To investigate the role of β-arrestin-1 in inhibition of endoxin-induced activation of MAPK signaling pathway in pulmonary microvascular endothelial cells (PMVECs) by penehyclidine hydrochloride (PHC).

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 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 (LPS group), PHC+ LPS+ empty plasmid transfection group (P+ LPS group), LPS+ β-arrestin-1 short hairpin RNA (shRNA) transfection group (LPS+ shRNA group), and PHC + LPS+ β-arrestin-1 shRNA transfection group (P+ LPS+ shRNA group). After the cells were transfected with empty plasmid 1.5 μg or with plasmid containing 15 nmol/L β-arrestin-1 shRNA, the cells were incubated for 24 h. At 24 h of incubation, LPS with the final concentration of 0.1 μg/ml was added, and the cells were then incubated for 1 h in LPS and LPS+ shRNA groups.In P+ LPS and P+ LPS+ shRNA groups, PHC with the final concentration of 2 μg/ml was added, and the cells were incubated for 1 h, and then LPS with the final concentration of 0.1 μg/ml was added, and the cells were incubated for 1 h. The expression of filamentous actin (F-actin) was detected by flow cytometry.The expression of myosin light chain kinase (MLCK) and vascular endothelial-cadherin (VE-cadherin) was detected by immunofluorescence.The expression of phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2) and phosphorylated c-Jun N-terminal kinase (p-JNK) was determined by Western blot.The expression of β-arrestin-1 mRNA was determined by real-time polymerase chain reaction.

Compared with group C, the expression of F-actin, VE-cadherin and β-arrestin-1 mRNA was significantly down-regulated, and the expression of MLCK, p-ERK1/2 and p-JNK was up-regulated in group LPS, and the expression of p-ERK1/2 and p-JNK was significantly up-regulated (P<0.05), and no significant change was found in the other parameters mentioned above in group P+ LPS (P>0.05). Compared with group LPS, the expression of F-actin, VE-cadherin and β-arrestin-1 mRNA was significantly up-regulated, and the expression of MLCK, p-ERK1/2 and p-JNK was down-regulated in group P+ LPS, and the expression of F-actin, VE-cadherin and β-arrestin-1 mRNA was significantly down-regulated, and the expression of MLCK and p-JNK was up-regulated in group LPS+ shRNA (P<0.05). Compared with group P+ LPS, the expression of F-actin, VE-cadherin and β-arrestin-1 mRNA was significantly down-regulated, and the expression of MLCK, p-ERK1/2 and p-JNK was up-regulated in group P+ LPS+ shRNA (P<0.05).

The mechanism by which PHC inhibits endoxin-induced activation of MAPK signaling pathway in PMVECs is partially related to up-regulation of β-arrestin-1 expression.

Arrestins; Cholinergic antagonists; Endotoxins; Lung; Capillaries; Endothelial cells; Extracellular signal-regulated MAP kinases; JNK mitogen-activated protein kinases