Supplementary Material for: Progression of Acute Lung Injury in Intratracheal LPS Rat Model: Efficacy of Fluticasone, Dexamethasone, and Pirfenidone

Introduction: We investigated the potential of LPS (10-300 µg/rat) administered i.t. to induce reproducible features of acute lung injury (ALI) and compared the pharmacological efficacy of anti-inflammatory glucocorticoids and antifibrotic to reduce disease. Additionally, we studied the time dependent progression of ALI in this LPS rat model. Methods: We conducted 1. Dose effect studies of LPS administered i.t. at 10, 30, 100, 300 µg/rat on ALI at 4 hr timepoint; 2. Pharmacological interventions using i.t. fluticasone (100, 300 µg/rat), i.t. pirfenidone (4000 µg/rat), and peroral dexamethasone (1 mg/kg) at 4 hr timepoint; 3. Kinetic studies at 0, 2, 4, 6, 8, 10 and 24 hr post LPS challenge. Phenotype or pharmacological efficacy was assessed using predetermined ALI features such as pulmonary inflammation, edema, and inflammatory mediators. Results: All LPS doses induced a similar increase of inflammation, edema, and inflammatory mediators e.g., IL6, IL1β, TNFα and CINC-1. In pharmacological intervention studies, we showed fluticasone and dexamethasone ameliorated ALI by inhibiting inflammation (>60-80%), edema (>70-100%), and the increase of cytokines IL6, IL1β, TNFα (≥70-90%). We also noticed some inhibition of CINC-1 (25-35%) and TIMP1 (57%) increase with fluticasone and dexamethasone. Conversely, pirfenidone failed to inhibit inflammation, edema, and mediators of inflammation. Lastly, in ALI kinetic studies, we observed progressive pulmonary inflammation and TIMP1 levels, which peaked at 6 hr and remained elevated up to 24 hr. Progressive pulmonary edema started between 2-4 hr and was sustained at later timepoints. On average, levels of IL6 (peak at 6-8 hr), IL1β (peak at 2-10 hr), TNFα (peak at 2 hr), CINC-1 (peak at 2-6 hr), and TGFβ1 (peak at 8 hr) were elevated between 2-10 hr and declined towards 24 hr post LPS challenge. Discussion/Conclusion: Our data show that 10 µg/rat LPS achieved a robust, profound, and reproducible experimental ALI phenotype. Glucocorticoids ameliorated key ALI features at the 4 hr timepoint but the antifibrotic pirfenidone failed. Progressive inflammation and sustained pulmonary edema were present up to 24 hr, whereas levels of inflammatory mediators were dynamic during ALI progression. This study data might be helpful in designing appropriate experiments to test the potential of new therapeutics to cure ALI.

引言：本研究探究了经气管内（intratracheal, i.t.）给予脂多糖（Lipopolysaccharide, LPS，10~300 μg/大鼠）以诱导可重复的急性肺损伤（Acute Lung Injury, ALI）模型的可行性，并对比了抗炎糖皮质激素与抗纤维化药物缓解该疾病的药理学功效。此外，本研究还在该LPS大鼠模型中考察了ALI的时间依赖性病程进展。

方法：本研究开展了三项实验：1. 于LPS攻毒后4小时时间点，开展气管内给予10、30、100、300 μg/大鼠剂量LPS的ALI剂量效应研究；2. 于攻毒后4小时时间点，通过气管内给予氟替卡松（100、300 μg/大鼠）、气管内给予吡非尼酮（4000 μg/大鼠）以及经口给予地塞米松（1 mg/kg）进行药理学干预实验；3. 于LPS攻毒后0、2、4、6、8、10及24小时开展动力学研究。本研究通过预设的ALI表征指标——包括肺部炎症、肺水肿及炎症介质水平，对疾病表型或药理学功效进行评估。

结果：所有LPS剂量组均可相似程度地升高炎症、水肿及炎症介质水平，例如IL6、IL1β、TNFα及CINC-1。在药理学干预实验中，氟替卡松与地塞米松可通过抑制炎症（抑制率>60%~80%）、肺水肿（抑制率>70%~100%）以及细胞因子IL6、IL1β、TNFα的升高（抑制率≥70%~90%）来改善ALI。同时本研究还观察到，氟替卡松与地塞米松对CINC-1（抑制率25%~35%）及TIMP1（抑制率57%）的升高也存在轻度抑制作用。与之相反，吡非尼酮无法抑制炎症、水肿及炎症介质的升高。在ALI动力学研究中，肺部炎症与TIMP1水平呈进行性升高，于6小时达到峰值并持续至24小时；进行性肺水肿于2~4小时开始出现，并在后续时间点持续存在。平均而言，IL6（峰值于6~8小时）、IL1β（峰值于2~10小时）、TNFα（峰值于2小时）、CINC-1（峰值于2~6小时）及TGFβ1（峰值于8小时）的水平在LPS攻毒后2~10小时内升高，并在24小时左右回落至基础水平。

讨论与结论：本研究数据表明，10 μg/大鼠的LPS即可构建出稳定、显著且可重复的实验性ALI表型。糖皮质激素可在攻毒后4小时时间点改善ALI的关键病理表征，但抗纤维化药物吡非尼酮则无此效果。进行性炎症与持续性肺水肿可持续至24小时，而炎症介质的水平在ALI病程进展中呈动态变化。本研究数据可为设计恰当的实验以测试新型治疗药物治愈ALI的潜力提供参考依据。