Wang Wei-Rong, Li Ting-Ting, Jing Ting, Li Yan-Xiang, Yang Xiao-Feng, He Yan-Hao, Zhang Wei, Lin Rong, Zhang Ji-Ye
Cell Physiol Biochem. 2017;41(2):569-582. doi: 10.1159/000457878. Epub 2017 Feb 3.
BACKGROUND/AIMS: Autophagy is a lysosomal degradation pathway that is essential for cellular survival, differentiation, and homeostasis. Sirtuin 1 (SIRT1), a NAD+-dependent deacetylase, plays a pivotal role in modulation of autophagy. Recent studies found that autophagy was involved in the regulation of inflammatory response. In this study, we aimed to determine the effect of SIRT1 on autophagy and inflammation, and whether autophagy can regulate the inflammatory response in vascular adventitial fibroblasts (VAFs).
Cell autophagy was evaluated by fluorescence microscope and transmission electron microscopy. The expression of protein and mRNA were determined by Western blot analysis and real time-PCR. The production of cytokine was detected by ELISA.
TNF-α induced autophagy and increased SIRT1 expression in VAFs. SIRT1 activator resveratrol enhanced TNF-α-induced VAF autophagy. In contrast, SIRT1 knockdown attenuated VAF autophagy. Both the Akt inhibitor MK2206 and mTOR inhibitor rapamycin further increased TNF-α-induced VAF autophagy. Furthermore, SIRT1 knockdown increased Akt phosphorylation and inhibited the autophagy in VAFs. However, MK2206 attenuated the effect of SIRT1 knockdown on VAF autophagy. In addition, ingenuity pathway analysis showed that there is a relationship between cell autophagy and inflammation. We found that SIRT1 knockdown increased the expression of NLRP3 and interleukin (IL)-6 and promoted the production of IL-1β in VAFs. Further study showed that autophagy activation decreased the expression of NLRP3 and IL-6 and inhibited the production of IL-1β, whereas autophagy inhibition increased the inflammatory response of VAFs. More importantly, our study showed that autophagy was involved in the degradation of NLRP3 through the autophagy-lysosome pathway.
SIRT1 not only regulates VAF autophagy through the Akt/mTOR signaling pathway but also suppresses the inflammatory response of VAFs through autophagy.
背景/目的：自噬是一种溶酶体降解途径，对细胞存活、分化和稳态至关重要。沉默调节蛋白1（SIRT1）是一种依赖烟酰胺腺嘌呤二核苷酸（NAD+）的脱乙酰酶，在自噬调节中起关键作用。最近的研究发现自噬参与炎症反应的调节。在本研究中，我们旨在确定SIRT1对自噬和炎症的影响，以及自噬是否能调节血管外膜成纤维细胞（VAFs）中的炎症反应。
通过荧光显微镜和透射电子显微镜评估细胞自噬。通过蛋白质印迹分析和实时聚合酶链反应（PCR）测定蛋白质和信使核糖核酸（mRNA）的表达。通过酶联免疫吸附测定（ELISA）检测细胞因子的产生。
肿瘤坏死因子-α（TNF-α）诱导VAFs自噬并增加SIRT1表达。SIRT1激活剂白藜芦醇增强TNF-α诱导的VAFs自噬。相反，SIRT1基因敲低减弱VAFs自噬。蛋白激酶B（Akt）抑制剂MK2206和雷帕霉素靶蛋白（mTOR）抑制剂雷帕霉素均进一步增加TNF-α诱导的VAFs自噬。此外，SIRT1基因敲低增加Akt磷酸化并抑制VAFs自噬。然而，MK2206减弱SIRT1基因敲低对VAFs自噬的影响。此外， Ingenuity通路分析表明细胞自噬与炎症之间存在关联。我们发现SIRT1基因敲低增加NLR家族含pyrin结构域蛋白3（NLRP3）和白细胞介素（IL）-6的表达，并促进VAFs中IL-1β的产生。进一步研究表明，自噬激活降低NLRP3和IL-6的表达并抑制IL-1β的产生，而自噬抑制增加VAFs的炎症反应。更重要的是，我们的研究表明自噬通过自噬-溶酶体途径参与NLRP3的降解。
SIRT1不仅通过Akt/mTOR信号通路调节VAFs自噬，还通过自噬抑制VAFs的炎症反应。
