The Role And Mechanism Of Ddvp Induced Autophagy In SK-N-SH Cells

Objective Study the effects and mechanisms of autophagy induced by dichlorvos(O,O-dimethyl-O-2,2-dichlorovinylphosphate,DDVP)on the neurotoxicity 0f SK-N-SH cells.By observing the effect of DDVP on oxidative damage in SK-N-SH cells,the way of oxidative protein's degradation in injured cells,explore the mutual relations between autophagy,calpain system and ubiquity proteasome pathway in the degradation process of oxidative damaged protein.Methods 1.SK-N-SH cells were counted to determine the population doubling time.treated with DDVP at different concentrations(0~120?M)for 48 hours,and then,the half maximal inhibitory concentration(IC50)were measured by MTT assay.2.cell morphological changes 0f SK-N-SH cells were observed under light microscopy by Pap staining and then the Superoxide dismutase(SOD)activity,catalase(CAT)content and intracellular malondialdehyde(MDA)content were detected.3.cofocal laser scanning to observe the changes of autophagy in SK-N-SH cells(treated with DDVP at different concentration or starvation for 48 h,MDC staining).Then,after the group of 3-MA(1 m M,PI3K/AKt VP34 signaling pathway inhibitor of autophagy),and the group of Calpastatin(500 n M,Calpain inhibitor)were pretreated by each other for 6h and 4h,SK-N-SH cells were treated with 100 ?M DDVP for 48 h,DMSO was the control group,the expression of Beclin1,LC3 II and LC3 I protein was detected by Western Blot.4.the activity of calpain in SK-N-SH cells(treated with 100 ?M DDVP for 48 h,or treated with 100 ?M DDVP for 48 h in the absence or presence of 20?M verapamil and 100?M 2-APB which were pretreated before DDVP 15 min)was measured by fluorospectrophotometer and the expression of Beclin1,LC3 II and LC3 I in the same group was analyzed by western blot.5.Sk-N-SH cells were treated with 0~100?M DDVP for 48 h,the effects of 26 S proteasome activities was measured by fluorospectrophotometer,the expression of P62,MAP-2 was analyzed by western blot.the ubiquity labeling of MAP-2 was analyzed by CO-IP.Results 1.The population doubling time of SK-N-SH cells is 36.28±1.46 hours.After exposure of DDVP for 24 or 48 hour,the cells have no significant changes in cell morphology.The IC50 of DDVP treatment to SK-N-SH cells for 48 hour is(105.22±5.80)?mol/L.2.With the increased concentration of DDVP treatment,intracellular MDA content gradually increased,while the activity of CAT and SOD gradually decreased(P<0.05),which showed significant dose-response relationship(P<0.05).3.Compared to control group,the number of autophagesomes in SK-N-SH cells treated with DDVP increased(P<0.05),just as the increased expression of Beclin1(P<0.05),LC3II(P<0.05).However,the increase can be inhibited by 3-MA(P<0.05)and Calpastatin(P<0.05).4.There isn't any significantly increase of Calpain activity in the SK-N-SH cells not or treated with DDVP that pretreated with the absence or presence of verapamil or 2-APB.But it's different in the expression of Beclin1,LC3 II and LC3 I.Compared to 100?M DDVP group,verapamil(P<0.05)inhibited the expression of Beclin1 and LC3 II induced by TOCP and verapamil+2-APB+TOCP group(P>0.05)was partly decreased,2-APB increased the expression(P>0.05)for 48 h,the expression of Beclin1 and LC3-II were significantly increase after a dose of 100?M DDVP for 48 h(P<0.05).5.Compared to control group,the levels of MAP-2(P<0.05)and p62(P<0.05)were decreased gradually by 0 ~ 100?M DDVP in SK-N-SH cells.The levels of ubiquities MAP-2 was increased gradually by DDVP.4.Compared to control group,the activity of 26 S proteasome CT-L(P<0.05),T-L(P<0.05)and PGPH(P>0.05)were decreased at different exposed doses by DDVP.Conclusions 1.DDVP can cause oxidative damage on human neuroblastoma SK-N-SH cells.2.Treated with DDVP would increase the Autophagy level of Sk-N-SH;PI3K/AKt signaling pathway,calpain system and ubiquity proteasome pathway participate in the process of increasing Autophagy and depredating oxidative damaged protein.3.DDVP induced SK-N-SH cells MAP-2 polyubiquitinated and inhibited the levels of p62.it is probably that p62 may mediate polyubiquitinated protein into autophagy-lysosome system to degraduate.