To analyze the protective effects and mechanisms of ophiopogonin D (OP-D) on acute lung injury (ALI) and intestinal mucosal barrier injury.

Forty-eight specific pathogen-free C57BL/6 mice were randomly divided into a shame operation group, a model group, a low-dose OP-D group, and a high-dose OP-D group, with 12 mice in each group. An ALI model was induced by intratracheal injection of 5 mg/kg lipopolysaccharide. After modeling, the dead mice were excluded. There were 12, 10, 9, and 11 mice in the shame operation group, the model group, the low-dose OP-D group, and the high-dose OP-D group respectively, which were included in the statistical analysis. The wet /dry (W/D) ratio of the lungs was detected to determine the degree of pulmonary tissue edema; the activity of myeloperoxidase (MPO) in the lung tissue was detected using a kit; The blood cell analyzers counted white blood cells and neutrophils; Hematoxylin-eosin staining was used to observe the pathological morphology of the lung and colon tissues; Western Blot and immunohistochemistry experiments were used to detect the expressions of proteins related to inflammation and barrier injury in the lung and colon.

The W/D ratio, MPO activity, and the numbers of white blood cells and neutrophils in the model group [(9.26±1.43), (1.06±0.15) U/g, (5.49±0.91)×10⁹/ml, and (40.83±6.75)%] were higher than those in the shame operation group [(4.92±0.68), (0.72±0.11)U/g, (2.35±0.54)×10⁹/ml, and (14.76±2.08)%] (P<0.05). Compared with the shame operation group, the alveolar walls of the lung tissue in the model group were thickened, with infiltration of inflammatory cells, and a large number of inflammatory cells infiltrated the mucosa of the colon tissue, and the crypts were damaged. The protein levels of inducible nitric oxide synthase (iNOS), tumor necrosis factor-alpha (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), toll-like receptor 4 (TLR4), phosphorylated c-Jun N-terminal kinase (p-JNK)/JNK, phosphorylated p38 mitogen-activated protein kinase (p-P38)/P38, phosphorylated nuclear factor-κB p65 subunit (p-P65)/P65, phosphorylated myosin light chain kinase (p-MLCK)/MLCK, and phosphorylated myosin light chain 2 (p-MLC2)/MLC2 in the model group were higher than those in the shame operation group, and the protein expressions of zonula occludens-1 (ZO-1) and claudin-1 were lower than those in the shame operation group (P<0.05). The W/D ratio, MPO activity, and the numbers of white blood cells and neutrophils in the high-dose OP-D group [(6.08±0.95), (0.81±0.13) U/g, (4.13±0.92)×10⁹/ml, and (26.47±3.84)%] were lower than those in the model group (P<0.05). Compared with the model group, the pathological damage of the lung and colon tissues in the high-dose OP-D group was significantly improved. The protein levels of iNOS, TNF-α, IL-1β, IL-6, TLR4, p-JNK/JNK, p-P38/P38, p-P65/P65, p-MLCK/MLCK, and p-MLC2/MLC2 in the lung and colon tissues of the high-dose OP-D group were lower than those in the model group (P<0.05). The protein expressions of ZO-1 and Claudin-1 in the lung tissue of the high-dose OP-D group [(0.95±0.16) and (0.98±0.15)] were higher than those in the model group [(0.42±0.07) and (0.39±0.06)] (P<0.05).

OP-D inhibits the inflammatory response and mucosal barrier injury in the lung and colon of ALI mice by regulating the TLR4/mitogen-activated protein kinase (MAPK)/NF-κB and MLCK/MLC2 signaling pathways.