对乙酰氨基酚耐受肝细胞株的建立及其作用机制研究
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摘要
对乙酰氨基酚(acetaminophen,APAP)是引起急性肝衰竭最主要的原因,但其确切的作用机制仍不明了。该研究建立了不同浓度的APAP耐药肝细胞,并对其耐药机制进行初步研究。克隆状密度培养的AML-12小鼠肝细胞浓度递增诱导建立APAP耐药细胞系;细胞计数检测细胞增殖能力;Mito Tracker和H_2DCF-DA分别检测线粒体膜电位和氧自由基水平;GSH/GSSH检测细胞抗氧化能力;q PCR检测细胞基因表达水平;Western blot检测蛋白质水平。成功建立了增殖和耐药稳定的1.25和2.50 mmol/L APAP耐药AML-12肝细胞。在相应浓度APAP处理后,与对照组相比,耐药组细胞增殖能力强,氧自由基水平低,线粒体膜电位水平高,GSH/GSSH值高。进一步研究结果显示,耐受组细胞抗氧化通路Nrf2及其靶基因表达活性提高,而凋亡相关信号通路JNK及其相关基因活性下降。肝细胞表型特征分析显示,耐药组细胞肝功能相关基因表达水平并未发生显著变化,但与染色体重塑相关的转录因子Foxa1和Foxa2 m RNA和蛋白质水平显著升高。该研究建立了增殖和耐药性状稳定的APAP耐药肝细胞,耐药性状的获得与抗氧化能力和抗凋亡能力的提高相关,并提示染色体重塑相关转录因子也可能参与这一过程,为深入研究耐药机制奠定了基础。
Acetaminophen(APAP) is the most important common cause of acute liver failure; however, the exact mechanism of toxicity of this analgesic drug is not elucidated. In the present study, we generated APAPresistant liver cell line and determined their basic properties. AML-12 mouse hepatocytes were cultured with clonal cell density to generate APAP-resistant cells by means of gradually increasing the concentration of the APAP drug. Cell proliferation was determined by cell counting. The levels of reactive oxygen species(ROS) and mitochondrial membrane potential(MMP) were determined by H2 DCF-DA and Mito Tracker detection, respectively. The levels of m RNA and protein were determined by q PCR and Western blot analysis, respectively. We successfully established 1.25 and 2.50 mmol/L APAP-resistant AML-12 cell lines(ARCs). Cell proliferation analysis revealed that, compared to the control group, ARCs had high proliferation capacity, low ROS levels, and high MMP and GSH/GSSH levels. Furthermore, Western blot and q PCR analysis showed that the expression level of the Nrf2 signaling pathway was up-regulated, whereas the level of the JNK signaling pathway was down-regulated. In addition, ARCs retained normal hepatocytic phenotypes, except for the high expressions of Foxa1 and Foxa2 genes, which were related to chromatin remodeling. We successfully established APAP-resistant hepatocyte lines with low oxidative stress levels but with retained normal hepatocytic phenotypes. Activation of Nrf2 and inactivation of JNK signaling pathway appeared to be involved in the protection of hepatocytes against APAP-induced cell apoptosis.
Acetaminophen(APAP) is the most important common cause of acute liver failure; however, the exact mechanism of toxicity of this analgesic drug is not elucidated. In the present study, we generated APAPresistant liver cell line and determined their basic properties. AML-12 mouse hepatocytes were cultured with clonal cell density to generate APAP-resistant cells by means of gradually increasing the concentration of the APAP drug. Cell proliferation was determined by cell counting. The levels of reactive oxygen species(ROS) and mitochondrial membrane potential(MMP) were determined by H2 DCF-DA and Mito Tracker detection, respectively. The levels of m RNA and protein were determined by q PCR and Western blot analysis, respectively. We successfully established 1.25 and 2.50 mmol/L APAP-resistant AML-12 cell lines(ARCs). Cell proliferation analysis revealed that, compared to the control group, ARCs had high proliferation capacity, low ROS levels, and high MMP and GSH/GSSH levels. Furthermore, Western blot and q PCR analysis showed that the expression level of the Nrf2 signaling pathway was up-regulated, whereas the level of the JNK signaling pathway was down-regulated. In addition, ARCs retained normal hepatocytic phenotypes, except for the high expressions of Foxa1 and Foxa2 genes, which were related to chromatin remodeling. We successfully established APAP-resistant hepatocyte lines with low oxidative stress levels but with retained normal hepatocytic phenotypes. Activation of Nrf2 and inactivation of JNK signaling pathway appeared to be involved in the protection of hepatocytes against APAP-induced cell apoptosis.
引文
1 Bunchorntavakul C,Reddy KR.Acetaminophen-related Hepatotoxicity.Clin Liver Dis 2013;17(4):587-607.
2 Herndon CM,Dankenbring DM.Patient perception and knowledge of acetaminophen in a large family medicine service.J Pain Palliat Care Pharmacother 2014;28(2):109-16.
3 Jaeschke H,Mc Gill MR,Ramachandran A.Oxidant stress,mitochondria,and cell death mechanisms in drug-induced liver injury:lessons learned from acetaminophen hepatotoxicity.Drug Metab Rev 2012;44(1):88-106.
