Objective: We employed a model in vitro that used PC12 cell line to test the concentration and time dependent relationship of Mn-treatment as well as the characteristics of MAPKs pathway under the same conditions, to explore the Neurotoxicity mechanisms of manganese.
Methods: PC12 cells in logarithm period incubated in culture media of 200, 400, 600, 800 micromol/L manganese (MnCl2) for 1 day, 2 days, 3 days, 4 days respectively. The neurotoxic concentration of manganese (MnCl2) on PC12 cells was screened by MTT and Plate clone forming tests. Cell growth curve was made in Typan-blue dying experiment. Western-blot was used to test p-Erk1/2 and p-p38.
Results: MTT and plate clone tests showed that 200, 400, 600, 800 micromol/L MnCl2 could suppress the proliferation of PC12 cells in dose and time-dependent trend during 1 d, 2 d, 3 d, 4 d respectively. The cell inhibited ratio on the fourth day in 600 micromol/L MnCl2 culture medium approached 50% or more. Western-blot tests showed that p-Erk2 of PC12 cells incubated in 600 micromol/L MnCl2 culture medium was decreasing gradually on the 1st, 2nd, 3rd and 4th day and on the 2nd day less than control group by 75% (n = 3, P < 0.05). With cells treated by 200, 400, 600 micromol/L MnCl2 for 4 days, p-Erk2 lost by degrees. On the 4th day, p-Erk2 of 400 micromol/L MnCl2-treated group was less 78% than that of control group (n = 3, P < 0.05). P-p38 of PC12 cells incubated in 600 micromol/L MnCl2 culture medium was increasing gradually on the 1st, 2nd, 3rd and 4th day and on the 3rd day 6.6 times higher than that of control group (n = 3, P < 0.05). P-p38 of PC12 cells enhanced by degrees in 200, 400, 600 micromol/L MnCl2 treated for 4 days and in 400 micromol/L MnCl2 treated group on the 4th day was 4.7 times higher than that of control group (n = 3, P < 0.05).
Conclusion: The decreased p-Erk2 and the increased p-p38 maybe co-worked to induce proliferation arrest and apoptosis in PC12 cells.