The cytotoxity of DOP on PC12 cells and it's effect on processing of APP-enzymolysis

Abstract

Objective: To study the toxicity of dioctyl phthalate (DOP) on adrenal pheochromocytoma (PC12) cells and its effect on processing of amyloid precursor protein (APP) -enzymolysis. Methods: In vitro experiments, PC12 cells were divided into blank control (CT) , low DOP (DOP1) , medium DOP (DOP2) , high DOP (DOP3) , low DOP+Aβ(25-35) (DOP1+Aβ) , medium DOP+Aβ(25-35) (DOP2+Aβ) , high DOP+Aβ(25-35) (DOP3+Aβ) , Aβ(25-35) (Aβ) , a total of 8 groups, each with 4 samples. The cell viability was measured by MTT assay, the contents of lactate dehydrogenase (LDH) , malondialdehyde (MDA) and nitric oxide (NO) were measured, and cysteine protease 3 (Caspase-3) was determined by Western blot. In the transfection experiment, the hamster ovary (CHO) cells were transfected with APP695 and treated with different concentrations of DOP. They were divided into V-Flag control (V-Flag) , APP695-Flag (APP695) , low DOP (DOP1+APP695) , medium DOP (DOP2+APP695) , high DOP (DOP3+APP695) , a total of 5 groups, each with 4 samples. Enzyme-linked immunosorbent assay (ELISA) was used to determine the content of Aβ(1-40) and the activity of γ-secretase. In vivo experiment, 50 male Kunming mice of SPF grade, weighing (20±2) g, were selected and randomly divided into control, lead (Pb) , low DOP (DOP1＇) , medium DOP (DOP2＇) , high DOP (DOP3＇) consisted of 5 groups, each with 10 mice, continuously gavage for 6 weeks. Morris water maze method was used to detect the effect of different concentrations of DOP on learning and memory in mice, and ELISA method was used to detect β-secretase, γ-secretase activity and Aβ(1-40) content in brain tissue. Results: Compared with the CT group, the cell viabilities of the DOP2 and DOP3 groups were decreased, and the contents of LDH, MDA, and NO were increased, and the differences were statistically significant (P<0.05) . Compared with the CT group, the cell viabilities of DOP1+Aβ, DOP2+Aβ and DOP3+Aβ groups were decreased, the contents of LDH, MDA, NO were increased, the differences were statistically significant (P<0.05) . Compared with the Aβ group, the cell viability of DOP3+Aβ group was decreased, the contents of LDH, MDA, NO were increased, the differences were statistically significant (P<0.05) . Compared with the Aβ group, the contents of LDH and NO in the DOP2+Aβ group were increased, and the differences were statistically significant (P<0.05) . Compared with the CT group, the expression levels of Caspase-3 in the DOP2 and DOP3 groups were increased, and the differences were statistically significant (P<0.05) . Compared with the Aβ group, the expression levels of Caspase-3 in the DOP2+Aβ and DOP3+Aβ groups were increased, and the differences were statistically significant (P<0.05) . Compared with the APP695 group, the contents of Aβ(1-40) and the activities of γ-secretase of the DOP2+APP695 and DOP3+APP695 groups were increased (P<0.05) . Compared with the control group, the activities of β-secretase, γ-secretase and the content of Aβ(1-40) in the brain tissue of DOP3＇group were increased, and the differences were statistically significant (P<0.05) . Compared with the Pb group, the activities of β-secretase, γ-secretase and the content of Aβ(1-40) of the DOP3＇group were increased, and the differences were statistically significant (P<0.05) . Compared with the control group, the target quadrant stay time and the number of crossings in the DOP2＇and DOP3＇groups were reduced, and the differences were statistically significant (P<0.05) . Conclusion: DOP has a certain toxic effect on PC12 cells, causing learning and memory impairment in mice, and may promote the pathological progression of Alzheimer＇s disease.