Establishing a Mouse Model of Acute Lung Injury and Pulmonary Fibrosis by Intermittent Lipopolysaccharide Intraperitoneal Injection_Chinese Journal of Respiratory and Critical Care Medicine

Authors：

HE Zhengyu , ZHU Yesen , JIANG Hong.

Department of Anesthesiology, Ninth People’s Hospital, Shanghai Jiaotong University School of Medicine. Shanghai, 200011,ChinaCorresponding Author: JIANG Hong, E-mail: jianghonjy@ yahoo. com. cn;

Corresponding author：

Keywords：

Acute lung injury; Pulmonary fibrosis; Lipopolysaccharide; Mouse; Animal model

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Objective To establish a mouse model of acute lung injury ( ALI) and pulmonary fibrosis by low dose lipopolysaccharide ( LPS) intermittent intraperitoneal injection, and to explore the pathogenesis of ALI and pulmonary fibrosis induced by endotoxin. Methods Forty C57BL/6 mice were randomly divided into a control group, a 3-days LPS group, a 2-weeks LPS group, and a 4-weeks LPS group,with 10 mice in each group. LPS was injected intraperitoneally at dose of 5 mg/ kg for three consecutive days
in the three LPS groups. Equivalent normal saline was injected by the same way in the control group. The mice lung tissues were obtained respectively 3 days ( the control group and 3-days LPS group) , 2 weeks ( the 2-weeks LPS group) , and 4 weeks ( the 4-weeks LPS group) after LPS or saline stimulation. HE staining,
Van-Gieson collagen staining, and Ashcroft fibrosis score assessment were applied to evaluate the development of inflammation and fibrosis in lung tissue at various stages of ALI after LPS-stimulation. The mRNA expression of type Ⅰ procollagen and alpha smooth muscle actin ( α-SMA) were detected by realtime PCR. The deposition of collagen and fibrosis in lung tissue were detected by hydroxyproline assay. The survival condition of each group was also recorded. Results Acute inflammation occurred in mice lung tissue 3 days after intraperitoneal injection of LPS. Collagen deposited in pulmonary interstitium2 weeks after
LPS-stimulation and formed typical pulmonary interstitial fibrosis 4 weeks later accompanying with increase of Ashcroft fibrosis score. Real-time PCR and hydroxyproline assay showed that the expression of collagen and α-SMA increased 3 days after LPS-stimulation and reached the peak 4 weeks later. The animals were all survived up to the endpoint of experiment. Conclusions Accompanying with inflammation, pulmonary fibrosis initiated at early stage of ALI induced by LPS. Intraperitoneal injection of LPS at dose of 5 mg/kg for three consecutive days was able to establish the mouse model of ALI and pulmonary fibrosis with high success
rate and low animal mortality, which provide an ideal experimental platform for further investigation.

Citation： HE Zhengyu,ZHU Yesen,JIANG Hong.. Establishing a Mouse Model of Acute Lung Injury and Pulmonary Fibrosis by Intermittent Lipopolysaccharide Intraperitoneal Injection. Chinese Journal of Respiratory and Critical Care Medicine, 2010, 9(1): 76-80. doi: Copy

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7.	Olman MA,White KE,Ware LB,,et al.Pulmonary edema fluid from patients with early lung injury stimulates fibroblast proliferation through IL-1 beta-induced IL-6 expression.J Immunol ,2004,172:2668-2677.
8.	Seki E,De Minicis S,Osterreicher CH,et al.TLR4 enhances TGF-beta signaling and hepatic fibrosis.Nat Med,2007,13:1324-1332.
9.	Vancheri C,Crimi N,Conte E,,et al.Human lung fibroblasts inhibit tumor necrosis factor-alpha production by LPS-activated monocytes.Am J Respir Cell Mol Biol,1996,15:460-466.
10.	He Z,Zhu Y,Jiang H.Toll-like receptor 4 mediates lipopolysaccharide-induced collagen secretion by phosphoinositide3-kinase-Akt pathway in fibroblasts during acute lung injury.J Recept Signal Transduct Res,2009,29:119-125.
11.	Marshall RP,Bellingan G,Webb S,et al.Fibroproliferation occurs early in the acute respiratory distress syndrome and impacts on outcome.Am J Respir Crit Care Med,2000,162:1783-1788.
12.	Armstrong L,Thickett DR,Mansell JP,et al.Changes in collagen turnover in early acute respiratory distress syndrome.Am J Respir Crit Care Med,1999,160:1910-1915.

1. Baumgarten G,Knuefermann P,Wrigge H,et al.Role of Toll-like receptor 4 for the pathogenesis of acute lung injury in Gram-negative sepsis.Eur J Anaesthesiol,2006,23:1041-1048.
2. Feng Y,Yang Q,Xu J,et al.Effects of HMGB1 on PMN apoptosis during LPS-induced acute lung injury.Exp Mol Pathol,2008,85:214-222.
3. 刘虹,安友仲,李芳芳,等.乌司他丁对脂多糖诱导大鼠急性肺损伤作用的研究.中国呼吸与危重监护杂志,2008,7:290-293.
4. 李琦,钱桂生,张青,等.递增剂量脂多糖致伤对大鼠SIRS-肺损伤的影响.第三军医大学学报,2001,23:1264-1267.
5. Zhang XY,Shimura S,Masuda T,et al.Antisense oligonucleotides to NF-kappaB improve survival in bleomycin-induced pneumopathy of the mouse.Am J Respir Crit Care Med,2000,162:1561-1568.
6. Meijerink J,Mandigers C,van de Locht L,et al.A novel method to compensate for different amplification efficiencies between patient DNA samples in quantitative real-time PCR.J Mol Diagn,2001,3:55-61.
7. Olman MA,White KE,Ware LB,,et al.Pulmonary edema fluid from patients with early lung injury stimulates fibroblast proliferation through IL-1 beta-induced IL-6 expression.J Immunol ,2004,172:2668-2677.
8. Seki E,De Minicis S,Osterreicher CH,et al.TLR4 enhances TGF-beta signaling and hepatic fibrosis.Nat Med,2007,13:1324-1332.
9. Vancheri C,Crimi N,Conte E,,et al.Human lung fibroblasts inhibit tumor necrosis factor-alpha production by LPS-activated monocytes.Am J Respir Cell Mol Biol,1996,15:460-466.
10. He Z,Zhu Y,Jiang H.Toll-like receptor 4 mediates lipopolysaccharide-induced collagen secretion by phosphoinositide3-kinase-Akt pathway in fibroblasts during acute lung injury.J Recept Signal Transduct Res,2009,29:119-125.
11. Marshall RP,Bellingan G,Webb S,et al.Fibroproliferation occurs early in the acute respiratory distress syndrome and impacts on outcome.Am J Respir Crit Care Med,2000,162:1783-1788.
12. Armstrong L,Thickett DR,Mansell JP,et al.Changes in collagen turnover in early acute respiratory distress syndrome.Am J Respir Crit Care Med,1999,160:1910-1915.

Chinese Journal of Respiratory and Critical Care Medicine

Establishing a Mouse Model of Acute Lung Injury and Pulmonary Fibrosis by Intermittent Lipopolysaccharide Intraperitoneal Injection

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