Role of β-arrestin-1 in penehyclidine hydrochloride-induced inhibition of LPS-caused increase in pulmonary microvascular permeability in human pulmonary microvascular endothelial cells / 中华麻醉学杂志

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 tableempty 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 pp38MAPK/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),and no significant change was found in the parameters mentioned above in group P+LPS (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-arrestin1 was up-regulated in group P +LPS,and p-p38MAPK/p38MAPK ratio was significantly increased (P＜0.05),and no significant change was found in the other parameters in group P+LPS+shRNA (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.