The effect of hydrochloric acid stimulation and mechanical stretch on epithelial-mesenchymal transition in human lung epithelial cells
Ying Pan, Pu Mao, Yongbo Huang, Jianchun Li, Zhiheng Xu, Xi Li, Sulong Wu, Ronghua Shi, Weiqun He, Xiaoqing Liu, Yimin Li
Abstract
ObjectiveTo investigate the effect of hydrochloric acid (HCl) stimulation and mechanical stretch on epithelial-mesenchymal transition (EMT) and hyaluronan (HA) production in human lung epithelial cells.
MethodsHuman lung epithelial cell line BEAS-2B was cultured in vitro, which was divided into phosphate-buffer saline (PBS) + static group, HCl + static group, PBS + stretch group, and HCl + stretch group respectively in the logarithmic phase. The BEAS-2B cells in two stretching groups were challenged by cyclic stretch with 20% amplitude, frequency of 0.33 Hz, sine wave of the FX-5000T system for 48 hours. The morphology changes in cells before and after stretch were observed with inverted microscope. The protein expressions of epithelial markers E-cadherin and cytokeratin-8 (CK-8) as well as mesenchymal markers vimentin and α-smooth muscle actin (α-SMA) were determined by Western Blot. The secretion of HA was determined by enzyme linked immunosorbent assay (ELISA).
Results① It was shown by microscopic observation that BEAS-2B cells displayed cobblestone morphology, linked closely and cell polarity in PBS + static group, which did not change obviously after HCl stimulation alone. Given purely mechanical stretch after 48 hours, the cells morphology changed from cobblestone shape into long spindle, and increased intercellular space obviously. Double hit of HCl and stretch changed the cells morphology more significantly. ② It was shown by Western Blot that compared with the PBS + static group, HCl alone or combined with purely mechanical stretch after 48 hours, the expressions of E-cadherin and CK-8 were decreased, while those of vimentin and α-SMA were increased, and it was more pronounced in HCl + stretch group [the expression quantity (gray value) as base 1 in PBS + static group, E-cadherin: 0.16±0.08 vs. 1, CK-8: 0.10±0.03 vs. 1, vimentin: 3.35±0.38 vs. 1, α-SMA: 3.10±0.45 vs. 1, all P < 0.01]. ③ It was shown by ELISA that both HCl stimulation and stretch could induce BEAS-2B cells secreting HA as compared with PBS + static group (μg/L: 55.763±0.687, 63.005±0.493 vs. 49.876±1.867), and the production of HA increased more remarkably after double hit (μg/L: 78.220±1.085 vs. 49.876±1.867, P < 0.01).
ConclusionsBoth HCl and mechanical stretch could induce EMT and increase HA secretion in human lung epithelial cells in vitro. Double hit of HCl stimulation and mechanical stretch induced EMT apparently, and further increased the production of HA.
Key words:
Mechanical stretch; Epithelial-mesenchymal transition; Hyaluronan; Lung fibrosis; Human lung epithelial cell
Contributor Information
Ying Pan
The First Affiliated Hospital of Guangzhou Medical University, Guangzhou Institute of Respiratory Disease, the State Key Laboratory of Respiratory Disease, Guangzhou 510120, Guangdong, China
Pu Mao
Department of Hospital Infection Control, the First Affiliated Hospital of Guangzhou Medical University, Guangzhou 510120, Guangdong, China
Yongbo Huang
The First Affiliated Hospital of Guangzhou Medical University, Guangzhou Institute of Respiratory Disease, the State Key Laboratory of Respiratory Disease, Guangzhou 510120, Guangdong, China
Jianchun Li
The First Affiliated Hospital of Guangzhou Medical University, Guangzhou Institute of Respiratory Disease, the State Key Laboratory of Respiratory Disease, Guangzhou 510120, Guangdong, China
Zhiheng Xu
The First Affiliated Hospital of Guangzhou Medical University, Guangzhou Institute of Respiratory Disease, the State Key Laboratory of Respiratory Disease, Guangzhou 510120, Guangdong, China
Xi Li
The First Affiliated Hospital of Guangzhou Medical University, Guangzhou Institute of Respiratory Disease, the State Key Laboratory of Respiratory Disease, Guangzhou 510120, Guangdong, China
Sulong Wu
The First Affiliated Hospital of Guangzhou Medical University, Guangzhou Institute of Respiratory Disease, the State Key Laboratory of Respiratory Disease, Guangzhou 510120, Guangdong, China
Ronghua Shi
The First Affiliated Hospital of Guangzhou Medical University, Guangzhou Institute of Respiratory Disease, the State Key Laboratory of Respiratory Disease, Guangzhou 510120, Guangdong, China
Weiqun He
The First Affiliated Hospital of Guangzhou Medical University, Guangzhou Institute of Respiratory Disease, the State Key Laboratory of Respiratory Disease, Guangzhou 510120, Guangdong, China
Xiaoqing Liu
The First Affiliated Hospital of Guangzhou Medical University, Guangzhou Institute of Respiratory Disease, the State Key Laboratory of Respiratory Disease, Guangzhou 510120, Guangdong, China
Yimin Li
The First Affiliated Hospital of Guangzhou Medical University, Guangzhou Institute of Respiratory Disease, the State Key Laboratory of Respiratory Disease, Guangzhou 510120, Guangdong, China
