The effect of pretreatment by metformin (0, 1, 2, 4 mM) was evaluated by the inflammatory response in the HMEECs exposed to LPS (10 ng/ml). For verifying the suppression effect of metformin on the inflammatory cytokines, tumor necrosis factor-alpha (TNF-α) was evaluated by real-time polymerase chain reaction, and COX-2 protein was assessed by western blotting. Intracellular reactive oxygen species (ROS) was measured using 2′, 7′-dichlorofluorescein diacetate (DCFHDA) fluorocytometer.
Stimulation by LPS 10 ng/ml concentration showed 12.4 folds increase the expression of TNF-α mRNA compared to control on HMEECs. Pretreatment of metformin dose dependently suppressed the expression of TNF-α mRNA induced by LPS (2 mM, p = 0.03). The amount of COX-2 protein production was significantly decreased by metformin pretreatment (4 mM, p = 0.01). The production of ROS was decreased significantly by pretreatment of metformin (p = 0.03).
These findings suggest that the inflammatory response and oxidative stress induced by LPS could be suppressed by metformin in HMEECs. Therefore, metformin may have a therapeutic potential for the treatment of the otitis media.