多肽基纳米粒子P12对急性肺损伤小鼠肺部炎症的抑制及其机制研究

Based on our previous success in the development of a novel peptide-based nanoparticle, P12, this research aims to investigate the protective effect and the mechanisms of action of P12 in a mouse model of acute lung injury (ALI). P12 was found to be able to potently inhibit several Toll-like receptor (TLR) signaling pathways and have multiple anti-inflammatory activities. In this study, LPS-induced ALI mouse models will be employed to first examine the efficacy of P12 on inhibiting the inflammatory responses in the lung and promoting the mouse survival under the severe ALI condition. The administration of P12 is through intratracheal instillation 1 h before, 4 h after or 24 h after LPS stimulation, and the results are analyzed at 24 h after LPS challenging. Secondly, multiple methodologies will be used to elucidate the mechanisms of action of P12 in vivo. The fluorescent P12 will be introduced to identify which types of cells P12 acts on in the lung, and study the corresponding inflammatory responses from these cells through immunofluorescence staining, immunohistochemistry, and flow cytometry analysis. High-throughput RNA-Seq analysis will be conducted to study the global impact of P12 on the gene expression, and find out the key signaling pathways regulated by P12. The identified potential molecular targets and signaling pathways will be further confirmed at mRNA and protein levels as well as validated in an ALI mouse model with specific small molecule inhibitors or RNAi. Lastly, the biodistribution and the excretion of the nanoparticles will be analyzed using a in vivo imaging system and inductively coupled plasma mass spectrometry (ICP-MS) in both healthy mice and the ALI mouse model; the results will provide preliminary pharmacokinetics data for future translation into clinical uses. The ultimate goal of this study is to supply a novel anti-inflammatory “nano-drug” and a new therapeutic strategy for effective pharmacological treatments of ALI and its severe form acute respiratory distress syndrome (ARDS).

拟在前期成功合成的具有抑制Toll样受体信号转导及多靶点抗炎功效的多肽基纳米粒子—P12的基础上，探讨其对急性肺损伤（ALI）小鼠模型的保护作用及其机制。本研究拟以LPS复制ALI小鼠模型，分别在LPS激发前1 h以及激发后4 h和24 h，采用气道内给药的方式进行治疗干预，观察P12对肺部炎症反应的抑制以及对小鼠存活率的影响；同时采用荧光标记P12和流式细胞分析、免疫荧光、免疫组化、RNA测序、小分子抑制剂、RNA干扰等技术方法，力求对P12在ALI小鼠体内发挥抗炎作用的主要效应细胞、信号通路及对相关炎症细胞的调控等机制进行阐明；此外，还将在健康小鼠和ALI小鼠模型上采用动物全身成像和电感耦合等离子体质谱方法对P12进入体内后的分布规律和排泄方式等药代动力学基本参数进行收集，以期为该抗炎药物向临床的转化提供实验依据，最终为新型抗炎纳米药物在ALI/ARDS预防及救治中的应用提供新的思路。

急性肺损伤/急性呼吸窘迫综合征（ALI/ARDS）是临床常见的呼吸系统危重症，病死率高达40%，至今仍缺乏有效的治疗药物。肺泡巨噬细胞（AM）及其Toll样受体（TLR）活化，在ALI/ARDS早期炎症爆发的启动阶段扮演重要角色。因此，针对AM并抑制其TLR信号通路的过度激活，极有可能对控制ALI/ARDS早期炎症反应的发生起到至关重要的作用。在课题组前期成功合成的具有抑制TLR信号转导及多靶点抗炎功效的多肽基纳米粒P12的基础上，本项目探讨了其对ALI小鼠模型的保护作用及其机制。主要结果为：（1）通过复制脂多糖（LPS）诱导ALI经典小鼠模型，验证了P12在体内的抗炎效果；发现气道滴注P12能有效缓解ALI小鼠肺部炎症损伤程度、减少肺部炎症细胞（尤其是中性粒细胞）的浸润、降低炎性趋化因子分泌水平、减轻肺泡出血水肿及蛋白渗漏等，表明P12可有效抑制ALI发生发展中至关重要的病理环节，对ALI小鼠具有较好的保护作用；同时，通过优化P12粒径大小、给药途径及表面修饰方法（微量香烟提取物），进一步提高了P12的抗炎活性和抗氧化能力。（2）采用氯磷酸二钠脂质体耗竭AM的方法，发现AM是P12在ALI小鼠中发挥抗炎保护作用的关键效应细胞；P12可通过抑制M1型巨噬细胞的炎症反应，并诱导AM向M2型极化，从而发挥体内抗炎与促修复的保护作用；还发现P12处理后小鼠肺泡内和中膈淋巴结中的调节性T细胞数量显著增高，表明P12在肺组织中发挥抗炎效用的机制与固有免疫及获得性免疫均相关联；在理解P12作用机制的基础上，我们找到了与其机制相似的临床药物（质子泵抑制剂）并将其制成靶向纳米剂型，促进该研究快速向临床转化。（3）通过分析P12的药代动力学还发现，大约91.5%的P12发挥抗炎效用后可在24小时内通过粪便和尿液有效地排出体外，在肝脾中蓄积较少（<0.03%）。本研究结合纳米技术优势、多重药理学干预策略与ALI炎性病理特征，为ALI/ARDS预防及救治提供新的思路和治疗方案。