Abstract
Introduction
Idiopathic Pulmonary Fibrosis (IPF) is an age-related disease characterized by the pathologic deposition of extracellular matrix (ECM) caused by chronic fibroblast activation and aberrant wound repair. There is no cure for IPF, and the disease carries a median survival of 3-5 years. While different cell lineages are implicated, lung fibrosis specifically is characterized by the over-accumulation and activation of fibroblasts, including senescent lung fibroblasts with a pathological senescence-associated secretory phenotype (SASP). The senescent cells drive IPF by disrupting lung epithelium, myofibroblast activation, ECM remodeling, and creating a pro-inflammatory environment through secretion of cytokines. In this project, we established an in vitro-cell model of bleomycin inducing cellular senescence in IMR90 human lung fibroblasts. The senescence markers p21, β-gal, and apoptosis markers BAX, XIAP were evaluated, as well as pro-fibrotic and inflammatory markers α-SMA, and IL-1B.
Methods
IMR90 human lung fibroblasts were exposed to bleomycin at 0, 10, 20, 30, 40, and 50 ug/mL for 24 hrs. Whole cell lysate was harvested 3 days following bleomycin exposure. Various markers to characterize the cell phenotypes were examined by Western blots. β-Gal staining was performed 3 days after exposure to bleomycin at 0, 10, and 40 ug/mL to examine cellular senescence.
Results
As bleomycin concentration increased, the expression of p21, a growth arrest marker; BAX and XIAP, markers of apoptosis; and α-SMA, a fibrotic marker, increased as well. The cell senescence marker, β-Gal expression, also increased with bleomycin concentration.
Conclusion
The established protein markers of senescence: p21 and β-Gal, inflammation: IL-1B, profibrotic: α-SMA, and apoptosis: BAX, XIAP, were evaluated by Western Blotting. Bleomycin exposure induced the upregulation of senescent, fibrotic, and apoptotic markers, with 10 ug/mL and 20 ug/mL being the most effective at inducing cellular senescence while limiting cell death. The effects of bleomycin on cellular proliferation are under investigation, along with the effects of antioxidants and epigenetic modifiers in the presence of genotoxic drugs. This project will further our understanding of underlying mechanisms of the development of IPF and possible therapies that could be used to treat the disease.