SIRT1/NICD/Smad3通路在放射性肺纤维化中的作用及分子机制

The Radiation Induced Pulmonary Fibrosis (RIPF) has high incidence and mortality rate, but has no effective prevention and treatment, because the pathogenesis remains unclear. The Epithelial-Mesenchymal Transition (EMT) mediated by Smad protein plays a critical role in the pathogenesis of RIPF. The SIRT1 deacetylase has been identified as a new intrinsic negative modulator of Notch1 intracellular domain (NICD). SIRT1 functions as a NICD deacetylase, which opposes the acetylation-induced NICD stabilization. Our preliminary data shows that SIRT1 also locates in human II alveolar epithelial cells (AEC), and the activation of NICD caused the phosphorylation of Smad3. And our previous clinical research also found that the single nucleotide polymorphism of the TGF-β1 gene is associated with the risk of RIPF in non-small-cell lung cancer patients treated with definitive radiotherapy. Based on our research background, we hypothesize the possible mechanism is: SIRT1 mutation is induced by radiation exposure and actives acetylation of NICD, triggers the phosphorylation of Smad3, incites EMT and leads to RIPF at last. In the in vitro experiment, we intend to construct SIRT1、NICD gene absented and high expression cell lines respectively by means of cell transfection technology. Western-bolt, qRT-PCR, immunoprecipitation, immunofluorescence, nick test and Transwell experiment will be empolyeed to investigate the key points of SIRT1/NICD/Smad signaling pathway and the phenomenon of EMT. In the in vivo experiment, the expression of the SIRT/NICD/Smad signaling pathway and downstream targets will be compared with radiological and pathological findings in the rat radiation-induced lung fibrosis model. We hope these findings may prove our initial hypothesis and ultimately provide a new strategy to prevent RIPF.

Smad3蛋白调控的上皮间充质转化（EMT）是放射性肺纤维化（RIPF）的关键机制之一。SIRT1是新发现的脐静脉血管内皮细胞中的Notch受体胞内域（NICD）的内源性负调控子。课题组前期研究发现人II型肺泡上皮细胞中存在SIRT1，NICD可磷酸化Smad3。据此,本研究假设：放射线诱导SIRT1突变、丧失去乙酰化功能，NICD激活，磷酸化Smad3，触发EMT，引起RIPF。本研究拟采用转染技术，分别构建SIRT1、NICD基因缺失或高表达细胞，采用qRT-PCR、蛋白印迹、免疫沉降、免疫荧光、划痕实验、Transwell实验研究SIRT1/NICD/Smad3通路的关键点调控和下游EMT效应，及在RIPF形成中的作用；利用大鼠放射性肺纤维化模型，行影像学和病理学对照研究，检测SIRT/NICD/Smad通路及下游蛋白在肺组织中的表达与结合。验证本假设，以期找到干预RIPF的分子靶点

Smad3蛋白调控的上皮间充质转化（EMT）是放射性肺纤维化（RIPF）的关键机制之一。SIRT1是新发现的脐静脉血管内皮细胞中的Notch受体胞内域（NICD）的内源性负调控子。课题组给予肺泡上皮细胞BEAS-2B和肺腺癌细胞A549 2Gy X线照射，照射后24小时，Notch1、Hes1、Hes2、p-Smad2/3、Vimentin表达升高，E-cadherin表达下降。在Notch1基因过表达肺泡上皮细胞BEAS-2B和肺腺癌细胞A549中，照射后24小时Notch1、Notch2、Hes1、Hes2、p-Smad2/3、Vimentin表达升高，E-cadherin表达下降。同时，课题组发现SIRT1激活后，通过AMPK/mTOR通路增加A549细胞放疗敏感性。课题组利用13Gy X线照射建立的C57BL/6小鼠放射性肺纤维化模型，发现全胸照射后小鼠肺组织Notch1、Hes1、Hes2表达升高，证实在小鼠放射性肺损伤模型中存在Notch通路激活。放疗后Notch信号的表达先降低，后升高，E-cadherin在放疗后1月即降低，3月回升，后持续降低；Vimentin在放疗后1月稍降低，后持续升高。课题组研究证实放射线激活肺上皮细胞Notch信号通路，并促进磷酸化Smad2/3表达，导致肺上皮细胞发生EMT；Notch1可激活肺上皮p-Smad2/3表达，并诱导EMT转化。.本研究创新性地发现Notch信号通路是放射性肺纤维化的新的分子机制，为寻找预防RIPF的途径提供了新依据和新策略。