Objective To evaluate the role of protein kinase C/extracellular signal-regulated kinase (PKC/ERK) signaling pathway in the proliferation of human embryonic neural stem cells regulated by paraquat.
Methods ReNcell CX immortalized cells cultured in vitro were exposed to paraquat at designed concentrations of 0, 5, 25, 50, and 100 μmol/L, respectively, for 24 h. Nestin, β-tubulin Ⅲ, and glial fibrillary acidic protein (GFAP) were detected by indirect immunofluorescence assay. Cell viability was determined by MTT assay. Lactate dehydrogenase (LDH) activity in supernatant was determined by spectrophotometry assay. Proliferation rate was evaluated by BrdU incorporation assay. Intracellular Ca2+ concentration was determined with flow cytometer. The protein expressions of PKCα and pERK1/2 were measured by Western blot.
Results Compared with the control group, cell viability decreased with increasing log-translated paraquat concentrations (25, 50, and 100 μmol/L) (r=-0.96, P < 0.05). The level of LDH significantly increased after the paraquat treatment at 50 and 100 μmol/L (P < 0.05). The results of immunofluorescent staining showed the BrdU positive cell counts decreased with the increasing paraquat concentrations; especially the paraquat treatment at 25, 50, and 100 μmol/L significantly inhibited ReNcell CX cell proliferation (P < 0.05). By flow cytometry, the paraquat treatment at 25, 50, and 100 μmol/L induced reduction in peak intracellular Ca2+ concentrations, and with the treatment concentration increasing, the Ca2+ fluorescence intensity was decreased (r=-0.97, P < 0.05). Meanwhile, the protein expressions of PKCα and pERK1/2 significantly decreased after treatment with various concentrations of paraquat (P < 0.05).
Conclusion Paraquat could decrease intracellular Ca2+ concentration and further reduce Ca2+-dependent PKCα activity and ERK1/2 phosphorylation. Ca2+/PKCα/ERK signaling pathway may be one of mechanisms that paraquat inhibits the proliferation of human embryonic neural stem cells.
DownLoad: