Role of PI3K/Akt signaling pathway in carbon monoxide-induced up-regulation of mitochondrial fusion proteins in endotoxin-challenged alveolar epithelial cells of rats

Abstract

Objective To evaluate the role of 1-phosphatidylinositol 3-kinase/serine threonine kinase(PI3K/Akt)signaling pathway in carbon monoxide(CO)-induced up-regulation of mitochondrial fusion proteins in endotoxin-challenged alveolar epithelial cells of rats. Methods The rat alveolar epithelial cells were cultured in a 5% CO2 cell culture incubator at 37 ℃ with F12K complete medium containing 10% fetal bovine serum and 1% green chain double antibody, and divided into 10 groups (n=5 each) according to the random number table method: control group (C group), endotoxin group (L group), lipopolysaccharide (LPS) plus exogenous CO-releasing agent CO-releasing-molecule-2 (CORM-2) group (L+ CO group), LPS plus PI3K inhibitor LY294002 group (L+ LY group), LPS plus CORM-2 plus LY294002 group (L+ CO+ LY group), LPS plus inactive CO releasing agent iCORM-2 group (L+ iCO group), LPS plus dimethyl sulfoxide (DMSO) group (L+ D group), CORM-2 group (CO group), LY294002 group (LY group) and CORM-2 plus LY294002 group (CO+ LY group). LPS 10 μg/ml was added to the culture medium in group L. CORM-2 100 μmol/L was added to the culture medium and 1 h later 10 μg/ml LPS was added in L+ CO group.LY294002 25 μmol/L was added to the medium, and 1 h later LPS 10 μg/ml was added in L+ LY group.In L+ CO+ LY group, 25 μmol/L LY294002 was added to the culture medium, CORM-2 100 μmol/L was added 1 h later, and then LPS 10 μg/ml was added 1 h later.iCORM 100 μmol/L was added to the culture medium and 1 h later LPS 10 μg/ml was added in L+ iCO group.In L+ D group, the equal concentration of DMSO was added to the culture medium and 1 h later LPS 1 μg/ml was added.CORM-2 100 μmol/L was added to the culture medium in CO group.LY294002 25 μmol/L was added to culture medium in LY group.LY294002 25 μmol/L was added to the culture medium, and 1 h later CORM-2 100 μmol/L was added in CO+ LY group.Cells were harvested after 24 h of incubation for measurement of the malondialdehyde (MDA) content, superoxide dismutase (SOD) activity and expression of heme oxygenase-1 (HO-1), phosphorylated Akt(p-Akt), mitochondrial fusion proteins mitofusion 1(Mfn1), Mfn2 and optic atrophy 1(OPA1) by Western blot. Results Compared with group C, the MDA content was significantly increased and SOD activity was decreased in L, L+ CO, L+ LY, L+ CO+ LY, L+ iCO, L+ D groups, and the expression of HO-1, Mfn1, Mfn2, OPA1 protein and p-Akt was significantly up-regulated in group L (P<0.05). Compared with group L, MDA content was significantly decreased and SOD activity was increased in group L+ CO, MDA content was significantly increased and SOD activity was decreased in group L+ LY , the expression of HO-1, Mfn1, Mfn2, OPA1 protein and p-Akt was significantly up-regulated in group L+ CO, and the expression of HO-1, Mfn1, Mfn2, OPA1 protein and p-Akt was significantly down-regulated in group L+ LY (P<0.05). Compared with group L+ CO, the MDA content was significantly increased, SOD activity was decreased, and the expression of HO-1, Mfn1, Mfn2, OPA1 and p-Akt was down-regulated in group L+ CO+ LY(P<0.05). Conclusion The mechanism by which CO up-regulates the expression of mitochondrial fusion proteins in endotoxin-challenged alveolar epithelial cells is related to activating PI3K/Akt signaling pathway in rats. Key words: 1-phosphoinositide 3-kinase; Protein-serine-threonine kinases; Carbon monoxide; Endotoxins; Pulmonary alveoli; Epithelial cells; Mitochondrial proteins