分析麦冬皂苷D(ophiopogonin D, OP-D)对急性肺损伤(acute lung injury, ALI)和肠黏膜屏障损伤的保护作用及机制。

选择48只无特定病原体C57BL/6小鼠随机分为假手术组、模型组、OP-D低剂量组和OP-D高剂量组，每组12只，使用气管推注5 mg/kg脂多糖诱导ALI模型。造模后剔除死亡小鼠，假手术组、模型组、OP-D低剂量组和OP-D高剂量组分别纳入12、10、9、11只。检测肺湿重/干重(wet/dry, W/D)；检测肺组织髓过氧化物酶(myeloperoxidase, MPO)活力、白细胞、中性粒细胞计数；观察肺和结肠组织病理形态；Western Blot和免疫组化检测肺和结肠中炎症和屏障损伤相关蛋白表达。

模型组W/D、MPO、白细胞和中性粒细胞数量[(9.26±1.43)、(1.06±0.15)U/g、(5.49±0.91)×10⁹/ml和(40.83±6.75)%]高于假手术组[(4.92±0.68)、(0.72±0.11)U/g、(2.35±0.54)×10⁹/ml和(14.76±2.08)%](P<0.05)；与假手术组相比，模型组肺组织肺泡壁增厚、炎性细胞浸润，结肠组织黏膜大量炎性细胞浸润和隐窝破坏；模型组诱导型一氧化氮合酶(inducible nitric oxide synthase, iNOS)、肿瘤坏死因子-α(tumor necrosis factor-alpha, TNF-α)、白细胞介素-1β(interleukin-1β，IL-1β)、白细胞介素-6(interleukin-6, IL-6)、Toll样受体4(toll-like receptor 4, TLR4)、磷酸化c-Jun氨基末端激酶(phosphorylated c-Jun N-terminal kinase, p-JNK)/JNK、磷酸化p38丝裂原活化蛋白激酶(phosphorylated p38 mitogen-activated protein kinase, p-P38)/P38、磷酸化核因子-κB p65亚基(phosphorylated nuclear factor-κB p65 subunit, p-P65)/P65、磷酸化肌球蛋白轻链激酶(phosphorylated myosin light chain kinase, p-MLCK)/MLCK、磷酸化肌球蛋白轻链2(phosphorylated myosin light chain 2, p-MLC2)/MLC2蛋白水平高于假手术组，紧密连接蛋白-1(zonula occludens-1, ZO-1)和闭合蛋白-1(Claudin-1)蛋白表达低于假手术组(P<0.05)。OP-D高剂量组W/D、MPO活力、白细胞和中性粒细胞数量[(6.08±0.95)、(0.81±0.13)U/g、(4.13±0.92)×10⁹/ml、(26.47±3.84)%]低于模型组(P<0.05)；与模型组相比，OP-D高剂量组肺和结肠组织病理损伤改善；OP-D高剂量组肺和结肠组织iNOS、TNF-α、IL-1β、IL-6、TLR4、p-JNK/JNK、p-P38/P38、p-P65/P65、p-MLCK/MLCK、p-MLC2/MLC2蛋白水平低于模型组(P<0.05)；OP-D高剂量组肺组织ZO-1(0.95±0.16)和Claudin-1蛋白表达(0.98±0.15)高于模型组ZO-1(0.42±0.07)、Claudin-1(0.39±0.06)(P<0.05)。

OP-D通过调控TLR4/丝裂原活化蛋白激酶(mitogen-activated protein kinase, MAPK)/NF-κB和MLCK/MLC2信号通路，抑制ALI小鼠肺和结肠的炎症反应及黏膜屏障损伤。

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.