Study On The Mechanism Of Autophagy In Inhibiting Differentiation Of Human Neuroblastoma SH-SY5Y Cells By Tri-ortho-cresyl Phosphate

Objective:To evaluate effects of autophagy in Tri-ortho-cresyl phosphate (TOCP)-induced delayed neurotoxicity in cells, and to probe the mechanisms and therelationship between autophagy,and its toxicity. The results determined theneurotoxicity mechanism in the progress of organophosphorus ester induceddelayed neurotoxicity (OPIDN), as while provided a theoretical basis of theprevention and clinical treatment for neurodegenerative diseases.Methods:1. SH-SY5Y cells were induced to differentiate by10mol/L all trans-Retinal(ATRA) for7days. During or after cell differentiating, SH-SY5Y cells wereincubated with TOCP in different concentration (0-0.6mmol/L). Cells were fixed inmethanol and stained with Coomassie brilliant blue R250. Percents of differentiatedcells were counted and length of the longest neurite on each differentiated cell wasmeasured under inverted phase-contrast microscopy. Trypan blue exclusion wasused to detect the antiproliferative effect on SH-SY5Y cells.2. Autophagic vacuole (AVs) labeled by monodansylcadaverin (MDC)staining in the SH-SY5Y cells were observed under inverted phase-contrastmicroscopy. Then SH-SY5Y cells were lysed after MDC staining, and the OD valuedetermined by fluorospectrophotometer (the wavelength of excitation light for380nm and the wavelength of emission light for525nm).3．The plasmid containing green fluorescent protein gene and LC3wastransfected into SH-SY5Y cells, the expression of LC3-GFP was observed.after3days’ ATRA-treatment. Indirect immunofluorescence was used to observe AVs andhigh molecular weight neurofilament (NF-H) have fusioned or not after1.0mmol/LTOCP-treatment for24h.4. SH-SY5Y cells were treated with TOCP (0–1.0mmol/L) for48h, cells were harvested and lysed. western blotting analysis was performed to detect NF-H，autophagy-related protein LC3and Beclin-1Results:1. ATRA could induce the morphological differentiation of axon growing ofSY5Y cells after7days’ treatment. At the same time and after, the SH-SY5Y cellswere incubated with TOCP in different concentrations. The result indicates asignificant difference, between every treatment group and the control group(P<0.05). The reducing of0.6mmol/L group is most efficacious.2. The Fluorescent inverted microscopy observation showed that monocytefluor particles in TOCP-treatment groups were more than that in control group. Andthe higher dose of TOCP begot the more fluorescent particles and the deeper colour.While the OD value (reflecting autophagy level) of TOCP-treatment groups inMDC stainging was significantly increased (P<0.05).3. The eukaryotic expression plasmids p-GFP have been constructed andexpressed in SH-SY5Y cells successfully. After1.0mmol/LTOCP treatment for24h,NF-H and AVs have fusioned in.4.The expression of NF-H and autophagy-related protein LC3and Beclin-1were changed after TOCP-treatment for48h.Conclusions:1.TOCP could inhibit the proliferation and differentiation of SH-SY5Y cell in atime-and dose-dependent manner.2. TOCP could cause autophagy in SH-SY5Y cells in a dose-dependent manner.3. Autophagy may be a way of NF-H protein reduction by TOCP in SH-SY5Ycells.