甲醛对HepG2细胞游离胆固醇代谢的影响
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摘要
目的:研究甲醛(FA)对人肝癌细胞株HepG2细胞游离胆固醇(FC)含量的影响及其作用机制。方法:分别采用0.004、0.02、0.1 mmol/L FA处理人肝癌细胞株HepG2细胞24和48 h,以完全培养基为阴性对照,过氧化物酶法测定HepG2细胞内FC含量;采用Western blot法检测HepG2细胞固醇调节元件结合蛋白-2(SREBP-2)、羟甲基戊二酸单酰辅酶A还原酶(HMGCR)、胆固醇酰基转移酶(ACAT)和低密度脂蛋白受体(LDLR)的蛋白表达水平。结果:与阴性对照组相比,各浓度的FA染毒24 h或0.02和0.1 mmol/L FA染毒48 h后,HepG2细胞内FC含量均明显增加(P均<0.05);FA染毒24和48 h后细胞内HMGCR蛋白表达水平均明显增加(P<0.05);FA染毒24 h后,ACAT和LDLR表达水平均明显降低(P均<0.05);FA染毒48 h,LDLR蛋白表达水平明显降低(P<0.05),ACAT蛋白表达水平在0.02和0.1 mmol/L FA组明显降低(P<0.05)。结论:甲醛染毒可使HepG2细胞内FC含量增加,可能与胆固醇的从头合成增加和胆固醇酯化水平降低有关。
OBJECTIVE:To explore effects of formaldehyde(FA) on intra-hepatocellular free cholesterolcontent in HepG2 cells.METHODS:HepG2 cells were treated with formaldehyde at 0.004,0.02 and 0.1mmol/L concentrations for 24 and 48 hours,respectively.The content of intra-hepatocellular free cholesterolwas detected by using free the cholesterol GPO-POD assay kit.Western blot was used to detect proteinexpression of the sterol regulatory element-binding protein(SREBP)-2,hydroxy mothylglutaryl coenzyme Areductase(HMGCR),acyl coenzyme A-cholesterol acyltransferase(ACAT) and low-density lipoprotein receptor(LDLR).RESULTS:Compared with negative control,the contents of intra-hepatocellular FC were increasedsignificantly after treatments with FA for 24 h and 0.02 and 0.1 mmol/L for 48 h(P<0.05).HMGCRexpression levels were significantly increased after exposure to formaldehyde for 24 and 48 h(P<0.05).Aftertreatment with FA for 24 h,expression levels of ACAT and LDLR were significantly down-regulated(P<0.05).After 48 h,expression levels of LDLR were decreased significantly,whereas the levels of ACAT weredecreased significantly after treatment with 0.02 and 0.1 mmol/L FA(P<0.05).CONCLUSION:FA exposureincreased intra-hepatocellular free cholesterol contents in HepG2 cells.The increase could be caused byelevated de novo synthesis of cholesterol and decreased cholesterol esterification.
OBJECTIVE:To explore effects of formaldehyde(FA) on intra-hepatocellular free cholesterolcontent in HepG2 cells.METHODS:HepG2 cells were treated with formaldehyde at 0.004,0.02 and 0.1mmol/L concentrations for 24 and 48 hours,respectively.The content of intra-hepatocellular free cholesterolwas detected by using free the cholesterol GPO-POD assay kit.Western blot was used to detect proteinexpression of the sterol regulatory element-binding protein(SREBP)-2,hydroxy mothylglutaryl coenzyme Areductase(HMGCR),acyl coenzyme A-cholesterol acyltransferase(ACAT) and low-density lipoprotein receptor(LDLR).RESULTS:Compared with negative control,the contents of intra-hepatocellular FC were increasedsignificantly after treatments with FA for 24 h and 0.02 and 0.1 mmol/L for 48 h(P<0.05).HMGCRexpression levels were significantly increased after exposure to formaldehyde for 24 and 48 h(P<0.05).Aftertreatment with FA for 24 h,expression levels of ACAT and LDLR were significantly down-regulated(P<0.05).After 48 h,expression levels of LDLR were decreased significantly,whereas the levels of ACAT weredecreased significantly after treatment with 0.02 and 0.1 mmol/L FA(P<0.05).CONCLUSION:FA exposureincreased intra-hepatocellular free cholesterol contents in HepG2 cells.The increase could be caused byelevated de novo synthesis of cholesterol and decreased cholesterol esterification.
引文
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[23]王晓微,高明明.酯酰辅酶A:胆固醇酰基转移酶的研究进展[J].中国动脉硬化杂志,2018,26(7):685-690.
[24]STOUDT,G.Cell toxicity induced by inhibition of acyl coenzyme A:Cholesterol acyltransferase and accumulation of unesterified cholesterol[J].J Biol Chem,1995,270(11):5772-5778.
[25]YAMASHITA M,KUMAZOE M,NAKAMURA Y,et al.The Combination of green tea extract and eriodictyol inhibited high-fat/high-sucrose diet-induced cholesterol upregulation is accompanied by suppression of cholesterol synthesis enzymes[J].J Nutr Sci Vitaminol,2016,62(4):249-256.
[26]GO G,MANI A.Low-density lipoprotein receptor(LDLR)family orchestrates cholesterol homeostasis[J].Yale J Biol Med,2012,85(1):19-28.
[27]金文媛,赵正言.低密度脂蛋白受体与代谢综合征的相关性研究进展[J].浙江大学学报(医学版),2015,44(1):101-107.
