内质网应激调控SPIONs激活NLRP3炎症小体的机制研究

Superparamagnetic iron oxide nanoparticles (SPIONs) are widely applied in biomedical field, and their biosafety attracts much attention. Inflammasome plays a key role in inflammation which is one of the adverse effects of nanomaterials. Our previous study found that SPIONs could trigger IL-1β secretion, induce endoplasmic reticulum stress (ERS) and activate the PERK pathway in unfolded protein response. We also found that BAT3, a molecule associated with ERS, interacted with NLRP3. Thus, we hypothesize that ERS regulates NLRP3 inflammasome activation induced by SPIONs through PERK and/or BAT3. In the present project, we plan to evaluate the NLRP3 inflammasome activation induced by SPIONs in mouse bone marrow-derived macrophages. Then, we will employ gene-knockdown technique and drugs to prove the regulation of ERS on NLRP3 inflammasome activation induced by SPIONs. Furthermore, PERK pathway and BAT3 will be studied to explore the mechanism of ERS in SPION-induced inflammasome activation. Finally, SPIONs and ERS inhibitors will be intraperitoneally injected into mice to verify the mechanism of NLRP3 inflammasome activation in vivo. This project will elucidate the role and mechanism of ERS in SPION-induced NLRP3 inflammasome activation and adverse inflammatory response, which will provide the scientific evidence and research strategy for the safe application of SPIONs and the biosafety assessment of nanomaterials.

超顺磁氧化铁纳米颗粒(SPIONs)广泛应用于生物医药领域，安全性备受关注。炎症小体在纳米材料引起的不良炎症反应中起重要作用。我们前期研究发现SPIONs可促进IL-1β分泌，诱导内质网应激(ERS)，激活未折叠蛋白反应中PERK通路，同时发现ERS相关蛋白BAT3与NLRP3互作，因此提出ERS通过PERK和/或BAT3调控SPIONs激活NLRP3炎症小体的假设。本项目拟用小鼠骨髓来源巨噬细胞，研究SPIONs激活NLRP3炎症小体效应；敲低和药物干预探明ERS在SPIONs激活NLRP3炎症小体中的作用，探讨ERS经PERK和/或BAT3调控SPIONs激活NLRP3炎症小体的分子机制；并利用小鼠腹膜炎模型进行体内验证。本项目预期阐明ERS调控SPIONs激活NLRP3炎症小体和引起不良炎症反应的机制，找到有效干预靶点，为SPIONs安全应用和纳米安全性评价提供科学依据和研究思路。

超顺磁氧化铁纳米颗粒(SPIONs)广泛应用于生物医药领域，根据临床报告和动物研究，SPIONs能够引起一些不良反应。SPIONs的免疫毒性，特别是对巨噬细胞的作用机制，以及不同包被、不同颗粒大小SPIONs产生效应的差异还没有得到充分阐明。. 本研究探索了PEI包被的SPIONs及10 nm和30 nm PEG包被的SPIONs对小鼠巨噬细胞免疫反应的影响。首先，我们发现SPIONs暴露将促进小鼠中性粒细胞迁移、浸润以及IL-6释放。SPIONs可刺激小鼠原代巨噬细胞产生IL-6和TNF-α；相比于10 nm SPIONs，30 nm能引起更强的炎症反应。通过抑制氧化应激或内质网应激(ERS)，能够减轻SPIONs诱导的急性炎症。. 其次，我们发现PEG包被的SPIONs可剂量依赖性的抑制RAW264.7巨噬细胞中LPS诱导的IL-6和TNF-α分泌，同时降低iNOS的mRNA和蛋白水平,该结果与TLR4表达的减少呈正相关。在人类来源的THP-1巨噬细胞中也发现了同样的现象，说明SPIONs可以通过下调TLR4表达，抑制小鼠和人类巨噬细胞中LPS引起的炎症反应。. 最后，我们还发现PEI包被的SPIONs可剂量依赖性地激活小鼠骨髓来源巨噬细胞中NLRP3炎症小体，诱导IL-1β分泌，激活ERS及未折叠蛋白反应中PERK通路。使用ERS及其下游PERK通路的抑制剂均能够降低PEI-SPIONs诱导的IL-1β水平。小鼠急性腹膜炎实验表明，ERS、NLRP3炎症小体抑制剂能够缓解PEI-SPIONs诱导的急性炎症。. 综上所述，本项目探索了两种不同包被的SPIONs对巨噬细胞及小鼠炎症反应的影响及机制。该结果将有助于了解SPIONs的免疫毒性，促进其在工程纳米材料或纳米药物中的安全应用，为纳米材料安全性评价提供新思路，具有重要的科学意义和实际应用价值。.