线粒体自噬功能障碍在高龄供肝缺血再灌注损伤中的作用及机制研究

Hepatic ischemia reperfusion injury (HIRI) fundamentally influences the performance of aged liver grafts. Aging constitutes a significant risk factor for several diseases that are specifically associated with declining mitophagy. In our previous studies, we found that aging aggravated HIRI and mitophagy was negatively correlated to HIRI and Rapamycin could alleviate HIRI through activation of mitophagy. The impairment or dysfunction of mitophagy was a causal mechanism for the age-dependent HIRI. Therefore, we hypothesized that aging may cause inefficient removal of damaged mitochondrion and aggravate HIRI through impairing the key steps of mitophagy influx based on our previous findings. In our present study, to identify the central players and the mechanisms by which aging impairs mitophagy, the key proteins and pathways of mitopahgy will be compared between the young and old mice both in vitro and in vivo during HIRI. The results of our present study will provide new insights into the influence of aging in HIRI and facilitate its clinical application in evaluation and improvement of aged graft undergoing HIRI in liver transplantation.

高龄供肝对肝脏缺血-再灌注损伤(HIRI)耐受不足是造成其移植后功能障碍的主要原因之一。已有研究表明线粒体自噬功能障碍与衰老及高龄疾病发生密切相关。我们前期研究发现高龄组小鼠HIRI较低龄组明显加重，且损伤程度与线粒体自噬（Mitophagy）水平负相关，增强高龄组小鼠的线粒体自噬水平可明显减轻高龄肝脏HIRI。据此推测线粒体自噬功能减弱导致损伤线粒体清除障碍可能是高龄肝脏对HIRI耐受不足的关键。故本项目以线粒体自噬为切入点，以自噬潮关键步骤为研究重点，通过体内外实验比较不同年龄组小鼠肝脏在缺血再灌注损伤过程中线粒体自噬相关蛋白及信号通路改变，从线粒体自噬新视角筛选出影响HIRI程度的关键因子，来进一步明晰高龄供肝发生HIRI的机制，为高龄供肝质量评估及治疗靶点提供理论依据。

高龄供肝对肝脏缺血再灌注损伤（Hepatic ischemia reperfusion injury，HIRI）耐受不足是造成其移植后功能障碍的主要原因。研究表明线粒体自噬（Mitophagy）功能障碍与衰老及高龄疾病发生密切相关。我们研究发现年老组小鼠HIRI较年轻组明显加重，且损伤程度与线粒体自噬水平负相关，增强年老组小鼠线粒体自噬可降低HIRI。据此我们推测线粒体自噬功能减弱，导致损伤线粒体清除障碍是高龄小鼠对HIRI耐受不足的关键。本课题对比不同年龄组小鼠发生HIRI时自噬关键蛋白表达差异，发现年老组小鼠肝脏Parkin表达较年轻组明显下降。干扰L02细胞Parkin的表达，体外模拟缺血再灌注，证实Parkin表达下降导致线粒体自噬功能降低，细胞内活性氧簇（Reactive oxygen species，ROS）增多，从而加重细胞损伤。体外研究进一步发现Parkin表达与内质网应激反应蛋白Eif2α磷酸化水平呈正相关，体内实验同样证实年老组小鼠Eif2α磷酸化水平明显低于年轻组小鼠。缺血前对年老组小鼠使用Eif2α磷酸酶抑制剂Salubrinal，使Eif2α磷酸化水平增高可上调Parkin表达，增强线粒体自噬，减轻HIRI。心死亡肝移植供肝复流后2小时标本行Parkin免疫组化染色发现，供体年龄与Parkin表达呈负相关，Parkin低表达患者术后一周内谷草转氨酶峰值明显升高。上述结果提示Eif2α磷酸化水平降低导致Parkin表达下降，引起线粒体自噬功能障碍最终导致老年小鼠HIRI加重，Parkin可作为评估高龄供肝耐受HIRI的潜在指标。