Analysis of cellular senescence induced by lipopolysaccharide in pulmonary alveolar epithelial cells
Introduction
Aging is a complex natural process potentially involving every molecule, cell and organ in the body. However, this definition is not always accurate and usually does not affect an individual's viability. To differentiate simple aging changes from those that increase the risk of disease, disability, or death, gerontologists usually use a more precise term, senescence (Dollemore, 2006).
Senescence is progressive deterioration of many bodily functions over time. Loss of such functions is accompanied by decreased fertility and increased risk of mortality as an individual gets older. Nevertheless, senescence is one of nature's least understood biological processes. Many theories have been proposed to explain the senescence process in nature. These theories are not mutually exclusive, because senescence is viewed as a combination of many processes, which are interactive and interdependent, determining an individual's lifespan and health (Austad, 1997, Dollemore, 2006).
Cellular senescence is originally described as the finite replicative lifespan of human somatic cells in culture. Senescent cells enter an irreversible growth arrest, exhibit a flattened and enlarged morphology, and express a different set of genes, including negative regulators of the cell cycle such as p53 and p16 (Minamino and Komuro, 2007). This hypothesis of cellular aging was first described by Hayflick in the 1960s (Hayflick, 1965). Cellular senescence is one key element that is tightly linked to aging-related and degenerative diseases (Yang and Fogo, 2010). However, the role of cellular senescence in acute and subacute diseases (e.g. acute respiratory distress syndrome and sepsis) has not attracted much attention in the past.
Lipopolysaccharide (LPS) is an endotoxin released mainly from gram negative bacteria during sepsis. LPS causes acute lung injury and acute respiratory distress syndrome. In LPS-induced lung injury, the polymorphonuclear leukocyte is a major causative agent and is responsible for excess production of superoxide anion in the lungs (Tsuji et al., 1998, Gebska et al., 2005). Also, macrophages of the lungs can produce the reactive oxygen materials (Laskin and Pendino, 1995, Sanders et al., 1999, Victor et al., 2004).
Several studies have shown that exposure of mammalian cells to oxidants can produce diverse results with respect to cell growth that are dependent on oxidant concentration. Thus, low levels of reactive oxygen species can be mitogenic rather than growth inhibitory (Burdon, 1995, Davis, 1999). Multiple sublethal exposures to oxidants such as hydrogen peroxide or tert-butyl hydroperoxide can induce cells to enter a state of premature cellular senescence (Chen and Ames, 1994, Dumont et al., 2000). Exposure to higher concentration of reactive oxygen species can result in cell death due to apoptosis or necrosis.
Lung alveolar epithelial cell replication, particularly the replication of type II pneumocytes, is a critical step in the tissue repair process leading to restoration of lung function following an injury to the epithelial lining (Adamson and Bowden, 1974). Hydrogen peroxide inhibited alveolar epithelial wound repair by inducing apoptosis in an in vitro wound repair assay (Geiser et al., 2004). Only few studies have shown that LPS induces the production of reactive oxygen species lung alveolar epithelial cells.
Therefore, we first analyzed the effects of LPS in lung epithelial cells. In this work, we examined the possibility of LPS causing cellular senescence in lung alveolar epithelial cells. Then, we studied how this cellular senescence phenomenon is associated with oxidative stress effect induced by LPS and whether antioxidants could inhibit reduced cellular viability by oxidant stress effect of LPS.
Section snippets
Cell culture and treatment of A549 cells with LPS
Human type II epithelial-like (A549) cells were used. Cell culture was performed in RPMI 1640 (RPMI; Welgene, Daegu, Korea) containing 10% fetal bovine serum (FBS; Welgene, Daegu, Korea), 100 U/ml penicillin and 100 μg/ml streptomycin (Welgene, Daegu, Korea). The cells were incubated for 24 h to allow cell adherence, and then the medium was removed and replaced with RPMI 1640 containing 1% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin. To determine the effect of LPS on A549 cells, cells were
Cellular morphologic findings by LPS treatment
At all time points there was no distinguishable morphological difference, as determined by phase-contrast microscopy, between control cells and cells treated with LPS at below 10 μg/ml. There was a slight morphological difference between control cells and cells treated with 10–20 μg/ml of LPS on day 2. On day 5, LPS at 10–20 μg/ml caused an increase in cell size and caused all cells to exhibit flattened and atrophic morphology. However, after day 5, control cells and LPS-treated cells grew and
Discussion
Although senescence theories are very complex, the characteristics of senescence phenotype are relatively well known. The senescent phenotype was characterized by an altered morphology, including an enlarged and flattened cellular appearance, increased lysosomal content, and an inability to respond to mitogenic stimuli (Ware and Matthay, 2000). In this work, LPS at various concentrations increased A549 cells size and caused all cells to exhibit atrophic morphology. In addition, A549 cells
Conflict of interest
There is no conflict of interest for all authors.
Acknowledgment
This paper was supported by Bumsuk Academic Research Fund in 2009.
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