从能量感受器AMPK揭示CR模拟剂限制失控性炎症反应的转录及翻译调控机制

Inflammation is an energy intensive molecular response. Modulating the energy metabolism is emerging as a novel strategy to limit the uncontrolled inflammation. Recently, we and other laboratories found that intervention of energy metabolism by the caloric restriction (CR) mimetics attenuated fulminant inflammatory injuries, but the underlying mechanisms remain unclear. Our following experiments found that inhibition of the energy senser AMP activated protein kinase (AMPK) could block the anti-inflammatory effects of CR mimetics and these were accompanied with alterations of the downstream targets of AMPK, including the altered activity of silent information regulation 2 homolog 1 (SIRT1) and the altered phosphorylation level of tuberous sclerosis complex 2 (TSC2). Therefore, AMPK might be the pivotal target to mediate the anti-inflammatory effects of CR mimetics. To comfirm this hypothesis and clarify the downstream molecular mechanisms, the following research plans are designed. Firstly, the crucial roles of AMPK in mediating the anti-inflammatory effects of CR mimetics will be confirmed in celluar models by molecular biological methods. And then, the molecular mechanisms through which CR mimetics modulate the acetylation status and activities of important transcription factor and suppress the transcription of inflammatory genes will be investiaged and the implications of SIRT1 will be studied. Additionally, the molecular mechanisms through which CR mimetics modulate the activities of translation regulators and suppress the translation of inflammatory mediators will be investiaged and the implications of TSC2 will be studied. Finally, the involvement of the above targets and the related transcriptional and translational regulatory mechanisms in the anti-inflammatory actions of CR mimetics will be verified in animal models with severe inflammation. This project is expected to reveal the pivotal targets and the downstream molecular mechanisms underlying the anti-inflammatory effects of CR mimetics, which might provide new data for the intervention of uncontrolled inflammation.

炎症是高耗能分子反应，调节能量代谢是限制失控性炎症的新思路。我们及其他实验室近期均发现采用热量限制（CR）模拟剂干预能量代谢可减轻爆发性炎症损伤，但其机制尚不清楚。我们随后发现抑制能量感受器AMPK可阻断CR模拟剂的抗炎效应，这伴随AMPK下游靶点SIRT1活性和TSC2磷酸化的改变。因而AMPK可能是介导CR模拟剂抗炎效应的关键靶点。为证实该假设并揭示下游分子机制，本项目拟首先在细胞模型采用分子生物学手段进一步证实AMPK在CR模拟剂抗炎效应中的关键作用；随后从SIRT1入手揭示CR模拟剂调节重要转录因子乙酰化及活性、抑制炎性基因转录的分子机制，从TSC2入手揭示CR模拟剂调节关键翻译调控因子活性、抑制炎症因子翻译的分子机制；最后在动物模型上验证CR模拟剂通过上述靶点及转录、翻译调控机制在体内发挥抗炎效应。这有望揭示CR模拟剂抗炎的关键靶点及下游分子机制，为失控性炎症防治提供新启示。

炎症是高耗能的分子反应，限制能量代谢有望成为炎症控制的新途径。葡萄糖是最主要的供能物质，2-脱氧葡萄糖（2-deoxyglucose, 2-DG）可通过抑制糖酵解限制能量代谢。我们发现2-DG预处理可抑制LPS诱导TNF-α和IL-6产生、减轻肺组织损伤并提升小鼠生存率。2-DG预处理可抑制LPS诱导的PKM2核聚集，采用工具药抑制PKM2核聚集也可下调炎症因子表达、减轻肺组织损伤。进一步研究发现PKM2可磷酸化STAT3，而STAT3抑制剂可发挥抗炎保护效应。更有趣的是，2-DG后处理仍可在LPS诱导的致死性炎症损伤中发挥保护效应，提示2-DG具有潜在的应用前景。AMPK是细胞内对能量状态高度敏感的代谢调节酶，在维持细胞能量稳态中发挥关键作用。由于炎症是高耗能的分子反应，AMPK也在炎症中发挥重要调控作用。AMPK可被代谢抑制剂2-DG激活，可能在2-DG的抗炎保护效应中发挥重要作用。研究发现，LPS暴露后AMPK磷酸化水平及其下游底物ACC磷酸化水平明显低于正常对照组，应用AMPK激活剂处理可恢复AMPK及ACC的磷酸化水平、降低炎症因子水平、减轻肺组织损伤程度、提升动物生存率。临床常用的AMPK激活剂二甲双胍也可发挥类似抗炎保护效果，且AMPK抑制剂可阻断二甲双胍的保护作用。LPS可下调肺内LC3-Ⅱ的水平并提升p62的含量，提示LPS可抑制自噬；AMPK激活剂则可逆转上述改变。自噬抑制剂可消除AMPK激活剂对自噬的增强作用及其抗炎保护作用。LPS可诱导mTORC1下游4E-BP-1及p70-S6K1的磷酸化水平升高，AMPK激活剂则可抑制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AMPK抑制剂则可消除二甲双胍对mTORC1的抑制效应。mTOR激活剂可消除AMPK激活剂及二甲双胍对肺损伤的保护效应。此外，ULK1抑制剂也可消除AMPK激活剂对自噬的增强作用及其对肺损伤的保护作用。由此可见，2-DG及AMPK激活剂在致死性炎症损伤的干预中有潜在应用价值。