Relationship between apoptosis and molecular mechanism of p-38 MAPKS activation in manganese-treated PC12 cells

  Objective   To employ PC12 cell line as a model in vitro to test the concentration and time of maganese on proliferation arrest, and to observe the changes of cell morphology, cell cycle and biochemical marker and further to study the relationships between above aspects and activiations of p38MA PKs pathway.PC cells in logarithm period incubated in culture media with 200, 400, 600, 800 μmol/L manganese(MnCl₂)for 1, 2, 3, 4 days respectively.

  Methods   Cell viability was examined by MTT.Cell cycle was monitored by FCM(flow cytometry).Morphological changes of PC12 cells was investigated by transmisssion electron microscope.Agarose gel electrophoresis was used to test the genomic DNA.Western-blot was used to test p-p38.

  Results   MTT revealed that 200, 400, 600, 800 μmol/L MnCl₂ could suppress the proliferation of PC12 cells in dose and time-dependent trend.The cell inhibited ratio on the fourth day in 600 μmol/L MnCl₂ approached 50% or more.FCM showed that the cell cycle of the PC12 cells could be inhibited in Speriod at the concentration of 600 μmol/L MnCl₂ on the 4th day and apoptosis was observed through FCM and transmisssion electron microscope Biochemical hallmark of DNA fragments was observed.Western-blottests showed the p-p38 in 600 μmol/L MnCl₂ culture medium was increasing gradually on the 1st, 2nd, 3rd and 4th day.the p-p38 on the 3rd day was 616 times higher than that of control group(n=3, P < 0.05).The p-p38 was enhanced by degrees in 200, 400, 600μmol/L MnCl₂-treated PC12 cells in 4 days.The p-p38 of 400 μmol/L MnCl₂ treated group on the 4th day was 4.7 times higher than that of control group(n=3, P < 0.05).

  Conclusion   Manganese upregulated p-p38 through MEK 3/6 downstream to induce proliferation arrest and apoptotic cell death.