Nan Li, Wu Yong, Bardag-Gorce Fawzia, Li Jun, French Barbaba A, Wilson La Toyia, French Samuel W
Department of Pathology, Harbor-UCLA Medical Center, 1000 W. Carson Street, Torrance, CA 90502, USA.
Exp Mol Pathol. 2005 Jun;78(3):198-206. doi: 10.1016/j.yexmp.2004.12.002. Epub 2005 Feb 9.
To determine if nuclear factor-kappaB (NF-kB) plays a role in Mallory body (MB) formation, quantitative real-time RT-PCR assay was used to measure liver NF-kappaB1/p105 mRNA levels in 4 different groups of mice. Group 1: mice given IP saline for 15 weeks; group 2: mice fed diethyl 1,4-dihydro-2,4,6,-trimethyl-3,5-pyridinedicarboxylate (DDC) for 10 weeks when MBs were formed; group3: mice fed DDC 10 weeks, then withdrawn 5 weeks when MBs disappeared; group 4: mice fed DDC 10 weeks, withdrawn 4 weeks, then fed DDC+chlormethiazole (CMZ) for 1 week when MBs again formed. The mRNA for p105 NF-kappaB expression was significantly increased in the livers of mice treated with DDC (group 2) and DDC+CMZ (group 4) compared with the control livers (group 1) as well as the drug-withdrawal livers (group 3). Primary cultures of hepatocytes from drug-primed mice (the group 4 mice were withdrawn for another 4 weeks when the MBs had disappeared) were studied. The hepatocytes from drug-primed mice were MB free when isolated and used for primary culture. MBs began to form spontaneously within their cytoplasm after 2-3 days of culture. The NF-kappaB inhibitor (NF-kappaBi), a cell-permeable quinazoline compound that acts as a potent inhibitor of NF-kappaB transcriptional activation, was added to the medium 3 h after planting the cultures of liver cells. No MBs formed in the cells treated with 10 microM, 1 microM, and 0.1 microM NF-kappaBi for 6 days. MBs still formed in the cells treated with 10 nM NF-kappaBi for 6 days. Both DDC-primed and normal control liver cells began to enlarge and elongate after a few hours of culture. In contrast, the cells treated with NF-kappaBi stayed polyhedral in shape just as they appeared prior to culturing. The level of NF-kappaB1/p105 mRNA significantly increased in DDC-primed hepatocytes after 24 h of culture and in normal control hepatocytes after 48 h of culture. In DDC-primed hepatocytes, NF-kappaBi 0.1 muM treatment for 6 days significantly decreased mRNA expression of Src, p105/NF-kappaB1, ERK1, MEKK1, and JNK1/2. In normal control liver cells, NF-kappaBi treatment decreased mRNA expression of Src and JNK1 and stimulated the mRNA expression of p105/NF-kappaB1 and Junk2. NF-kappaBi treatment significantly decreased the total ERK1/2 protein and further decreased the phosphorylated (activated) form of ERK1/2 in the cultured hepatocytes. The results indicate that the p105 NF-kappaB pathway which putatively regulates ERK at both the transcriptional and post-translational levels regulates MB formation by way of changes in gene expression.
为了确定核因子-κB（NF-κB）是否在马洛里小体（MB）形成中发挥作用，采用定量实时RT-PCR分析法检测4组不同小鼠肝脏中NF-κB1/p105 mRNA水平。第1组：腹腔注射生理盐水15周的小鼠；第2组：喂食二乙基1,4-二氢-2,4,6-三甲基-3,5-吡啶二羧酸酯（DDC）10周，此时形成MB的小鼠；第3组：喂食DDC 10周，然后停药5周，此时MB消失的小鼠；第4组：喂食DDC 10周，停药4周，然后喂食DDC+氯美噻唑（CMZ）1周，此时MB再次形成的小鼠。与对照肝脏（第1组）以及停药肝脏（第3组）相比，用DDC处理的小鼠（第2组）和DDC+CMZ处理的小鼠（第4组）肝脏中p105 NF-κB表达的mRNA显著增加。对用药物预处理的小鼠（第4组小鼠在MB消失后再停药4周）的肝细胞进行原代培养研究。从用药物预处理的小鼠分离得到的肝细胞在用于原代培养时没有MB。培养2 - 3天后，MB开始在其细胞质中自发形成。在接种肝细胞培养物3小时后，将NF-κB抑制剂（NF-κBi）添加到培养基中，NF-κBi是一种可渗透细胞的喹唑啉化合物，作为NF-κB转录激活的有效抑制剂。用10μM、1μM和0.1μM NF-κBi处理6天的细胞中未形成MB。用10 nM NF-κBi处理6天的细胞中仍形成MB。DDC预处理的肝细胞和正常对照肝细胞在培养数小时后开始增大并伸长。相比之下，用NF-κBi处理的细胞在培养前就保持多面体形状。培养24小时后，DDC预处理的肝细胞中NF-κB1/p105 mRNA水平显著增加，培养48小时后，正常对照肝细胞中NF-κB1/p105 mRNA水平显著增加。在DDC预处理的肝细胞中，0.1μM NF-κBi处理6天显著降低Src、p105/NF-κB1、ERK1、MEKK1和JNK1/2的mRNA表达。在正常对照肝细胞中，NF-κBi处理降低Src和JNK1的mRNA表达，并刺激p105/NF-κB1和Junk2的mRNA表达。NF-κBi处理显著降低培养肝细胞中总ERK1/2蛋白水平，并进一步降低ERK1/2的磷酸化（激活）形式。结果表明，推测在转录和翻译后水平调节ERK的p105 NF-κB途径通过基因表达变化调节MB形成。
