内质网应激在乙型重型肝炎(肝衰竭)中的作用研究

Study of endoplasmic reticulum stress role in hepatic failure induced by severe hepatitis B

目的探讨内质网应激在乙型肝炎病毒(HBV)感染后引发的重型肝炎(肝衰竭)(重型肝炎肝衰竭)过程中的作用及其相关机制。方法收集2009—2011年首都医科大学附属北京佑安医院就诊患者中慢性乙型肝炎患者(12例,慢性乙型肝炎患者组)和HBV感染所引发的重型肝炎肝衰竭患者(12例,乙型重型肝炎肝衰竭组)以及正常人(8名,对照组)的肝组织标本和临床资料。对各组临床检测指标进行统计分析;选用透射电镜对肝组织内质网结构进行观察;用蛋白质印迹法和qRT-PCR检测内质网应激和凋亡相关因子葡萄糖调节蛋白(Grp)、C/EBP环磷酸腺苷反应元件结合转录因子同源蛋白(CHOP)等的表达;制备冰冻切片进行免疫荧光检测。所有数据以均数±标准差(±s)表示,用LSD-t检验进行组间比较,P<0.05为差异有统计学意义。结果透射电镜检测结果表明,慢性乙型肝炎组和重型肝炎肝衰竭组内质网形态结构均受到破坏,且重型肝炎肝衰竭组更为严重;蛋白质印迹法和qRT-PCR结果显示Grp78、Grp94、胱天蛋白酶4在正常组和慢性乙型肝炎组表达较高,蛋白相对表达量分别为1.20±0.13和0.78±0.11、0.90±0.06和0.11±0.01、0.15±0.02和0.22±0.04;在重型肝炎肝衰竭组表达减弱(蛋白相对表达量分别为0.01±0、0.01±0、0.11±0.02),且与正常组差异有统计学意义,P值均<0.05;而CHOP表达与免疫荧光结果一致,随着损伤的加重而增强。结论内质网应激在乙型肝炎重症化过程中失调:慢性乙型肝炎阶段引发轻度内质网应激,而在重型肝炎肝衰竭阶段引发重度内质网应激。因此,内质网应激在重型肝炎肝衰竭发病过程中起到重要而又复杂的作用。

Objective To investigate the endoplasmic reticulum stress (ERS) role in the course of liver failure induced by severe hepatitis B virus (HBV) infection and its related mechanism.Methods Liver tissue samples and clinical data [chronic hepatitis B patients (12 cases,chronic hepatitis B group),hepatic failure induced by severe hepatitis B virus (12 cases,severe hepatitis B virus liver failure group),and normal subjects (8 cases,control group)] were collected from the Beijing You'an Hospital affiliated to Capital Medical University between 2009 to 2011.Statistical analysis was performed on the clinical indicators of each group.The structure of endoplasmic reticulum in liver tissue was observed by transmission electron microscopy.Western blot and qRT-PCR were used to detect the expression of endoplasmic reticulum stress and apoptosis-related factors,including glucose-regulated protein (Grp),and C/EBP homologous protein (CHOP).Frozen sections of liver tissues were prepared for immunofluorescence test.All data were expressed as mean ± standard deviation.LSD-t test was used to compare the results between groups.A p value < 0.05 was considered as statistically significant.Results Transmission electron microscopy showed that the morphological structure of the endoplasmic reticulum was damaged in both groups (chronic hepatitis B and liver failure induced by severe hepatitis B virus),and liver failure induced by severe hepatitis B virus group was more critical.Western blot and qRT-PCR showed that Grp78,Grp94 and Caspase-4 were highly expressed in normal group and chronic hepatitis B group,and the relative protein expressions were 1.20 ± 0.13 and 0.78 ± 0.11,0.90 ± 0.06 and 0.11 ± 0.01,0.15 ± 0.02 and 0.22 ± 0.04,respectively.The expression of protein was weakened in liver failure induced by severe hepatitis B virus group (relative protein expression was 0.01 ± 0,0.01 ± 0,and 0.11 ± 0.02,respectively).There was a statistically significant difference between the two groups (P < 0.05).The expression of CHOP was consistent with the results of immunofluorescence,and increased with the stressing of injury.Conclusion During the course of severe hepatitis B infection,dysregulated endoplasmic reticulum stress activated mild stress in chronic hepatitis B group,while severe stress in hepatic failure induced by severe hepatitis B virus group.Therefore,endoplasmic reticulum stress plays an important and complex role in the pathogenesis of hepatic failure induced by severe hepatitis B virus.