miR-122在D-氨基半乳糖联合脂多糖诱导急性肝衰竭小鼠体内的表达及其意义

目的 通过监测急性肝功能衰竭小鼠体内miR-122的表达,探讨miR-122与小鼠急性肝衰竭疾病程度和进展之间的关系,为肝功能衰竭的早期诊断提供新的生物学标志物. 方法将BALB/C小鼠随机分为4组,实验组用D-氨基半乳糖(D-GalN,900 mg/kg)联合脂多糖(LPS,10 μg/kg)腹腔注射建立肝衰竭模型,对照组3组,分别予以D-GalN(900 mg/kg),LPS(10 μg/kg)和等渗盐水腹腔注射,在不同时间点观察小鼠病死率、肝脏组织学变化,给药后0、1、3、5、7、9 h分别留取血清、肝脏组织标本,实时定量逆转录多聚酶链反应检测小鼠体内miRNA-122和炎症因子的表达,LNA(锁核酸)-Northern blot验证miRNA-122的表达,生化分析仪检测血清中ALT、AST水平,酶联免疫吸附法检测血清中炎症因子水平.组间均数比较用two-WayANOVA方差分析,相关性分析采用Pearson和Spearman相关分析.结果 D-GalN/LPS给药24 h,小鼠病死率率达80%以上,而3个对照组则无一只小鼠死亡;肝脏特异性miR-122在正常小鼠肝脏内含量丰富(ct≈14),D-GalN/LPS诱导后1 h,miR-122即发生了明显的变化(P=0.013),表现为上调,之后随疾病的进展,miR-122表达进行性下降,9 h下调最为明显(ct≈15,P=0.002);ALT/AST于给药1 h无明显变化,3 h后呈明显上升趋势,7 h达高峰,之后ALT/AST急剧下降;对miR-122和ALT的表达对比,发现在该模型中miR-122比ALT变化快,且持久;肝衰竭相关炎症因子肿瘤坏死因子(TNF)α和白细胞介素(IL)-6在肝组织和血清中的变化一致,均上调(P<0.05); miR-122和ALT、TNFα和IL-6的相关性分析显示miR-122与以上三项指标均呈良好的相关性(相关系数分别为-0.505、0.493和0.674、).结论 肝衰竭小鼠体内肝脏特异性miR-122和ALT呈负相关关系,但又较ALT更敏感,更持久地反映肝细胞损伤程度,且miR-122表达变化与肝脏炎症损伤相关因素TNF α、IL-6均具良好的相关性,推测miR-122有望成为判断急性肝衰竭肝细胞损伤程度的一个新的分子生物学标志物.

The Role of miRNA-122 expression during the acute liver failure in mice induced by D-GalN/LPS

Objective To investigate the expression of miR-122 and its relationship with progression and development of acute liver failure in mice induced by D-GalN/LPS,and to explore new biomarker(s)for early diagnosis of acute liver failure.Methods BALB/C mice were randomly divided into four groups:the mice were given D-GalN(900 mg/kg body weight)and LPS(10 μg/kg body weight)intraperitoneally(i.P.)to construct the acute liver model;whereas the control groups were given D-GalN(900 mg/kg),LPS (10 μg/kg)and normal saline respectively.All biochemical and histological indexes were determined at 0,1,3,5,7 and 9h respectively after administration.Real-time RT-PCR were used to detect the expression of miR-122 and pro-inflammatory cytokines,furthermore,the expression of miR-122 was verified by LNA (lock nucleic acid)-Northern-blot.ALT and AST levels were tested by biochemistry analyzer.Serum proinflammatory cytokine levels were tested by ELISA.Results The mortality rate was about 80% at 24h after D-GalN/LPS treatment,but no mortality was observed in the other three control groups.Liver special miRNA miR-122 was highly expressed in liver tissue of normal mice(ct≈14),it was up-regulated significantly(P=0.013)at first hour after treatment then down-regulated according to the development of acute liver failure,the change was more obvious at 9h(ct≈15,P=0.002).ALT and AST levels increased obviously at 3h after treatment and reached peak at 7 hours then they were declined sharply.It was found that the expression of miR-122 was faster and more durable than ALT.Pro-inflammatory cytokines related to acute liver failure including TNFα and IL-6 were all up-regulated in serum as well as liver tissue(P<0.05).Correlation analysis showed that miR-122 had anegative correlation with ALT (correlation coefficients-0.505)and positive correlations with TNF α and IL-6(correlation coefficients were 0.493 and 0.674 respectively).Conclusions Liver-specific miR-122 supposed be a new marker molecule for early diagnosis of liver cells injury in the acute liver failure.