Time-dependent changes of autophagy and apoptosis in lipopolysaccharide-induced rat acute lung injury

Document Type : Original Article


1 School of Basic Medicine, Hubei University of Science and Technology, Xianning 437100, P.R. China

2 Hubei Province Key Laboratory on Cardiovascular, Cerebrovascular, and Metabolic Disorders, Hubei University of Science and Technology, Xianning


Objective(s): Abnormal lung cell death including autophagy and apoptosis is the central feature in acute lung injury (ALI). To identify the cellular mechanisms and the chronology by which different types of lung cell death are activated during lipopolysaccharide (LPS)-induced ALI, we decided to evaluate autophagy (by LC3-II and autophagosome) and apoptosis (by caspase-3) at different time points after LPS treatment in a rat model of LPS-induced ALI.
Materials and Methods: Sprague-Dawley rats were randomly divided into two groups: control group and LPS group. ALI was induced by LPS intraperitoneal injection (3 mg/kg). The lung tissues were collected to measure lung injury score by histopathological evaluation, the protein expression of LC3-II and caspase-3 by Western blot, and microstructural changes by electron microscopy analysis.
Results: During ALI, lung cell death exhibited modifications in the death process at different stages of ALI. At early stages (1 hr and 2 hr) of ALI, the mode of lung cell death started with autophagy in LPS group and reached a peak at 2 hr. As ALI process progressed, apoptosis was gradually increased in the lung tissues and reached its maximal level at later stages (6 hr), while autophagy was time-dependently decreased.
Conclusion:These findings suggest that activated autophagy and apoptosis might play distinct roles at different stages of LPS-induced ALI. This information may enhance the understanding of lung pathophysiology at the cellular level during ALI and pulmonary infection, and thus help optimize the timing of innovating therapeutic approaches in future experiments with this model.


