Role of caveolin-1 in penehyclidine hydrochioride-induced inhibition of lipopolysaccharide-induced activation of TLR4/p38 MAPK signaling pathway in macrophages of mice

Objective To evaluate the role of caveolin-1 (Cav-1) in penehyclidine hydrochioride(PHC)-induced inhibition of lipopolysaccharide(LPS)-induced activation of Toll-like receptor 4 (TLR4)/p38 mitogen-activated protein kinase (p38 MAPK) signaling pathway in macrophages of mice. Methods Macrophages of mice were seeded in 6 cm diameter dishes (5 ml per dish) and divided into 5 groups (n=20 each) using a random number table: Scr-siRNA group (S group), Scr-siRNA+ LPS group (LPS group), Scr-siRNA+ LPS + PHC group (LPS+ P group), Cav-1-siRNA+ LPS group (C+ LPS group) and Cav-1-siRNA+ LPS+ PHC group (C+ LPS+ P group). Macrophages were transfected with Scr-siRNA for 24 h in S, LPS and LPS+ P groups and with Smart pool Cav-1 siRNAs for 24 h in C+ LPS and C+ LPS+ P groups.LPS at the final concentration of 1 μg/ml was added after the end of transfection, and macrophages were then incubated for 2 h in LPS, LPS+ P, C+ LPS and C+ LPS+ P groups.In LPS+ P and C+ LPS+ P groups, PHC at the final concentration of 2 μg/ml was added at 2 h of incubation with LPS, and macrophages were then incubated for 2 h. The expression of Cav-1 and TLR4 was detected by Western blot.The expression of p38 MAPK was determined by immunofluorescence.The level of tumor necrosis factor-alpha (TNF-α) in the culture medium was determined by enzyme-linked immunosorbent assay.The activity of myeloperoxidase (MPO) in macrophages was measured by colorimetry. Results Compared with group S, the expression of TLR4 and p38 MAPK was significantly up-regulated, and the concentration of TNF-α in the culture medium and activity of MPO were increased in the other four groups, the expression of Cav-1 was significantly down-regulated in LPS and C+ LPS groups (P 0.05). Compared with group LPS, the expression of Cav-1 was significantly up-regulated, the expression of TLR4 and p38 MAPK was down-regulated, and the concentration of TNF-α in the culture medium and activity of MPO were decreased in group LPS+ P, and the expression of Cav-1 was significantly down-regulated, the expression of TLR4 and p38 MAPK was up-regulated, and the concentration of TNF-α in the culture medium and activity of MPO were increased in group C+ LPS (P<0.05). Compared with group LPS+ P, the expression of Cav-1 was significantly down-regulated, the expression of TLR4 and p38 MAPK was up-regulated, and the concentration of TNF-α in the culture medium and activity of MPO were increased in group C+ LPS+ P (P<0.05). Compared with group C+ LPS, the expression of Cav-1 was significantly up-regulated, the expression of TLR4 and p38 MAPK was down-regulated, and the concentration of TNF-α in the culture medium and activity of MPO were decreased in group C+ LPS+ P (P<0.05). Conclusion The mechanism by which PHC inhibits LPS-induced activation of TLR4/p38 MAPK signaling pathway in macrophages is related to up-regulating Cav-1 expression in mice. Key words: Caveolin 1; Cholinergic antagonists; Lipopolysaccharides; Toll-like receptor 4; p38 Mitogen-activated protein kinases; Macrophages