LncRNA-Cox2在肝脏热缺血－再灌注损伤中对TLR4激活调控机制研究

Activation of Toll-like receptor 4 (TLR4) is a key point in hepatic ischemia reperfusion (I/R) injury. However, the mechanism of the regulation of TLR4 activation is unclear. On the experiment in vitro, it was confirmed that the expression of long non-coding RNA Cox2 (LncRNA-Cox2) could be induced after stimulation of TLR4. On the contrary, the activation of LncRNA-Cox2 could repress the transcriptions of inflammatory genes. In our preliminary experiment, the expressions of TLR4 and LncRNA-Cox2 increased after hepatic warm I/R in mice. Whereas the mechanism of action should be investigated further. Therefore, we raised the hypothesis that the activation of TLR4 could induce the expression of LncRNA-Cox2, whereas LncRNA-Cox2 inhibites the activation of TLR4 by the degenerative manner in hepatic warm I/R. For the validation of this hypothesis, we will investigate the role of LncRNA-Cox2 in hepatic warm I/R injury in many ways including molecular, cell, tissue and animal. In this study, the model of cellular hypoxia reoxygenation and the model of hepatic warm I/R in mice will be used. The research methods include real-time PCR, western blot, transfection of viral plasmid, RNA interference, reporter gene experiment, chromatin immunoprecipitation, RNA immunoprecipitation, etc. This investigation will identify the degenerative effect of LncRNA-Cox2 on the activation and signal transmission of TLR4, and reveal the mechanism of TLR4 gene transcription by the regulation of LncRNA-Cox2. From new visual point of LncRNA, this investigation will raise new mechanism of the regulation of TLR4 activation in hepatic I/R injury. The research results will suggest new thread for the prevention and therapy for hepatic I/R injury in clinical setting.

在肝脏缺血－再灌注（I/R）损伤中TLR4激活调控机制还未明了。体外实验证实TLR4激活可诱导LncRNA-Cox2表达，但是LncRNA-Cox2表达可抑制炎症基因转录。预实验结果观察到小鼠肝脏热I/R后LncRNA-Cox2和TLR4均表达增加，但LncRNA-Cox2调控TLR4调节机制还有待进一步探讨。为此，我们提出假说：肝脏热I/R中TLR4激活可诱导LncRNA-Cox2表达，但预先诱导LncRNA-Cox2表达可能抑制TLR4激活。为了验证这一假说，我们将通过细胞系、细胞缺氧－复氧、小鼠肝脏热I/R模型，从分子、细胞、组织以及动物整体水平多方面探讨肝脏热I/R中LncRNA-Cox2对TLR4激活的负反馈效应，揭示LncRNA-Cox2调控TLR4基因转录的机制。本研究将从LncRNA这个新视点揭示肝脏热I/R损伤中TLR4激活机制，为临床肝脏I/R的防治提供新的思路。

巨噬细胞激活是肝脏缺血/再灌注（I/R）损伤重要病理生理过程。本项目采用小鼠全肝I/R模型以及体外细胞模型，结合CRISPR基因编辑、巨噬细胞嵌合体模型、流式细胞术、小鼠活体成像以及荧光素酶报告基因等技术，深入探究了双向调控炎症反应的长链非编码RNA LncRNA-Cox2在肝脏全肝I/R损伤过程中的时空分布以及其对巨噬细胞激活的调控作用。研究发现：（1）LncRNA-Cox2在全肝I/R损伤早期在巨噬细胞中激活。（2）巨噬细胞特异敲除LncRNA-Cox2的嵌合体小鼠在全肝I/R模型中巨噬细胞极化受阻，肝脏I/R损伤表型受到影响。（3）Toll样受体下游关键接头蛋白MyD88介导LncRNA-Cox2在炎症状态下的激活。（4）LncRNA-Cox2阻碍巨噬细胞中NFkB通路激活。本研究项目的结果说明LncRNA-Cox2在肝脏巨噬细胞激活与极化中起到重要作用，有望解释LncRNA-Cox2对炎症的双向调控作用以及肝脏巨噬细胞在I/R损伤中的双刃剑作用，具有相当的科学意义。