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Abstract
Background
Sepsis still has a high mortality rate despite adequate supportive care. Newer therapeutic modalities have been developed but they have generally ended in failure. Recently, insulin was reported to have an anti-inflammatory effect by inhibiting the IκB/NF-κB pathway, and may have therapeutic potential in sepsis. However, the precise mechanism of the anti-inflammatory effect of insulin is unclear. This study examined the role of insulin in activating IκB/NF-κB in macrophage.
Methods
Raw 264.7 cells, a murine macrophage cell line, were used in this experiment. Western blotting using IκB Ab and phosphor-specific IκB Ab was performed to evaluate the degradation and phosphorylation of IκB cells. For the IκB Kinase (IKK) activity, an immune complex kinase assay was performed. The level of interleukin-6 (IL-6) was measured by ELISA to determine the level of proinflammatory cytokine.
Results
IκBα degradation began 30 min after lipopolysaccharide (LPS) treatment. However, an insulin pretreatment suppressed the IκBα degradation caused by the LPS treatment. The phosphorylation of IκBα and IKK activity was also inhibited by the insulin pretreatment. Finally, the insulin pretreatment showed a tendency to suppress the induction of IL-6 by LPS.
Figures and Tables
Figure 1
Insulin suppresses lipopolysaccharide (LPS)-induced IκBα degradation in macrophage cell line. (A) Raw 264.7 cells were pre-treated with 10 µg/mL of insulin for 6 hours and then treated with 1 µg/mL of LPS for 0, 0.5, 2, 4 hours. (B) Raw 264.7 cells were pre-treated with 10 µg/mL of insulin for 0, 2, 4, 24 hours and then treated with 1 µg/mL of LPS for 30 minutes. IκBα and actin expressions were evaluated by Western blot. Results are representative of three distinct experiments.
Figure 2
Insulin suppresses lipopolysaccharide (LPS)-induced IκBα phosphorylation in macrophage cell line. Raw 264.7 cells were pre-treated with 10 µg/mL of insulin for 6 hours and then treated with 1 µg/mL of LPS for 0, 5, 10, 30 minutes. Phosphorylated IκBα and actin expressions were evaluated by Western blot. Results are representative of three distinct experiments.
Figure 3
Insulin attenuates lipopolysaccharide (LPS)-induced p65 in macrophage cell line. Raw 264.7 cells were pre-treated with 10 µg/mL of insulin for 6 hours and then treated with 1 µg/mL of LPS for 0, 10, 30 minutes. Phosphorylated p65 and actin expressions were evaluated by Western blot. Results are representative of three distinct experiments.
Figure 4
Insulin inhibits lipopolysaccharide (LPS)-induced IκB kinase (IKK) activation in macrophage cell line. Raw 264.7 cells were pre-treated with 10 µg/mL of insulin for 6 hours and then treated with 1 µg/mL of LPS for 5 minutes. IKK activity was evaluated by in vitro immune complex kinase assay. Results are representative of three distinct experiments.
Figure 5
Insulin attenuates lipopolysaccharide (LPS)-induced interleukin-6 (IL-6) secretion dose-dependently in macrophage cell line. Raw 264.7 cells were pre-treated with 0, 10, 20, 30, 100 µg/mL of insulin for 6 hours and then treated with 1 µg/mL of LPS for 24 hours. Levels of secreted IL-6 were evaluated by ELISA. Results are representative of three distinct experiments.
References
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