4 Jaeschke H,Mc Gill MR.Cytochrome P450-derived versus mitochondrial oxidant stress in acetaminophen hepatotoxicity.Toxicol Lett 2015;235(3):216-7.
5 Ramachandran A,Jaeschke H.Mechanisms of acetaminophen hepatotoxicity and their translation to the human pathophysiology.J Clin Transl Res 2017;3(Suppl 1):157-69.
6 Shinohara M,Ybanez MD,Win S,Than TA,Jain S,Gaarde WA,et al.Silencing glycogen synthase-3beta inhibits acetaminophen hepatotoxicity and attenuates JNK activation and loss of glutamate cysteine ligase and myeloid cell leukemia sequence 1.J Biol Chem 2010;285(11):8244-55.
7 M i s h r a R,B a r t h w a l M K,S o n d a r v a G,R a n a B,Wo n g L,Chatterjee M,et al.Glycogen synthase kinase-3beta induces neuronal cell death via direct phosphorylation of mixed lineage kinase 3.J Biol Chem 2007;282(42):30393-405.
8 Nakagawa H,Maeda S,Hikiba Y,Ohmae T,Shibata W,Yanai A,et al.Deletion of apoptosis signal-regulating kinase 1attenuates acetaminophen induced liver injury by inhibiting c-Jun N-terminal kinase activation.Gastroenterology 2008;135(4):1311-21.
9 Wang W,Guan C,Sun X,Zhao Z,Li J,Fu X,et al.Tanshinone IIA protects against acetaminophen-induced hepatotoxicity via activating the Nrf2 pathway.Phytomedicine 2016;23(6):589-96.
10 Ahmed MM,Wang T,Luo Y,Ye S,Wu Q,Guo Z,et al.Aldo-keto reductase-7A protects liver cells and tissues from acetaminopheninduced oxidative stress and hepatotoxicity.Hepatology 2011;54(4):1322-32.
11 Mc Gill MR,Williams CD,Xie Y,Ramachandran A,Jaeschke H.Acetaminophen-induced liver injury in rats and mice:comparison of protein adducts,mitochondrial dysfunction,and oxidative stress in the mechanism of toxicity.Toxicol Appl Pharmacol2012;264(3):387-94.
12 Tamai S,Iguchi T,Niino N,Mikamoto K,Sakurai K,Sayama A,et al.A monkey model of acetaminophen-induced hepatotoxicity;phenotypic similarity to human.J Toxicol Sci 2017;42(1):73-84.
13 Godoy P,Hewitt NJ,Albrecht U,Andersen ME,Ansari N,Bhattacharya S,Bode JG,Bolleyn J,et al.Recent advances in 2D and 3D in vitro systems using primary hepatocytes,alternative hepatocyte sources and non-parenchymal liver cells and their use in investigating mechanisms of hepatotoxicity,cell signaling and ADME.Arch Toxicol 2013;87(8):1315-30.
14 Toyoda Y,Kashikura K,Soga T,Tagawa YI.Metabolomics of an in vitro liver model containing primary hepatocytes assembli ng around an endothelial cell network:comparative study on the metabolic stability and the effect of acetaminophen treatment.J Toxicol Sci 2017;42(4):445-54.
15 A h m e d M M,A l-O b o s i J A,O s m a n H M,S h a y o u b M E.Overexpression of Aldose Reductase Render Mouse Hepatocytes More Sensitive to Acetaminophen Induced Oxidative Stress and Cell Death.Indian J Clin Biochem 2016;31(2):162-70.
16 Zhang H,Cook J,Nickel J,Yu R,Stecker K,Myers K,Dean NM.Reduction of liver Fas expression by an antisense oligonucleotide protects mice from fulminant hepatitis.Nat Biotechnol 2000;18(8):862-7.
17 Besnard V,Wert SE,Hull WM,Whitsett JA.Immunohistochemical localization of Foxa1 and Foxa2 in mouse embryos and adult tissues.Gene Expr Patterns 2004;5(2):193-208.
18 Fu X,Jeselsohn R,Pereira R,Hollingsworth EF,Creighton CJ,Li F,et al.FOXA1 overexpression mediates endocrine resistance by altering the ER transcriptome and IL-8 expression in ERpositive breast cancer.Proc Natl Acad Sci USA 2016;113(43):E6600-9.
19 Tanaka K,Tokunaga E,Yamashita N,Sagara Y,Ohi Y,Taguchi K,et al.The relationship between the expression of FOXA1 and GATA3 and the efficacy of neoadjuvant endocrine therapy.Breast Cancer 2017;24(3):384-92.
20 Zhao JC,Fong KW,Jin HJ,Yang YA,Kim J,Yu J.FOXA1 acts upstream of GATA2 and AR in hormonal regulation of gene expression.Oncogene 2016;35(33):4335-44.
2 Herndon CM,Dankenbring DM.Patient perception and knowledge of acetaminophen in a large family medicine service.J Pain Palliat Care Pharmacother 2014;28(2):109-16.
