Dai L, Jin Y Y, Qu B Y, Xu Y J
Department of Preventive Medicine, Medical College Yanbian University, Yanji 133000, China.
Zhonghua Lao Dong Wei Sheng Zhi Ye Bing Za Zhi. 2020 Jul 20;38(7):490-494. doi: 10.3760/cma.j.cn121094-20190709-00288.
To study the toxicity of dioctyl phthalate (DOP) on adrenal pheochromocytoma (PC12) cells and its effect on processing of amyloid precursor protein (APP) -enzymolysis. 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. 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 (<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 (<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 (<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 (<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 (<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 (<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 (<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 (<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 (<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 (<0.05) . 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.
研究邻苯二甲酸二辛酯（DOP）对肾上腺嗜铬细胞瘤（PC12）细胞的毒性作用及其对淀粉样前体蛋白（APP）酶解加工的影响。体外实验中，将PC12细胞分为空白对照（CT）、低剂量DOP（DOP1）、中剂量DOP（DOP2）、高剂量DOP（DOP3）、低剂量DOP + Aβ（25 - 35）（DOP1 + Aβ）、中剂量DOP + Aβ（25 - 35）（DOP2 + Aβ）、高剂量DOP + Aβ（25 - 35）（DOP3 + Aβ）、Aβ（25 - 35）（Aβ）共8组，每组4个样本。采用MTT法检测细胞活力，测定乳酸脱氢酶（LDH）、丙二醛（MDA）和一氧化氮（NO）含量，通过蛋白质免疫印迹法检测半胱天冬酶3（Caspase - 3）。转染实验中，用APP695转染仓鼠卵巢（CHO）细胞并用不同浓度的DOP处理。分为V - Flag对照（V - Flag）、APP695 - Flag（APP695）、低剂量DOP（DOP1 + APP695）、中剂量DOP（DOP2 + APP695）、高剂量DOP（DOP3 + APP695）共5组，每组4个样本。采用酶联免疫吸附测定（ELISA）法测定Aβ（1 - 40）含量和γ - 分泌酶活性。体内实验中，选取50只体重（20±2）g的SPF级雄性昆明小鼠，随机分为对照、铅（Pb）、低剂量DOP（DOP1＇）、中剂量DOP（DOP2＇）、高剂量DOP（DOP3＇）5组，每组10只，连续灌胃6周。采用Morris水迷宫法检测不同浓度DOP对小鼠学习记忆的影响，用ELISA法检测脑组织中β - 分泌酶、γ - 分泌酶活性及Aβ（1 - 40）含量。与CT组相比，DOP2组和DOP3组细胞活力降低，LDH、MDA和NO含量升高，差异有统计学意义（<0.05）。与CT组相比，DOP1 + Aβ、DOP2 + Aβ和DOP3 + Aβ组细胞活力降低，LDH、MDA、NO含量升高，差异有统计学意义（<0.05）。与Aβ组相比，DOP3 + Aβ组细胞活力降低，LDH、MDA、NO含量升高，差异有统计学意义（<0.05）。与Aβ组相比，DOP2 + Aβ组LDH和NO含量升高，差异有统计学意义（<0.05）。与CT组相比，DOP2组和DOP3组Caspase - 3表达水平升高，差异有统计学意义（<0.05）。与Aβ组相比，DOP2 + Aβ和DOP3 + Aβ组Caspase - 3表达水平升高，差异有统计学意义（<0.05）。与APP695组相比，DOP2 + APP695和DOP3 + APP695组Aβ（1 - 40）含量及γ - 分泌酶活性升高（<0.05）。与对照组相比，DOP3＇组脑组织中β - 分泌酶、γ - 分泌酶活性及Aβ（1 - 40）含量升高，差异有统计学意义（<0.05）。与Pb组相比，DOP3＇组β - 分泌酶、γ - 分泌酶活性及Aβ（1 - 40）含量升高，差异有统计学意义（<0.05）。与对照组相比，DOP2＇组和DOP3＇组目标象限停留时间和穿越次数减少，差异有统计学意义（<0.05）。DOP对PC12细胞有一定毒性作用，可导致小鼠学习记忆障碍，可能促进阿尔茨海默病的病理进程。
