Role of PKCα/HO-1 signaling pathway in endotoxin-induced damage to alveolar macrophages of rats: the relationship with mitofusin-1

Abstract

Objective To evaluate the role of protein kinase C α (PKCα)/heme oxygenase-1 (HO-1) signaling pathway in endotoxin-induced damage to alveolar macrophages and the relationship with mitofusin-1 (Mfn1) in rats. Methods Rat alveolar macrophages NR8383 cells cultured in vitro were seeded in 96-well plates at a density of 1×104 cells/ml.NR8383 cells were divided into 5 groups (n=15 each) using a random number table: control group (group C), endotoxin challenge model group (group E), PKCα inhibitor Go6976 group (group G), PKCα agonist PMA group (group P) and dimethyl sulfoxide group (group D). NR8383 cells were stimulated with 10 μg/ml lipopolysaccharide (LPS) to establish the model of endotoxin challenge in alveolar macrophages.In G, P and D groups, cells were pretreated with 5 μmol/L Go6976, 100 nmol/L PMA and 0.1% dimethyl sulfoxide, respectively, for 30 min starting from 30 min before stimulation with LPS, and 10 μg/ml LPS was then given.The cells were collected after 24 h of incubation for measurement of malondialdehyde (MDA) and reactive oxygen species (ROS) contents, superoxide dismutase (SOD) activity and expression of PKCα, HO-1 and Mfn1 protein and mRNA (by fluorescent quantitative polymerase chain reaction or Western blot). Results Compared with group C, MDA and ROS contents were significantly increased, the SOD activity was decreased, the expression of PKCα and HO-1 protein and mRNA was up-regulated, and the expression of Mfn1 protein and mRNA was down-regulated in E, G, P and D groups (P 0.05). Conclusion Promotion of Mfn1 expression following PKCα/HO-1 signaling pathway activation is the endogenous protective mechanism of endotoxin-induced damage to alveolar macrophages of rats. Key words: Protein kinase C-alpha; Heme oxygenase-1; Endotoxins; Mitochondrial proteins; Membrane fusion proteins