Roles of β-arrestin-1 in inhibition of endotoxin-induced activation of NF-κB in human pulmonary microvascular endothelial cells by penehyclidine hydrochloride

Abstract

Objective To evaluate the role of β-arrestin-1 in inhibition of endotoxin-induced activation of nuclear factor kappa B (NF-κB) in human pulmonary microvascular endothelial cells (HPMVECs) by penehyclidine hydrochloride (PHC). Methods HPMVECs were seeded in 6-well plates (2 ml/hole) or in culture flasks (4 ml/flask) at the density of 1×105/ml, and were randomly divided into 5 groups(n=20 each) using a random number table: empty plasmid transfection group (group C), lipopolysaccharide (LPS) + empty plasmid transfection group (group LPS), PHC + LPS + empty plasmid transfection group (group P+ LPS), LPS + β-arrestin-1 gene-shRNA transfection group (group LPS+ shRNA) and PHC + LPS + β-arrestin-1 gene-shRNA transfection group (group P+ LPS+ shRNA). HPMVECs were transfected with empty plasmid 1.5 μg or with plasmid containing 15 nmol/L β-arrestin-1gene-shRNA.At 24 h of incubation, PHC with the final concentration of 2 μg/ml was added, the cells were incubated for 1 h, LPS with the final concentration of 0.1 μg/ml was then added, and the cells were continuously incubated for another 1 h. The supernatant was collected to measure the activity of lactic dehydrogenase (LDH). The cell suspension was collected for determination of vascular cell adhesion molecule-1 (VCAM-1) expression and NF-κB activities and NF-κB inhibitor I-κB and β-arrestin-1expression. Results Compared with group C, the activities of LDH in supernatant were increased, VCAM-1 expression was up-regulated, NF-κB activity was significantly increased, and I-κB and β-arrestin-1 expression was down-regulated in LPS and LPS+ shRNA groups.Compared with group LPS, the activities of LDH in supernatant were decreased, VCAM-1 expression was down-regulated, NF-κB activity was significantly decreased, and I-κB and β-arrestin-1 expression was up-regulated in group P+ LPS, and no significant change was found in the parameters mentioned above in group P+ LPS+ shRNA.Compared with group P+ LPS, the activities of LDH in supernatant were increased, VCAM-1 expression was up-regulated, NF-κB activity was significantly increased, and I-κB and β-arrestin-1 expression was down-regulated in group P+ LPS+ shRNA. Conclusion PHC inhibits endotoxin-induced activation of NF-κB in HPMVECs completely through up-regulating β-arrestin-1 expression. Key words: Arrestins; Cholinergic agents; NF-kappa B; Endothelial cells; Capillaries; Lung; Endotoxemia