Effect of penehyclidine hydrochloride on β-arrestin-1 expression in lipopolysaccharide-induced human pulmonary microvascular endothelial cells

Abstract

β-arrestins are expressed proteins that were first described, and are well-known, as negative regulators of G protein-coupled receptor signaling. Penehyclidine hydrochloride (PHC) is a new anti-cholinergic drug that can inhibit biomembrane lipid peroxidation, and decrease cytokines and oxyradicals. However, to date, no reports on the effects of PHC on β-arrestin-1 in cells have been published. The aim of this study was to investigate the effect of PHC on β-arrestin-1 expression in lipopolysaccharide (LPS)-induced human pulmonary microvascular endothelial cells (HPMEC). Cultured HPMEC were pretreated with PHC, followed by LPS treatment. Muscarinic receptor mRNAs were assayed by real-time quantitative PCR. Cell viability was assayed by the methyl thiazolyl tetrazolium (MTT) conversion test. The dose and time effects of PHC on β-arrestin-1 expression in LPS-induced HPMEC were determined by Western blot analysis. Cell malondialdehyde (MDA) level and superoxide dismutase (SOD) activity were measured. It was found that the M3 receptor was the one most highly expressed, and was activated 5 min after LPS challenge. Furthermore, 2 μg/mL PHC significantly upregulated expression of β-arrestin-1 within 10 to 15 min. Compared with the control group, MDA levels in cells were remarkably increased and SOD activities were significantly decreased in LPS pretreated cells, while PHC markedly decreased MDA levels and increased SOD activities. We conclude that PHC attenuated ROS injury by upregulating β-arrestin-1 expression, thereby implicating a mechanism by which PHC may exert its protective effects against LPS-induced pulmonary microvascular endothelial cell injury.

Pleiotropic effects and pharmacological properties of penehyclidine hydrochloride

“…PHC has a great advantage in the treatment of sepsis regardless of being induced by cecal ligation and puncture or lipopolysaccharide. 4 , 25 The possible mechanisms are anti-oxidation by attenuating ROS injury, upregulating β-arrestin-1 expression against pulmonary microvascular endothelial cell injury, 26 and anti-inflammation through the inhibition of NF-κB activation via inhibition of the p38 mitogen-activation protein kinase (MAPK) and ERK signaling pathways ( Figure 3 ). The findings by Wu et al indicated that different doses of PHC, especially, a medium dose could prevent LPS-induced ALI in rats.…”

“…PHC can improve the tolerance of cells to ischemia and hypoxia, stabilize the subcellular structure of lysosomes and mitochondria, reduce the release of lysosomes, and inhibit the production of arachidonic acid metabolites and the formation of shock factors. 25 – 26 However, it is necessary to increase the dose of PHC in the treatment of shock. The dosage is 0.04 mg/kg, q12 h. After blood pressure returns to a normal range in 12 hours, the dosage reduces to once a day.…”