鼻腔滴注与气管滴注脂多糖建立小鼠急性肺损伤模型的比较研究

Comparison of Acute Lung Injury Mice Model Established by Intranasal and Intratracheal Instillation of Lipopolysaccharide

目的:通过比较鼻腔滴注与气管滴注脂多糖(lipopolysaccharide,LPS)建立的急性肺损伤(acute lung injury,ALI)小鼠模型在不同时间点肺组织损伤情况,为探讨ALI的发病机制及治疗研究提供动物模型。方法:小鼠以LPS 5 mg/kg鼻腔滴注和气管滴注20μL/20 g复制ALI模型,分别在造模后6、12和24 h取材,通过检测支气管肺泡灌洗液(bronchoalveolar lavage fluid,BALF)中白细胞数量、总蛋白浓度、白介素-1β(IL-1β)、IL-6、肿瘤坏死因子-α(TNF-α)和巨噬细胞炎性蛋白-2(MIP-2)含量,以及肺组织湿/干质量(W/D)比、髓过氧化物酶(MPO)活力、HE染色、免疫荧光染色和TLR4蛋白表达来反映中性粒细胞浸润、炎症反应以及肺微血管损伤情况。结果:与正常对照组相比,鼻腔滴注和气管滴注LPS后BALF中白细胞数目和总蛋白浓度逐渐升高(P<0.01),12 h时白细胞数量分别为(3.007±0.121)×10^(5)个/mL、(3.459±0.258)×10^(5)个/mL,总蛋白浓度分别为(0.502±0.037)mg/mL和(0.592±0.006)mg/mL;滴注LPS后6 h BALF中IL-1β和TNF-α含量升高,12 h BALF中IL-6和MIP-2活力升高;12 h和24 h肺组织W/D比值、MPO活力、Ly-6G表达、中性粒细胞浸润及炎症反应显著升高(P<0.01);气管滴注后6 h TLR4蛋白表达显著上调,而鼻腔滴注后12 h TLR4蛋白表达显著上调(P<0.01)。结论:鼻腔与气管滴注LPS均可制备小鼠ALI模型,与鼻腔滴注法相比,气管滴注法激活TLR4时间较早,肺组织炎症反应较高。

Objective:To compare the lung injury of acute lung injury(ALI)mouse models established by intranasal and intratracheal instillation of lipopolysaccharide(LPS)at different time points,so as to provide an animal model for exploring the pathogenesis and therapeutic method of ALI.Methods:Mouse model was established with 20μL/20 g LPS(5 mg/kg)through intranasal and intratracheal instillation,respectively,and samples were collected at 6,12 and 24 h after modeling,separately.The white blood cell count,total protein concentration,content of interleukin(IL)-1β,IL-6,tumor necrosis factor(TNF)-α,and macrophage inflammatory protein(MIP)-2 in bronchoalveolar lavage fluid(BALF),lung wet-to-dry(W/D)weight ratio,myeloperoxidase(MPO)activity,and Toll-like receptor 4(TLR4)protein expression were detected and hematoxylin-eosin(HE)staining and immunofluorescence staining were performed to examine the neutrophil infiltration,inflammatory reaction,and pulmonary microvascular injury.Results:White blood cell count and total protein concentration in BALF increased gradually after intranasal and intratracheal instillation of LPS compared with those in the control.At 12 h,the white blood cell count was(3.007±0.121)×10^(5)/mL and(3.459±0.258)×10^(5)/mL,separately,while the total protein concentration was(0.502±0.037)mg/mL and(0.592±0.006)mg/mL,respectively.The levels of IL-1βand TNF-αwere high in BALF 6 h after LPS instillation,while those of IL-6 and MIP-2 were high 12 h after the instillation.Lung W/D weight ratio,MPO content,and Ly-6 G expression were increased significantly and neutrophil infiltration and inflammatory reaction of lung tissue were enhanced significantly at 12 h and 24 h.The expression of TLR4 protein was significantly up-regulated 6 h after intratracheal instillation and 12 h after intranasal instillation.Conclusion:Both intranasal and intratracheal instillation of LPS are feasible for the establishment of ALI mouse model.Compared with intranasal instillation,intratracheal instillation activates TLR4 early and induces strong inflammatory reaction of lung tissue.