IL-18通路在体外循环术后肝损伤中的作用及瑞芬太尼对该损伤的保护作用研究

Cardiopulmonary bypass (CPB)-induced acute liver injury is very common in patients after cardiac surgery. While the injury is thought to be associated with systemic inflammatory response syndrome (SIRS) and ischemia/reperfusion injury (IRI), the underlying molecular mechanism is not known yet. IL-18 pathway plays an important role in the development of both SIRS and IRI, indicating the possibility that this is the critical pathway mediating the CPB-induced liver injury. Biological activities of IL-18 are modulated and suppressed by its natural antagonist, IL-18 binding protein (IL-18BP). The regulation of IL-18BP expression occurs mainly at transcriptional level, with transcription factors STAT1 and C/EBP playing an essential role in this progress. Previous studies demonstrate that u opioid receptor agonists are able to induce activation of STAT1 and C/EBP in various situations. Therefore, we hypothesize that these agonists can upregulate the transcription of IL-18BP gene and increase its expression levels, thereby attenuating IL-18-mediated tissue injuries. The present research project will utilize a rat CPB model to investigate the role of IL-18 signaling pathway in the development of CPB-induced liver injury in vivo, and to study the protective effects of remifentanil, a novel u opioid receptor agonist, in this progress. We will then culture human hepatic epithelial cells to further explore the mechanisms underlying remifentanil induction of IL-18BP gene transcription using molecular biology techniques such as luciferase reporter assay and CHIP. This project will help expanding our understandings of the molecular mechanisms of CPB-induced liver injury and the pharmacological effects of remifentanil, providing important implications for preventive and therapeutic interventions during the perioperative periods.

体外循环(CPB)术后急性肝损伤在临床上十分常见。该损伤的发生与CPB期间全身炎症反应综合征(SIRS)和肝脏缺血再灌注损伤(IRI)等病理生理过程有关，但其分子生物学机制尚未明。IL-18通路在SIRS和IRI的发生发展中均起重要作用，提示其很可能是介导CPB术后肝损伤的关键通路。IL-18的生物活性受其结合蛋白(IL-18BP)有效抑制,后者的表达主要受STAT1和C/EBP等转录因子活性调控。已知u阿片受体激动剂具有激活上述转录因子的作用，因此我们推测该类药物能够诱导IL-18BP基因转录和表达，从而中和IL-18介导的组织损伤。本课题将应用大鼠CPB模型，在体研究IL-18通路在CPB术后肝损伤中的作用并检测新一代u受体激动剂瑞芬太尼对该损伤的保护作用；接着培养人肝脏上皮细胞，体外深入研究瑞芬太尼诱导IL-18BP基因转录的机制。以期为CPB围术期肝脏保护策略的制定提供理论依据。

肝缺血-再灌注（I/R）损伤是肝脏手术后一个常见的病理生理现象，严重影响了手术的成功率和患者的愈后。已有报道瑞芬太尼作为临床常用的麻醉镇痛药在心、脑等器官的I/R损伤中有明显的保护作用，但在肝脏I/R损伤中的作用尚不明确。. 本课题第一部分探讨瑞芬太尼对肝I/R损伤的作用及其与IL-18信号通路的关系。结果发现，肝脏I/R损伤6h导致大鼠肝脏组织IL-18、IL-18BP mRNA和蛋白及下游炎症因子IFN-γ、TNF-α 、IL-1β均明显升高，肝脏组织出现明显中性粒细胞浸润；瑞芬太尼预处理显著降低了肝损伤程度，表现为血清转氨酶降低、肝组织病理学损伤减轻。对肝脏IL-18通路的检测提示，瑞芬太尼明显抑制了I/R导致的IL-18 mRNA和蛋白水平升高，并进一步上调肝脏中IL-18BP mRNA和蛋白水平。它对IL-18/ IL-18BP平衡的调节伴随着IL-18下游炎症因子IFN-γ、TNF-α 、IL-1β表达的降低和肝组织中性粒细胞浸润减轻。进一步的研究发现，阿片受体拮抗剂纳洛酮显著抑制了瑞芬太尼介导的IL-18降低，部分阻断了瑞芬太尼的肝保护作用，但对IL-18BP的表达没有明显影响，提示瑞芬太尼依赖阿片受体的保护作用是通过抑制IL-18表达实现的，而上调IL-18BP可能是其非依赖阿片受体的保护机制之一。. 在课题的第二部分，我们探讨了瑞芬太尼上调IL-18BP表达的机制。在正常大鼠肝脏及培养的正常人肝脏上皮细胞LO2细胞中，我们均发现，瑞芬太尼能够明显上调IL-18BP表达。通过应用双重荧光素酶报告基因系统、EMSA和siRNA干扰技术，我们进一步确定，瑞芬太尼诱导IL-18BP表达的机制与激活转录因子STAT1和CEBP β、上调IL-18BP基因转录有关。. 这些实验结果为瑞芬太尼的临床应用提供了更丰富的理论基础，提示瑞芬太尼不仅有麻醉镇痛作用，还有肝组织保护作用，因此在与肝脏IL-18信号通路激活相关的手术如肝移植或肝叶切除等的临床应用上拥有独特的优势。本课题以LO2细胞为载体，对瑞芬太尼上调IL-18BP表达的机制的研究进一步阐明了瑞芬太尼的作用靶点，为其在与IL-18相关的免疫性、炎症性疾病的治疗提供了新思路。