Verhaag Esther M, Buist-Homan Manon, Koehorst Martijn, Groen Albert K, Moshage Han, Faber Klaas Nico
Department of Gastroenterology and Hepatology, Center for Liver, Digestive, and Metabolic Diseases, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands.
Department of Laboratory Medicine, Center for Liver, Digestive, and Metabolic Diseases, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands.
PLoS One. 2016 Mar 7;11(3):e0149782. doi: 10.1371/journal.pone.0149782. eCollection 2016.
Cholestasis is characterized by accumulation of bile acids and inflammation, causing hepatocellular damage. Still, liver damage markers are highest in acute cholestasis and drop when this condition becomes chronic, indicating that hepatocytes adapt towards the hostile environment. This may be explained by a hormetic response in hepatocytes that limits cell death during cholestasis.
To investigate the mechanisms that underlie the hormetic response that protect hepatocytes against experimental cholestatic conditions.
HepG2.rNtcp cells were preconditioned (24 h) with sub-apoptotic concentrations (0.1-50 μM) of various bile acids, the superoxide donor menadione, TNF-α or the Farsenoid X Receptor agonist GW4064, followed by a challenge with the apoptosis-inducing bile acid glycochenodeoxycholic acid (GCDCA; 200 μM for 4 h), menadione (50 μM, 6 h) or cytokine mixture (CM; 6 h). Levels of apoptotic and necrotic cell death, mRNA expression of the bile salt export pump (ABCB11) and bile acid sensors, as well as intracellular GCDCA levels were analyzed.
Preconditioning with the pro-apoptotic bile acids GCDCA, taurocholic acid, or the protective bile acids (tauro)ursodeoxycholic acid reduced GCDCA-induced caspase-3/7 activity in HepG2.rNtcp cells. Bile acid preconditioning did not induce significant levels of necrosis in GCDCA-challenged HepG2.rNtcp cells. In contrast, preconditioning with cholic acid, menadione or TNF-α potentiated GCDCA-induced apoptosis. GCDCA preconditioning specifically reduced GCDCA-induced cell death and not CM- or menadione-induced apoptosis. The hormetic effect of GCDCA preconditioning was concentration- and time-dependent. GCDCA-, CDCA- and GW4064- preconditioning enhanced ABCB11 mRNA levels, but in contrast to the bile acids, GW4064 did not significantly reduce GCDCA-induced caspase-3/7 activity. The GCDCA challenge strongly increased intracellular levels of this bile acid, which was not lowered by GCDCA-preconditioning.
Sub-toxic concentrations of bile acids in the range that occur under normal physiological conditions protect HepG2.rNtcp cells against GCDCA-induced apoptosis, which is independent of FXR-controlled changes in bile acid transport.
胆汁淤积的特征是胆汁酸积累和炎症，导致肝细胞损伤。然而，肝损伤标志物在急性胆汁淤积时最高，而在病情转为慢性时下降，这表明肝细胞会适应这种不利环境。这可能是由于肝细胞中的一种应激反应，在胆汁淤积期间限制细胞死亡。
研究保护肝细胞免受实验性胆汁淤积条件影响的应激反应的潜在机制。
用亚凋亡浓度（0.1 - 50 μM）的各种胆汁酸、超氧化物供体甲萘醌、肿瘤坏死因子-α或法尼醇X受体激动剂GW4064对HepG2.rNtcp细胞进行预处理（24小时），随后用诱导凋亡的胆汁酸甘氨鹅去氧胆酸（GCDCA；200 μM，处理4小时）、甲萘醌（50 μM，6小时）或细胞因子混合物（CM；6小时）进行刺激。分析凋亡和坏死细胞死亡水平、胆盐输出泵（ABCB11）和胆汁酸传感器的mRNA表达以及细胞内GCDCA水平。
用促凋亡胆汁酸GCDCA、牛磺胆酸或保护性胆汁酸（牛磺）熊去氧胆酸预处理可降低GCDCA诱导的HepG2.rNtcp细胞中的caspase - 3/7活性。胆汁酸预处理在GCDCA刺激的HepG2.rNtcp细胞中未诱导出显著水平的坏死。相反，用胆酸、甲萘醌或肿瘤坏死因子-α预处理会增强GCDCA诱导的凋亡。GCDCA预处理特异性地减少了GCDCA诱导的细胞死亡，而不是CM或甲萘醌诱导的凋亡。GCDCA预处理的应激效应具有浓度和时间依赖性。GCDCA、CDCA和GW4064预处理提高了ABCB11 mRNA水平，但与胆汁酸不同，GW4064并未显著降低GCDCA诱导的caspase - 3/7活性。GCDCA刺激强烈增加了这种胆汁酸的细胞内水平，而GCDCA预处理并未使其降低。
正常生理条件下出现的亚毒性浓度胆汁酸可保护HepG2.rNtcp细胞免受GCDCA诱导的凋亡，这与法尼醇X受体控制的胆汁酸转运变化无关。
