported that inosine also plays an important trophic role in the CNS. This study aimed to explore the possible effects of inosine on neuroprotection and neurite outgrowth of rat pheochromocytoma (PC 12) cells, an acknowledged model of neuron.In the first part of this study, we injured the PC 12 cells by the high concentration of zinc sulfate, and after administering inosine, the morphological changes of the PC 12 cells were observed and the mortality was numbered. We found that inosine at various concentration strikingly attenuated the mortality induced by zinc sulfate in a dose-dependent manner.Because the nucleoside phosphorylase (PNP) is known to be absent in PC 12 cells, inosine is not able to replenish the ATP pool through its classic pathway. Therefore we postulated that the neuroprotective effect of inosine might be realized by a nuclear enzyme-poly (ADP-ribose) polymerase. In the second part, the PC 12 cells were injured by directly blowing off (instead of being digested with trypsin) from the flask wall, and then further mechanically deprived of their neurites by repetitive aspiration in the media through a Pasteur pipette. After adding inosine, we 1) observed the morphological changes of PC 12 cells and quantified the cell number according to the length of neurites; 2) immunostained the cells with antibodies against GAP-43 and MAPK. Our results showed that at different time points, the number of cells with long neurite or branching neurite in the inosine groups was much more than that in the control groups. And so did the case of the immunocytochemical positive cells. The mechanism that inosine enhances the neurite outgrowth is discussed, and possible involvement of intracellular MAPK is analysed.Our study reveals that inosine plays important roles in protecting the PC 12 cells against zinc-induced injury in a dose-dependant manner, prompting the neurite outgrowth and branching, and increasing the expression of neurite outgrowth-associated proteins.