鼠三型肝炎病毒诱导暴发性肝衰竭小鼠模型中程序性坏死的肝脏损伤机制

Mechanism of necroptosis of liver injury in murine hepatitis virus strain 3(MHV-3)induced fulminant hepatic failure model

目的:探讨鼠三型肝炎病毒(MHV-3)诱导暴发性肝衰竭小鼠模型中程序性坏死的肝脏损伤机制。方法:将48只雌性SPF级Balb/c小鼠随机分为程序性坏死抑制剂Necrostatin-1(Nec-1)预处理组和生理盐水组,每组24只,腹腔注射200μl含100 PFU MHV-3的无菌生理盐水诱导暴发性肝衰竭小鼠模型。观察记录小鼠存活状况,蛋白免疫印迹法检测程序性坏死关键蛋白受体相互作用蛋白1(RIP1)、受体相互作用蛋白3(RIP3)表达水平,测定血清转氨酶水平、HE染色观察肝脏病理改变并比较坏死面积百分比,实时荧光定量PCR法检测肝脏炎性因子表达水平。另46只雌性SPF级Balb/c小鼠建立暴发性肝衰竭小鼠模型,只用于生存分析。结果:在暴发性肝衰竭小鼠模型中,随着感染时间的延长,Nec-1预处理组和生理盐水组小鼠全部死亡,生存率无显著性差异。Nec-1预处理组小鼠肝脏RIP1、RIP3表达出现明显下调,而生理盐水组小鼠肝脏RIP1表达随时间未出现明显变化。随着感染时间的延长,Nec-1预处理组和生理盐水组小鼠肝脏病理切片均出现大片坏死,组间坏死情况未发现明显差异。Nec-1预处理组和生理盐水组小鼠血清ALT和AST水平均显著升高,差异无统计学意义。Nec-1预处理组和生理盐水组间肝脏炎性细胞因子水平无明显差异。结论:程序性坏死不是导致MHV-3诱导暴发性肝衰竭小鼠模型肝脏损伤的主要机制,针对程序性坏死的实验性治疗无法改善肝脏损伤。

Objective:The aim of this study was to investigate the role of necroptosis in liver injury in murine hepatitis virus strain 3(MHV-3)induced fulminant hepatic failure model.Methods:Forty-eight female Balb/c mice were randomly divided into two groups:Necrostatin-1(Nec-1)preconditioning group and normal saline group.Fulminant hepatic failure was induced by injecting 200μl of sterile saline containing 100 PFU MHV-3.The survival status of mice was observed and recorded.The expression levels of necroptosis key proteins receptor-interacting protein 1(RIP1)and receptor-interacting protein 3(RIP3)were detected by Western blot.HE staining was used to observe the pathological changes of the liver and the percentage of necrotic area was compared.Real-time fluorescent quantitative PCR was used to detect the expression levels of inflammatory factors in the liver.Another 46 female SPF Balb/c mice were used to establish a mouse model of fulminant hepatic failure and were used only for survival analysis.Results:In MHV-3-induced fulminant hepatic failure model,all mice of the Nec-1-pretreated group and normal saline group died over time,and we found no significant difference in the survival.After Nec-1 pretreatment,the expression of RIP1 and RIP3 decreased markedly,but the protein expressions of RIP1 was not found significant elevation post infection in normal saline group.With the MHV-3 infection prolonged,the histopathology of liver tissue showed extraordinary injury in both Nec-1-pretreated group and normal saline group,but Nec-1 pretreatment could not alleviate the liver injury,as proved by the similar necrosis area fractions and serum transaminase levels of both group.The serum levels of ALT and AST were significantly increased in NEC-1 preconditioning group and saline group,but there was no significant difference between the two groups.Meanwhile,the inflammatory factors levels of the Nec-1-pretreated group and normal saline group showed no significant difference.Conclusion:Necroptosis is not the main mechanism of liver injury in MHV-3-induced fulminant hepatic failure model,and targeted inhibition of necroptosis cannot relieve liver injury in MHV-3-induc-ed fulminant hepatic failure model.