Critical Care Medicine
Role of M3 receptor in penehyclidine hydrochloride-induced reduction of increased permeability of human pulmonary microvascular endothelial cells caused by endotoxin: the relationship with MAPK signaling pathway
Shen Shiwen, Liu Qiangsheng, Zheng Fei, Yuan Qinghong, Wang Yipeng, Zhang Zongze, Chen Kai, Wang Yanlin, Zhan Jia
Published 2017-12-20
Cite as Chin J Anesthesiol, 2017,37(12): 1529-1532. DOI: 10.3760/cma.j.issn.0254-1416.2017.12.030
Abstract
ObjectiveTo evaluate the role of M3 receptor in penehyclidine hydrochloride(PHC)-induced reduction of increased permeability of human pulmonary microvascular endothelial cells(PMVECs)caused by endotoxin and the relationship with mitogen-activated protein kinase(MAPK)signaling pathway.
MethodsHuman PMVECs 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 randomly divided into 6 groups(n=5 each): control group(group C), M3 receptor shRNA transfection group(group shRNA), lipopolysaccharide(LPS)group, penehyclidine plus LPS group(group P+ LPS), LPS plus M3 receptor shRNA transfection group(group LPS+ shRNA)and PHC plus LPS plus M3 shRNA transfection group(group P+ LPS+ shRNA). The cells were transfected with shRNA plasmid containing 2.5 nmol/L M3 receptors in shRNA, LPS+ shRNA and P+ LPS+ shRNA groups.LPS at the final concentration of 0.1 μg/ml was added at 24 h of incubation and then cells were incubated for 1 h in LPS and LPS+ shRNA groups.PHC at the final concentration of 2 μg/ml was added at 24 h of incubation, cells were incubated for 1 h, then LPS at the final concentration of 0.1 μg/ml was added, and cells were incubated for another 1 h in P+ LPS and P+ LPS+ shRNA groups.The permeability of PMVECs was measured using Transwell assay.The expression of phosphorylated p38 MAPK(p-p38 MAPK)and phosphorylated extracellular signal-regulated protein kinase 1/2(p-ERK1/2)was detected by Western blot, the expression of heat shock protein 27(HSP27)using immunofluorescent staining, and the expression of M3receptor mRNA by real-time polymerase chain reaction.
ResultsCompared with group C, M3 receptor mRNA expression was significantly down-regulated in group shRNA, and the permeability of cells was significantly increased, and the expression of p-p38 MAPK, p-ERK1/2, HSP27 and M3 receptor mRNA was up-regulated in group LPS(P<0.05). The permeability of cells was significantly decreased, and the expression of p-p38 MAPK, p-ERK1/2, HSP27 and M3 receptor mRNA was down-regulated in P+ LPS, LPS+ shRNA and P+ LPS+ shRNA groups as compared with group LPS, and in group P+ LPS+ shRNA as compared with group LPS+ shRNA(P<0.05).
ConclusionThe mechanism by which PHC reduces endotoxin-caused increased permeability of human PMVECs is related to inhibiting activation of MAPK signaling pathway after down-regulating M3 receptor.
Key words:
Receptor, muscarinic M3; Cholinergic agent; Endothelial cells; Blood capillary; Lung; Endoxemia; p38 MAPKs; Extracellular Signal-Regulated MAP Kinases
Contributor Information
Shen Shiwen
Department of Anesthesiology, Zhongnan Hospital, Wuhan University, Wuhan 430071, China
Liu Qiangsheng
Department of Anesthesiology, Zhongnan Hospital, Wuhan University, Wuhan 430071, China
Zheng Fei
Department of Anesthesiology, Zhongnan Hospital, Wuhan University, Wuhan 430071, China
Yuan Qinghong
Department of Anesthesiology, Zhongnan Hospital, Wuhan University, Wuhan 430071, China
Wang Yipeng
Department of Anesthesiology, Zhongnan Hospital, Wuhan University, Wuhan 430071, China
Zhang Zongze
Department of Anesthesiology, Zhongnan Hospital, Wuhan University, Wuhan 430071, China
Chen Kai
Department of Anesthesiology, Zhongnan Hospital, Wuhan University, Wuhan 430071, China
Wang Yanlin
Department of Anesthesiology, Zhongnan Hospital, Wuhan University, Wuhan 430071, China
Zhan Jia
Department of Anesthesiology, Zhongnan Hospital, Wuhan University, Wuhan 430071, China
