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目的探讨内毒素(即脂多糖)诱导D-半乳糖胺致敏大鼠急性肝衰竭肝细胞凋亡情况及肝损伤发生机制。方法48只Wistar大鼠进行随机对照分组实验,分为6 h、24 h和48 h取材3大组(各16只),每个大组再分为处理组和对照组(各8只)。处理组大鼠以脂多糖(50μg／kg)+D-半乳糖胺(300 mg／kg),用1 ml无菌生理盐水溶解后腹腔内注射,对照组动物仅腹腔内注射1 ml生理盐水。在相应时间点,门静脉或下腔静脉采血查丙氨酸氨基转移酶(ALT);肝组织切片分别行透射电镜检查和脱氧核糖核苷酸末端转移酶介导的缺口末端标记分析(TUNEL分析);基因表达通过逆转录。聚合酶链反应(RT-PCR)的方法检测。结果所有处理组大鼠ALT水平均显著高于对照组(66 U／L±3 U／L),而6 h组ALT水平(399 U／L±83 U／L)显著低于24 h组(3178 U／L±63 U／L)和48 h组(1506 U／L±56U／L)。48 h组ALT水平则低于24 h组。透射电镜检查见正常对照组肝组织内罕有凋亡细胞,脂多糖／D-半乳糖胺处理6 h组凋亡的肝细胞明显较多,且多表现为早期细胞凋亡的特征,24 h和48 h组的肝细胞凋亡明显多于6 h组,并多表现为晚期凋亡的特征。肝组织的TUNEL分析显示,对照组可见少量凋亡的肝细胞(凋亡指数为2．6％±1．1％),6 h组凋亡的肝细胞明显增多(凋亡指数为7．3％±1.5％),24 h和48 h组肝组织内则可见大量肝细胞凋亡(凋亡指数分别为71．8％±10．3％和68．2％±11．9％)。iNOS mRNA在正常对照组无表达,脂多糖／D-半乳糖胺作用后早期(6 h)有高水平的表达,24 h和48 h则显著较低;p53基因在对照组和6 h组有低水平表达,在24 h和48 h组表达明显较高;p21waf1/cip1基因在对照组无表达,6 h出现低水平表达,24 h mRNA表达水平达峰值,在48 h则迅速下降到0。结论小剂量脂多糖可诱导D-半乳糖胺致敏大鼠发生急性肝衰竭;细胞凋亡是其重要的病理形态学改变;肝衰竭肝损伤的发生与iNOS基因早期高水平的表达有密切关系。
Objective To investigate the apoptosis of liver cells induced by endotoxin (LPS) -induced D-galactosamine-induced acute liver failure and the mechanism of hepatic injury. Methods Forty - eight Wistar rats were randomly divided into three groups (6 rats at 6 h, 24 h, 48 h). Each group was divided into treatment group and control group (n = 8). The rats in the treatment group were injected intraperitoneally with lipopolysaccharide (50μg / kg) and D-galactosamine (300mg / kg) after intraperitoneal injection with 1 ml of sterile saline. The control animals were injected intraperitoneally with 1 ml of normal saline. At the corresponding time points, alanine aminotransferase (ALT) was detected in the portal vein or inferior vena cava; the liver sections were examined by transmission electron microscopy and DNA nick end labeling (TUNEL) Gene expression is reversed by reverse transcription. Polymerase chain reaction (RT-PCR) method of detection. Results The levels of ALT in all treatment groups were significantly higher than those in control group (66 U / L ± 3 U / L), while those in 6 h group were significantly lower than those in 24 h group (399 U / L ± 83 U / L) 3178 U / L ± 63 U / L) and 48 h group (1506 U / L ± 56 U / L). The level of ALT in 48 h group was lower than that in 24 h group. Transmission electron microscopy showed that there were many apoptotic cells in normal liver tissue. LPS / D-galactosamine treatment significantly increased the number of apoptotic hepatocytes at 6 h, The apoptosis of hepatocytes in 48 h group was significantly more than that of 6 h group, and more often showed the characteristics of late apoptosis. TUNEL analysis of liver tissue showed that a small amount of apoptotic hepatocytes were observed in the control group (apoptotic index 2.6% ± 1.1%), and apoptotic hepatocytes significantly increased after 6 hours (apoptotic index 7.3 % ± 1.5%). A large number of hepatocytes were found in the 24 h and 48 h groups (apoptosis index 71.8% ± 10.3% and 68.2% ± 11.9%, respectively). There was no expression of iNOS mRNA in the normal control group, and the expression of iNOS mRNA was high in the early stage (6 h) after lipopolysaccharide / D-galactosamine treatment, but significantly lower at 24 and 48 h The expression of p21waf1 / cip1 gene was not observed in the control group at 6 h. The mRNA expression of p21waf1 / cip1 peaked at 6 h and peaked at 24 h, then decreased rapidly to 0 at 48 h. Conclusion Low-dose lipopolysaccharide can induce acute liver failure in D-galactosamine-sensitized rats. Apoptosis is an important pathomorphological change. The occurrence of liver injury in liver failure is closely related to the early high-level expression of iNOS gene .