AMPK-SIRT1通路介导的转录沉默：急性肺损伤凝血紊乱调控新机制

Inflammation and inflammation-induced coagulopathy is the central mechanisms underlie the development of acute lung injury (ALI). The anti-inflammatory AMP-activated protein kinase (AMPK)-sirtuin 1 (SIRT1) pathway was recently found to alleviate ALI through suppressing the transcription of inflammatory genes. We also found that this pathway attenuated the expression of tissue factor (TF) induced by inflammatory stimuli, suggesting that AMPK-SIRT1 pathway might be involved in the regulation of coagulopathy in ALI. To further investigate the possible mechanisms, we plan to determine the modulatory effects of AMPK-SIRT1 pathway on the abberant coagulation, anti-coagulation and fibrinolysis activities in ALI. And then, the crucial regulatory role of AMPK-SIRT1 pathway on the expression of key coagulation-related molecules, such as TF and plasminogen activator inhibitor 1 (PAI-1), wound be confirmed in an in vitro model with the combined administration of pharmacological reagents, small interfering RNA and over-expression vectors. In addition, the modulatory effects of AMPK-SIRT1 pathway on the acetylation level of histones in the promoter region ,the activities of important coagulation-related transcriptional factors, and the binding of RNA polymerase II will be determined. And finally, activators enhancing the function of AMPK-SIRT1 pathway wound be administration into animal models with ALI and the possible therapeutic benefits wound be monitored. In this project,we would like to reveal some novel mechanisms participating in the regulation of coagulopathy in ALI and provide some new experimental data for exploring new therapeutic strategy for the treatment of ALI.

炎症及其诱发的凝血紊乱是急性肺损伤（ALI）发病的关键机制。新近发现的抗炎性AMPK-SIRT1通路可通过抑制炎症基因的转录而减轻ALI，而我们近期发现该通路还可下调致炎刺激诱导的组织因子表达，提示该通路可能对ALI凝血紊乱也具有调节效应。为进一步探讨相关机制，我们将首先在动物模型中探明AMPK-SIRT1对ALI中异常凝血、抗凝、纤溶功能的调节效应；进而在细胞模型中通过应用工具药、siRNA、过表达载体等手段明确AMPK-SIRT1通路在调节组织因子、纤溶酶原激活物抑制物-1等关键凝血相关分子表达中的作用，并从启动子区组蛋白乙酰化水平、转录因子活性、RNA聚合酶Ⅱ结合情况等方面揭示相关基因转录调控的分子机制；最后尝试应用激活剂增强AMPK-SIRT1通路的活性，观察其对肺内凝血紊乱的防治疗效。本研究旨在揭示ALI 中凝血紊乱调控的新机制并为ALI的防治提供新思路。

AMPK是维持能量稳态的重要代谢调节酶，也参与调节炎症等高耗能分子反应。本研究发现：LPS暴露后肺组织内AMPK磷酸化水平明显降低。应用AMPK激活剂A-769662矫正AMPK失活可减轻肺组织损伤并提升动物生存率，这伴随炎性因子TNF-α、IL-6及凝血相关因子TF、PAI-1的mRNA及蛋白水平的降低。应用AMPK间接激活剂二甲双胍也有类似效应，而AMPK抑制剂compound C可消除二甲双胍的作用。AMPK可抑制mTORC1的活性，本研究发现：LPS暴露可诱导mTORC1下游4E-BP-1及p70-S6K1的磷酸化水平升高，激活AMPK可显著抑制LPS诱导的4E-BP-1及p70-S6K1磷酸化。换用二甲双胍，也获得了类似的结果，而联合应用AMPK抑制剂则可消除二甲双胍对mTORC1的抑制效应。联合应用mTOR激活剂3BDO可消除A-769662及二甲双胍对肺损伤的保护效应。mTOR是调控自噬的关键靶点，本研究还发现，LPS暴露可下调肺组织内LC3-Ⅱ的水平并提升p62的含量；A-769662处理则可显著逆转上述改变；联合应用自噬抑制剂3-MA可抑制A-769662对自噬的增强作用，这伴随肺内炎症损伤的增强；联合应用mTOR激活剂3BDO也可消除AMPK激活剂对自噬的增强作用及其对肺损伤的保护作用；联合应用ULK1抑制剂MRT68921也可消除AMPK激活剂对自噬的增强作用及其对肺损伤的保护作用。此外，本研究还发现，SIRT1抑制剂可明显减轻肺内炎症反应和凝血激活，这伴随有mTORC1下游4E-BP-1磷酸化受抑；联合应用mTOR激活剂3BDO消除SIRT1抑制剂的抗炎抗凝保护效应。本研究提示：炎症刺激可诱导AMPK失活，这可解除mTORC1的抑制作用、抑制自噬并激活炎症和凝血相关因子的表达，促进炎症的发生发展。本研究揭示了急性肺损伤发生发展的新机制，为AMPK激活剂在急性肺损伤中的应用提供了发病学基础。