ROCK/PTEN信号通路对脂多糖诱导的巨噬细胞炎症反应的调控作用及机制研究

Sepsis is associated with overwhelming production of proinflammatory and anti-inflammatory cytokines, which may lead to serious systemic complications such as microcirculatory dysfunction, tissue damage, and septic shock with a high mortality. During sepsis, macrophages are the main effectors of innate immunity because of their capacity to ingest microbes, their antimicrobial activity, and their role as a main source of inflammatory mediators. They also contribute to adaptive immunity as antigen-presenting cells..Recent studies have suggested that ROCK might play an important role in the pathogenesis of sepsis-induced organ dysfuction by regulating inflammatory cell migration and adhesion, and endothelial cell permeability. But whether ROCK mediates LPS-induced inflammatory response in macrophages has not been addressed. Our previous study found that inhibition of Rho kinase strongly reduced LPS-induced inflammation, attenuated lung injury, and improved survival in murine sepsis models. Moreover, Rho kinase inhibitor upregulated expression of p-Akt in lung tissue of LPS-treated mice, indicating that PTEN may be involved in the effect of Rho kinase on Akt signaling..The objectives of this study are to investigate the effects and its mechanisms of ROCK/PTEN pathway on LPS-induced inflammatory response in macrophages by using gene silencing(RNA interference) technology. The significance of this study is to establish an experimental foundation for further researches on the role of ROCK gene in immune response in sepsis, and to explore a new strategy for sepsis immunomodulation therapy.

脓毒症的发生发展过程中，过量促炎和抗炎介质的释放是引起感染性休克和器官功能障碍的重要原因。巨噬细胞在脓毒症的先天免疫、获得性免疫应答以及组织器官损伤中均扮演着重要角色。近年来的研究发现，ROCK可通过调节炎性细胞粘附、迁移以及增加内皮细胞通透性等机制在脓毒症的器官功能损伤中发挥作用，但目前尚无ROCK对巨噬细胞炎症反应调控方面的研究。我们以往的研究发现，应用ROCK特异性抑制剂可以降低脓毒症小鼠全身炎症反应，缓解肺损伤，提高脓毒症小鼠的生存率；而且，ROCK抑制剂能够上调脓毒症小鼠肺组织p-Akt的表达，从而提示PTEN可能参与ROCK对Akt蛋白磷酸化的调控。本课题组拟通过基因沉默等技术深入探讨ROCK对脂多糖诱导的巨噬细胞炎症反应的影响以及ROCK是否通过调节PTEN活性发挥上述作用，这为进一步研究ROCK在脓毒症免疫应答中的功能、机制及其临床意义奠定基础，为脓毒症的免疫治疗开辟新思路

脓毒症的发生发展过程中，过量促炎和抗炎介质的释放是引起感染性休克和器官功能障碍的重要原因。巨噬细胞在脓毒症的先天免疫、获得性免疫应答以及组织器官损伤中均扮演着重要角色。我们以往的研究发现，应用ROCK特异性抑制剂可以降低脓毒症小鼠全身炎症反应，缓解肺损伤，提高脓毒症小鼠的生存率，但是ROCK通过何种机制发挥上述作用尚不十分清楚。本课题通过内毒素血症动物模型和LPS刺激的巨噬细胞模型，利用RT-PCR，ELISA，Western-Blot，EMSA，免疫组化和免疫荧光等技术，深入探讨ROCK对内毒素血症小鼠脏器功能损伤以及脂多糖诱导的巨噬细胞炎症反应的影响，并进一步探究ROCK是否通过调节PI3K/PTEN/Akt/NF-κB信号通路发挥上述作用。我们的研究结果如下：1. ROCK和PI3K抑制剂均能够降低内毒素血症小鼠的肝损伤和/或肺损伤；2.ROCK能够调控PI3K/PTEN/Akt/NF-κB信号通路，但是这一信号通路在ROCK参与脓毒症免疫调节和脏器功能损伤中的作用还有待于进一步研究。本课题的实施揭示了ROCK参与脓毒症脏器功能损伤以及炎症反应的新机理，为今后开展以ROCK为靶点治疗脓毒症提供新的理论依据。