Effects of Isoprocarb Exposure on Differentiation, Migration and Neurite Outgrowth of the Key Cell During Neural Development in Vitro
|School||Anhui Medical University,|
|Keywords||isoprocarb mouse neural stem cells migration differentiation neurite outgrowth|
Objective The primary culture of mouse embryonic neural stem cells(mNSC)、C6 glioma cells and SH-SY5Y neural tumor cells were used for research model.To investigate the effect of isoprocarb on the migration and differentiation of mNSC and the neurite outgrowth of C6 and SH-SY5Y cells in vitro.Methods The mNSC、C6 and SH-SY5Y cells were exposed to isoprocarb (5、25、50、100 mg/L) for 24h and/or 48h. The cytotoxicity of on differentiating cells were deceted by MTT assay and LIVE/DEAD assay (only LIVE/DEAD assay was used to assay the cytotoxicity of C6 and SH-SY5Y cells). Then Immunofluorescence staining was used to determine the migration and differentiation of mNSC, and the neurite outgrowth of differentiating C6 and SH-SY5Y cells was examined by Coomassie blue dye binding assays.Statistical treatment The data were analyzed with SPSS 11.0.Results MTT results:the differentiating mNSC showed significant cytocoxicity by 50 mg/L isoprocarb treatment for 24 and 48 h.With the increase of isoprocarb dose,it strengthened cytotoxicity of differentiating mNSC in a dose-dependen(t24 h, rs=-0.820; 48 h, rs=-0.950; all P<0.01). MTT and LIVE/DEAD results both showed: After 48 h treatment, 50 mg /L isoprocarb caused the significant cytotoxicity of the differentiating SH-SY5Y cells, while the differentiating C6 cells had some differences in cytotoxicity only in 100 mg /L dose. As the increase of isoprocarb concentration , cell viability decreased in a dose-response relationship (C6: MTT, rs=-0.862; LIVE/DEAD, rs=0.818; SH-SY5Y: MTT, rs=-0.796 ; LIVE/DEAD, rs=0.906; all P<0.05).Immunofluorescence results showed that , the migration of mNSC was inhibited by isoprocarb in a dose-response relationship(Aa/Ab, rs=-0.998; Dm/Db, rs=-0.995, all P<0.01) and 25 mg/L isoprocarb had affected mNSC migration. the rate of the GFAP- positive and TUJ-positive cells were both reduced(8.34%±1.78%,1.97%±0.35%)by 50 mg/L isoprocarb and had significant differences. 25 mg/L isoprocarb had decressed the number of GFAP-positive cells’neurite and the length of the TUJ-positive cells’neurite. To 100 mg/L isoprocarb, this phenomenon is more pronounced.Coomassie blue dye binding results:the rate of C6 cells’neurite extension was inhibited by 50 mg/L isoprocarb, but 25 mg/L isoprocarb inhibited the rate of SH-SY5Y cells’neurite extension. Isoprocarb inhibited the rate of C6 and SH-SY5Y cells’neurite extension in a dose-dependent manner(C6, rs=-0.806; SH-SY5Y, rs=-0.975; all P<0.01).Conclusions Under the condition of no significant cytotoxicity, isoprocarb inhibited the migration and differentiation of mNSC, especially the astroglias were more vulnerable to isoprocarb than neurons in the mNSCs differentiation stage. And then, it also could affect neurite outgrowth of glial cells(C6) and neurons(SH-SY5Y). Therefore, isoprocarb interfered the key events (cell migration,differentiation and neurite outgrowth) of neural development system,suggesting isoprocarb may have developmental neurotoxicity.