急性肝衰竭小鼠线粒体分裂蛋白DRP1在肝脏和大脑皮层的表达变化

Changes of expressions of mitochondria dynamin-related protein 1 (DRP1) in the liver and cerebral cortex of acute liver failure mice

目的:观察C57小鼠急性肝衰竭(acute liver failure,ALF)过程中线粒体分裂蛋白(dynamic-related protein 1,DRP1)在肝脏和大脑皮层中的变化及与肝衰竭的相关性。方法:C57/BL小鼠,随机分为对照组、ALF1d、4 d、7 d组。腹膜腔内注射硫代乙酰胺(TAA)建立ALF模型,利用高架十字和旷场实验测定行为学,取血清检测谷氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)水平;之后取肝组织,HE染色观察肝组织的病理变化;用Western Blot和RT-q PCR检测DRP1在肝脏和大脑皮层中蛋白及mRNA水平的表达变化。结果:(1)行为学结果显示:ALF各组小鼠自发活动及探索行为均明显降低(P<0.05);(2)肝功检测ALF组ALT、AST表达水平明显升高(P<0.05);(3)肝组织病理学检查ALF组1 d时肝细胞出现大面积坏死,7 d时肝细胞坏死较1 d、4 d缓解;(4)Western Blot检测DRP1在ALF全肝组织和肝线粒体中明显降低(P<0.05);而在大脑皮层全组织蛋白中明显升高(P<0.05),但在提取的线粒体中则无明显改变(P>0.05);(5)RT-q PCR检测DRP1在肝组织中ALF各组中表达明显降低(P<0.05);而在大脑皮层组织中1 d时表达增多,持续到4 d,在7 d时恢复(P<0.05)。结论:DRP1在急性肝衰竭小鼠的肝脏和大脑皮层中对线粒体形态学发挥着重要的作用。

Objective： The changes of mitochondria dynamic related protein （ DRP1 ） expressions in the liver and cerebral cortex of acute liver failure （ALF） mice were observed. Methods： Fourty-eight male C57/BL mice were randomly divided into four groups： control group, ALF1 d, ALF 4 d and ALF 7 d. ALF groups received a peritoneal cavity injection of TAA. Elevated plus maze （EPM） and open field （OF） experiments were valued in all groups. The levels of serum aspartate amino transferase （AST） and alanine aminotransferase （ALT） were analyzed. The liver tissue samples were harvested and pathomorphological changes of samples were examined under a microscope after HE staining. Western Blot was used to detect the expression of DRP1 protein in both whole tissue and purified mitochondria tissue from liver and cortex of all groups. RT-qPCR was used to detect the expression of DRP1 at the mRNA level in liver and cortex. Results：（ 1 ） The results of EPM and OF showed that ALF mice had damaged spontaneous and exploratory activities, compared with the control mice （ P 〈 0.05 ）. （2） The levels of ALT and AST in three ALF groups were significantly higher than those in the control mice （ P 〈 0.05 ）. （3） HE staining showed a mass of necrosis of hepatocytes in three ALF groups （ 1 d, 4 d and 7 d） and the damage was the most evident in ALF groups at 1 d and 4 d. （4） Western Blot results showed that the expression of DRP1 in the extracted protein from liver whole tissue and liver purified mitochondria at three time points reduced significantly, compared with those in the control group （ P 〈 0.05 ）. On the contrary, the expression of DRP1 in the extracted protein from the whole cortex tissue increased significantly in three ALF groups, compared with those in the control group （ P 〈 0.05 ）. Interestingly, the expression of DRP1 in the purified mitochondria tissue from the cortex in ALF groups had no detectable changes, compared with those in the control group （ P 〉 0.05 ）. （5） The results of RT-qPCR indicated that the levels of DRP1 mRNAs in the liver reduced significantly at in ALF groups compared with those in the control group （ P 〈 0.05 ） ; however, the results of the levels of DRP1 mRNAs in the cortex increased from 1 d after ALF, lasted at 4 d, but recovered at 7 d. Conclusion： Mitochondrial fission related protein DRP1 may play an important role for mitochondrial dynamic changes in the liver and the cortex after acute liver failure injury.