肝刺激因子对线粒体分裂蛋白Drp1的SUMO化调节及其对于脂肪肝缺血-再灌注损伤的影响

Ischemia-reperfusion (IRI) is a pathophysiological process occurring in ischemic tissue after restoration of blood supply. Although extensively investigated, the precious mechanism of IRI remains far from fully understood. Mitochondrial dynamics plays a cardinal role in regulation of cell death, therefore, contribution of balance-homeostasis in mitochondrial dynamic to IRI is an interesting topic to be investigated. Many factors, including Drp1, have been identified to regulate mitochondrial dynamics. Hepatic stimulator substance (HSS) is believed to protect hepatocytes from a variety of toxic injuries. The hepatic protection provided by HSS is probably related with its effect on stabilityon of mitochondrial membrane. However, whether HSS protecting hepatocytes is related with Drp1 is completely unknown. In this study, fatty liver IRI model will be prepared in HSS-KI/KO mice and the relationship between HSS and IRI will be investigated. Alternations in mitochondrial dynamics regulated by Drp1 during IRI are to be primarily evaluated. As to SUMOylaiotn of Drp1, the Drp1 conjugate to SUMO and their interacting bases will be further analyzed. DeSUMOylation of Drp1 resulted from SENP and its relevant effect to IRI will be also addressed. Finally, the regulation of HSS on SUMO expression during IRI and candidate molecules involved are at priority to be elucidated. In all, this project, if well handled and comprehensively carried out, will be aiming to expand viewing the IRI pathogensis and be benefit for clinical therapeutics.

缺血再灌注损伤（IRI）是恢复血供后引发缺血组织严重受损的病理过程，其机制不甚明了。近来发现，Drp1参与调节线粒体(mito）动态平衡，引发mito分裂和细胞凋亡，推测mito稳态失衡可能与IRI发生有关。肝刺激因子（HSS)可保护mito，但是否与调节Drp1活性有关？作用机制如何？都还未知。取HSS-KI/KO小鼠，制备脂肪肝和IRI模型，观察IRI与HSS表达之关系；检测mito形态/功能改变对IRI影响，明确mito稳态失衡与IRI之关系；检测Drp1分子SUMO化，筛查两者互作位点及机制，明确SUMO化Drp1对mito稳态影响；检测SENP调节Drp1去SUMO化对mito稳态和IRI作用；检测IRI肝组织SUMO表达，分析HSS是否调节其表达，通过表达谱分析，明晰HSS调节SUMO表达及可能机制。.本课题旨在扩展/认识IRI发病新机制，为临床防治提供可借鉴的科学依据。

缺血/再灌注损伤(IRI)是外科手术常见的并发症，因其发病机制复杂，迄今临床干预效果不佳。近年研究显示，IRI与线粒体损害有关。肝再生增强因子（ALR或HSS），是一种定位于线粒体膜间隙的巯基氧化酶，可保护线粒体，减轻肝细胞凋亡，但作用机制未明。本课题证明，IRI后移植肝脏线粒体分裂蛋白Drp1磷酸化和SUMO化增强，线粒体断裂加重。尔后，在IRI小鼠肝脏及细胞缺氧-复氧模型上发现同样结果。转染ALR基因可抑制小鼠肝Drp1磷酸化和SUMO化，减轻线粒体破碎，缓解细胞损伤，提示，ALR可通过抑制Drp1磷酸化/SUMO化修饰，减轻IRI。生信分析显示，转录因子YY1能与UBA2（SUMO活化酶E1）启动子区域结合并激活其转录。ALR通过与转录因子YY1互作，阻止后者入核及其与UBA2启动子结合，持续下调UBA2转录表达，有效阻截Drp1的SUMO化。本研究首次发现, ALR可交互式调节Drp1磷酸化与SUMO化修饰，有效地阻止Drp1的线粒体移位，维持线粒体稳态，抵抗IRI损伤。尔后，我们更加关注ALR抗IRI损伤的实际应用。线粒体自噬在肝脏IRI过程中发挥重要作用。通过线粒体自噬可选择性清除受损线粒体，维持肝细胞稳态，抵御肝IRI损伤。我们推测ALR是否也能通过调节线粒体自噬发挥保肝作用。利用大鼠在体原位肝移植方法，我们证明，在肝脏冷IRI过程中，ALR通过促进线粒体自噬减轻肝脏损伤。进一步在ALR敲减小鼠热IRI模型和细胞缺氧-复氧模型上又证明，ALR可以通过调节线粒体相关蛋白PINK1/Parkin介导线粒体自噬，减轻线粒体功能障碍和细胞损伤。机制上，我们证实，ALR促进线粒体自噬的作用与线粒体融合蛋白Mfn2表达密切相关。敲减Mfn2后，ALR诱导线粒体自噬作用受到抑制，线粒体损伤加重；相反，回补Mfn2，线粒体自噬增加，ALR保肝作用得以再现。本课题解释了ALR对抗I/R损伤的细胞与分子生物学机制，为防治肝移植引发的I/R损伤提供理论支撑。最后，我们在成体肝脏前体细胞（LEPCs）还发现，结果显示：ALR通过抑制Drp1的S616位点磷酸化激活，减少线粒体过度分裂，从而维持成体肝脏前体细胞 “干性”并促进细胞存活。这将有助于阐明干细胞治疗急性肝病的机制，有利于该技术向实际应用拓展。