小鼠肝再生过程中ROS及线粒体代谢变化规律的研究

Study on the Metabolic Changes of ROS and Mitochondria during the Process of Liver Regeneration in Mice

目的:肝脏是维持人体发挥功能的重要器官,同时肝脏再生能力十分强大。本文通过部分肝切除术后小鼠肝再生模型,观察肝再生过程中氧化应激及线粒体代谢变化规律,以期为将来的调控肝再生提供新的干预靶点。方法:选择雄性健康体重均匀的Balb/c小鼠,采用经典70%肝切除模型,随机分为假手术对照组(Sham组)以及70%肝切除组(70%PH组)。肝切除术后6 h、1d、2 d、3 d、5 d、7 d不同时间点取肝组织,制备冰冻切片检测活性氧(ROS)水平,Western blot分别检测细胞增殖相关蛋白PCNA、Cyclin D1;氧化应激相关蛋白SOD1、SOD2、CAT、GPX1;以及线粒体代谢相关蛋白PGC-1α、Nrf1、TFAM、Drp1、Fis1、Mfn1、Mfn2、OPA1的表达并分析其变化规律。结果:70%肝切除术后小鼠肝脏增长迅速,细胞增殖关键蛋白PCNA和Cyclin D1表达显著增加;在此过程中细胞ROS水平呈现先升高后降低的变化,细胞主要抗氧化酶SOD1、SOD2、CAT、Gpx1与ROS相一致出现先升高后降低的变化。线粒体生物合成调控因子PGC-1α、Nrf1、TFAM呈现先降低后升高的趋势,而线粒体分裂蛋白Drp1和Fis1呈现先降低后显著升高的趋势,线粒体融合相关蛋白Mfn1、Mfn2和OPA1总体为先降低后恢复至正常水平。结论:在小鼠70%肝切除再生过程中,存在着明显的氧化应激,线粒体生物合成增加,线粒体分裂/融合平衡偏向分裂,并且这些变化呈现具有一定的时间变化规律,这些变化及规律很可能作为将来调控肝再生的重要的潜在干预靶点。

Objective： The liver plays an important role in human body. At the same time, the liver has strong ability of regeneration. In the present study, we observed the changes of reactive oxygen species（ROS） and mitochondrial metabolism during liver regeneration after liver partial hepatectomy in order to provide new targets for future regulation of liver regeneration. Methods： Using the classic mouse 70 % partial hepatectomy model, the male healthy Balb/c mice were randomly divided into Sham group and 70 % partial hepatectomy group（70 % PH）. The liver tissues were harvested at 6 h, 1 d, 2 d, 3 d, 5 d and 7 d. The frozen sections were used to detect the level of ROS. The expression of the cell proliferation-related proteins PCNA, Cyclin D1; the cell antioxidant enzymes related proteins SOD1, SOD2, CAT, GPX1, and the mitochondrial biogenesis and dynamics regulators PGC-1α, Nrf1, TFAM, Drp1, Fis1, Mfn1, Mfn2,OPA1 were determinated by Western blot respectively. Results： The liver of mice regenerated rapidly after 70 % hepatectomy, and the expression of the cell proliferation-related proteins PCNA and Cyclin D1 increased significantly. During the process, the level of ROS firstly increased and then decreased. The protein levels of antioxidant enzymes SOD1, SOD2, CAT, and Gpx1 were consistent with the change of ROS level. The mitochondrial biogenesis regulatory factors PGC-1α, Nrf1 and TFAM firstly decreased and then increased. The mitochondrial fission protein levels of Drp1 and Fis1 decreased firstly and then increased significantly with the mitochondrial fusion protein levels of Mfn1, Mfn2 and OPA1 decreased and then restored to the normal level during the regeneration process. Conclusions： In the regeneration process after 70 % liver hepatectomy in mice, the ROS level increased accompanied with the high levels of antioxidant proteins. The mitochondrial biogenesis increased while the mitochondrial fission/fusion balance moved towards fission. These changes are likely to be important potential intervention targets for the regulation of liver regeneration in the future.