需肌醇酶1介导的内质网应激在暴发性肝功能衰竭肝细胞凋亡中的作用及其意义

Effect of inositol requiring enzyme 1-mediated endoplasmic reticulum stress in liver cell apoptosis ofexperimental fulminant hepatic failure and its significance

目的探讨需肌醇酶1（IREl）介导的内质网应激肝细胞凋亡途径在暴发性肝功能衰竭中的作用及意义。方法雄性Wistar大鼠共60只，暴发性肝功能衰竭模型组30只腹腔注射口氨基半乳糖（D-GaIN）＋脂多糖（LPS），对照组30只腹腔注射0．9％氯化钠溶液，应用流式细胞技术观察两组大鼠肝细胞凋亡情况，分别采用免疫组织化学法及RT—PCR法动态观察肝组织中Caspase-12、IREl蛋白及mRNA表达情况。多个独立样本采用Kruskal—wallisH检验，同一时间点两两比较采用Mann-WhitneyU检验，相关性分析采用秩相关。结果模型组2、4、8和12h肝细胞凋亡率随着给药时间延长呈上升趋势（X2=25．475，P％0．01），明显高于对照组（U=0，P＜0．01）；模型组随着给药时间的延长肝细胞胞质Caspase-12、IREla蛋白表达增多，对照组无表达；模型组Caspase-12、IRElamRNA表达随着给药时间的延长而增加，8h达高峰，以后逐渐下降，各时间点的差异均有统计学意义（X2=23．983，X2=24．820；均P〈0．01），明显高于对照组（U=0，P〈0．01）；IREl与Caspase-12、细胞凋亡率均呈正相关（r=0．733，P〈0．01；r=0．715，P〈0．01），Caspase-12与肝细胞凋亡率呈正相关（r=0．586，P〈O．01）。结论IREl介导的内质网应激肝细胞凋亡与暴发性肝功能衰竭的发生发展密切相关，早期干预内质网应激途径对防治肝功能衰竭可能具有保护作用。

Objective To study the role of inositol requiring enzyme 1 （IRE1）-mediated endoplasmic reticulum stress on hepatocyte apoptosis of experimental fulminant hepatic failure （FHF）. Methods Thirty male depuratory Wistar rats were manufactured to be FHF model by peritoneal injection of D-galactosamine （D-GalN） and lipopolysaccharide （LPS）, and 30 rats were injected peritoneally with 0.9 V00 sodium chloride solution as controls. The apoptosis of liver cells was detected by flow cytometry. The protein and mRNA expressions of Caspase-12 and IRE1 in liver tissues were detected by immunohistochemistry and reverse transcriptation-polymerase chain reaction （RT-PCR）. The independent samples were compared by Kruskal-Wallis H test. The comparison between two groups at the same time point was done by Mann-Whitney U test. The correlation analysis was done by rank correlation. Results The apoptotic rates of liver cells at 2, 4, 8 and 12 hours were increased over time in model group X2 = 25. 475, P = 0.01）, which were higher than control group （U= 0, P〈0.01）. The expressions of Caspase-12 and IRE1 proteins in liver tissues were upregnlated in mode[ group, while the expressions were not detected in control group. The expressions of Caspase-12 and IRE1 mRNA in model group were also increased over time and peaked at 8 h, then graduallydecreased; the differences among different time points were statistically significant （X2 = 23. 983, X2 = 24. 820; both P〈（O. 01）, and all higher than control group （U=0, P〈0.01）. IRE1 was positively correlated with both Caspase-12 and hepatocellular apoptotic rate （r=0. 733 and 0. 715, respectively; both P〈0.01）. Caspase-12 was positively correlated with hepatocellular apoptotic rate （r= 0. 586, P〈0. 01）. Conclusions IRE1 mediated endoplasmic reticulum stress on hepatocyte apoptosis is closely related to the development of FHF. The earlier intervention on endoplasmic reticulum stress pathway, the more protective effect in liver failure.