对乙酰氨基酚诱导的急性肝损伤和肝衰竭模型中线粒体基因组转录改变

摘要/Abstract

摘要： 目的通过探索对乙酰氨基酚(APAP)诱导的急性肝损伤和急性肝衰竭模型中线粒体基因组转录水平的改变与疾病进展之间的关联,为AILI预后的预测提供新的生物标记。方法将90只小鼠随机分为3组:对照组、APAP导致的DILI组(AILI,300 mg/kg)和APAP导致的急性肝衰竭组(AILF,750 mg/kg)。禁食16 h后,腹腔注射体积相近的0.9%氯化钠溶液和不同剂量APAP,在0、1、3、6、12 h等不同时间点时,每组随机选取6只小鼠处死,留取小鼠的血浆和肝脏组织。检测每只小鼠ALT、AST、ROS变化水平; 提取肝脏总RNA,采用RT-PCR技术检测线粒体基因组基因的转录水平。结果与对照组相比,腹腔注射APAP后,两组均可见转氨酶明显升高,AILI组在6-12 h时ALT达到峰值(5 000~10 000 U/L);AILF组12 h时ALT水平超过10 000 U/L,明显高于AILI组(P<0.05)。APAP处理后,两组小鼠均可见典型的3区微泡性脂肪变,AILF组可见典型的急性肝衰竭的大块性坏死。与对照组相比,AILI和AILF组肝脏中ROS从1 h开始,各时间点均可见显著升高,且1 h时,AILF组ROS的生成量约为AILI组的2.5倍(P<0.05)。AILI组COX1在6 h时显著升高(P<0.05),而对照组和AILF组均未见显著升高。与对照组相比,3 h时,AILI组和AILF组可见CYTB、COX2和ATP8显著降低(P<0.05);6 h时,AILI组和AILF组COX1、ND1、ND5和ATP8转录水平显著降低(P<0.05);12 h时,AILI组和AILF组中NADH各亚基均可见显著降低(P<0.05)。AILI组与AILF组相比, 6 h时,AILF组中 ATP6的转录水平显著低于AILI组和对照组(P<0.05);12 h时,AILF组的肝脏中CYTB、COX2、ATP8以及NADH的2、3、5和6亚基的转录水平显著低于AILI组(P<0.05)。结论线粒体基因组除COX1在AILI组明显上调,其他存在明显改变的基因均出现降低趋势,且AILF组变化更明显。线粒体基因组转录水平改变对于预测AILI预后有潜在价值。

关键词: 对乙酰氨基酚, 肝损伤, 肝衰竭, 线粒体, 转录

Abstract: Objective To explore the relationship between transcription level of mitochondrial DNA (mtDNA) and disease progression in the mice models of acetaminophen (APAP)- induced acute liver injury (AILI) and acute liver failure (ALF), and to find the relative new biomarkers for outcome.Methods Ninety mice were randomly divided into three groups, including control group, AILI group (300 mg/kg) and ALF group (750 mg/kg). After fasting 16 h, all mice were intraperitoneally injected with same volume of saline or different doses of APAP. At different time points of 0, 1, 3, 6 and 12 h, 6 mice randomly selected from each group were sacrificed for blood and liver, respectively. Plasma alanine aminotransferase (ALT), aspartate aminotransferase (AST) and reactive oxygen (ROS) levels were detected, and liver total RNA was extracted for RT-PCR to detect changes in transcription of mitochondrial genome. Results Compared with the control group, ALT and AST levels in AILI and ALF group were significantly increased. In AILI group, ALT peaked at 6-12 h (5000-10000 IU/L), and ALT exceeded 10000 IU/L at 12 h in ALF group, which showed significantly difference (P<0.05). Microbubbles steatosis in three zones was observed in both AILI and ALF groups, and massive hepatic necrosis (MHN) were found merely in ALF group. In 1 h after APAP injection, ROS in ALF group was about 2.5 times as much as that in AILI group, which significantly increased at all time points in both group. Contrasting with control and ALIF groups, COX1 transcriptional level in AILI group increased significantly at 6 h. In AILI and ALF group, CYTB, COX2 and ATP8 reduced significantly at 3 h (P<0.05), COX1,ND1,ND5 and ATP8 significantly decreased at 6 h (P<0.05), and transcription level of other subunits of NADH significantly decreased at 12 h (P<0.05) comparing with those in control group. Furthermore, ATP6 at 6h in ALF group was obviously lower than that in AILI and control group (P<0.05). At 12 h, the majority of mtDNA (CYTB, COX2, ATP8, ND2, ND3, ND5 and ND6 ) had significant differences between AILI and ALF group.Conclusion There are significant difference in mtDNA transcription between AILI and ALF groups. Besides COX1, other mtDNA showed significant decreases in AILI and ALF group compared with those in control group, especially in ALF group. mtDNA transcriptional changes may have great potential as biological makers to predict outcomes of AILI.

Key words: APAP, Liver injury, Liver failure, Mitochondrial, Transcription

明雅南, 李春敏, 张静怡, 刘晓琳, 茅益民. 对乙酰氨基酚诱导的急性肝损伤和肝衰竭模型中线粒体基因组转录改变[J]. 肝脏, 2016, 21(6): 447-451.

MING Ya-nan, LI Chun-min, ZHANG Jing-yi, LIU Xiao-lin, MAO Yi-Min. The transcriptional change of mitochondrial genome in mice models for acetaminophen-induced acute liver injury and failure[J]. Chinese Hepatolgy, 2016, 21(6): 447-451.

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