缺血后处理对边缘性供肝的影响及HMGB1在其中的作用机制研究

The only effective method to treat liver disease in final stage is liver transplantation. Due to lack of donor, more and more margin liver donors were adopted, especially fatty liver donors. Fatty donor livers expand the source of donor liver graft, but increase the risk of poor early graft function (PEGF) and primary non-function (PNF) on liver allograft. Hepatic ischemia reperfusion (IR) injury is a common pathological process of liver transplantation, which is considered as the primary cause of PEGF and PNF. Therefore, it is of great interest to elucidate the underlying pathomechanisms and to develop protective strategies for attenuating the IR injury of the margin donor livers. High mobility group box-1 (HMGB1) is a capital mediator in the early inflammatory reaction after IR injury, and it exerts important accommodation function in the priming and developing process of immune reaction after injury. The effect of HMGB1 in the IR injury after fatty liver transplantation is still not studied. Ischemic postconditioning (IPostC), which consists of several very short circles of ischemia and reperfusion applied immediately at the onset of reperfusion, offers an attractive and amenable approach to organism protection. In our early study, we found IPostC could attenuate IR injury in orthotopic liver transplantation (OLT) model of normal rats. In this study, OLT model of rats with fatty liver will be adopted, the relevance among different degrees of fatty degeneration in liver grafts, degree of IR injury and expression level of HMGB1 will be explored, the effect of IPostC on fatty liver allografts suffering IR injury after OLT will be revealed, and through strengthening and inhibiting HMGB1, the related mechanisms with HMGB1 in the effect of IPostC on fatty liver graft will be elucidated. We hope we can provide new pathway to attenuate the IR injury of margin donor livers.

以脂肪肝为主的边缘性供肝扩大了供肝来源，但增加了术后早期移植肝功能不良和原发性移植肝无功能的风险，而脂肪肝对缺血再灌注损伤（IRI）的高易感性被认为是其主要原因。脂肪肝移植IRI的具体机制尚不明了，并缺乏有效的保护方法；研究表明高迁移率族蛋白-1（HMGB1）在机体损伤后免疫反应中发挥重要的调节作用，是IRI中关键的早期炎症反应介质，但在脂肪肝移植IRI中的作用尚无研究。本课题组在前期研究中发现，缺血后处理（IPostC）能够显著减轻正常供肝移植后的IRI。本项目通过建立大鼠脂肪肝移植模型，检测不同脂肪变性程度供肝与移植后IRI程度和HMGB1表达水平的相关性，观察IPostC对脂肪供肝移植后IRI是否具有保护作用，并通过特异性增强和抑制HMGB1，研究HMGB1在脂肪肝移植后IRI中的作用，揭示IPostC影响脂肪供肝移植后IRI的分子机制，为减轻边缘性供肝移植后的IRI提供新的途径。

背景：供肝短缺是目前临床肝移植的关键限制，因此许多边缘性供肝如脂肪变供肝将越来越多的应用于临床。以脂肪肝为主的边缘性供肝扩大了供肝来源，但增加了术后早期移植肝功能不良和原发性移植肝无功能的风险，而脂肪变供肝对缺血再灌注损伤（ischemic reperfusion injury，IRI）的高易感性被认为是其主要诱因，开展对脂肪变供肝IRI 进行干预的实验研究是非常必要的。HMGB1 作为功能强大的炎症介质，在组织缺血再灌注后释放到细胞外，在多个水平介导组织器官IRI，但其在脂肪变性供肝的IRI 中研究甚少。主要研究内容：本项目通过建立大鼠脂肪肝模型并对肝脏脂肪变性程度进行分级，检测不同脂肪变性程度肝脏对部分肝脏IRI耐受性的差异和HMGB1表达水平的相关性；以改良“二袖套”法建立大鼠肝移植模型，并以脂肪肝作为供肝建立大鼠脂肪肝移植模型，研究缺血后处理对脂肪肝移植后IRI 是否具有保护作用，并观察后处理对HMGB1、ATP水平、过氧化产物、抗氧化酶水平以及相关炎性因子的影响，探索缺血后处理可能涉及的内源性保护机制，以期为减轻脂肪肝移植后IRI提供新的途径和实验依据。结果：实验结果表明，不同脂肪变性程度的肝脏对缺血再灌注损伤的耐受性不同，脂肪变性程度越高的肝脏其肝损害的表现越重并且HMGB1的表达水平越高，HMGB1与脂肪肝的IRI程度呈线性关系；结束无肝期后给予3个循环的30s再通-30s阻断的后处理方案能够明显减轻脂肪肝移植后的IRI，后处理对脂肪供肝缺血再灌注损伤的保护作用可能是通过阻断HMGB1表达、提高抗氧化酶活力、提高ATP保存、减轻中性粒细胞浸润反应和降低TNF-a表达水平等内源性保护机制来发挥的。意义：本研究首次证明HMGB1是脂肪肝缺血再灌注损伤中的重要介质，缺血后处理能够通过内源性保护机制减轻脂肪肝移植后的缺血再灌注损伤，为减轻脂肪变供肝移植后IRI提供新的途径和实验依据。