邻苯二甲酸单乙基己基酯对HepG2细胞内三酰甘油合成的影响

Effects of mono(2-ethylhexyl) phthalate on triglyceride synthesis in HepG2 cells

[目的]邻苯二甲酸盐是一类公认的环境内分泌干扰物,其作为塑化剂的普遍应用性使得人们可通过各种途径广泛接触,并越来越关注其健康危害。尽管现有的文献报道了邻苯二甲酸盐的肝脏毒性,但是具体机制还需进一步研究。本研究通过观察邻苯二甲酸单乙基己基酯(MEHP)对Hep G2细胞内三酰甘油(TG)合成的影响,从而进一步探讨MEHP对脂肪代谢的影响。[方法]分别用不同浓度的MEHP(0、0.8、4.0、20.0、100.0μmol/L)以及0.05 mmol/L油酸(OA,阳性对照)对HepG2细胞染毒24、48 h,用化学-酶法测定HepG2细胞内以及细胞培养液上清中TG含量。用不同浓度的MEHP(0、0.01、1.0、10.0、100.0μmol/L)以及1 mmol/L OA (阳性对照)对HepG2细胞进行染毒6、24、48 h,采用实时定量PCR法检测细胞内TG合成相关基因GPAM、AGPAT2、LPIN2、DGAT2的mRNA表达水平。[结果]与阴性对照组比较:染毒24 h,仅0.5 mmol/L OA可使Hep G2细胞上清TG含量增加,20.0～100.0μmol/L MEHP和0.5 mmol/L OA可使Hep G2细胞内TG含量增加(P<0.05);染毒48 h,100.0μmol/L MEHP和0.5 mmol/L OA可使Hep G2细胞上清TG含量增加,20.0～100.0μmol/L MEHP和0.5 mmol/L OA可使Hep G2细胞内TG含量增加(P<0.05)。与阴性对照组比较:1.0～100.0μmol/L MEHP染毒6 h,100.0μmol/L MEHP染毒24 h以及10.0～100.0μmol/L MEHP染毒48 h均可使Hep G2细胞内GPAM mRNA的表达水平升高(P<0.05);不同浓度MEHP染毒6、24、48 h,AGPAT2 mRNA的表达水平均升高(P<0.05);染毒6、24、48 h,LPIN2 mRNA表达水平在10.0、100.0μmol/L MEHP剂量组均升高(P<0.05);染毒6 h,10.0、100μmol/L MEHP使细胞内DGAT2 mRNA的表达水平升高(P<0.05);染毒24 h,仅有100.0μmol/L MEHP可使DGAT2 mRNA表达水平升高(P<0.05);染毒48 h,MEHP染毒各剂量组均可使DGAT2 mRNA表达水平升高(P<0.05)。[结论]高剂量MEHP染毒可引起HepG2细胞内TG含量增加,导致肝细胞内脂质代谢紊乱。

[Objective]Phthalates is a kind of recognized environmental endocrine disruptors,and its universal applications as a plasticizer allow various exposure pathways and have raised increasing health concerns.Although available studies have reported its hepatotoxicity,the specific mechanism requires further investigation.This study is designed to observe the effects of mono(2-ethylhexyl)phthalate(MEHP)on triglyceride(TG)synthesis in HepG2 cells,and to further assess the effects of MEHP on lipid metabolism.[Methods]HepG2 cells were treated with various concentrations of MEHP(0,0.8,4.0,20.0,and 100.0μmol/L)and 0.5 mmol/L oleic acid(OA,positive control)for 24 h and 48 h,respectively,and detected for intra-and extra-hepatocellular TG contents by chemical-enzymatic method.HepG2 cells were treated with various concentrations of MEHP(0,0.01,1.0,10.0,and 100.0μmol/L)and 1 mmol/L OA(positive control)for 6 h,24 h,and 48 h,respectively,and measured for the mRNA expression levels of genes related to TG synthesis by real-time quantitative PCR,including GPAM,AGPAT2,LPIN2,and DGAT2.[Results]Compared with the negative control group,after 24 h treatment,only the 0.5μmol/L OA significantly increased the extra-hepatocellular TG contents,and the 20-100μmol/L MEHP and the 0.5μmol/L OA significantly increased the intra-hepatocellular TG contents(P<0.05);after 48 h treatment,the 100.0μmol/L MEHP and the 0.5μmol/L OA significantly increased the extra-hepatocellular TG contents,and the 20.0-100.0μmol/L MEHP and 0.5μmol/L OA significantly increased the intra-hepatocellular TG contents(P<0.05).Compared with the negative control group,the mRNA expression levels of GPAM were increased in HepG2 cells treated with 1.0-100.0μmol/L MEHP for 6 h,100.0μmol/L MEHP for 24 h,and 10.0-100.0μmol/L MEHP for 48 h(P<0.05);the mRNA expression levels of AGPAT2 were increased after exposure to all designed concentrations of MEHP for 6,24,and 48 h(P<0.05);the mRNA expression levels of LPIN2 were increased after 10.0-100.0μmol/L MEHP exposure for 6,24,and 48 h(P<0.05);the mRNA expression levels of DGAT2 were increased after being treated with 10.0-100.0μmol/L MEHP for 6 h,100.0μmol/L MEHP for 24 h,and 0.01-100.0μmol/L MEHP for 48 h(P<0.05).[Conclusion]High concentrations of MEHP can increase TG contents in HepG2 cells,leading to lipid metabolism disorder.