3 Jaeschke H,Mc Gill MR,Ramachandran A.Oxidant stress,mitochondria,and cell death mechanisms in drug-induced liver injury:lessons learned from acetaminophen hepatotoxicity.Drug Metab Rev 2012;44(1):88-106.
4 Jaeschke H,Mc Gill MR.Cytochrome P450-derived versus mitochondrial oxidant stress in acetaminophen hepatotoxicity.Toxicol Lett 2015;235(3):216-7.
5 Ramachandran A,Jaeschke H.Mechanisms of acetaminophen hepatotoxicity and their translation to the human pathophysiology.J Clin Transl Res 2017;3(Suppl 1):157-69.
6 Shinohara M,Ybanez MD,Win S,Than TA,Jain S,Gaarde WA,et al.Silencing glycogen synthase-3beta inhibits acetaminophen hepatotoxicity and attenuates JNK activation and loss of glutamate cysteine ligase and myeloid cell leukemia sequence 1.J Biol Chem 2010;285(11):8244-55.
7 M i s h r a R,B a r t h w a l M K,S o n d a r v a G,R a n a B,Wo n g L,Chatterjee M,et al.Glycogen synthase kinase-3beta induces neuronal cell death via direct phosphorylation of mixed lineage kinase 3.J Biol Chem 2007;282(42):30393-405.
8 Nakagawa H,Maeda S,Hikiba Y,Ohmae T,Shibata W,Yanai A,et al.Deletion of apoptosis signal-regulating kinase 1attenuates acetaminophen induced liver injury by inhibiting c-Jun N-terminal kinase activation.Gastroenterology 2008;135(4):1311-21.
9 Wang W,Guan C,Sun X,Zhao Z,Li J,Fu X,et al.Tanshinone IIA protects against acetaminophen-induced hepatotoxicity via activating the Nrf2 pathway.Phytomedicine 2016;23(6):589-96.
10 Ahmed MM,Wang T,Luo Y,Ye S,Wu Q,Guo Z,et al.Aldo-keto reductase-7A protects liver cells and tissues from acetaminopheninduced oxidative stress and hepatotoxicity.Hepatology 2011;54(4):1322-32.
11 Mc Gill MR,Williams CD,Xie Y,Ramachandran A,Jaeschke H.Acetaminophen-induced liver injury in rats and mice:comparison of protein adducts,mitochondrial dysfunction,and oxidative stress in the mechanism of toxicity.Toxicol Appl Pharmacol2012;264(3):387-94.
12 Tamai S,Iguchi T,Niino N,Mikamoto K,Sakurai K,Sayama A,et al.A monkey model of acetaminophen-induced hepatotoxicity;phenotypic similarity to human.J Toxicol Sci 2017;42(1):73-84.
13 Godoy P,Hewitt NJ,Albrecht U,Andersen ME,Ansari N,Bhattacharya S,Bode JG,Bolleyn J,et al.Recent advances in 2D and 3D in vitro systems using primary hepatocytes,alternative hepatocyte sources and non-parenchymal liver cells and their use in investigating mechanisms of hepatotoxicity,cell signaling and ADME.Arch Toxicol 2013;87(8):1315-30.
14 Toyoda Y,Kashikura K,Soga T,Tagawa YI.Metabolomics of an in vitro liver model containing primary hepatocytes assembli ng around an endothelial cell network:comparative study on the metabolic stability and the effect of acetaminophen treatment.J Toxicol Sci 2017;42(4):445-54.
15 A h m e d M M,A l-O b o s i J A,O s m a n H M,S h a y o u b M E.Overexpression of Aldose Reductase Render Mouse Hepatocytes More Sensitive to Acetaminophen Induced Oxidative Stress and Cell Death.Indian J Clin Biochem 2016;31(2):162-70.
16 Zhang H,Cook J,Nickel J,Yu R,Stecker K,Myers K,Dean NM.Reduction of liver Fas expression by an antisense oligonucleotide protects mice from fulminant hepatitis.Nat Biotechnol 2000;18(8):862-7.
17 Besnard V,Wert SE,Hull WM,Whitsett JA.Immunohistochemical localization of Foxa1 and Foxa2 in mouse embryos and adult tissues.Gene Expr Patterns 2004;5(2):193-208.
18 Fu X,Jeselsohn R,Pereira R,Hollingsworth EF,Creighton CJ,Li F,et al.FOXA1 overexpression mediates endocrine resistance by altering the ER transcriptome and IL-8 expression in ERpositive breast cancer.Proc Natl Acad Sci USA 2016;113(43):E6600-9.
19 Tanaka K,Tokunaga E,Yamashita N,Sagara Y,Ohi Y,Taguchi K,et al.The relationship between the expression of FOXA1 and GATA3 and the efficacy of neoadjuvant endocrine therapy.Breast Cancer 2017;24(3):384-92.
20 Zhao JC,Fong KW,Jin HJ,Yang YA,Kim J,Yu J.FOXA1 acts upstream of GATA2 and AR in hormonal regulation of gene expression.Oncogene 2016;35(33):4335-44.