Role of M3 receptors in penehyclidine hydrochloride-induced reduction of endotoxin-induced injury to human pulmonary microvascular endothelial cells

Objective To evaluate the role of M3 receptors in penehyclidine hydrochloride (PHC)-induced reduction of lipopolysaccharide (LPS)-induced injury to human pulmonary microvascular endothelial cells (PMVECs). Methods Human PMVECs transfected with M3 shRNA were seeded in 6-well plates (2 ml/hole) or in culture flasks (4 ml/flask) at the density of 1×105 cells/ml and divided into 5 groups (n=5 each) using a random number table method: control group (group C), LPS group, PHC plus LPS group (group P+ LPS), LPS plus M3 shRNA transfection group (group LPS+ shRNA), and PHC plus LPS plus M3 shRNA transfection group (group P+ LPS+ shRNA). Group C received no mediation, and LPS was added at the final concentration of 0.1 μg/ml in the other groups.PHC 2 μg/ml was added at 1 h before adding LPS in P+ LPS and P+ LPS+ shRNA groups.The cells were transfected with plasmid containing 2.5 nmol/L M3 receptor shRNA in LPS+ shRNA group and P+ LPS+ shRNA group.Contents of filamentous actin (F-actin) in endothelial cells were measured by flow cytometry at 1 h after adding LPS.The expression of myosin light chain kinase (MLCK) and VE-cadherin protein was examined by immunofluorescence.The expression of nuclear factor kappa B (NF-κB) p65 and IκB was detected by Western blot.Contents of tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) were determined by enzyme-linked immunosorbent assay.The M3 receptor mRNA transcription was detected by real-time polymerase chain reaction at 10, 30 and 60 min after adding LPS. Results Compared with group C, F-actin content was significantly decreased, the expression of VE-cadherin and IκB was down-regulated, the contents of TNF-α and IL-6 were increased, and the expression of MLCK and NF-κB p65 was up-regulated in LPS and P+ LPS groups (P 0.05). Compared with group LPS, F-actin content was significantly increased, the expression of VE-cadherin and IκB was up-regulated, the contents of TNF-α and IL-6 were decreased, and the expression of MLCK, NF-κB p65 and M3 receptor mRNA was down-regulated in group P+ LPS and group LPS+ shRNA (P<0.05). Compared with group P+ LPS, F-actin content was significantly increased, the expression of VE-cadherin and IκB protein was up-regulated, TNF-α and IL-6 contents were decreased, and the expression of MLCK, NF-κB p65 and M3 receptor mRNA was down-regulated in group P+ LPS+ shRNA (P<0.05). Conclusion PHC reduces LPS-induced injury to human PMVECs through interfering with M3 receptors and inhibiting NF-κB-mediated inflammatory responses. Key words: Receptor, muscarinic M3; Cholinergic antagonists; Endotoxins; Endothelial cells