摘要
目的分析麦冬皂苷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。方法选择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。结果模型组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)×10^(9)/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)×10^(9)/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)×10^(9)/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)。结论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。
Objective To analyze the protective effects and mechanisms of ophiopogonin D(OP-D)on acute lung injury(ALI)and intestinal mucosal barrier injury.Methods 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.Results 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)×10^(9)/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)×10^(9)/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)×10^(9)/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).Conclusion 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.
作者
谭宜
许新才
万秋风
曹博威
李春兴
郭杨超
Tan Yi;Xu Xincai;Wan Qiufeng;Cao Bowei;Li Chunxing;Guo Yangchao(Gastrointestinal(tumor)surgery,The First Affiliated Hospital of Xinjiang Medical University,urumqi,Xinjiang,830054;Respiratory intensive care unit,The First Affiliated Hospital of Xinjiang Medical University,urumqi,Xinjiang,830054)
出处
《中华肺部疾病杂志(电子版)》
2025年第4期527-533,共7页
Chinese Journal of Lung Diseases(Electronic Edition)
基金
吴阶平医学基金会临床科研专项资助基金(320.6750.2024-7-10)。
关键词
小鼠
急性肺损伤
麦冬皂苷D
肺及结肠病理形态
黏膜屏障
Mouse
Acute lung injury
Ophiopogonin D
Pathological morphology of the lung and colon
Mucosal barrier
