肝缺血再灌注(IR)损伤是导致肝手术预后不佳和移植肝功能不全的重要原因，但临床仍缺乏有效防治药物。本课题组前期通过高通量蛋白质组学分析发现病人肝IR损伤与肝实质细胞中环指蛋白RNF2表达下降有关；RNF2与乙酰化酶p300直接作用，并促进NF-κB亚基RelA的乙酰化。故我们推测RNF2作用于p300促进RelA的乙酰化，使NF-κB调控的抗凋亡蛋白表达增加，保护肝IR损伤。本项目拟应用肝实质细胞特异性RNF2敲除小鼠研究肝IR的表型变化；体内外验证RNF2通过p300/RelA调节肝细胞凋亡及增殖，以RNF2/p300相互作用和RelA的乙酰化为切入点，着重阐明肝IR过程中RNF2调控NF-κB的关键分子及翻译后修饰的精确分子机制；探讨以RNF2为靶点的突变体和小分子抑制剂/激活剂逆转肝IR损伤；并将上述研究结果进行临床相关验证。本项目的开展为以RNF2为靶点治疗肝IR损伤提供实验依据。

Liver diseases have been serious threats to people’s health in China. Hepatic ischemia/reperfusion (IR) injury represents a major risk factor of hepatectomy and liver transplantation, is related to graft dysfunction and acute/chronic rejection. It is becoming urgent to elucidate the mechanisms of liver IR injury. To explore molecular mechanisms during hepatic IR, we collected human liver biopsies during hepatectomy before and after Pringle maneuver and conducted a TMT-based quantitation high-throughput mass spectrometry technology. We found that RNF2, a member of the polycomb group (PcG), was significantly down-regulated upon IR treatment. Our preliminary data indicated that the decreased levels of RNF2 were companied with higher level of hepatic apoptosis. Immunoprecipitation (IP) and GST-pull down revealed that RNF2 directly interacted with p300 and facilitated the acetylation of RelA.Futhermore, double luciferase reporter assay showed that RNF2 over-expression increased NF-κB activity. RNF2 over-expression in primary hepatocytes induced the mRNA expressions of NF-κB targeted anti-apoptosis genes to protect hepatocytes against IR injury. Strikingly, our preliminary results also found RNF2 was expressed only in hepatocytes but not kupffer and other cells. Therefore, our central hypothesis is that the interaction between RNF2 and p300 facilitates acetylation of RelA to protect against hepatic ischemia/reperfusion injury. We will uncover the mechanism of RNF2/p300 interaction and NF-κB signal transduction during hepatic ischemia/reperfusion. We propose to define the interaction domain of RNF2/p300 and illuminate the mechanisms by using recombinant RNF2 viruses and the hepatocyte-specific RNF2 knockout (RNF2ΔHep) mice. Moreover, the correlation of RNF2 expression and the outcomes of hepatectomy underwent hepatic IR will be investigated in blood test and liver biopsies. The systematic studies proposed in this project will reveal the novel key factor and its molecular mechanisms in hepatic IR and provide experiment basis of targeted therapy for liver IR injury.