ω-3PUFA通过AMP/LKB1/AMPK信号通路调控肝切除术后肝再生的分子机制

Mortality after partial hepatectomy was associated with severe acute liver failure. Recently, we revealed that ω-3 polyunsaturated fatty acids (ω-3PUFA) have a strong promotional effect on liver regeneration. Hepatic tight junctions (TJs) not only lie between hepatocytes but also localize along the bile canaliculi, bile secretion, and cell polarity. Furthermore, hepatic TJs are composed of using molecules including Occludin, Claudins, and zonula occludens. Construction of TJs is a vital event during liver regeneration leading to the reorganization of the bile canaliculi and the repolarization of hepatocytes, thus, maintaining liver function. Several studies have revealed that ω-3 polyunsaturated fatty acids (ω-3 PUFA) reduce the inflammatory cytokine release and modulate of lipid mediators synthesis, which then suppress hepatocyte apoptosis and promote cell proliferation. Therefore, ω-3 PUFA may show protective effects on hepatic TJs. We sought to experience changes in the expression and structure of hepatic TJs, the level of liver regeneration, and liver function after ω-3 PUFA treatment. Restoration of liver function during regeneration is determined by the hepatocyte repolarization with the formation of polarity based on the integrity of TJ Structures. Previous studies have revealed that during liver regeneration hepatocyte polarization parallels changes in cellular energy metabolism. Among multiple signaling pathways within energy metabolism, activation of the AMP (adenosine monophosphate) -liver kinase B1 (LKB1)-adenosine monophosphate-activated protein kinase (AMPK) signaling pathway can reduce the consumption of cellular energy having a protective effect on cellular structures. Activation of AMPK promotes the formation of TJs and modulates the polarity of various types of cells. Moreover, the AMP-LKB1-AMPK signaling pathway has recently been implicated in the formation of the bile canalicular network that requires hepatocyte polarization. Therefore, AMP-LKB1-AMPK signaling pathway is a crucial cascade in cellular energy metabolism may affecting expression, structure, and function of TJs in the liver. In addition, activation of the AMP-LKB1-AMPK signaling pathway promotes hepatocyte proliferation. Because dietary ω-3 PUFA can enhance AMPK activity in rats, it has been proposed that this supplement may restore hepatic function after liver injury. In this study, we focused on the role of ω-3 PUFA to improve expression, structure, and function of hepatic TJs as well as hepatocyte proliferation in isolated and cultured hepatocytes from C56BL6 mice. To investigate the underlying molecular mechanism，we perform the 70% PH using AMPKα1α2LS?/?knockout mice. Finally, we hypothesized that ω-3 PUFA contributed to liver regeneration and energy metabolism pathways maintaining liver function by protecting hepatic TJs.

肝切除技术是治疗肝脏肿瘤的有效方法，术后保护残肝功能，促进肝再生至关重要。研究显示ω-3多不饱和脂肪酸（ω-3PUFA）具有改善肝切除术后肝功能，促进肝再生的作用。本项目拟从分子、细胞水平阐明ω-3PUFA通过AMP/LKB1/AMPK信号通路调控肝细胞能量代谢，促进紧密连接（TJ）蛋白表达，维护肝细胞极性的机制。在此基础上，采用AMPK 基因敲除小鼠70%肝切除模型，进一步揭示ω-3PUFA通过AMP/LKB1/AMPK信号通路影响细胞能量代谢，调控肝细胞TJ，保护肝功能，促进肝再生的作用。研究结果有助于证实ω-3PUFA通过AMP/LKB1/AMPK通路，调控肝切除术后肝再生的科学假设，为添加ω-3PUFA的营养支持方案在肝切除术围手术期的应用提供实验和理论依据。

肝切除技术是治疗巨大肝脏肿瘤的有效方法，术后保护残肝功能，促进肝再生至关重要。我们前期研究显示：ω-3多不饱和脂肪酸（ω-3PUFA）具有改善极限性肝切除术后肝功能，促进肝再生的作用。本项目采用小鼠原代肝细胞三维立体培养模型，加入ω-3PUFA后，检测胆小管的形成情况。为了验证LKB1/AMPK信号通路在胆小管形成中的作用，我们构建了LKB1和AMPK的DN-negative腺病毒，感染原代肝细胞后，再次检测胆小管的形成。我们还采用高效液相色谱检测ω-3PUFA对肝细胞内AMP、ADP和ATP水平的影响。在动物实验上，我们采用了口服AMPK激动剂AICAR、AMPK抑制剂Compound C联合ω-3PUFA，检测小鼠肝切除术后肝功能以及肝脏紧密连接的形成情况。研究结果显示：在小鼠原代肝细胞三维立体培养模型中，加入ω-3PUFA DHA能够显著促进胆小管的形成，并且激活LKB1/AMPK信号通路。用腺病毒抑制LKB1或AMPK功能后，胆小管的形成速度显著减缓。高效液相色谱检测结果显示：ω-3PUFA显著提高了肝细胞内AMP/ATP水平。在小鼠肝切除模型中，口服ω-3PUFA能够促进肝脏紧密连接的形成，提高肝脏/体重比，降低血清转氨酶和胆红素。利用Compound C抑制AMPK活性后，小鼠肝再生延迟、紧密连接形成减少、肝功能降低。本课题从体内和体外阐述了ω-3PUFA通过LKB1/AMPK信号通路调控肝细胞紧密连接，诱导肝再生的机理。研究结果有助于证实ω-3PUFA通过提高AMP水平调控LKB1/AMPK通路，影响肝细胞TJ，调控肝切除术后肝再生的科学假设，为添加ω-3PUFA的营养支持方案在肝切除术围手术期的应用提供实验和理论依据。