Thy-1表达缺失诱导整合素αvβ3变构及活化：脂多糖抑制肺成纤维细胞自噬的机制

The inhibited autophagy of lung fibroblast is the precondition for lung fibroblast proliferation and pulmonary fibrosis. Our previous studies revealed that lipopolysaccharide (LPS) could induce Thy-1 gene deletion and autophagy inhibition in lung fibroblast, but the underlying mechanism remains unknown. Thy-1 deletion was reported to change the conformational alternation of integrin αvβ3. Here we hypothesize that LPS-induced deletion of Thy-1 can cause the conformational change of integrinαvβ3, which result in inhibited autophagy of lung fibroblast though activation of PI3K-Akt-mTOR pathway. In this study, under the condition of three-dimensional polymerized collagen matrices, we aim to elucidate the possible mechanism of LPS-induced inhibition of lung fibroblast autophagy and to clarify the role of Thy-1 deletion, integrinαvβ3 activation and PI3K-Akt-mTORsignaling pathway activation play in this process though inhibiting/over-expressing Thy-1gene/integrinαvβ3 or blocking PI3K-Akt-mTOR pathway. Meanwhile, based on the mouse model of LPS-induced pulmonary fibrosis, we plan to up-regulate Thy-1 expression by establishing the pulmonary-specific Thy-1 gene knock-in mouse though CRISPR/Cas9 genome-editing technique, and to promote autophagy by autophagy inducer administration in order to inhibit lung fibroblast proliferation and pulmonary fibrosis. This study is expected to explore the mechanism of LPS-induced pulmonary fibrosis from the perspective of synergistic interaction between bio-macromolecules and to lay the foundation for its countermeasures explorer.

肺成纤维细胞（LF）自噬抑制是LF增殖与肺纤维化发生的前提。申请者发现脂多糖（LPS）能引起LFThy-1表达缺失并抑制LF自噬，但机制未明。整合素αvβ3分子变构与Thy-1表达缺失相关，申请者推测LPS可诱导LFThy-1表达缺失，改变整合素αvβ3构象，使其活化并激活PI3K-Akt-mTOR通路从而抑制LF自噬。本课题在胶原蛋白三维培养环境下研究LPS抑制LF自噬的机制，通过抑制或过表达Thy-1和整合素、阻断PI3K-Akt-mTOR通路，明确Thy-1表达缺失、整合素αvβ3活化和PI3K-Akt-mTOR通路活化的作用。同时建立LPS诱导小鼠肺纤维化模型，采用CRISPR/Cas9技术构建肺组织特异性Thy-1过表达小鼠，使用自噬诱导剂促进肺组织自噬过程以抑制LF增殖和肺纤维化。本研究有望从生物大分子间相互作用的角度完善LPS诱导肺纤维化的机理，为探索该病的防治手段奠定基础。

脂多糖（LPS）诱导肺成纤维细胞自噬抑制是肺成纤维细胞增殖与肺纤维化发生的重要因素，对相关机制进行研究具有重要意义。本课题重点研究LPS抑制肺成纤维细胞自噬并促进细胞增殖的相关机制，通过抑制或过表达Thy-1和整合素β3、阻断PI3K-Akt-mTOR通路，明确Thy-1表达缺失、整合素β3活化和PI3K-Akt-mTOR通路活化在LPS诱导肺成纤维细胞自噬抑制、细胞增殖和肺纤维化过程中的作用。细胞实验发现：LPS刺激肺成纤维细胞可抑制细胞自噬并促进细胞增殖，伴Thy-1表达下降、Thy-1与整合素β3相互作用减弱、整合素β3表达和活性增高以及PI3K-Akt-mTOR通路活化。抑制整合素β3表达和活性、过表达Thy-1能逆转LPS诱导的PI3K-Akt-mTOR通路活化及细胞自噬抑制；在LPS缺失时，抑制Thy-1表达能模拟LPS刺激所诱导的PI3K-Akt-mTOR通路活化及细胞自噬抑制的相关效应；而使用自噬抑制剂能模拟LPS刺激所诱导的细胞增殖过程。抑制PI3K-Akt-mTOR通路可逆转LPS诱导的细胞自噬抑制和增殖过程。动物实验证实LPS刺激能引起小鼠肺纤维化反应，伴肺组织Thy-1表达下调、整合素β3表达增高和自噬抑制；在整体水平过表达小鼠肺组织Thy-1基因或者抑制整合素β3活性后能逆转LPS诱导的小鼠肺组织Thy-1表达下调、整合素β3表达增高和自噬抑制过程进而抑制LPS诱导的小鼠肺纤维化进程。综上所述，本课题研究证实LPS可诱导肺成纤维细胞Thy-1基因表达缺失并引起整合素β3表达活化以及PI3K-Akt-mTOR通路活化，进而抑制肺成纤维细胞自噬并促进细胞增殖和肺纤维化进程。对Thy-1、整合素β3以及PI3K-Akt-mTOR通路进行有效干预，有望为脓毒症相关性肺纤维化的临床治疗提供一种基于调控细胞自噬状态而抑制肺纤维化进程的治疗策略。