霉酚酸酯（MMF）对脓毒症肝损伤的保护作用及机制研究

Hepatic injury during sepsis seriously affect prognosis of patients. Effective management of hepatic injury during sepsis is an urgent problem in clinical medicine. TLR4 signaling pathway contributes to inflammatory injury of hepatic during sepsis and accumulating evidence has indicated that autophagy plays important roles in liver physiology and pathology. However, the research of mycophenolate mofetil(MMF), a kind of immunosuppressive agents, in the field of sepsis associated hepatic injury and autophagy has been less reported. On the preliminary experiment, it was confirmed that MMF can induce autophagy, reduce hepatic injury, at the same time decrease expression of TLR4. Therefore, we raised the hypothesis that MMF can reduce hepatic injury during sepsis through inducing autophagy and inhibiting TLR4 inflammatory response. For the validation of this hypothesis, we will investigate liver protect role of MMF and the mechanism through MMF induces autophagy. In this study the model of hepatocytes stimulated by LPS, and cecal ligation and puncture (CLP) will be used. The research methods include real-time PCR, western blot, shRNA, transfection of viral plasmid, laser-scanning confocal microscope etc.. This investigation will identify the liver protective effect of MMF during sepsis and reveal the mechanism of autophagy induced by MMF. From new visual point of autophagy and TLR4, this investigation will raise new mechanism through which MMF induces hepatic injury during sepsis. The research results will suggest new thread for the prevention and therapy for sepsis associated hepatic injury in clinical setting.

脓毒症引起的肝损伤严重影响患者预后，是目前临床亟待解决的问题。脓毒症肝损伤时TLR4参与炎症反应，而肝细胞内自噬的发生起重要调节作用。近年来自噬在脓毒症中的作用备受关注。免疫抑制剂霉酚酸酯（MMF）在脓毒症肝损伤及自噬方面的研究没有报道。预实验结果提示MMF可减轻脓毒症引起的肝损伤，同时发现MMF可抑制TLR4表达并诱导自噬增加。为此我们提出假说：MMF可能通过诱导自噬减轻脓毒症肝损伤，同时抑制TLR4炎症反应。为了验证这一假说，我们将通过小鼠肝脏细胞LPS刺激模型、盲肠结扎穿孔（CLP）脓毒症模型，采用Real TimePCR、Western blot质粒病毒转染、激光扫描共聚焦显微镜等手段从分子、细胞和整体水平等多方面探讨MMF对脓毒症肝损伤的保护作用及其诱导自噬的机制，本研究将从TLR4和自噬这一新视点揭示MMF减轻脓毒症肝损伤的机制，为临床上脓毒症肝损伤的防治提供新的思路。

全球每年因脓毒症导致的死亡病例超过600万，且流行病学调查提示脓毒症的发病率仍呈上升趋势。尽管近年来多种诊断及治疗方法纷纷得到应用，脓毒症患者的病死率并未显著下降，成为重症医学领域的重大挑战。大量研究已发现机体在感染状态下产生的失控炎症反应是导致患者不良预后的主要原因，过度的炎症反应造成脏器功能损伤，而随后的免疫抑制则导致继发感染加重。此前大量研究集中在抗炎症反应这一领域，且未能取得成功。本课题组既往发现抗移植排斥药物霉酚酸酯（MMF）可减轻脓毒症小鼠肝损伤，但具体机制不详。为验证MMF在脓毒症治疗中的潜在作用，我们首先验证了其对脓毒症小鼠生存率的影响。在脓毒症造模后使用60mg/kg的MMF治疗小鼠，观察发现实验组小鼠7日生存率（38%）较对照组（25%）显著提高，同时其肺、心、肝病理损伤较对照组显著减轻。血清及腹腔灌洗液的ELISA分析表明MMF显著降低了小鼠体内多种细胞因子（IL-1β，IL-6， TNF-α，MCP-1，TGF-β及IL-10等）的水平。同时，通过对血清及腹腔灌洗液的定量培养，发现MMF降低了脓毒症小鼠体内的病原体负荷，这提示MMF可能减轻了免疫抑制。针对巨噬细胞的流式细胞学分析表明，MMF降低了巨噬细胞的PD-1表达，同时改善了巨噬细胞的免疫活性。以上研究表明MMF可能通过抑制细胞因子的产生减轻机体炎症反应。同时通过下调PD-1减轻了巨噬细胞的免疫抑制，最终改善了脓毒症小鼠的预后。这项研究为揭示脓毒症的发病机制提供了新的论据，并为脓毒症治疗提供了新的潜在方法。