| Objective: To study the effects of mono(2-ethylhexyl)phthalate(MEHP)on the synthesis of fatty acids and triglycerides in HepG2 cells,and to explore the mechanism of liver fat degeneration.Methods: HepG2 cells were treated with MEHP(0.8,4,20,100 ?mol/L)and 0.05 mmol/L OA(positive control)at different concentrations for 24 h and 48 h,respectively.The contents of triglyceride(TG)in intra and extra-hepatocellular were detected by POD chemicalenzymatic method.HepG2 cells were treated with different concentrations of MEHP(0,0.01,1,10,100 ?mol/L)and 1 mmol/L OA(positive control)for 48 h,the fat accumulation in cells was observed by oil red O staining.HepG2 cells were respectively treated with different concentrations of MEHP(0,0.01,1,10,100 ?mol/L)and 1 mmol/L OA(positive control)for 6 h,24 h,48 h,q RT-PCR was used to detect the expressions of TG related genes,such as Sterol regulatory element binding protein(SREBP1),Carbohydrate response element binding protein(Ch REBP1),Acetyl-Co A carboxylase(ACC1),Fatty acid synthase(FASN),Steroyl-Co A desaturase(SCD),Long-chain acyl-Co A synthetase(ACSL5),Glycerol-3-phosphate acyltransferase mitochondrial(GPAM),1-acylglycerol-3-phosphate acyltransferase(AGPAT),Lipid phosphate phosphohydrolase(LIPIN),Diacylglycerol acyltransferase(DGAT),Peroxisome proliferator-activated receptor ?(PPAR?),Carnitine palmitoyltransferase IA(CPT1A).Western blot was used to detect the expression of fat acid synthesis related proteins,such as Sterol regulatory element binding protein(SREBP-1),Carbohydrate response element binding protein(Ch REBP1),Acetyl-Co A carboxylase(ACC1),Fatty acid synthase(FASN),Steroyl-Co A desaturase(SCD).Results: 1.Oil red O staining showed that 48 h after MEHP exposure,compared with the negative control group,red lipid droplets appeared in all dose groups,and accompanied by lipid droplets fusion phenomenon.2.After MEHP exposure for 24 h,compared with the negative control group,the TG content in extracellular triglyceride level showed no significant change,while the TG content in intracellular was increased significantly in the 20?100 ?mol/L MEHP group.After exposure for 48 h,100 ?mol/L MEHP increased the TG content in extracellular,20,100 ?mol/L MEHP significantly increased the intracellular TG content.3.Compared with the negative control group,the expression of SREBP-1c m RNA was increased by 100 ?mol/L after MEHP exposure for 6 h,the expression of SREBP-1c m RNA was increased by 1?100 ?mol/L MEHP for 24 h,and the expression of SREBP-1c m RNA was decreased by 10 ?mol/L MEHP after exposure for 48 h.Compared with the negative control group,the expression of Ch REBP1 m RNA was increased in all dose group after MEHP exposure for 6 h,the expression of Ch REBP1 m RNA was increased by 1?100 ?mol/L MEHP after 24 h,and the expression of Ch REBP1 m RNA was decreased by 10,100 ?mol/L MEHP after exposure for 48 h.Compared with the negative control group,after 6 h intervention,the expression of ACC1 m RNA increased in each dose group of MEHP.The expression was no significant difference in all dose groups after 24 h exposure.For 48 h,ACC1 m RNA expression decreased in the 100 ?mol/L group.The FASN m RNA expression in all dose groups increased except the 0.01 group treated with MEHP after treatment for 6 h.The expression of FASN m RNA in all dose groups decreased after 24 h exposure.However,there was no significant difference in the expression of FASN m RNA in all dose group of MEHP at 48 h.The expression of SCD m RNA increased in each dose group of MEHP for 6 h after exposure,after 24 h treatment of MEHP,the expression of SCD m RNA in MEHP group was 10,100 ?mol/L group both showed a decreasing trend,the expression of SCD m RNA in 100 ?mol/L MEHP decreased for 48 h.4.Compared with the negative control group,the expression of ACSL5 m RNA in HepG2 cells was significantly increased in 10,100 ?mol/L MEHP after exposure for 6 h,24 h and 48 h.The expression of GPAM m RNA in HepG2 cells were increased by 1?100 ?mol/L MEHP exposure for 6 h,100 ?mol/L MEHP exposure for 24 h,and 10,100 ?mol/L MEHP exposure for 48 h.The expression of AGPAT2 m RNA was increased in all dose groups at 6 h,24 h and 48 h after MEHP exposure.LIPIN2 m RNA expression were increased at 10 and 100 ?mol/L MEHP at 6 h,24 h and 48 h after MEHP exposure.The expression of DGAT2 m RNA was increased by 10 and 100 ?mol/L MEHP at 6 h,and only increased by 100 ?mol/L MEHP at 24 h,and increased in all dose groups after exposure to MEHP for 48 h.5.Compared with the negative control group,there was no significant difference in the protein expression level of SREBP-1c in all dose group of MEHP for 24 h and 48 h.The expression of Ch REBP1 protein was significantly increased in 10,100 ?mol/L MEHP groups at 24 h,48 h.The expression of ACC1 protein was increased in 1?100 ?mol/L MEHP after exposure for 24 h,but the expression of ACC1 protein was decreased in 1?100 ?mol/L MEHP after exposure for 48 h.Compared with the negative control group,the expression of FASN protein was increased in 1?100 ?mol/L MEHP after exposure for 24 h,and the expression of FASN protein was decreased after exposure to 48 h in 100 ?mol/L MEHP.The expression of SCD protein was increased by only 100 ?mol/L MEHP for 24 h,and the expression of SCD protein was decreased at 48 h in 0.01?100 ?mol/L MEHP.6.Compared with negative control group,the expression of P53 m RNA was increased in 10?100 ?mol/L MEHP after exposure for 24 h,the expression of P53 m RNA in HepG2 cells was increased by only 100 ?mol/L MEHP at 48 h.The expression of PPAR? m RNA was decreased in 10,100 ?mol/L MEHP at 24 h,the expression of PPAR? m RNA was decreased in 1?100 ?mol/L MEHP after exposure for 48 h.The expression of CPT1 A m RNA in HepG2 cells was decreased in 10,100 ?mol/L MEHP after exposure for 24 h,48 h.Conclusions: 1.The high concentration of MEHP exposure can lead to fat accumulation in HepG2 cells,resulting in abnormal lipid metabolism.2.The fat accumulation induced by MEHP exposure in HepG2 cells may be related to the increased synthesis of triglycerides,the inhibited fatty acid ?-oxidation. |
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