MD2-IN-1 is a Myeloid differentiation protein 2 (MD2) inhibitor with a KD of 189  μM for the recombinant human MD2 (rhMD2).

MD2 (recombinant human):189 μM (KD)

不同剂量的MD2-IN-1预处理能够剂量依赖性地降低FITC-LPS与细胞表面膜上MD2的结合，其中10μM的剂量可使平均荧光强度的抑制率达到65%。MD2-IN-1的预处理还能剂量依赖性地阻断LPS诱导的MPMs中MAPK磷酸化。与仅用载体处理相比，单独LPS处理显著增加了TLR4/MD2复合物的数量，而MD2-IN-1的预处理则能抑制TLR4/MD2复合物数量的增加，使其恢复至仅载体处理的水平。SPR分析表明，MD2-IN-1能够以剂量依赖的方式与rhMD2蛋白发生显著结合，KD值为189μM；而黄腐醇与MD2结合的KD值为460μM。

在LPS处理组中，肺部湿/干重量比显著高于对照组，而MD2-IN-1的治疗能降低LPS引起的肺水肿。MD2-IN-1的给药明显减少了LPS导致的BALF中蛋白质浓度增加。LPS还引起了可观察的肺组织病理变化，包括炎症浸润区域、出血、间质性水肿、肺泡壁增厚和肺组织破坏。这些病理学变化在MD2-IN-1治疗组中得到了改善。

Mouse RAW264.7 macrophages are starved for 3?h before experimentation. Cells are incubated with or without FITC-LPS (50?μg/mL) in the presence or absence of MD2-IN-1 (0.1, 1 and 10?μM) for 30?min. After incubation, macrophages are fixed with paraformaldehyde for 10?min at 4°C and washed with PBS before being analyzed by flow cytometry.

Male Sprague Dawley (SD) rats are randomly divided into three groups,designated "control" (5 rats,only receive the vehicle of 0.9% saline),"LPS" (7 rats,receive 5?mg/kg LPS alone) and "MD2-IN-1 (20)?+?LPS" (6 rats,receive both MD2-IN-1 and 5?mg/kg LPS).Prior to LPS-induced Acute lung injury (ALI),the MD2-IN-1+LPS group rats are treated intragastrically with MD2-IN-1 at a dosage of 20?mg/kg/day continuously for one week.Under ether anesthesia,all the rats are exposed their trachea and challenged with intratracheal instillation of 50?μL of LPS,while the control group challenged with intratracheal instillation of 50?μL of 0.9% saline.Rats are then euthanized with ketamine after 6?h of LPS induction.