Effect Of DAPT Blocking Notch Signaling Pathway On TOCP Induced Neurotoxicity In SH-SY5Y Cells

ObjectiveIn order to investigate the relationship of Notch signaling pathway in Tri-ortho-cresyl phosphate(TOCP) induced neurotoxicity in SH-SY5 Y neuroblastoma cells,further elaborates the mechanism of TOCP neurotoxicity. Methods Methods1.One experiment group with different concentrations TOCP(0,0.25,0.50,1.0m M) treated SH-SY5 Y cells,another group of first with DAPT pretreatment, then using TOCP(0,0.25,0.50,1.0m M) exposure treatment 48 h, examine the effect of the two treatments SH-SY5 Y cell viability by MTT method; Western Blot detected the TOCP treatment or by DAPT pretreated cells, then TOCP treatment 24 h,andeffect on Notch signaling pathway in cells.2. DMSO as the control group, DAPT pretreatment SH-SY5 Y cells 30 min, and then exposed to 0.50 m M TOCP 24 h, cells were collected, and was detected by Western Blot autophagy protein Beclin1 and LC3 ? / ? protein expression;Cell staining was carried out with the staining agent MDC, and the changes of the vacuoles in each group were observed.3. DAPT pretreatment with undifferentiated and differentiated SH-SY5 Y cells 30 min, and TOCP 0.50 m M exposure to 24 h, DMSO as the control group, Western Blot detection of NF-H, tau, p-tau protein expression.4. DAPT pretreated SH-SY5 Y cells and sh-Beclin1 cells were treated with 30 min, and then 0.50 m M TOCP was exposed to 24 h. Western Blot was used to detect the expression of apoptosis related genes Bcl-2, BAX, and Casepase-3 in two groups of cell lines. Results1. MTT results showed that with TOCP concentration increased, and compared to the control group,TOCP treatment group and DAPT pretreatment exposed to TOCP group,cell survival rate decreased;while the use of DAPT pretreatment blocking Notch, then 0.25 m M,0.5m M TOCP cells exposed group of cell survival rate than TOCP group high(P<0.05), and 1.0m M TOCP, two groups of survival rate did not differ significantly.2.TOCP exposure group and DAPT pretreatment exposed to TOCP group inhibited SH-SY5 Y cells Notch signaling pathway related protein NIC1 and Hes1 protein expression(P<0.05)3.DAPT pretreated SH-SY5 Y then TOCP exposed groups and 0.50 m M TOCP exposed group compared with the control group, in the DAPT pretreatment group,Beclin1 and LC3 expression was inhibited(P<0.05). MDC staining showed that DAPT pretreatment group generated by autophagy bubble number less than that of the TOCP treatment group.4.Compared with the control group, after DAPT pretreatment undifferentiated and differentiated SH-SY5 Y cells exposed TOCP groups rejection ratio 0.50 m M TOCP group, to some extent, the degradation of NF-H(P< 0.05), but DAPT preprocessing group, tau further degradation(P< 0.05), and p-tau in 0.50 m M TOCP treatment group expression was the lowest(P< 0.05).5.Pretreatment of DAPT SH-SY5 Y cells and sh-Beclin1 cells exposed to 0.50 m M TOCP, using DMSO as control group. Western blot was used to detect the apoptosis related proteins; in SH-SY5 Y cells,Bcl-2/Bax ratio without significant changed(P< 0.05) and Cleaved-caspase-3 expression had no significant difference(P < 0.05). In sh-Beclin1 cells, compared with the control, the DAPT pretreatment group,bcl-2/bax ratio was higher than that of 0.50 m M TOCP(P<0.05). Conclions1.TOCP could inhibit the expression of NIC1 and Hes1 protein in notch signal pathway.2.After blocked the notch signaling pathway by DAPT, autophagy induced by TOCP was inhibited.3.TOCP decreased the level of NF-H protein in undifferentiated and differentiated cells, but inhibited the decreasing of NF-H protein after DAPT blocked notch signaling pathway.