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Role of PI3K/Akt signaling pathway in carbon monoxide-induced up-regulation of mitofusin-1 expression in endotoxin-challenged rat alveolar macrophages

Zhen Li, Jia Shi, Jianbo Yu, Dan Wang, Shuan Dong, Lirong Gong, Yuan Zhang

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Abstract

Objective To evaluate the role of phosphatidylinositol 3-kinase/serine-threonine kinase(PI3K/Akt)signaling pathway in carbon monoxide(CO)-induced up-regulation of the mitofusin-1(Mfn1)expression in endotoxin-challenged rat alveolar macrophages. Methods Alveolar macrophages obtained from the rats aged 12-20 weeks were subcultured and seeded in 96 well plates at a density of 4×104 cells/ml.After being cultured for 24 h, the cells were divided into 4 groups(n=10 each)using a random number table: control group(group C), endotoxin group(group L), lipopolysaccharide(LPS)+ CO-releasing molecule-2(CORM-2)group(group L+ C)and LPS+ CORM-2+ PI3K inhibitor LY294002 group(group L+ C+ LY). Cells were cultured normally in group C. Cells were stimulated by using LPS 10 μg/ml in L, L+ C and L+ C+ LY groups.In group L+ C, CORM-2 100 μmol was given at 1 h before stimulation with LPS.In group L+ C+ LY, LY294002 20 μg and CORM-2 100 μmol were given at 1.5 and 1.0 h before stimulation with LPS, respectively.The cells were continuously incubated for 24 h after the end of treatment.The concentrations of tumor necrosis factor-α(TNF-α)and interleukin-10(IL-10)in the supernatant were determined by enzyme-linked immunosorbent assay.The expression of PI3K, phosphorylated Akt(p-Akt)and Mfn1 in cells was measured by real-time polymerase chain reaction and Western blot. Results Compared with group C, the concentration of TNF-α was significantly increased, and the IL-10 concentration was decreased in L, L+ C and L+ C+ LY groups(P<0.05). Compared with group L, the concentration of IL-10 was significantly increased, the TNF-α concentration was decreased, and the expression of PI3K, p-Akt and Mfn1 was up-regulated in group L+ C(P<0.05). Compared with group L+ C, the concentration of IL-10 was significantly decreased, the TNF-α concentration was increased, and the expression of PI3K, p-Akt and Mfn1 was down-regulated in group L+ C+ LY(P<0.05). Conclusion PI3K/Akt signaling pathway is involved in CO-induced up-regulation of Mfn1 expression in endotoxin-challenged rat alveolar macrophages. Key words: Carbon monoxide; Mitochondrial proteins; 1-Phosphatidylinositol 3-kinase; Protein-serine-threonine kinases

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Objective To evaluate the role of phosphatidylinositol 3-kinase/serine-threonine kinase(PI3K/Akt)signaling pathway in carbon monoxide(CO)-induced up-regulation of the mitofusin-1(Mfn1)expression in endotoxin-challenged rat alveolar macrophages. Methods Alveolar macrophages obtained from the rats aged 12-20 weeks were subcultured and seeded in 96 well plates at a density of 4×104 cells/ml.After being cultured for 24 h, the cells were divided into 4 groups(n=10 each)using a random number table: control group(group C), endotoxin group(group L), lipopolysaccharide(LPS)+ CO-releasing molecule-2(CORM-2)group(group L+ C)and LPS+ CORM-2+ PI3K inhibitor LY294002 group(group L+ C+ LY). Cells were cultured normally in group C. Cells were stimulated by using LPS 10 μg/ml in L, L+ C and L+ C+ LY groups.In group L+ C, CORM-2 100 μmol was given at 1 h before stimulation with LPS.In group L+ C+ LY, LY294002 20 μg and CORM-2 100 μmol were given at 1.5 and 1.0 h before stimulation with LPS, respectively.The cells were continuously incubated for 24 h after the end of treatment.The concentrations of tumor necrosis factor-α(TNF-α)and interleukin-10(IL-10)in the supernatant were determined by enzyme-linked immunosorbent assay.The expression of PI3K, phosphorylated Akt(p-Akt)and Mfn1 in cells was measured by real-time polymerase chain reaction and Western blot. Results Compared with group C, the concentration of TNF-α was significantly increased, and the IL-10 concentration was decreased in L, L+ C and L+ C+ LY groups(P<0.05). Compared with group L, the concentration of IL-10 was significantly increased, the TNF-α concentration was decreased, and the expression of PI3K, p-Akt and Mfn1 was up-regulated in group L+ C(P<0.05). Compared with group L+ C, the concentration of IL-10 was significantly decreased, the TNF-α concentration was increased, and the expression of PI3K, p-Akt and Mfn1 was down-regulated in group L+ C+ LY(P<0.05). Conclusion PI3K/Akt signaling pathway is involved in CO-induced up-regulation of Mfn1 expression in endotoxin-challenged rat alveolar macrophages. Key words: Carbon monoxide; Mitochondrial proteins; 1-Phosphatidylinositol 3-kinase; Protein-serine-threonine kinases