[2]KIM K H,JAHAN S A,LEE J T.Exposure to formaldehyde and its potential human health hazards[J].JEnviron Sci Health C Environ Carcinog Ecotoxicol Rev,2011,29(4):277-299.
[3]SAITO Y,NISHIO K,YOSHIDA Y,et al.Cytotoxic effect of formaldehyde with free radicals via increment of cellular reactive oxygen species[J].Toxicology,2005,210(2/3):235-245.
[4]张艺滨,吴传楠,杨慧敏,等.甲醛毒性作用的研究进展[J].吉林医药学院学报,2008,29(4):232-235.
[5]HIDIR P,NERIMAN C C,DSMAIL Z,et al.The effect of melatonin hormone on formaldehyde-induced liver injury:A light microscopic and biochemical study[J].Firat Tip Dergisi,2008,13(2):92-97.
[6]KU R H,BILLINGS R E.Relationships between formaldehyde metabolism and toxicity and glutathione concentrations in isolated rat hepatocytes[J].Chem Biol Interact,1984,51(1):25-36.
[7]王凤玲,隋小芳,索树珍,等.胆固醇代谢紊乱及其相关疾病的研究进展[J].中国老年保健医学,2015,13(1):13-15.
[8]MUSSO G,GAMBINO R,CASSADER M.Cholesterol metabolism and the pathogenesis of non-alcoholic steatohepatitis[J].Prog Lipid Res,2013,52(1):175-191.
[9]KERR T A,DAVIDSON N O.Cholesterol and NAFLD:Renewed focus on an old villain[J].Hepatology,2012,56(5):1995-1998.
[10]BAI J Y,WANG P,LIU Y F,et al.Formaldehyde alters triglyceride synthesis and very low-density lipoprotein secretion in a time-dependent manner[J].Environ Toxicol Pharmacol,2017,56:15-20.
[11]郑健.甲醛与人体健康[J].大学化学,2009,24(1):52-56.
[12]BAAN R,GROSSE Y,STRAIF K,et al.A review of human carcinogens-part F:chemical agents and related occupations[J].Lancet Oncol,2009,10(12):1143-1144.
[13]TENG S,BEARD K,POURAHMAD J,et al.The formaldehyde metabolic detoxification enzyme systems and molecular cytotoxic mechanism in isolated rat hepatocytes[J].Chem Biol Interact,2001,130/132(1/2/3):285-296.
[14]薛来俊,晓开提·依不拉音,张彦红.甲醛染毒小鼠肝脏,肾脏,脾脏的病理变化[J].中国职业医学,2007,34(3):197-198.
[15]NAKAMUTA M,FUJINO T,YADA R,et al.Impact of cholesterol metabolism and the LXRalpha-SREBP-1c pathway on nonalcoholic fatty liver disease[J].Int J Mol Med,2009,23(5):603-608.
[16]WANG X H,TIAN Y,GUO Z J,et al.Cholesterol metabolism and expression of its relevant genes in cultured steatotic hepatocytes[J].J Dig Dis,10(4):310-314.
[17]DENG X,PAN X,CHENG C,et al.Regulation of SREBP-2 intracellular trafficking improves impaired autophagic flux and alleviates endoplasmic reticulum stress in NAFLD[J].Biochim Biophys Acta Mol Cell Biol Lipids,2016,1862(3):337-350.
[18]KRYCER J R,PHAN L,BROWN R J.A key regulator of cholesterol homoeostasis,SREBP-2,can be targeted in prostate cancer cells with natural products[J].Biochem J,2012,446(2):191-201.
[19]NESS G C,CHAMBERS C M.Feedback and hormonal regulation of hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase:the concept of cholesterol buffering capacity[J].Proc Soc Exp Biol Med,2000,224(1):8-19.
[20]BROWN M S,GOLDSTEIN J L,BROWN M S,et al.Multivalent feedback regulation of HMG CoA reductase,a control mechanism coordinating isoprenoid synthesis and cell growth[J].J Lipid Res,1980,21(5):505-517.
[21]REYNOLDS G A,GOLDSTEIN J L,BROWN M S.Multiple mRNAs for 3-hydroxy-3-methylglutaryl coenzyme A reductase determined by multiple transcription initiation sites and intron splicing sites in the 5'-untranslated region[J].J Biol Chem,1985,260(18):10369-10377.
[22]SHIMANO H,HORTON J D,HAMMER R E,et al.Overproduction of cholesterol and fatty acids causes massive liver enlargement in transgenic mice expressing truncated SREBP-1a[J].J Clin Invest,1996,98(7):1575-1584.
[23]王晓微,高明明.酯酰辅酶A:胆固醇酰基转移酶的研究进展[J].中国动脉硬化杂志,2018,26(7):685-690.
[24]STOUDT,G.Cell toxicity induced by inhibition of acyl coenzyme A:Cholesterol acyltransferase and accumulation of unesterified cholesterol[J].J Biol Chem,1995,270(11):5772-5778.
[25]YAMASHITA M,KUMAZOE M,NAKAMURA Y,et al.The Combination of green tea extract and eriodictyol inhibited high-fat/high-sucrose diet-induced cholesterol upregulation is accompanied by suppression of cholesterol synthesis enzymes[J].J Nutr Sci Vitaminol,2016,62(4):249-256.
[26]GO G,MANI A.Low-density lipoprotein receptor(LDLR)family orchestrates cholesterol homeostasis[J].Yale J Biol Med,2012,85(1):19-28.
[27]金文媛,赵正言.低密度脂蛋白受体与代谢综合征的相关性研究进展[J].浙江大学学报(医学版),2015,44(1):101-107.