李耀福, 白剑英, 贺真, 姜雪霞, 夏娜, 秦小江. 邻苯二甲酸二(2-乙基己基)酯对HepG2细胞脂质代谢的影响[J]. 环境与职业医学, 2018, 35(3): 234-240. DOI: 10.13213/j.cnki.jeom.2018.17576
引用本文: 李耀福, 白剑英, 贺真, 姜雪霞, 夏娜, 秦小江. 邻苯二甲酸二(2-乙基己基)酯对HepG2细胞脂质代谢的影响[J]. 环境与职业医学, 2018, 35(3): 234-240. DOI: 10.13213/j.cnki.jeom.2018.17576
LI Yao-fu, BAI Jianying, HE Zhen, JIANG Xue-xia, XIA Na, QIN Xiao-jiang. Effects of di(2-ethylexyl)phthalate on cellular lipid metabolism in HepG2 cells[J]. Journal of Environmental and Occupational Medicine, 2018, 35(3): 234-240. DOI: 10.13213/j.cnki.jeom.2018.17576
Citation: LI Yao-fu, BAI Jianying, HE Zhen, JIANG Xue-xia, XIA Na, QIN Xiao-jiang. Effects of di(2-ethylexyl)phthalate on cellular lipid metabolism in HepG2 cells[J]. Journal of Environmental and Occupational Medicine, 2018, 35(3): 234-240. DOI: 10.13213/j.cnki.jeom.2018.17576

邻苯二甲酸二(2-乙基己基)酯对HepG2细胞脂质代谢的影响

Effects of di(2-ethylexyl)phthalate on cellular lipid metabolism in HepG2 cells

  • 摘要: 目的 观察邻苯二甲酸二(2-乙基己基)酯(DEHP)及其代谢产物邻苯二甲酸(2-乙基己基)酯(MEHP)对人肝癌细胞株HepG2细胞脂肪代谢的影响,及其致肝损伤的作用机制。

    方法 分别用不同浓度的DEHP(31.25、62.5、125、250、500、1 000 μmol/L)、MEHP(3.125、6.25、12.5、25、50、100、250 μmol/L)、完全培养基(阴性对照)、0.1%(体积分数)二甲基亚砜(溶剂对照)处理HepG2细胞24 h、48 h和72 h,采用MTT试剂盒检测细胞活力。不同浓度的DEHP(10.0、250.0 μmol/L)、MEHP(4.0、100.0 μmol/L)、完全培养基(阴性对照)、0.1%(体积分数)二甲基亚砜(溶剂对照)、0.5 mmol/L油酸(阳性对照)处理HepG2细胞48 h,油红O染色法观察HepG2细胞脂肪累积。不同浓度的DEHP (2.0、10.0、50.0、250.0 μmol/L)、MEHP (0.8、4.0、20.0、100.0 μmol/L)、完全培养基(阴性对照)、0.5 mmol/L油酸、0.5 μg/mL布雷德菌素A处理HepG2细胞24 h和48 h,Western blot法检测细胞内固醇元件调节蛋白(SREBP-1c)、肉毒碱棕榈酰基转移酶(CPT1A)、丙二酰辅酶A脱羧酶(MLYCD)和脂肪组织三酰甘油水解酶(ATGL)的蛋白表达。

    结果 与阴性对照组相比,250.0~1 000.0 μmol/L DEHP及50.0~250.0 μmol/L MEHP染毒组HepG2细胞存活率降低,差异具有统计学意义(P < 0.05)。染毒48 h后,10.0、250.0 μmol/L DEHP染毒组与4.0、100.0 μmol/L MEHP染毒组HepG2细胞内可见红色脂滴,且伴有脂滴融合现象。与阴性质对照组相比,250.0 μmol/L DEHP或100.0 μmol/L MEHP染毒HepG2细胞24 h,50.0~250.0μmol/L DEHP或4.0~100.0μmol/L MEHP染毒HepG2细胞48 h,使CPT1A表达降低,差异具有统计学意义(P < 0.05);10.0~250.0 μmol/L DEHP或4.0~100.0 μmol/L MEHP染毒HepG2细胞48 h使MLYCD表达降低,差异具有统计学意义(P < 0.05);250.0 μmol/L DEHP与20.0~100.0 μmol/L MEHP染毒HepG2细胞24 h,10.0~250.0 μmol/L DEHP与0.8~100.0μmol/L MEHP染毒HepG2细胞48 h,可使ATGL表达降低,差异具有统计学意义(P < 0.05)。

    结论 DEHP及MEHP染毒可引起HepG2细胞发生脂肪堆积,其过程可能与脂肪酸β-氧化受阻及三酰甘油水解受到抑制有关。

     

    Abstract: Objective To observe the effects of di(2-ethylhexyl)phthalate (DEHP) and its metabolite mono(2-ethylhexyl)phthalate (MEHP) on lipid metabolism of human hepatoma cell line HepG2 cells, and explore the mechanism of relevant liver injury.

    Methods HepG2 cells were treated with different concentrations of DEHP (31.25, 62.5, 125, 250, 500, and 1 000 μmol/L), MEHP (3.125, 6.25, 12.5, 25, 50, 100, and 250 μmol/L), complete medium (negative control), and 0.1% DMSO (solvent control) for 24 h, 48 h, and 72 h, respectively, to detect the cell viability by MTT assay. HepG2 cells were treated with different concentrations of DEHP (10.0 and 250.0 μmol/L), MEHP (4.0 and 100.0 μmol/L), complete medium (negative control), 0.1% DMSO (solvent control), and 0.5 mmol/L oleic acid (positive control) for 48 h, respectively, to observe the lipid accumulation of HepG2 cells by oil red O staining. HepG2 cells were treated with different concentrations of DEHP (2.0, 10.0, 50.0, and 250.0 μmol/L), MEHP (0.8, 4.0, 20.0, and 100.0μmol/L), complete medium (negative control), 0.5 mmol/L oleic acid, and 0.5μg/mL Brefeldin A for 24 h and 48 h, respectively, to detect the protein expressions of sterol regulatory element binding protein 1c (SREBP-1c), carnitine palmitoyltransferase 1A (CPT1A), malonyl-CoA decarboxylase (MLYCD), and adipose tissue triglyceride hydrolase (ATGL) by Western blot.

    Results The DEHP (250.0-1 000.0 μmol/L) and MEHP (50.0-250.0 μmol/L) treatments decreased the cell viability of the experimented HepG2 cells compared with the negative control group (P < 0.05). After exposure for 48 h, the 10.0 and 250.0 μmol/L DEHP groups and the 4.0 and 100.0 μmol/L MEHP groups showed visible lipid droplets accompanied by lipid droplet fusion. Compared with the negative control group, the expression of CPT1A was down-regulated in the HepG2 cells treated with 250.0μmol/L DEHP or 100.0μmol/L MEHP for 24h and 50.0-250.0μmol/L DEHP or 4.0-100.0μmol/L MEHP for 48h compared with the negative control group (P < 0.05); the expression of MLYCD was down-regulated in the HepG2 cells treated with 10.0-250.0 μmol/L DEHP or 4.0-100.0μmol/L MEHP for 48 h (P < 0.05); the expression of ATGL was down-regulated in the HepG2 cells treated with 250.0μmol/L DEHP or 20.0-100.0μmol/L MEHP for 24 h and 10-250.0μmol/L DEHP or 0.8-100.0μmol/L MEHP for 48 h (P < 0.05).

    Conclusion DEHP and MEHP can cause lipid accumulation in HepG2 cells, which may be related to the inhibition of fatty acid beta-oxidation and triglyceride hydrolysis.

     

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