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Available abstract

Objective To evaluate the role of phosphatidylinositol 3-kinase/serine-threonine kinase(PI3K/Akt)signaling pathway in carbon monoxide(CO)-induced up-regulation of the mitofusin-1(Mfn1)expression in endotoxin-challenged rat alveolar macrophages. Methods Alveolar macrophages obtained from the rats aged 12-20 weeks were subcultured and seeded in 96 well plates at a density of 4×104 cells/ml.After being cultured for 24 h, the cells were divided into 4 groups(n=10 each)using a random number table: control group(group C), endotoxin group(group L), lipopolysaccharide(LPS)+ CO-releasing molecule-2(CORM-2)group(group L+ C)and LPS+ CORM-2+ PI3K inhibitor LY294002 group(group L+ C+ LY). Cells were cultured normally in group C. Cells were stimulated by using LPS 10 μg/ml in L, L+ C and L+ C+ LY groups.In group L+ C, CORM-2 100 μmol was given at 1 h before stimulation with LPS.In group L+ C+ LY, LY294002 20 μg and CORM-2 100 μmol were given at 1.5 and 1.0 h before stimulation with LPS, respectively.The cells were continuously incubated for 24 h after the end of treatment.The concentrations of tumor necrosis factor-α(TNF-α)and interleukin-10(IL-10)in the supernatant were determined by enzyme-linked immunosorbent assay.The expression of PI3K, phosphorylated Akt(p-Akt)and Mfn1 in cells was measured by real-time polymerase chain reaction and Western blot. Results Compared with group C, the concentration of TNF-α was significantly increased, and the IL-10 concentration was decreased in L, L+ C and L+ C+ LY groups(P<0.05). Compared with group L, the concentration of IL-10 was significantly increased, the TNF-α concentration was decreased, and the expression of PI3K, p-Akt and Mfn1 was up-regulated in group L+ C(P<0.05). Compared with group L+ C, the concentration of IL-10 was significantly decreased, the TNF-α concentration was increased, and the expression of PI3K, p-Akt and Mfn1 was down-regulated in group L+ C+ LY(P<0.05). Conclusion PI3K/Akt signaling pathway is involved in CO-induced up-regulation of Mfn1 expression in endotoxin-challenged rat alveolar macrophages. Key words: Carbon monoxide; Mitochondrial proteins; 1-Phosphatidylinositol 3-kinase; Protein-serine-threonine kinases

Key concepts: PI3K/AKT/mTOR pathway, Protein kinase B, Molecular biology, Chemistry, Lipopolysaccharide, LY294002, Western blot, Tumor necrosis factor alpha

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Role of PI3K/Akt signaling pathway in carbon monoxide-induced up-regulation of mitofusin-1 expression in endotoxin-challenged rat alveolar macrophages — Research Paper | ScholarLens