胆酸-FXR通路在休克肝脏乳酸代谢障碍中的作用及机制研究

Lactate metabolism disorder is an important cause of death in shock. Hepatic ischemia-reperfusion injury may play an important role in lactate metabolism disorder of shock and the mechanism is still unclear. In previous studies, we accidentally found biliary tract external drainage increased liver injury in shock. Some articles reported that FXR (Farnesoid X receptor), a bile acid receptor, plays an important role in the process of liver repair. In our preliminary experiments, we found that the transcription and expression level of FXR in liver decreased significantly after biliary tract external drainage in ischemia-reperfusion rat models. So we speculated that bile acids may alleviate hepatic ischemia-reperfusion injury through the activation of FXR pathway. In this way, bile acids may improve lactate clearance rate and reduce the mortality of shock. To test this hypothesis, we will investigate the influence of bile acids to liver lactate metabolism disorder in shock from aspects of molecules, cells and tissues. In this study, NCTC1469 mice hepatocyte and mice model of ischemia-reperfusion will be used. The research methods will include transfection of viral plasmid, gene knock out, TUNEL, RT-PCR, Western blot, immunohistochemistry, histopathologic grading etc. From new visual point of FXR, this investigation will raise new mechanism through which bile acids reduce lactate metabolism disorder in shock. The research results will suggest new thread for the therapy of shock in clinical setting.

乳酸代谢障碍是休克患者死亡的重要原因。缺血再灌注引起的肝脏损伤很可能在乳酸代谢能力的大幅降低中起重要作用，其机制仍不明确。在前期研究中我们意外发现休克大鼠行胆汁外引流后肝脏的病理损伤加重，而有文献报道胆酸受体FXR（Farnesoid X receptor）在肝脏修复中起到重要作用。我们的预实验提示休克大鼠行胆汁外引流后，肝脏FXR的表达显著下降。据此我们提出假说，胆酸可能通过激活FXR通路减轻肝缺血再灌注损伤、提高乳酸清除率，从而有可能降低休克患者死亡率。为了验证这一假设，我们将通过小鼠NCTC1469肝细胞、小鼠休克缺血再灌注模型，采用质粒病毒转染、基因敲除、TUNEL、RT-PCR、Western blot、免疫组化、组织病理评分等手段从分子、细胞、组织水平多方面探讨胆酸对休克中肝脏乳酸代谢障碍的影响，从FXR这一新视点揭示休克时胆酸减轻肝脏乳酸代谢障碍的机制，为休克治疗提供新思路。

背景：乳酸代谢障碍是休克患者死亡的重要原因。缺血再灌注引起的肝脏损伤导致乳酸代谢能力的大幅降低。Farnesoid X receptor（FXR）起着肝脏保护的作用。我们设计了本实验来探讨熊去氧胆酸（ursodeoxycholic acid，UDCA）对休克缺血再灌注（ischemia-reperfusion，IR）时肝脏乳酸代谢的影响及其与FXR的关系。.方法：1.低氧下，将肝细胞、沉默FXR的肝细胞培养在添加10 mM乳酸的培养基中，然后将细胞暴露于100 μM UDCA中，或不添加(对照) 2、4、6、8小时后，测定乳酸浓度。2.将18只成年雄性C57BL/6小鼠随机分为3组:Sham组;IR组和IR + UDCA组。将6只FXR(-)成年雄性C57BL小鼠分为FXR (-) + IR + UDCA组。IR模型平均动脉压通过放血维持在40±5 mmHg 1小时。UDCA组鼻饲UDCA (300mg/kg)。RT-PCR、Western Blotting检测肝脏中caspase-3、bax、LC3Ⅰ、LC3Ⅱ、bcl2、beclin1的表达。TUNEL法检测肝细胞凋亡。测定血浆谷丙转氨酶(ALT)、天冬氨酸转氨酶(AST)、乳酸浓度。肝组织用苏木精和伊红染色并进行病理学评分。3.将18只成年雄性C57BL/6小鼠随机分为3组: IR组，IR + UDCA组和IR + GW4064组。方法同前，GW4064组鼻饲GW-4064 (30mg/kg)。.结果：低氧培养下，UDCA提高了肝细胞清除乳酸的能力。沉默FXR后，UDCA的作用减弱。IR时，UDCA处理后肝脏中caspase-3和bax的表达水平显著降低，LC3Ⅰ、LC3Ⅱ、bcl2和beclin1的表达水平显著升高。IR时，UDCA处理后肝脏TUNEL阳性百分率明显降低。IR时，UDCA处理后血浆ALT、AST、乳酸水平明显降低。IR时，UDCA处理后肝脏组织病理评分明显降低。在FXR(-)的情况下，UDCA的以上影响被抑制。FXR激动剂GW4064可以起到UDCA类似的作用。.结论：UDCA通过FXR通路促进肝细胞乳酸代谢。UDCA通过FXR通路降低休克IR时肝细胞凋亡，降低ALT、AST、乳酸的水平，减轻肝损伤。FXR激动剂GW4064可以起到UDCA类似的作用。