Home > Browse Issues > Vol.40 No.5
Establishment of Acetaminophen-Resistant Liver Cell Line and Its Possible Mechanism
Cao Huanhuan1,2, Mao Yulei1,3, Chen Juanhui1, Chen Jiayuan1, Zheng Ruipan1, Cen Mengjiao1, Ni Jian1, Jin Lifang1,2*
1College of Life Science, Shaoxing University, Shaoxing 31200, China; 2Hangzhou Precision Medicine Research Center, Hangzhou 310018, China; 3Affiliated Hospital of Shaoxing University, Shaoxing 312000, China
Abstract: 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 H2DCF-DA and MitoTracker detection， respectively. The levels of mRNA and protein were determined by qPCR 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 qPCR 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.
CN
EN