ALDH2在DCD供肝缺血再灌注损伤中的作用及机制

Donor shortage has hampered the development of liver transplantation in China. Donation after cardiac death (DCD) will effectively expand the donor source, while hepatic ischemia-reperfusion injury (IRI) has severe influence on the prognosis of DCD liver graft. However, there is no ideal method to prevent it..A major damaging event upon reperfusion of ischemic tissue is a burst of ROS production, among which the role of mitochondrial ROS upon initiation of reperfusion seems to be critical. ROS can induce production of some kinds of toxic aldehydes, such as 4-hydroxynonenal (4HNE), which exacerbate the burst of ROS and aggravate hepatic IRI. Mitochondrial aldehyde dehydrogenase 2 (ALDH2) is an allosteric tetrameric enzyme responsible for the metabolism or detoxification of acetaldehyde and other toxic aldehydes, such as 4HNE, as well as reactive oxygen species (ROS) generation. ALDH2 activity can affect the cellular response to oxidative stress both in vitro and in vivo. Accumulating evidence shows that ALDH2, especially ALDH2 activation or overexpression is closely related to liver liver cirrhosis and cardioprotection against ischemia IR injury. However, the ALDH2-mediated mechanism in hepatic IR has not been elucidated..The ALDH2 enzyme is mainly located in mitochondria and is encoded by the ALDH2 gene on chromosome 12. The ALDH2 gene is composed of 13 exons. Exon 12 contains a G-to-A missense mutation, whereby glutamate at position 504 is replaced by lysine (Glu504Lys); hence, two ALDH2 alleles exist (Glu504 and Lys504, also named ALDH2*1 and ALDH2*2). The Glu504Lys single nucleotide polymorphism in the ALDH2 gene exists mainly in East Asians. Around 40% of East Asia carry the ALDH2*2 allele of ALDH2, the activity of which decline significantly compared to ALDH2*1. Recently, studies in East Asia demonstrated a close relationship between ALDH2 Glu504Lys polymorphism and some diseases such as alcoholic liver cirrhosis, hypertension and coronary artery disease. These findings suggest that the ALDH2 Glu504Lys polymorphism may play a functional role in hepatic IRI. However, the association of ALDH2 Glu504Lys polymorphism and hepatic IR, and the potential mechanisms involved have not been examined in Han Chinese..Mitophagy, a selective form of autophagy, is a specific process for degradation of dysfunctional or damaged mitochondria to maintain a healthy mitochondria population and mitochondrial quality. Hypoxemia and ROS may trigger mitophagy, which was found associated with several forms of myocardial IRI. ALDH2 has been revealed to have a dual regulatory paradox in cardioprotection against IR injury via mitophagy. Nevertheless, the precise role of mitophagy in hepatic IR injury and the influence of ALDH2 on mitophagy is unclear..The aim of the study is to investigate the relationship between ALDH2 Glu504Lys polymorphism and the risk of DCD donor liver IRI in Han Chinese of our hospital and to explore the potential involvement and function of toxic aldehydes and mitophagy in the ALDH2-eliciting effect on hepatic IR injury in vivo and in vitro. Our research is important for further understanding the molecular mechanisms of hepatic IR injury and searching new methods to prevent DCD donor hepatic IR injury.

肝缺血再灌注损伤（IRI）是导致心脏死亡捐献（DCD）供肝移植术后并发症增加的最主要原因。活性氧（ROS）是再灌注早期最重要的损伤因素，而氧化应激过程中产生的4-羟基壬烯醛、丙二醛等活性醛类物质，可进一步增加ROS生成，导致恶性循环。线粒体乙醛脱氢酶2（ALDH2）是参与醛类代谢的关键酶，其编码基因外显子上存在功能性单核苷酸多态性，使40%的东亚人群ALDH2酶活性显著下降。我们的预实验已发现ALDH2激动剂可减轻大鼠DCD供肝移植IRI，因此我们推测，ALDH2的基因多态性可能通过对醛类物质灭活的影响，成为DCD供肝IRI的重要影响因素；ALDH2可能通过调控肝巨噬细胞线粒体自噬影响ROS生成。本课题拟结合临床和基础研究，观察ALDH2基因多态性与DCD供肝移植患者术后肝损伤的关系，研究ALDH2对DCD供肝IRI的影响及醛类物质、线粒体自噬等在其中的作用，为防治肝IRI提供新的途径。

（一）课题组在研究工作中，通过大鼠DCD供肝缺血再灌注损伤模型和肝细胞缺氧复氧损伤模型，采用激动剂和抑制剂调控ALDH2的活性，验证了ALDH2对DCD供肝缺血再灌注损伤的影响及醛类物质在其中的作用。 .1）动物实验：建立大鼠DCD供肝缺血再灌注损伤模型，动物分组：A组：假手术组；B组：I/R组；C组：ALDH2激动剂处理+I/R组；D组：激动剂+抑制剂处理+ I/R组。研究结果发现加ALDH2活性可减轻DCD供肝缺血再灌注损伤，表现为使肝损伤减轻，肝组织ROS水平下降，醛类物质减少。而ALDH2抑制剂可阻断这一保护作用。 .2）体外实验：建立肝细胞缺氧复氧损伤模型，细胞分组：A组：正常对照组；B组：空白处理+缺氧/复氧组（H/R组）；C组：ALDH2激动剂处理+ H/R组；D组：空白处理+ 4-HNE组; E组：HR + 4-HNE组；F组：HR+ 激动剂 + 4-HNE组。研究结果发现ALDH2激动剂可通过增加ALDH2活性，减轻肝细胞缺氧复氧损伤，使肝细胞ROS水平下降，醛类物质减少，线粒体膜电位恢复。.（二）在肝缺血再灌注损伤机制的研究方面，本课题组还获得了以下进展： .1）TRPM2在肝缺血再灌注损伤中的作用及机制. TRPM2已经被证明与药物导致的肝损伤相关，但是TRPM2在肝脏缺血再灌注损伤中的作用依然未知。本研究构建了TRPM敲除小鼠，并进行肝脏缺血再灌注处理，结果发现敲除TRPM2基因可以抑制缺血再灌注导致的肝损伤。在动物实验中发现，TRPM2的敲除鼠可能通过促进缺血再灌注中自噬的发生，而缓解缺血再灌注造成的肝损伤。为了进一步明确TRPM2、自噬及缺氧缺血肝损伤的关系，本研究在OGD/R处理细胞中加入自噬激动剂雷柏霉素，发现其缓解了细胞OGD/R处理导致的细胞活力下降。此外，雷柏霉素的处理还可以抑制TRPM2的上调。另一方面，发现干扰TRPM2可促进自噬的发生。同时，OGD/R中干扰TRPM2可以抑制NLRP3炎症小体通路。而自噬又可负调控NLRP3通路。.2）HMGB1相关坏死性凋亡和库普弗细胞M1极化导致肠缺血/再灌注诱导的大鼠远隔肝损伤. 在本研究中，大鼠肠缺血/再灌注（I/R）损伤模型中再灌注6小时后发现明显的远隔肝功能障碍，阻断HMGB1可通过抑制肝细胞坏死性凋亡、组织炎症、肝KC和循环巨噬细胞M1极化，恢复肝功能。