叉头状转录因子O1/p38丝裂原活化蛋白激酶信号通路在脂多糖致急性肺损伤中的作用

Role of the forkhead transcription factor O1/p38 mitogen activated protein kinase signaling pathway in acute lung injury induced by lipopolysaccharide

目的:探讨叉头状转录因子O1(FoxO1)/p38丝裂原活化蛋白激酶(p38 MAPK)信号通路在脂多糖致急性肺损伤中的作用。方法:按照随机数字表法将20只雄性无特定病原体(SPF)级C57BL/6J小鼠分为正常组及模型组,每组10只,模型组采用脂多糖气管滴注小鼠复制急性肺损伤模型,正常组给予等量生理盐水。分类计数小鼠支气管肺泡灌洗液(BALF)中的细胞,肺损伤评分及肺湿/干重比,苏木精-伊红(HE)染色检测肺组织病理形态,酶联免疫吸附实验(ELISA)检测肺组织中肿瘤坏死因子-α(TNF-α)、白细胞介素(IL)-1β、IL-6含量,免疫印迹法检测FoxO1及pp38 MAPK表达。将小干扰RNA(siRNA)NC和siRNA FoxO1转染至BEAS-2B人正常肺上皮细胞中,实时荧光定量聚合酶链反应(RT-qPCR)检测FoxO1 mRNA表达,免疫印迹法检测FoxO1及pp38 MAPK表达,ELISA检测细胞中TNF-α、IL-1β、IL-6含量。组间比较采用t检验。结果:正常组小鼠肺泡完整;模型组小鼠肺泡断裂,大量炎性细胞浸润,肺泡间隙增宽并充血水肿严重。模型组病理评分(13.02±1.09比0.64±0.02,t=6.316,P<0.01)、湿重/干重比(18.52±1.85比1.89±0.19,t=5.987,P<0.01)、总蛋白含量[(252.46±25.25)g/L比(45.28±4.53)g/L,t=6.326,P<0.01]、中性粒细胞[(8.35±0.83)×10^(8)/L比(2.73±0.28)×10^(8)/L,t=5.326,P<0.01]、白细胞总数[(13.29±1.33)×10^(8)/L比(4.50±0.45)×10^(8)/L,t=6.127,P<0.01]、TNF-α[(14.24±1.38)μg/L比(1.41±0.14)μg/L,t=6.124,P<0.01]、IL-1β[(280.31±28.03)pg/ml比(25.34±2.53)pg/ml,t=5.684,P<0.01]、IL-6[(263.35±20.63)ng/ml比(73.41±7.34)ng/ml,t=5.654,P<0.01]含量、FoxO1(0.41±0.02比0.12±0.01,t=6.421,P<0.01)及pp38 MAPK(0.89±0.03比0.21±0.01,t=5.864,P<0.01)蛋白表达量高于正常组。干扰组FoxO1(0.35±0.02比1.00±0.07,t=6.851,P<0.01)mRNA表达量、FoxO1(0.46±0.10比0.98±0.05,t=6.147,P<0.01)、pp38 MAPK(1.87±0.01比0.97±0.06,t=5.840,P<0.01)蛋白表达量、TNF-α[(1.23±0.11)μg/L比(8.35±0.45)μg/L,t=5.987,P<0.01]、IL-1β[(2.65±0.16)pg/ml比(29.46±1.54)pg/ml,t=5.743,P<0.01]及IL-6[(2.49±0.16)ng/ml比(24.37±1.29)ng/ml,t=5.671,P<0.01]含量低于对照组。结论:FoxO1/pp38 MAPK信号通路能参与脂多糖诱导的小鼠急性肺损伤。

Objective:To role of the forkhead transcription factor O1(FoxO1)/p38 mitogen activated protein kinase(p38 MAPK)signaling pathway in acute lung injury induced by lipopolysaccharide(LPS).Methods:Twenty male specific pathogen free(SPF)grade C57BL/6J mice were divided into a normal group and a model group,10 per group.The model group received intratracheal instillation lipopolysaccharide to replicate an acute lung injury model.The normal group was given an equal amount of physiological saline.The cells in bronchoalveolar lavage fluid(BALF),lung injury score,and lung wet/dry weight ratio was detected.The pathological morphology of lung tissue was detected by hematoxylin and eosin(HE)staining.The level of tumor necrosis factor-alpha(TNF-α),interleukin(IL)-1β,IL-6 in lung tissue was detected by enzyme-linked immunosorbent assay(ELISA).The expression of FoxO1 and pp38 MAPK was detected by Western blotting.Transfection small interfering RNA(siRNA)NC and siRNA FoxO1 into BEAS-2B human normal lung epithelial cells,the expression of FoxO1 mRNA was detected by real-time fluorescent quantitative polymerase chain reaction(RT-qPCR).The expression of FoxO1 and pp38 MAPK was detected by western blot.The level of TNF-α,IL-1β,IL-6 was detected yb ELISA.Group t test was used to analyze differences between groups.Results:Mices had intact alveoli in normal group.Mices in model group had alveolar rupture,extensive infiltration of inflammatory cells,widened alveolar spaces,and severe congestion and edema.Pathological score(13.02±1.09比0.64±0.02,t=6.316,P<0.01),W/D(18.52±1.85 vs.1.89±0.19,t=5.987,P<0.01),the total protein content[(252.46±25.25)g/L vs.(45.28±4.53)g/L,t=6.326,P<0.01],neutrophils number[(8.35±0.83)×10^(8)/L vs.(2.73±0.28)×10^(8)/L,t=5.326,P<0.01],the total leukocyte number[(13.29±1.33)×10^(8)/L vs.(4.50±0.45)×10^(8)/L,t=6.127,P<0.01],the level of TNF-α[(14.24±1.38)μg/L vs.(1.41±0.14)μg/L,t=6.124,P<0.01],IL-1β[(280.31±28.03)pg/ml vs.(25.34±2.53)pg/ml,t=5.684,P<0.01],IL-6[(263.35±20.63)ng/ml vs.(73.41±7.34)ng/ml,t=5.654,P<0.01],the expression of FoxO1(0.41±0.02 vs.0.12±0.01,t=6.421,P<0.01)and pp38 MAPK(0.89±0.03 vs.0.21±0.01,t=5.864,P<0.01)in model group was higher than that in normal group.The expression of FoxO1(0.35±0.02 vs.1.00±0.07,t=6.851,P<0.01)mRNA,FoxO1(0.46±0.10 vs.0.98±0.05,t=6.147,P<0.01),pp38 MAPK(1.87±0.01 vs.0.97±0.06,t=5.840,P<0.01),protein level of TNF-α[(1.23±0.11)μg/L vs.(8.35±0.45)μg/L,t=5.987,P<0.01],IL-1β[(2.65±0.16)pg/ml vs.(29.46±1.54)pg/ml,t=5.743,P<0.01]and IL-6[(2.49±0.16)ng/ml vs.(24.37±1.29)ng/ml,t=5.671,P<0.01]was lower than that in control group.Conclusion:FoxO1/pp38 MAPK signaling pathway could participate in lipopolysaccharide induced acute lung injury in mice.