Critical Care Medicine
Role of β-arrestin-1 in penehyclidine hydrochloride-induced inhibition of LPS-caused increase in pulmonary microvascular permeability in human pulmonary microvascular endothelial cells
Qinghong Yuan, Xuetao Yan, Fei Zheng, Yipeng Wang, Zongze Zhang, Kai Chen, Yanlin Wang, Jia Zhan
Published 2017-07-20
Cite as Chin J Anesthesiol, 2017, 37(7): 869-873. DOI: 10.3760/cma.j.issn.0254-1416.2017.07.029
Abstract
ObjectiveTo 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).
MethodsHuman 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.
ResultsCompared 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-arrestin-1 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).
ConclusionThe 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
Contributor Information
Qinghong Yuan
Department of Anesthesiology, Zhongnan Hospital, Wuhan University, Wuhan 430071, China
Xuetao Yan
Department of Anesthesiology, Maternal and Child Care Service Centre of Shenzhen Bao′an District, Shenzhen 518133, China
Fei Zheng
Department of Anesthesiology, Zhongnan Hospital, Wuhan University, Wuhan 430071, China
Yipeng Wang
Department of Anesthesiology, Zhongnan Hospital, Wuhan University, Wuhan 430071, China
Zongze Zhang
Department of Anesthesiology, Zhongnan Hospital, Wuhan University, Wuhan 430071, China
Kai Chen
Department of Anesthesiology, Zhongnan Hospital, Wuhan University, Wuhan 430071, China
Yanlin Wang
Department of Anesthesiology, Zhongnan Hospital, Wuhan University, Wuhan 430071, China
Jia Zhan
Department of Anesthesiology, Zhongnan Hospital, Wuhan University, Wuhan 430071, China