Effect Of Glutathione And N-acetylcysteine Against Glutamate Cytotoxicity

Massage transmitter turbulence, abnormal receptor activation, intracellular Ca2+ overload, oxidative stress, et al are believed to play important roles in many diseases of central nervous system (CNS), such as stroke, epilepsy, chronic neurodegenerative diseases. Besides as a free radical scavenger, glutathione may also acts as neuropeptide in CNS considering its peculiar composing and structure. It is rather deserved to investigate whether glutathione could do good to diseases of CNS. In this paper, we observed the effects of Ghithion, reduced glutathione (GSH) , and cysteine donor, N-acetylcysteine (NAC), on neuron death and intracellular Ca2+ induced by glutamate (Glu) and analysed their mechanism. We hope it can offer evidence and guidance to applying these drugs in CNS diseases.Glutamate cytotoxicity model of primary culture neurons from neonatal Wistar rat hippocampal was used to evaluate the effects of different treatment of glutathione and NAC on neuron damage caused by Glu of different concentration and compare with each other. Terminal deoxynucleotidyl transferase mediated nick end (TUNE) labeling and trypan blue dye were used to detect cultured neuron apoptosis and death respectively. Intracellular Ca2+ current was determined by Laser scanning confocal microscopy (LSCM) . The results were as follows:1. At the 11-12th cultured day, the primary culture neurons are multiform with strong refraction and third dimension. The nucleolus in cells was clear and the slender processes were thick. It was proved that almost 95 percent of the cells in culture were neurons that expressed neurospectific enolase (NSE).2. It was obviously that Glu have toxicity to neurons at the 12th cultured day. Noncompetitive NMDA receptor antagonist, MK-801, could reduce ratio of neuron death and apoptosis induced by Glu. No matter pre-treatment or post-treatment of GSH or NAC had no obvious protective effect on neuron death caused by 500 uM Glu, but NAC of ImM could markedly reduce the ratio of neuron apoptosis induced by 500 u M Glu. GSH or NAC could prevent neuron death and apoptosis induced by Glu of 100 u M. The ratio of surviving neurons of GSH post-treatment group was higher than the pre-treat one, while the NAC pre-treatment group was superior. The protective effect of GSH post-treatment and NAC post-treatment at 1mM were as same as MK-801 group.3. GSH and NAC at 1mM cultured with neurons for 3 days could reduce basal level of intracellular Ca2+, and inhibite Ca2+ influx induced by Glu of 100u M. NAC was more efficiency than MK-801 of 10 u M. 1mM GSH treated instantly could induce Ca2+ influx, while NAC at the same concentration had no such effect.4. The ratio of surviving neurons cultured with GSH or NAC at 1mM and 10mM for 3 days was not affected. GSH at 100mM could cause neurons death and collapse. Although the configuration of cultured neurons was integrity after treated with NAC of lOOmM, all the cells were stained with trypan blue; membrane of neuron was crimpled under Atomic Force Microscopy scan; cultures marked with Fluo-3 (AM) had no fluorescence under LSCM and neurons had no response to Glu.Conclusion:1. Either pre-treatment or post-treatment both Gluthion and NAC can protect neurons against light Glu damage. The difference between Gluthion and NAC showed that Gluthion is fit for emergency use while NAC is more fit for prevention. The mechanism of Gluthion against light Glu cytotoxicity is antioxidation and decreasing Ca2+ influx induced by Glu.2. Because both Gluthion and NAC exhibit toxicity at the concentration which is much higher than that in vivo, which indicates an useful clinical purpose. It is the first report that NAC at higher concentration can fix tissue.3. GSH and NAC have effects on basal intracellular Ca2+ of neurons, but the exact mechanism still under further consideration.