对乙酰氨基酚诱导急性肝衰竭小鼠动物模型的建立

摘要/Abstract

摘要： 目的通过腹腔注射高剂量对乙酰氨基酚(APAP)构建稳定的用于研究药物导致急性肝衰竭的动物模型。方法本研究首先进行生存率实验,取60只C57BL/6小鼠随机分为4组,每组15只,分别腹腔注射0.9%氯化钠溶液及不同剂量(300 mg/kg、500 mg/kg 、750 mg/kg)APAP后,观察不同组别72 h内小鼠的精神、活动状态和生存率。根据生存率分析结果,另选取180只C57BL/6小鼠,随机分为3组,分别腹腔注射0.9%氯化钠溶液、低剂量(300 mg/kg)和高剂量(750 mg/kg)APAP,每组分别在 0、1、3、6、12 h等时间点随机选取12只小鼠,留取血清和肝组织,验证小鼠的生化和组织学是否符合急性肝衰竭的表现。结果生存率实验结果显示,0.9%氯化钠溶液组、300 mg/kg以及500 mg/kg组72 h内无小鼠死亡;750 mg/kg组72 h死亡率为100%,推测750 mg/kg组可能为急性肝衰竭导致的死亡。生化和组织学验证实验结果发现,对照组各时间点转氨酶均无明显升高;APAP处理的两组动物模型ALT 3 h时开始升高,6 h时低剂量组ALT升高达到峰值[(6766.5±2001.27) IU/L],而高剂量组ALT于12 h达到(11707.58±1882.45) U/L(P<0.01)。从HE病理染色来看,0.9%氯化钠溶液组各时间点肝脏形态结构正常。APAP处理的两组动物模型的肝组织学,主要表现为以中央静脉为中心的肝细胞变性坏死,并随时间延长,损伤范围逐渐扩大。低剂量组坏死周围界限清楚,汇管区肝细胞结构形态正常,12 h可见坏死区周围肝细胞增生表现。高剂量组表现为典型的急性大片状坏死特点,仅汇管区残存少量变性的肝细胞,细胞快速坏死后,留下空的网状纤维支架,肝窦淤积大量红细胞,未见肝细胞增生。HAI评分结果显示,高剂量组的HAI得分(7.33±1.5)显著高于低剂量组(5.25±2.26,P<0.05)。结论 C57BL/6小鼠腹腔注射高剂量APAP(750 mg/kg)后,生化和组织学改变与急性肝衰竭相似,本研究构建的动物模型对于探索APAP导致的AHF的发病及进展机制研究具有潜在的应用价值。

关键词: 对乙酰氨基酚, 肝衰竭, 动物模型

Abstract: Objective To establish a stable animal model of drug-induced acute hepatic failure (AHF) with different doses of acetaminophen (APAP) by intraperitoneal injection.Methods Sixty mice, which were randomly divided into four groups (n=15), were intraperitoneally injected with saline and different doses of APAP (300 mg/kg, 500 mg/kg and 750 mg/kg), respectively. Mental status, activity and survival rates in different groups were observed within 72 hours. According to the analysis of survival rates, another 180 mice were divided into three groups randomly (n=60) with injection of saline, low (300 mg/kg) and high dose (750 mg/kg) of APAP, respectively. To detect the biochemical and pathological changes of AHF, 12 mice randomly selected from each group were sacrificed for serum and liver tissues collection at 0 h, 1 h, 3 h, 6 h and 12 h after injection, respectively.Results No mice died within 72 h in the control group, APAP (300 mg/kg and 500 mg/kg group) , while the mortality of APAP 750 mg/kg group was 100%. In control group, aminotransferase (ALT) level showed no significant increase at all time points. However, ALT levels in two APAP groups (300 mg/kg and 750 mg/kg) began to increase at 3 h, and reached to peak at 6 h (6766.5±2001.27 IU/L) or 12 h (11707.58±1882.45 IU/L) in low-dose or high-dose APAP group, respectively. Additionally, ALT level in high-dose APAP group was significantly higher than that in low-dose APAP group at 12 h (P<0.01). In view of haematoxylin-eosin (HE) staining, control group displayed normal liver structure. In APAP group, degeneration and necrosis of hepatocytes mainly occurred around central vein, and damage extent gradually expanded over time. In low-dose group, boundaries of necrotic zones were clear with normal liver cell morphology in portal areas, and visible hepatocytes proliferation around the boundaries was observed at 12 h. In high-dose group, typical acute massive hepatic necrosis was found and few of degenerated hepatocytes stayed alive at portal areas. After rapid necrosis of hepatocytes, empty fiber mesh stent remained with large red blood cells deposited in sinusoids and no proliferation of hepatocytes. At 12 h, histological activity index (HAI) score of high-dose group (7.33±1.5) was higher than that of low-dose group (5.25±2.26), which showed statistically significant differences (P<0.05).Conclusion C57BL/6 mice injected with high dose of APAP (750 mg/kg) have similar biochemical and pathological changes with AHF, which might be a reliable AHF model for investigating the role of APAP in pathogenesis and development of liver failure.

