The suppressive effects of metformin on inflammatory response of otitis media model in human middle ear epithelial cells.
OBJECTIVE
Metformin is a well-known anti-diabetic agent, but its mechanism is unclear. Recently, many reports have described the anti-inflammatory effects of metformin on various cell types, including human vascular smooth muscle cells and endothelial cells. This study was designed to investigate the anti-inflammatory effect of metformin on lipopolysaccharide (LPS) induced inflammation in human middle ear epithelial cell lines (HMEECs).


METHODS
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.


RESULTS
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).


CONCLUSIONS
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.
Department of Otolaryngology-Head and Neck Surgery, Korea University College of Medicine, Seoul, South Korea.
Jae Gu
JG
Cho
Department of Otolaryngology-Head and Neck Surgery, Korea University College of Medicine, Seoul, South Korea.
Jae Jun
JJ
Song
Department of Otolaryngology-Head and Neck Surgery, Korea University College of Medicine, Seoul, South Korea.
June
J
Choi
Department of Otolaryngology-Head and Neck Surgery, Korea University College of Medicine, Seoul, South Korea.
Gi Jung
GJ
Im
Department of Otolaryngology-Head and Neck Surgery, Korea University College of Medicine, Seoul, South Korea.
Hak Hyun
HH
Jung
Department of Otolaryngology-Head and Neck Surgery, Korea University College of Medicine, Seoul, South Korea. Electronic address: koreaschae@gmail.com.
Sung Won
SW
Chae
D015153:Blotting, Western; D002460:Cell Line; D051546:Cyclooxygenase 2 / Q000187:drug effects / Q000378:metabolism; D016207:Cytokines / Q000187:drug effects* / Q000378:metabolism; D004432:