机械牵张微环境诱导肺成纤维细胞代谢重编程致糖代谢稳态失衡：机械通气促进脓毒症相关性肺纤维化的机制研究

Mechanical ventilation could exacerbate LPS-induced sepsis-associated pulmonary fibrosis, however the underlying mechanism remains unknown. Our previous studies revealed that LPS could induce the formation of focal adhesion(FA) through activation of integrin β3, the mechanical stretch receptor of lung fibroblast. Mechanical stretch was reported to activate lung fibroblast focal adhesion kinase(FAK), which was associated with the activation of PKM2, the key enzyme of aerobic glycolysis. Therefore, we speculate that LPS may activate integrin β3 to induce FA formation in lung fibroblast; Meanwhile, mechanical ventilation could regulate cellular metabolism thorough mechanical stretch microenvironment alteration, which initiates glycometabolic reprogramming by FAK activation and induce glycometabolic homeostasis imbalance to promote lung fibroblast differentiation and pulmonary fibrosis. In this study, based on the mechanical stretch model of LPS-pretreatment mice lung fibroblast and mechanical ventilation model of mice sepsis-associated pulmonary fibrosis, we aim to explore the mechanism through which mechanical ventilation exacerbate LPS-induced lung fibroblast differentiation and promote sepsis-associated pulmonary fibrosis. Through intervening the expression and activation states of integrin β3, FAK, PKM2 in mice lung fibroblast and establishing the CRISP/Cas9 genome-editing technique-based integrin β3 gene knockout mouse to inhibit the expression of integrin β3 in mice lung tissue, we especially focus on the role of activation of integrin β3 and FA as well as cell glycometabolic homeostasis imbalance play in above process. This study aim to explore the mechanism of sepsis-associated pulmonary fibrosis from the perspective of mechanical stretch microenvironment regulate the process of cellular metabolism.

机械通气（MV）可加速LPS诱导的脓毒症相关性肺纤维化，但机制不明。申请者发现LPS能诱导肺成纤维细胞（LF）机械牵张感受器整合素β3表达活化，促进黏着斑形成。有研究表明机械牵张可活化黏着斑激酶（FAK），后者可活化糖有氧酵解关键酶PKM2。申请者推测LPS可诱导LF整合素β3表达活化，促进黏着斑形成；MV可改变机械牵张微环境而调控细胞代谢，通过活化FAK启动糖代谢重编程致糖代谢稳态失衡，加速LF分化并促进肺纤维化。本课题利用LPS预处理小鼠LF机械牵张模型及小鼠脓毒症相关性肺纤维化MV模型，通过干预整合素β3、FAK和PKM2表达活化，以CRISPR/Cas9技术构建整合素β3基因敲除小鼠研究MV加速LPS诱导LF分化并促进脓毒症相关性肺纤维化的机制，明确整合素和黏着斑活化、细胞糖代谢稳态失衡在其中的作用，由此从探讨机械牵张微环境调控细胞代谢过程的角度完善脓毒症相关性肺纤维化的发病机制。

机械通气（MV）已成为临床一线治疗措施广泛应用于急性呼吸窘迫综合征或肺纤维化合并呼吸衰竭患者的救治。然而，机械通气在发挥积极治疗作用的同时可以作为“二次打击”因素诱发机械通气相关性肺损伤或机械通气相关性肺纤维化（MVPF）。但目前尚未明确机械通气是否会进一步加重脓毒症相关性肺纤维化进程，最终形成脓毒症-机械通气相关性肺纤维化（S-MVPF），也未明确机械通气诱导MVPF或S-MVPF发病的相关机制。作为机械牵张的重要感受器，整合素β3可能在MVPF或S-MVPF发病过程中发挥重要作用。本研究旨在探讨整合素β3在MVPF或S-MVPF发病过程中的作用及机制，重点明确整合素β3-M2型丙酮酸激酶（PKM2）通路调控肺成纤维细胞糖代谢重编程，以及整合素β3通路组成成分黏着斑激酶（FAK）调控肺成纤维细胞硬度在该过程中的作用。研究证实在脓毒症-机械通气相关性肺纤维化过程中，机械通气可激活整合素β3-PKM2通路，增强肺组织有氧酵解而促进MVPF，并加速脓毒症相关性肺纤维化进程而形成S-MVPF。机械牵张微环境促进肺成纤维细胞FAK表达与黏着斑的形成，导致肺成纤维细胞硬度增加从而促进MVPF的发生。通过对整合素β3-PKM2通路及FAK的有效调控，可能改善肺组织代谢状态和细胞硬度，从而抑制MVPF与S-MVPF进程。本研究从探讨机械牵张微环境变化调控细胞代谢过程的角度进一步丰富和完善机械通气促进脓毒症相关性肺纤维化进展的机理，有望为脓毒症-机械通气相关性肺纤维化的临床治疗提供一种基于调控细胞代谢状态和细胞硬度而抑制纤维化进程的全新治疗策略。