1.   Phua J, Badia JR, Adhikari NK, Friedrich JO, Fowler RA, Singh JM, et al. Has mortality from acute respiratory distress syndrome decreased over time? A systematic review. Am J Respir Crit Care Med 2009; 179: 220-227.
2.   Tang PS, Mura M, Seth R and Liu M. Acute lung injury and cell death: how many ways can cells die? Am J Physiol 2008; 294: L632-L641.
3.   Neff TA, Guo R-F, Neff SB, Sarma JV, Speyer CL, Gao H, et al. Relationship of Acute Lung Inflammatory Injury to Fas/FasL System. Am J Pathol 2005; 166: 685-694.
4.   Quadri SM, Segall L, De Perrot M, Han B, Edwards V, Jones N, et al. Caspase Inhibition Improves Ischemia-Reperfusion Injury After Lung Transplantation. Am J Transplant 2005; 5: 292-299.
5.   Gao M, Liu D, Du Y, Sun R and Zhao L. Autophagy facilitates ventilator-induced lung injury partly through activation of NF-kappaB pathway. Med Sci Monit 2013; 19: 1173-1175.
6.   Zhang J, Wang J-S, Zheng Z-K, Tang J, Fan K, Guo H, et al. Participation of autophagy in lung ischemia-reperfusion injury in vivo. J Surg Res 2013; 182: e79-e87.
7.   Tanaka A, Jin Y, Lee S-J, Zhang M, Kim HP, Stolz DB, et al. Hyperoxia-Induced LC3B Interacts with the Fas Apoptotic Pathway in Epithelial Cell Death. Am J Respir Cell Mol Biol 2012; 46: 507-514.
8.   Matute-Bello G, Winn RK, Jonas M, Chi EY, Martin TR and Liles WC. Fas (CD95) induces alveolar epithelial cell apoptosis in vivo: implications for acute pulmonary inflammation. Am J Pathol 2001; 158: 153-161.
9.   Kabeya Y, Mizushima N, Ueno T, Yamamoto A, Kirisako T, Noda T, et al. LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J 2000; 19: 5720-5728.
10. Shintani T and Klionsky DJ. Autophagy in health and disease: a double-edged sword. Science 2004; 306: 990-995.
11. Gustafsson AB and Gottlieb RA. Autophagy in Ischemic Heart Disease. Circ Res 2009; 104: 150-158.
12. Nakahira K and Choi AMK. Autophagy: a potential therapeutic target in lung diseases. Am J Physiol 2013; 305: L93-L107.
13. Ryter SW, Lee SJ and Choi AM. Autophagy in cigarette smoke-induced chronic obstructive pulmonary disease. Expert Rev Respir Med 2010; 4: 573-584.
14. Lo S, Yuan SS, Hsu C, Cheng YJ, Chang YF, Hsueh HW, et al. Lc3 over-expression improves survival and attenuates lung injury through increasing autophago-somal clearance in septic mice. Ann Surg 2013; 257: 352-363.
15. Yen YT, Yang HR, Lo HC, Hsieh YC, Tsai SC, Hong CW, et al. Enhancing autophagy with activated protein C and rapamycin protects against sepsis-induced acute lung injury. Surgery 2013; 153: 689-698.
16. Perl M, Chung C-S, Lomas-Neira J, Rachel T-M, Biffl WL, Cioffi WG, et al. Silencing of Fas, but Not Caspase-8, in Lung Epithelial Cells Ameliorates Pulmonary Apoptosis, Inflammation, and Neutrophil Influx after Hemorrhagic Shock and Sepsis. Am J Pathol 2005; 167: 1545-1559.
17. Messer MP, Kellermann P, Weber SJ, Hohmann C, Denk S, Klohs B, et al. Silencing of fas, fas-associated via death domain, or caspase 3 differentially affects lung inflammation, apoptosis, and development of trauma-induced septic acute lung injury. Shock 2013; 39: 19-27.
18. Kawasaki M, Kuwano K, Hagimoto N, Matsuba T, Kunitake R, Tanaka T, et al. Protection from Lethal Apoptosis in Lipopolysaccharide-Induced Acute Lung Injury in Mice by a Caspase Inhibitor. Am J Pathol 2000; 157: 597-603.
19. Xie K, Yu Y, Huang Y, Zheng L, Li J, Chen H, et al. Molecular Hydrogen Ameliorates Lipopolysaccharide-Induced Acute Lung Injury in Mice Through Reducing Inflammation and Apoptosis. Shock 2012; 37: 548-555.
20. Ma X, Xu D, Ai Y, Ming G and Zhao S. Fas inhibition attenuates lipopolysaccharide-induced apoptosis and cytokine release of rat type II alveolar epithelial cells. Mol Biol Rep 2010; 37: 3051-3056.
21. Thambiayya K, Wasserloos K, Kagan VE, Stoyanovsky D and Pitt BR. A critical role for increased labile zinc in
reducing sensitivity of cultured sheep pulmonary artery endothelial cells to LPS-induced apoptosis. Am J Physiol 2012; 302: L1287-L1295.
22. Vernooy JHJ, Dentener MA, van Suylen RJ, Buurman WA and Wouters EFM. Intratracheal Instillation of Lipopolysaccharide in Mice Induces Apoptosis in Bronchial Epithelial Cells. Am J Respir Cell Mol Biol 2001; 24: 569-576.
23. Kitamura Y, Hashimoto S, Mizuta N, Kobayashi A, Kooguchi K, Fujiwara I, et al. Fas/FasL-dependent Apoptosis of Alveolar Cells after Lipopolysaccharide-induced Lung Injury in Mice. Am J Respir Crit Care Med 2001; 163: 762-769.
24. Lim SK, Jeong YW, Kim DI, Park MJ, Choi JH, Kim SU, et al. Activation of PRMT1 and PRMT5 mediates hypoxia- and ischemia-induced apoptosis in human lung epithelial cells and the lung of miniature pigs: The role of p38 and JNK mitogen-activated protein kinases. Biochem Biophys Res Commun 2013; 440: 707-713.
25. Kuwano K. Epithelial cell apoptosis and lung remodeling. Cell Mol Immunol 2007; 4: 419-429.
26. Ryter SW and Choi AM. Autophagy in the lung. Proc Am Thorac Soc 2010; 7: 13-21.
27. Pan H, Zhang Y, Luo Z, Li P, Liu L, Wang C, et al. Autophagy mediates avian influenza H5N1 pseudotyped particle-induced lung inflammation through NF-kappaB and p38 MAPK signaling pathways. Am J Physiol 2014; 306: L183-195.
28. Li L, Wu W, Huang W, Hu G, Yuan W and Li W. NF-kappaB RNAi decreases the Bax/Bcl-2 ratio and inhibits TNF-alpha-induced apoptosis in human alveolar epithelial cells. Inflamm Res 2013; 62: 387-397.
29. Lee KY, Oh S, Choi YJ, Oh SH, Yang YS, Yang MJ, et al. Activation of autophagy rescues amiodarone-induced apoptosis of lung epithelial cells and pulmonary toxicity in rats. Toxicol Sci 2013; 136: 193-204.
30. Wu G, Li H, Ji Z, Jiang X, Lei Y and Sun M. Inhibition of autophagy by autophagic inhibitors enhances apoptosis induced by bortezomib in non-small cell lung cancer cells. Biotechnol Lett 2014; 36: 1171-1178.