Role of β-arrestin-1 in penehyclidine hydrochloride-induced inhibition of LPS-caused increase in pulmonary microvascular permeability in human pulmonary microvascular endothelial cells

Abstract

Objective To evaluate the role of β-arrestin-1 in penehyclidine hydrochloride(PHC)-induced inhibition of lipopolysaccharide(LPS)-caused increase in pulmonary microvascular permeability in human pulmonary microvascular endothelial cells(PMVECs). Methods Human PMVECs were seeded in 6-well plates(2 ml/well)or in culture flasks(4 ml/flask)at the density of 1×105 cells/ml and divided into 5 groups(n=15 each)using a random number table: empty plasmid transfection group(group C), LPS plus empty plasmid transfection group(LPS group), PHC plus LPS plus empty plasmid transfection group(P+ LPS group), LPS plus β-arrestin-1 short hairpin RNA(shRNA)transfection group(LPS+ shRNA group)and PHC plus LPS plus β-arrestin-1 shRNA transfection group(P+ LPS+ shRNA group). In LPS and LPS+ shRNA groups, the cells were transfected with empty plasmid 1.5 μg or with plasmid containing 15 nmol/L β-arrestin-1 shRNA, LPS with the final concentration of 0.1 μg/ml was added at 24 h of incubation, and the cells were then incubated for 1 h. In P+ LPS and P+ LPS+ shRNA groups, the cells were transfected with empty plasmid 1.5 μg or with plasmid containing 15 nmol/L β-arrestin-1 shRNA, PHC with the final concentration of 2 μg/ml was added at 24 h of incubation, LPS with the final concentration of 0.1 μg/ml was added at 1 h of incubation, and the cells were then incubated for 1 h. The cell permeability was measured using Transwell chambers.The expression of heat shock protein(HSP27)was detected by immunofluorescence.The expression of β-arrestin-1, p38 mitogen-activated protein kinase(p38MAPK)and phosphorylated p38MAPK(p-p38MAPK)was detected by Western blot.The ratio of p-p38MAPK/p38MAPK was calculated. Results Compared with group C, the cell permeability was significantly increased, the expression of HSP27 was up-regulated, p-p38MAPK/p38MAPK ratio was increased, and the expression of β-arrestin-1 was down-regulated in LPS, LPS+ shRNA and P+ LPS+ shRNA groups(P 0.05). Compared with group LPS, the cell permeability was significantly decreased, the expression of HSP27 was down-regulated, p-p38MAPK/p38MAPK ratio was decreased, and the expression of β-arrestin-1 was up-regulated in group P+ LPS, and p-p38MAPK/p38MAPK ratio was significantly increased(P 0.05). Compared with group P+ LPS, the cell permeability was significantly increased, the expression of HSP27 was up-regulated, p-p38MAPK/p38MAPK ratio was increased, and the expression of β-arrestin-1 was down-regulated in group P+ LPS+ shRNA(P<0.05). Conclusion The mechanism by which PHC inhibits LPS-induced increase in pulmonary microvascular permeability is totally related to β-arrestin-1 in human PMVECs. Key words: Arrestins; Cholinergic antagonists; Endotoxins; Lung; Capillary permeability