SIRT6在多壁碳纳米管致肺纤维化中的作用及机制研究

The potential risk of multi-walled carbon nanotubes (MWCNT) to induce lung fibrosis in humans has been demonstrated by in vivo animal study. Due to the lack of effective treatment for lung fibrosis in clinics, it is imperative to study the underling mechanism. Recently it has been shown that TGF-β/Smad signaling pathway plays an important role in MWCNT-induced lung fibrosis, but the key molecule(s) that modulate TGF-β/Smad signaling pathway and if there is other mechanism or pathway involved have yet to be identified. Our previous study revealed that overexpression of SIRT6 in human alveolar epithelial A549 cells significantly inhibited MWCNT-induced TGF-β1, epithelial-mesenchymal transition and fibroblast phenotype. We hypothesize that SIRT6 inhibited MWCNT-induced lung fibrosis by inactivating TGF-β/Smad pathway. Animal, cell culture and 3D fibroblastic models will be established by adeno-associated virus or adenovirus transfection to overexpress or knockdown SIRT6. We will investigate the role of SIRT6 in MWCNT-induced fibroblast-myofibroblast transition and epithelial-mesenchymal transition. We will also study the impacts of SIRT6 on TGF-β/Smad pathway by immunoprecipitation and dual luciferase reporter gene assay. This study will further elucidate the targets and molecular mechanism of MWCNT-induced lung fibrosis.

多壁碳纳米管（MWCNT）致人肺纤维化潜在风险已被动物实验证实，临床上缺乏有效疗法，其机制研究十分紧迫。近期研究表明TGF-β/Smad是MWCNT致肺纤维化的重要信号通路，但调控TGF-β/Smad的关键分子及是否存在其它机制、通路仍不清楚。申请人前期发现：在人肺泡上皮细胞A549中过表达SIRT6可显著抑制MWCNT诱导的TGF-β1、上皮间质转化及成纤维细胞表型转化，据此假设SIRT6可通过削弱TGF-β/Smad通路抑制MWCNT诱导的肺纤维化。本项目拟通过腺病毒/腺相关病毒转染建立SIRT6过表达或沉默的动物、细胞和3D肺纤维化模型，研究SIRT6在MWCNT诱导的肺成纤维细胞向肌成纤维细胞表型转化和上皮间质转化中的作用，并采用免疫共沉淀和双荧光素酶报告基因等方法研究SIRT6对TGF-β/Smad通路的作用，为进一步揭示MWCNT致肺纤维化的作用靶点和分子机制提供一定理论依据。

多壁碳纳米管（multi-walled carbon nanotubes，MWCNTs）致人肺纤维化潜在风险已被动物实验证实，但临床上缺乏有效疗法，其机制研究十分紧迫。本项目主要研究SIRT6在MWCNTs致肺纤维化中的作用及SIRT6是否通过削弱TGF-β/Smad信号通路抑制上皮间质转化（epithelial to mesenchymal transition，EMT）和成纤维细胞向肌成纤维细胞表型转化，从而削弱或逆转MWCNTs致肺纤维化的进程。研究结果表明，SIRT6能够抑制MWCNTs诱导的人支气管上皮BEAS-2B细胞EMT；SIRT6通过失活TGF-β1/Smad2信号通路抑制MWCNTs诱导的EMT，且该过程依赖SIRT6的去乙酰化酶活性。基于SIRT6在MWCNTs诱导EMT中的作用，我们还在经典的TGF-β1诱导EMT的细胞模型和博来霉素诱导的小鼠肺纤维化模型中进一步验证了SIRT6对EMT和肺纤维化的抑制作用，明确了SIRT6可通过直接抑制TGF-β1/Smad3信号通路，抑制Smad3与转录因子Snail1结合，以及抑制EMT相关转录因子的表达这三个途径调控EMT。此外，SIRT6还可通过抑制TGF-β1/Smad2和NF-κB信号通路削弱TGF-β1诱导的肺成纤维细胞向肌成纤维细胞转化，从而抑制肺纤维化。该研究结果不仅为进一步揭示MWCNTs致肺纤维化的作用靶点和分子机制提供理论依据，同时也为肺纤维化的预防和治疗提供科学依据。