邻苯二甲酸二异壬酯对HepG2细胞脂质代谢的影响

Effects of diisononyl phthalate on lipid metabolism in HepG2 cells

[背景]邻苯二甲酸二异壬酯(DINP)是一种与代谢性疾病有关的内分泌干扰物,广泛应用于塑料制品中。暴露于DINP与包括非酒精性脂肪性肝病(NAFLD)在内的几种肝脏不良健康结局的发展有关。[目的]探讨DINP暴露对人肝癌细胞(HepG2细胞)脂质代谢的影响及其可能的分子机制。[方法]首先,对HepG2细胞进行不同时间(24、48和72 h)、不同剂量(0、0.003、0.01、0.03、0.1、0.3、1、3、10、30和100 mmol·L^(-1))的DINP处理,采用细胞计数试剂盒8(CCK8)检测细胞活力;通过油红O染色及脂质含量测定细胞内的脂质沉积情况,并进一步检测甘油三酯(TG)和胆固醇(TC)含量;最后通过荧光定量PCR检测脂肪酸合成相关基因乙酰辅酶A羧化酶α(Accα)、脂肪酸合成酶(Fasn)、丙二酰辅酶A脱羧酶(Mlycd)、固醇调节元件结合蛋白1(Srebp1),以及β-氧化相关基因过氧化物酶体增殖活化受体α(Pparα)、腺苷酸活化蛋白激酶(Ampk)、肉毒碱棕榈酰基转移酶1A(Cpt-1a)、线粒体转录因子A(Tfam)、核呼吸因子1(Nrf1)、过氧化物酶体增殖活化受体γ共激活因子1α(Pgc1-α)的mRNA表达水平。[结果]与对照组(0 mmol·L^(-1))相比,DINP暴露24、48和72 h后,HepG2细胞活力的未观察到有害作用水平(NOAEL)分别为0.3、0.1和0.1 mmol·L^(-1),观察到有害作用最低水平(LOAEL)分别为1、0.3和0.3 mmol·L^(-1)(P<0.05)。30 mmol·L^(-1)和100 mmol·L^(-1)DINP染毒24 h后,与对照组相比,细胞内脂质含量增加且出现明显的脂质沉积,TG和TC水平也明显上升(P<0.01)。与对照组相比,100 mmol·L^(-1)DINP染毒24 h可使细胞内脂肪酸合成相关基因Mlycd、Srebp1、Fasn、Accα的mRNA表达水平下降,而30 mmol·L^(-1)剂量组Mlycd mRNA表达上调;β-氧化相关基因Ampk、Pparα、Tfam在100 mmol·L^(-1)DINP暴露后mRNA表达水平均出现上调,而Cpt-1a mRNA表达下降(P<0.05)。[结论]30 mmol·L^(-1)和100 mmol·L^(-1)DINP暴露24 h后可干扰HepG2细胞脂质代谢过程中的脂肪酸合成和β-氧化过程,导致脂质沉积。

[Background]Exposure to diisononyl phthalate(DINP),an endocrine disruptor associated with metabolic diseases and widely used in plastic products,has been linked to the development of several adverse health outcomes in the liver,including non-alcoholic fatty liver disease(NAFLD).[Objective]To investigate the effects and the possible molecular mechanisms of DINP exposure on lipid metabolism in human hepatocellular carcinoma cells(HepG2 cells).[Methods]First,HepG2 cells were treated with DINP at three time spots(24,48,and 72 h)and eleven doses(0,0.003,0.01,0.03,0.1,0.3,1,3,10,30,and 100 mmol·L^(-1)).Cell viability were detected using cell counting kit 8(CCK8).Intracellular lipid deposition was determined by oil red O staining and lipid content detection,and triglyceride(TG)and cholesterol(TC)were further detected.Finally,the mRNA expression levels were detected by fluorescence quantitative PCR,including fatty acid synthesis related genes[acetyl-CoA carboxylase alpha(Accα),fatty acid synthase(Fasn),malonyl-CoA decarboxylase(Mlycd),and sterol regulatory element binding protein 1(Srebp1)]and β-oxidation related genes[peroxisome proliferator activated receptor alpha(Pparα),AMP-activated protein kinase(Ampk),carnitine palmitoyltransferase 1A(Cpt-1a),transcription factor A,mitochondrial(Tfam),nuclear respiratory factor 1(Nrf1),and peroxisome proliferator-activated receptor gamma and coactivator 1 alpha(Pgc1-α)].[Results]Compared with the control group(0 mmol·L^(-1)),the no observed adverse effect levels(NOAEL)of HepG2 cell viability were 0.3,0.1,and 0.1 mmol·L^(-1)after 24,48,and 72 h exposure to DINP,respectively,and the corresponding lowest observed adverse effect levels(LOAEL)were 1,0.3,and 0.3 mmol·L^(-1),respectively(P<0.05).After exposure to 30 mmol·L^(-1)and 100 mmol·L^(-1)DINP for 24 h,the intracellular lipid content,lipid deposition,TG,and TC levels were increased significantly compared with the control group(P<0.01).Compared with the control group,the mRNA expression levels of genes related to fatty acid synthesis,such as Mlycd,Srebp1,Fasn,and Accα,were downregulated after the 100 mmol·L^(-1)DINP exposure for 24 h,while the mRNA expression level of Mlycd was up-regulated in the 30 mmol·L^(-1)group.The β-oxidation related genes such as Ampk,Pparα,and Tfam were up-regulated significantly after the 100 mmol·L^(-1)DINP exposure,while Cpt-1a mRNA expression level was down-regulated(P<0.05).[Conclusion]Exposure to DINP at 30 mmol·L^(-1)and 100 mmol·L^(-1)can interfere with fatty acid synthesis andβ-oxidation in lipid metabolism of HepG2 cells,resulting in lipid deposition.