Effects Of Sodium Para-amino Salicylate On Manganese-induced Injury In Rat Primary Hippocampal Neurons Cultures

Objective To explore the effect of Manganese (Mn) and intervenient role of sodium para-amino salicylate (PAS-Na) on Mn-induced injury of hippocampal neurons of rats in primary culture. Materials and Methods 8-day-old cultured neurons in hippocampus of rat were randomly exposed to (1) 0,25,50,100,200μM MnCl2 for 24h, (2) 0,0.5,5,50,100,500μM PAS-Na for 24h ; morpHology of neurons was observed,and the effect of MnCl₂ or PAS-Na on the survival of hippocampal neurons was determined by MTT analysis. (3) Hippocampal neurons were randomly divided into the control, Mn-exposed, low, mid and high dose PAS-Na (L-PAS, M-PAS and H-PAS) intervenient groups. The control neurons were cultured for 48h only with medium; Mn-exposed neurons were exposed to medium with 50μM MnCl2 for 24h,and cultured by medium for 24h. Neurons of PAS intervenient groups were firstly exposed to medium with 50μM MnCl2 for 24h, followed by the cultured mediums with PAS-Na (50, 500 and 5000μM as the L-PAS, M-PAS and H-PAS groups, respectively) for another 24h. MorpHology of hippocampal neurons was observed under pHase-contrast microscope.Cell viability, apoptosis ratio,mean of fluorescence intensity in mitochondria and DNA damage of neurons were respectively determined by MTT, Annexin V- FITC apoptosis assay, Rhodarnine123 and SCGE. Results (1)Manganese induced the morphology injury and the concentration- dependently descended cell viability of of neurons. (2) Morphology and cell viability of neurons in PAS-Na exposure groups are similar to those of the control group. (3) Neurons of Mn-exposed group shrunk , with decreased synapses. Compared with Mn-exposed group, L-,M-PAS groups had similar shrunk neurons,with more synapses, while H-PAS group had more shrunk neurons ,with less synapses. Cells viability of Mn-exposed group was significantly lower than that of the control group (P<0.05). Apoptosis rate in neuron and mean of fluorescence intensity in mitochondria of Mn-exposed group were respectively higher than those of the control group(P<0.05). However, no significant change of cell viability, apoptosis ratio and mean of fluorescence intensity in mitochondria in L-PAS, M-PAS and H-PAS groups was found to compare respectively with Mn-exposed group(P>0.05). Compared with the control group, Mn-exposed group had lower fluorescent comet head, of which tail DNA percentage, Olive tail moment were higher (P <0.05).Compared with Mn-exposed group, L-, M-and H-PAS intervention group had similar fluorescence intensity of comet head, and of which the percentage of DNA tail and Olive tail moment both decreased (P <0.05). Conclusion (1) Mn induced hippocampal neurons injury. (2) 0.5-500μM PAS-Na wasn't toxic to these neurons. (3) The intervention effect of 50-5000μM PAS-Na on the DNA damage of Mn-exposed hippocampal neurons was observed.