Drp1-ROS信号调控M1型极化巨噬细胞炎症启动的机制研究

The phenotype polarization and inflammation initiation of macrophage is the basic pathological mechanism of inflammatory diseases. Previous studies indicate that inflammation response of M1 phenotype macrophage is linked to oxidative stress and mitochondrial damage, but the mechanism involved is far from elucidated. Our previous study found that Drp1 may act as novel regulatory factor for inflammation activation in M1 phenotype macrophage. Inhibition of Drp1 can significantly suppress the mitochondrial damage, oxidative stress and inflammatory cytokines expression induced by Lipopolysaccharide (LPS). Based on the previous research and literature progress, we make the following conclusion: Drp1 expression and mitochondrial fission are enhanced in M1 phenotype activation of macrophages induced by LPS, which will cause large dose of ROS generation due to abnormal mitochondrial structure function. Meanwhile, ROS will negatively down-regulate Drp1 activity and aggravate mitochondrial division and oxidative stress. The interaction between them becomes a vicious circle, which will ultimately initiate the inflammatory response through activating inflammatory transcription factors and NLRP3 inflammasom. In the present study, we intend to establish Drp1 knockdown cell and animal models and induced M1-type polarization of macrophage. Then, we will investigate the interaction effect between Drp1 and ROS, as well as the possible role of Drp1 in regulating the inflammation of M1 macrophage. We will also investigate the key inflammatory transcriptional genes that are sensitive to ROS signaling. These works can provide theoretical and practical evidences for the insight into inflammatory diseases.

巨噬细胞局部浸润与M1型极化是机体炎症相关疾病的基本病理特征。研究提示，氧化应激和线粒体损伤与巨噬细胞炎症过程密切相关，但其作用机制远未阐明。我们前期研究发现，线粒体分裂动力蛋白1（Drp1）可能是新的炎症调控因子，抑制Drp1可阻断脂多糖诱导的M1型巨噬细胞发生线粒体损伤和氧化应激，并下调炎症因子表达。基于前期研究及文献进展我们推论：在巨噬细胞M1型极化过程中，Drp1表达和线粒体分裂增强，并促进线粒体结构功能异常和大量ROS生成，ROS负反馈调节Drp1表达活性，加重线粒体分裂和氧化应激，二者交互作用形成恶性循环，最终导致炎症转录通路和炎症小体激活，启动炎症反应。本项目拟构建Drp1敲减的细胞和动物模型，采用脂多糖诱导巨噬细胞M1型极化，探讨Drp1与ROS间的交互作用，重点研究二者在巨噬细胞炎症启动中的关键作用，并筛选ROS信号敏感的炎症转录基因，以期为机体炎症疾病的治疗提供新策略。

M1型巨噬细胞促炎分化不仅是生命科学领域的重大问题，也是脓毒症等疾病治疗的新靶点。线粒体是细胞内能量代谢中心和信号平台，在细胞分化和免疫调控中发挥至关重要作用。本项目旨在探讨巨噬细胞由静息态向M1型转化时线粒体稳态变化规律，阐明其在炎症调控中作用及分子机制，具体结果如下。1.我们采用LPS刺激不同类型的巨噬细胞系和原代培养细胞，发现M1型巨噬细胞线粒体稳态显著重塑，主要表现为数目增多、长度变短和嵴结构减少。而M1型巨噬细胞Δψm并非升高，而是降低，流式检测Δψm结果可能受线粒体数目影响。2.LPS刺激巨噬细胞中线粒体数目和上清促炎因子呈现正相关，而线粒体缺失RAW-ρ0细胞在LPS诱导下促炎因子生成明显降低。RNAseq筛选发现，Stat2和Drp1可能是M1型巨噬细胞线粒体分裂和稳态重塑的核心因子。3.Drp1是线粒体分裂的关键调控基因， LPS刺激Drp1表达和S616磷酸化增强，敲减或抑制Drp1可阻断M1型巨噬细胞线粒体稳态重塑，降低NFκB及下游促炎因子表达。而Drp1S616失活突变可阻断上述过程，而自激活突变Drp1S616E可直接促进巨噬细胞线粒体稳态重塑，启动促炎反应。此外，Drp1抑制剂Mdivi-1能有效阻断脓毒症小鼠腹腔巨噬细胞促炎分化，改善动物炎症反应。4.首次证实Stat2是一个巨噬细胞促炎调控因子，敲减Stat2能降低Drp1S616磷酸化，阻断线粒体重塑和促炎反应。具体机制为Stat2可通过磷酸化Drp1增强线粒体分裂，并上调线粒体生物合成，进而促使线粒体稳态发生重塑，进而启动巨噬细胞促炎反应。5.发现M1型巨噬细胞中糖代谢发生重编程，且主要功能由合成ATP转变为生成ROS，是细胞内ROS的主要来源。进一步敲减Stat2和Drp1可阻断M1型巨噬细胞糖代谢重编程，促进ATP合成，降低ROS产生，表明Stat2-Drp1通过重塑线粒体稳态调控ROS生成。MitoQ是一种mtROS特异性清除剂，可显著抑制LPS诱导NFκB磷酸化及核转位，降低TNF-α和IL-6的转录表达。因此，线粒体稳态重塑是促进巨噬细胞由静息向促炎分化的关键因素，Stat2-Drp1通过增强线粒体分裂和生物合成调控线粒体稳态，生成大量mtROS，进而启动NFκB及下游促炎信号，为脓毒症等炎症疾病的防治提供新思路。