肝再生增强子通过调节线粒体MCU钙流减轻肝脏缺血-再灌注损伤的研究

缺血-再灌注损伤(ischemia-reperfusion injury, IRI)为临床常见疾病，发病机制复杂，临床干预效果不佳。近报，线粒体钙通道(MCU)失活导致线粒体钙超载与IRI有关。我们前期证实，肝再生增强子(ALR)保护肝脏抵御IRI，而这一作用是否与ALR维持MCU活性和钙稳态相关，值得深究。本课题选用ALR肝特异敲除小鼠和肝细胞，分别制备IRI和H/R模型，探究ALR缺失对MCU亚单位-MICU1 (mitochondrial calcium uptake 1, MICU1)调节及线粒体钙流的影响；鉴定ALR与MICU1互作位点；研究位点突变对MICU1结构、钙流和线粒体功能影响；建立ALR-KO动物IRI挽救模型，探究回补ALR减轻肝IRI、恢复MCU活性、保护线粒体的可能性。本课题旨在揭示ALR通过阻止线粒体钙超载减轻IRI的机制，为ALR潜在应用提供理论依据。

Ischemia-reperfusion injury (IRI) is the tissue damage worsen by restoring blood supply to tissue which undergoes a period of ischemia or lacks of oxygen (anoxia or hypoxia). The IRI is one of common clinical syndromes with complicated pathogenesis. Recently, it has been reported that mitochondrial calcium overload is apparently attributed to IRI development. It is known that augmenter of liver regeneration (ALR) protects liver from IRI, however, whether this protection is associated with attenuating mitochondrial calcium overload encountered during IRI remains unrevealed. Mitochondrial calcium uniporter (MCU) is an essential regulator which plays key role for controlling Ca2+ influx to mitochondria. This study is aiming to depict the regulation of ALR on the activity of MICU1, a subunit of MCU complex and mitochondrial Ca2+ stability. The IRI model is prepared in ALR-liver specific KO mice and the MICU1 activity in ALR-lacking mice will be assessed under condition of IRI challenge. The potential protein interaction between ALR and MICU1 will be investigated to identify the regulatory basis of ALR on MICU1, and the predictably interactive motifs are subjected to mutation. The mitochondrial Ca2+ levels and subsequent IR impair in ALR-deleted cells are determined on the basis of motif mutation. Finally，rescuing experiment will be employed by re-implanted a viral-plasmid carrying ALR-gene via tail vein into ALR-liver specific KO mice or MICU1-KO mice to ensure that ALR-regulation on MCU can be functionally recovered and ALR-attenuating mitochondrial impairment can be obtained. The purpose of this study is to understand the ALR regulation on MCU and its contribution to improve IRI injury.