Key words: Acetaminophen; Liver failure; Animal model

明雅南, 李春敏, 张静怡, 刘晓琳, 茅益民. 对乙酰氨基酚诱导急性肝衰竭小鼠动物模型的建立[J]. 肝脏, 2016, 21(5): 351-354.

MING Ya-nan, LI Chun-min, ZHANG Jing-yi, LIU Xiao-lin, MAO Yi-min. Establishment of acetaminophen-induced acute hepatic failure model in mice[J]. Chinese Hepatolgy, 2016, 21(5): 351-354.

参考文献

[1] Larsen FS, Wendon J. Understanding paracetamol-induced liver failure. Intensive Care Med, 2014, 40:888-890.
[2] Rahman TM, Selden AC, Hodgson HJ. A novel model of acetaminophen-induced acute hepatic failure in rabbits. J Surg Res, 2002, 106:264-272.
[3] Kelly JH, Koussayer T, He DE, et al. An improved model of acetaminophen-induced fulminant hepatic failure in dogs. Hepatology, 1992, 15:329-335.
[4] Francavilla A, Makowka L, Polimeno L, et al. A dog model for acetaminophen-induced fulminant hepatic failure. Gastroenterology, 1989, 96:470-478.
[5] Rumbeiha WK, Lin YS, Oehme FW. Comparison of N-acetylcysteine and methylene blue, alone or in combination, for treatment of acetaminophen toxicosis in cats. Am J Vet Res, 1995, 56:1529-1533.
[6] Liu P, McGuire GM, Fisher MA, et al. Activation of Kupffer cells and neutrophils for reactive oxygen formation is responsible for endotoxin-enhanced liver injury after hepatic ischemia. Shock, 1995, 3:56-62.
[7] Laskin DL, Gardner CR, Price VF, et al. Modulation of macrophage functioning abrogates the acute hepatotoxicity of acetaminophen. Hepatology, 1995, 21:1045-1050.
[8] Kofman AV, Morgan G, Kirschenbaum A, et al. Dose- and time-dependent oval cell reaction in acetaminophen-induced murine liver injury. Hepatology, 2005, 41:1252-1261.
[9] Ramachandran A, McGill MR, Xie Y, et al. Receptor interacting protein kinase 3 is a critical early mediator of acetaminophen-induced hepatocyte necrosis in mice. Hepatology, 2013, 58:2099-2108.
[10] 李继强. 急性肝衰竭的病理表现. 肝脏, 1997, 2:93-94.
[11] Numata K, Kubo M, Watanabe H, et al. Overexpression of suppressor of cytokine signaling-3 in T cells exacerbates acetaminophen-induced hepatotoxicity. J Immunol, 2007, 178:3777-3785.
[12] Yang R, Zhang S, Cotoia A, et al. High mobility group B1 impairs hepatocyte regeneration in acetaminophen hepatotoxicity. BMC Gastroenterol, 2012, 12:45.
[13] Zaher H, Buters JT, Ward JM, et al. Protection against acetaminophen toxicity in CYP1A2 and CYP2E1 double-null mice. Toxicol Appl Pharmacol, 1998, 152:193-199.
[14] Mitchell JR, Jollow DJ, Potter WZ, et al. Acetaminophen-induced hepatic necrosis. IV. Protective role of glutathione. J Pharmacol Exp Ther, 1973, 187:211-217.
[15] Davis DC, Potter WZ, Jollow DJ, et al. Species differences in hepatic glutathione depletion, covalent binding and hepatic necrosis after acetaminophen. Life Sci, 1974, 14:2099-2109.
[16] McGill MR, Williams CD, Xie Y, et al. Acetaminophen-induced liver injury in rats and mice: comparison of protein adducts, mitochondrial dysfunction, and oxidative stress in the mechanism of toxicity. Toxicol Appl Pharmacol, 2012, 264:387-394.
[17] McGill MR, Jaeschke H. Metabolism and disposition of acetaminophen: recent advances in relation to hepatotoxicity and diagnosis. Pharm Res, 2013, 30:2174-2187.
[18] Saracyn M, Zdanowski R, Brytan M, et al. D-Galactosamine Intoxication in Experimental Animals: Is it Only an Experimental Model of Acute Liver Failure? Med Sci Monit, 2015, 21:1469-1477.
[19] Hanawa N, Shinohara M, Saberi B, et al. Role of JNK translocation to mitochondria leading to inhibition of mitochondria bioenergetics in acetaminophen-induced liver injury. J Biol Chem, 2008, 283:13565-13577.
[20] Kon K, Kim JS, Jaeschke H, et al. Mitochondrial permeability transition in acetaminophen-induced necrosis and apoptosis of cultured mouse hepatocytes. Hepatology, 2004, 40:1170-1179.