小鼠暴发性肝衰竭肝细胞凋亡形态学及其调控机制

Morphology and regulatory mechanism of hepatocyte apoptosis in experimental fulminant hepatic failure

目的:研究在实验性暴发性肝衰竭(fulminant hepatic failure,FHF)中肝细胞凋亡的形态学变化以及一氧化氮(nitric oxide,NO)、Fas和Bcl-2对肝细胞凋亡的调控作用．方法:脂多糖(LPS)和D-氨基半乳糖(D-GalN)联合应用制备FHF小鼠模型;采用免疫组化方法检测肝组织Fas 及Bcl-2表达,分别采用硝酸还原酶法和RT-PCR法检测血清NO水平及肝组织iNOS mRNA表达;TUNEL法检测肝细胞凋亡;在用药后动态观察Fas及Bcl-2表达、血清NO水平及肝组织iNOS mRNA表达及肝细胞凋亡的变化,并对模型鼠给予iNOS的抑制剂L-NMMA,动态观察上述指标的变化．结果:在FHF模型小鼠中,用药后2 h开始血清NO水平及iNOs mRNA的表达即升高,于4 h达高峰;用药后2 h 开始Fas有少量表达,至8 h和12 h表达均明显增多,与 2 h组比较差异显著(100％ vs 20％,P<0．01),与4 h组比较差异也比较显著(100％ vs 40％,P<0．05)．模型组2 h Bcl-2有较多表达,4 h表达最多,4 h与2 h比较差异显著 (90％ vs 60％,P<0．05),与8,12h比较差异非常显著(90％ vs 20%,均P<0．01)．8 h可出现典型的肝细胞凋亡表现．与模型组各时间点比较,给予iNOS的抑制剂L-NMMA 后血清NO水平及iNOs mRNA的表达均为正常水平, Fas及Bcl-2表达均无显著差异(P>0．05),阻断后8 h亦可见典型的肝细胞凋亡表现,阻断NO可使病变较模型组更为严重．结论:在FHF中,Fas及Bcl-2的表达均增加,Fas的表达与肝细胞凋亡相一致,而Bcl-2的表达与肝细胞凋亡呈负相关．单纯应用NO拮抗剂对肝细胞凋亡及肝损伤无保护作用．

AIM： To study the morphological changes and the regulation of nitric oxide （NO）, Fasand Bcl-2 on hepatocyte apoptosis in mouse model of experimental fulminant hepatic failure （FHF）. METHODS： Mouse model of experimental FHF was established by combination of lipopolysaccharide （LPS） and D-galactosamin （D-GalN）. The expression of Fas and Bcl-2 in the liver tissues was tested by immunohistochemistry. The level of serum NO and iNOS mRNA expression in liver were tested by nitrate reductase method and reverse transcription polymerase chain reaction （RT-PCR）, respectively. The hepatocyte apoptosis was examined by TUNEL method. In addition, the changes of the above items were observed after pretreatment with L-NMMA, an inhibitor of iNOS. RESULTS： The level of serum NO and expression of iNOS mRNA in the liver tissues were increased at 2 h in model group, reaching the peak at 4 h. There was a little Fas expression at 2 h in model group. The expression of Fas was increased significantly at 8 and 12 h,which was distinctly higher than that at 2 h （100% vs 20%, P 〈 0.01） and 4 h （100% vs 40%, P 〈 0.05）. The expression of Bcl-2 started to increase at 2 h, reaching the peak at 4 h, which was markedly higher than that at 2 h （90% vs 60%, P 〈 0.05）. The expression of Bcl-2 at 4 h was also significantly higher than that at 8 or 12 h （90% vs 20%, both P 〈 0.01 ）. Typical features of hepatocyte apoptosis were observed at 8 h. The level of serum NO and liver iNOS mRNA expression were normal and the Fas, Bcl-2 expression did not change notably after L-NMMA administration in comparison with those in model group （P 〉 0.05）. Typical hepatocyte apoptosis was also observed at 8 h after L-NMMA administration, and the pathological changes of the liver tissues were more severe. CONCLUSION： Both expression of Fas and Bcl-2 are increased in FHF. Fas expression is consistent with hepatocyte apoptosis, while Bcl-2 expression is negatively correlated with hepatocyte apoptosis. Single administration of iNOS inhibitor can not protect hepatocytes against apoptosis and injury.