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.
目的： 研究邻苯二甲酸二辛酯（DOP）对肾上腺嗜铬细胞瘤（PC12）细胞的毒性以及对淀粉样前体蛋白（APP）酶解的影响。 方法： 体外试验中将PC12细胞分为空白对照（CT）组、低浓度DOP（DOP1）组、中浓度DOP（DOP2）组、高浓度DOP（DOP3）组、低浓度DOP+Aβ(25-35)（DOP1+Aβ）组、中浓度DOP+Aβ(25-35)（DOP2+Aβ）组、高浓度DOP+Aβ(25-35)（DOP3+Aβ）组、Aβ(25-35)（Aβ）组共8组，每组4个样本；采用MTT法测定细胞活力，测定乳酸脱氢酶（LDH）、丙二醛（MDA）、一氧化氮（NO）含量，采用蛋白免疫印迹（Western blot）法测定半胱氨酸天冬氨酸蛋白酶3（Caspase-3）表达。转染实验用仓鼠卵巢（CHO）细胞转染APP695，使用不同浓度的DOP进行处理，分为转染空载体V-Flag对照（V-Flag）组、转染APP695-Flag模型（APP695）组、低浓度DOP（DOP1+APP695）组、中浓度DOP（DOP2+APP695）组、高浓度DOP（DOP3+APP695）组共5组，每组4个样本；采用酶联免疫分析（ELISA）法测定Aβ(1-40)含量以及γ-分泌酶活力。体内实验选择SPF级雄性昆明种小鼠50只，体重为（20±2）g，随机分为对照组、铅（Pb）组、低DOP浓度（DOP1’）组、中DOP浓度（DOP2’）组、高DOP浓度（DOP3’）组共5组，每组10只，连续灌胃6周；使用Morris水迷宫方法检测不同浓度的DOP对小鼠学习记忆的影响，并用ELISA法检测脑组织中β和γ-分泌酶活力及Aβ(1-40)含量。 结果： 与CT组比较，DOP2、DOP3组细胞活力降低，LDH、MDA、NO含量增加，差异均有统计学意义（P<0.05）；与CT组比较，DOP1+Aβ、DOP2+Aβ、DOP3+Aβ组细胞活力下降，LDH、MDA、NO含量增高，差异均有统计学意义（P<0.05）；与Aβ组比较，DOP3+Aβ组细胞活力下降，LDH、MDA、NO含量增加，差异均有统计学意义（P<0.05）；与Aβ组比较，DOP2+Aβ组LDH、NO含量增加，差异均有统计学意义（P<0.05）。与CT组比较，DOP2、DOP3组Caspase-3表达水平增高，差异均有统计学意义（P<0.05）；与Aβ组比较，DOP2+Aβ、DOP3+Aβ组Caspase-3表达水平增高，差异均有统计学意义（P<0.05）。与APP695组比较，DOP2+APP695、DOP3+APP695组Aβ(1-40)含量及γ-分泌酶活力增高，差异均有统计学意义（P<0.05）。与对照组比较，DOP3’组小鼠脑组织β和γ-分泌酶活力及Aβ(1-40)含量增加，差异均有统计学意义（P<0.05）；与Pb组比较，DOP3’组β和γ-分泌酶活力及Aβ(1-40)含量增加，差异均有统计学意义（P<0.05）。与对照组比较，DOP2’和DOP3’组小鼠目标象限停留时间、穿台次数减少，差异均有统计学意义（P<0.05）。 结论： DOP对PC12细胞具有一定的毒性作用，引起小鼠学习记忆障碍，可能对阿尔茨海默病的病理进展起促进作用。.
Keywords: Amyloid; Amyloid precursor protein (APP); Cytotoxity; Dioctyl phthalate (DOP); PC12 cells.