论文部分内容阅读
目的探讨Caspase-12在D-氨基半乳糖(D-Gal)/脂多糖(LPS)诱导小鼠急性肝功能衰竭发生发展过程中表达水平的变化及Caspase-12介导的内质网应激肝细胞凋亡途径在急性肝功能衰竭中的作用。方法以D-Gal/LPS联合腹腔注射诱导小鼠急性肝功能衰竭建立实验模型,在不同时间点动态检测血清氨基转移酶水平和观察肝组织病理变化,评估肝细胞凋亡和肝坏死演变过程;应用琼脂糖凝胶电泳检测肝细胞DNA凋亡条带;用半定量逆转录聚合酶链反应检测肝组织中Caspase-12 mRNA表达水平;west- ern blot检测Caspase-12、Bip/GRP78蛋白表达。结果药物诱导5h时肝组织中典型凋亡细胞增多,血清丙氨酸氨基转移酶(ALT)、天冬氨酸氨基转移酶(AST)开始升高,Caspase-12 mRNA表达明显增加, Caspase-12蛋白量表达反而减少;7h镜下出现大量肝细胞凋亡和坏死,ALT、AST水平达高峰,分别为(2564±1384)U/L和(1198±497)U/L,正常对照组为(59±17)U/L和(135±12)U/L,Caspase- 12 mRNA表达仍增加,Caspase-12蛋白表达量继续降低;9h 肝细胞坏死明显,伴散在凋亡细胞,ALT、AST水平明显下降,Caspase-12 mRNA表达较7 h下降。Bip/GRP78蛋自表达从5 h开始,至7 h逐渐增加。结论D-Gal/LPS诱导小鼠急性肝功能衰竭早期Caspase-12 mRNA表达水平逐渐升高,后期(7-9 h)降低,与肝细胞凋亡发生的时相一致;Caspase-12蛋白酶因内质网应激而被大量活化,提示Caspase-12介导的内质网应激肝细胞凋亡参与炎症性急性肝功能衰竭的发生发展,是急性肝功能衰竭中肝细胞损伤的重要机制之一,提示早期干预Caspase-12表达及活化对急性肝功能衰竭可能具有保护作用。
Objective To investigate the expression of caspase-12 in the development of acute liver failure induced by D-galactose / lipopolysaccharide (LPS) and the effect of Caspase-12-mediated endoplasmic reticulum stress Apoptosis pathway in acute liver failure. Methods The experimental model of acute liver failure was induced by D-Gal / LPS combined with intraperitoneal injection in rats. The level of serum aminotransferase and the pathological changes of liver were observed at different time points to evaluate the evolution of hepatocyte apoptosis and hepatic necrosis. The apoptotic DNA bands of hepatocytes were detected by agarose gel electrophoresis. Caspase-12 mRNA expression was detected by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). Caspase-12 and Bip / GRP78 protein expressions were detected by western blot. Results The number of typical apoptotic cells increased after 5h of drug induction. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) began to increase and the expression of Caspase-12 mRNA increased significantly. Caspase-12 (P <0.05) .At the same time, a large amount of apoptosis and necrosis of hepatocytes were observed under 7h microscopy. The levels of ALT and AST peaked at 2564 ± 1384 U / L and 1198 ± 497 U / L, respectively. The normal control group was ( 59 ± 17 U / L and 135 ± 12 U / L respectively. The expression of Caspase-12 mRNA still increased and the expression of Caspase-12 protein continued to decrease. Obviously decreased, Caspase-12 mRNA expression decreased compared with 7 h. Bip / GRP78 self-expression began from 5 h, gradually increased to 7 h. Conclusions The expression of Caspase-12 mRNA in early stage of acute liver failure induced by D-Gal / LPS is gradually increased in the late stage (7-9 h), which is consistent with the appearance of hepatocyte apoptosis. Caspase- It is suggested that caspase-12-mediated endoplasmic reticulum stress-induced hepatocyte apoptosis is involved in the occurrence and development of inflammatory acute liver failure, which is one of the important mechanisms of hepatocyte injury in acute liver failure , Suggesting that the early intervention of Caspase-12 expression and activation of acute liver failure may have a protective effect.