Inhalation of panaxadiol alleviates lung inflammation via inhibiting TNFA/TNFAR and IL7/IL7R signalling [Human]. Inhalation of panaxadiol alleviates lung inflammation via inhibiting TNFA/TNFAR and IL7/IL7R signalling [Human]

In order to study the effects and mechanisms of PD inhalation drug delivery, models of LPS-induced murine holistic pneumonia, isolated macrophage inflammation, and epithelial cell co-culture are utilized. For the evaluation of efficacy, pathology and molecular assays have been utilized. Mechanisms of action and targets were screened and validated by molecular means using transcriptome sequencing. Efficacy and mechanism of action were ultimately validated using human BALF cell models. Also studied were pharmacokinetic parameters of inhaled PD. Overall design: Human monocytic THP-1 and A549 were obtained from the Shanghai Institute of Cell Biology (Shanghai, China). THP-1 cells were cultured in RPMI 1640 (GIBCO, Grand Island, NY) containing 10% FBS (GIBCO, Grand Island, NY) in 5% CO2 at 37 °C. Before further treatment, THP-1 cells (1×106 cells/mL) were inoculated into 6-well plates and treated with 500 nM PMA for 4 h to induce macrophage differentiation. Next, cells were washed 3 times with phosphate-buffered saline (PBS) and incubated for 24 h to exclude PMA interference. THP-1 cells treatment was divided into 3 groups: a control group with no treatment, a model group with LPS at 1 μg/mL, a PD group with 1 μg/mL of LPS and 10 μM of PD.

为研究吸入型PD的药物递送效应与作用机制，本研究采用脂多糖（LPS）诱导的小鼠全身性肺炎模型、离体巨噬细胞炎症模型以及上皮细胞共培养模型。在疗效评估环节，采用病理学检测与分子生物学实验手段。通过转录组测序的分子生物学方法筛选并验证了其作用靶点与机制。最终借助人类支气管肺泡灌洗液（BALF）细胞模型验证了其疗效与作用机制。此外，本研究还对吸入型PD的药代动力学参数进行了系统探究。

实验整体设计如下：人单核细胞THP-1与A549细胞系均购自中国科学院上海细胞生物学研究所。THP-1细胞于添加10%胎牛血清（FBS，GIBCO，美国纽约格兰德艾兰）的RPMI 1640培养基中，于37℃、5%二氧化碳（CO₂）培养箱内培养。待开展后续实验处理前，将THP-1细胞以1×10⁶个/mL的密度接种于6孔板，使用500 nM佛波酯（PMA）处理4小时以诱导巨噬细胞分化。随后用磷酸盐缓冲液（PBS）洗涤细胞3次，继续孵育24小时以消除PMA的残留干扰。THP-1细胞实验分为3组：空白对照组（未施加任何处理）、模型组（给予1 μg/mL LPS）、给药组（同时给予1 μg/mL LPS与10 μM PD）。