肺成纤维细胞有氧酵解促进致纤维化微环境形成：脓毒症相关性肺纤维化早期肺成纤维细胞增殖活化的新机制

The formation of fibrotic microenvironment is crucial to pulmonary fibrosis initiation, however the underlying mechanism remains unknown. Our previous studies revealed that LPS could induce lung fibroblast proliferation and activation through activation of PI3K-Akt-mTOR pathway and secretion of endogenous HMGB1. mTOR was also reported to activate PFKFB3, the key enzyme of aerobic glycolysis. Moreverover, our preliminary experiment further confirms that lactate, the metabolite of aerobic glycolysis, could induce secretion of HMGB1. Therefore, we speculate that LPS can activate PFKFB3 through PI3K-Akt-mTOR pathway to initiate lung fibroblast aerobic glycolysis and lactate production, which could induce HMGB1 secretion from macrophage and lung fibroblast, contribute to the formation of fibrotic microenvironment and eventually facilitate the proliferation and activation of lung fibroblast. In this study, based on the co-culture cellular model of lung fibroblast and macrophage and the mice model of LPS-induced sepsis-associated pulmonary fibrosis, we aim to study the mechanism that LPS initiates lung fibroblast aerobic glycolysis, which promote the formation of fibrotic microenvironment and consequently promote lung fibroblast proliferation and activation. We especially focus on the role of PI3K-Akt-mTOR pathway, PFKFB3 and lactate play in above process. In addition, through inhibiting the expression or activity of PFKFB3 by establishing the CRISPR/Cas9 genome-editing technique-based PFKFB3 gene conditional knockout mouse and application of PFKFB3 inhibitor 3PO, we also attempt to restrain the initiation of aerobic glycolysis to inhibit the process of pulmonary fibrosis. This study is expected to explore the mechanism of LPS-induced sepsis-associated pulmonary fibrosis from the perspective of aberrant cellular metabolism-facilitated fibrotic microenvironment formation and to lay foundation for the countermeasures exploration of sepsis-associated pulmonary fibrosis.

致纤维化微环境形成是肺纤维化启动的关键，但机制未明。申请者发现LPS可诱导肺成纤维细胞（LF）PI3K-Akt-mTOR通路活化及HMGB1分泌而促进LF增殖活化。有研究发现mTOR可活化有氧酵解关键酶PFKFB3，而预实验证实乳酸能促进HMGB1分泌。由此推测LPS经PI3K-Akt-mTOR通路活化PFKFB3而启动LF有氧酵解，通过生成乳酸促进巨噬细胞及LF分泌HMGB1并形成致纤维化微环境而促进LF增殖活化。本课题利用细胞共培养模型和肺纤维化模型研究LPS启动LF有氧酵解促使致纤维化微环境形成并促进LF增殖活化的机制，明确PI3K-Akt-mTOR、PFKFB3及乳酸的作用；采用CRISPR/Cas9技术构建PFKFB3基因敲除小鼠，或以PFKFB3抑制剂阻止有氧酵解启动而抑制肺纤维化。本课题有望从细胞异常代谢调控致纤维化微环境形成的角度探讨肺纤维化发生的机制，为该病防治奠定基础。

脂多糖（LPS）诱导肺成纤维细胞有氧酵解是肺成纤维细胞增殖活化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启动肺成纤维细胞有氧酵解过程，促使肺组织致纤维化微环境形成并促进肺成纤维细胞增殖活化的详细机制，重点明确PI3K-Akt-mTOR通路活化、PFKFB3表达活化、有氧酵解产物乳酸生成以及致纤维化微环境主要成分TGF-β1、HMGB1以及TNF-α在以上过程中的重要作用。研究证实在LPS诱导的脓毒症相关性肺纤维化过程中，LPS通过PI3K-Akt-mTOR通路上调PFKFB3表达，引起肺成纤维细胞有氧酵解。巨噬细胞及肺成纤维细胞可构成细胞互作网络，通过分泌TGF-β1、HMGB1以及TNF-α等炎性介质/细胞因子而形成致纤维化微环境，加速肺组织有氧酵解过程和肺成纤维细胞增殖活化而促进肺纤维化进程。通过对肺组织有氧酵解启动和致纤维化微环境形成关键环节（如PI3K-Akt-mTOR通路、PFKFB3、TGF-β1、HMGB1以及TNF-α等）的有效调控，能有效抑制肺成纤维细胞增殖活化和肺纤维化进程。本研究从LPS通过启动肺成纤维细胞异常代谢过程调控致纤维化微环境形成的角度探讨LPS诱导肺成纤维细胞增殖活化过程的精准调控机制，进一步丰富和完善肺成纤维细胞异常细胞生物学行为在肺纤维化发病过程中的作用和机制研究，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致纤维化微环境形成和有氧酵解启动过程的全新的治疗策略。