肝再生刺激因子(HSS)通过调节线粒体合成/分裂关键酶Drp1对肝再生的影响

Liver possess an extraordinary capacity to restore its structure or function if subjected to various liver injuries. This capacity, referred to liver regeneration, plays often an important role in clinic for the prognoses of patients with liver diseases. Hepatic stimulator substance (HSS), also name augmenter of liver regeneration (ALR), is newly identified growth factor that can protect hepatocytes and stimulate liver regeneration. Recently it is shown that HSS inhibits cell apoptosis and helps maintain intracellular APT level which is required for hepatocyte survival and their regenerations as well. However, the precious mechanism remains unclear. In this study, liver regeneration via a partial hepatoectomy is employed in mice and the role of HSS with connection to mitochondrial dynamics (fusion/fission) will be investigated. The research items mainly include: 1) observation of HSS expression with alternations of mitochondrial dynamics-related factors such as Mfn1，Mfn2 and Drp1; 2) the potential interaction of HSS with Drp1 is primarily explored to check if HSS inhibition on cell apoptosis is associated with Drp1; 3 ) the mechanisms of HSS interactive to Drp1 with regards to aspects of the transcriptional regulation, the cytoplasmic recruitment, the translocation onto mitochondria and the putative inactivation of enzymology; 4) in light of genetic modeling, mice with HSS-/- mutant or HSS-siRNA knockdown are employed to elucidate characteristic of liver regeneration under condition of HSS deficiency. This investigation will be probably providing clear illustration on HSS regulation on liver regeneration in coordination with mitochondrial dynamics.

受损后的肝脏表现出超强恢复能力，谓之肝再生。肝损伤后如何有效其再生，对于挽救患者生命至关重要。肝刺激因子（HSS），也称肝再生增强子（ALR）是新近发现具有保护肝脏，促进肝再生的因子。晚近报道，HSS可抑制细胞凋亡，稳定细胞内ATP含量，但作用机制仍不明了。线粒体作为重要的细胞器，其合成与分裂保持动态平衡，是维持肝细胞存活及促进肝再生的关键所在。本课题制备小鼠肝切/肝再生模型，研究1）肝再生中HSS表达及与线粒体稳态相关分子（Mfn1，Mfn2，Drp1等）表达的相互关系；2）以Drp1为代表，研究HSS是否与Drp1相互作用，从而阻止线粒体分裂，抑制细胞凋亡；3）探究HSS抑制Drp1的机制，如基因转录、分子招募、线粒体定向转运、调节酶结构等环节；4）利用HSS敲减/敲除动物模型，研究HSS与Drp1互作与肝再生的关系。本课题旨在阐明HSS通过线粒体途径促肝再生的细胞与分子机制。

线粒体分裂相关凋亡异常引起的缺血/再灌注损伤是肝移植中的一个主要问题。我们已经证明肝刺激因子（HSS）能够有效抵抗细胞凋亡，保护肝脏免受缺血/再灌注损伤。然而，其具体机制仍不清楚。本课题研究HSS在体外和体内通过调节动力相关蛋白1（Drp1）的转运和激活，调节肝脏缺血/再灌注损伤时线粒体的分裂和肝细胞凋亡。利用小鼠缺血/再灌注肝损伤模型，我们发现HSS敲减型小鼠（Gfer+/-）血清转氨酶水平升高，细胞结构破坏程度和细胞凋亡水平都高于野生型小鼠（Gfer+/+），肝损伤加重。HSS的降低导致细胞周期蛋白依赖性激酶1（CDK1）和Bax的表达增加，伴有磷酸化Drp1的升高和细胞色素c的释放。在体外实验中，我们发现HSS能够抑制CDK1的表达，从而抑制CDK1/CyclinB复合体对Drp1的ser 616的磷酸化，进而减少Drp1在线粒体中的积累，减轻Drp1介导的线粒体分裂程序的激活，抑制肝细胞凋亡。相反，HSS基因敲除后，CDK1和Drp1的磷酸化增加，加重肝细胞凋亡。深入机制研究表明，HSS通过调节miR-410-3p、miR-490-3p、miR-582-5p等多种microRNAs的表达，降低CDK1 mRNA的稳定性和翻译。本课题揭示了HSS调节线粒体裂变的新机制，为缓解肝损伤提供一种新途径。.此外，本课题延伸探索了HSS与线粒体自噬的关系。已受损的线粒体能够产生活性氧（ROS），引发氧化应激，诱导细胞凋亡，线粒体自噬即清除受损的线粒体，达到保护细胞的作用。本课题用线粒体解偶联剂CCCP诱导肝细胞线粒体去极化，检测HSS过表达细胞和对照组细胞内自噬、线粒体自噬的标志蛋白、磷酸化AKT的表达变化，用western blotting和共聚焦激光扫描显微镜检测PINK1和Parkin的移位情况。结果发现，HSS能够促进细胞自噬和线粒体自噬，其机制可能是通过抑制AKT磷酸化，促进PINK1的表达，依赖PINK1/Parkin信号通路促进线粒体自噬，从而维持正常的线粒体功能，保护肝细胞。本研究进一步为HSS通过保护线粒体缓解肝损伤提供了理论依据。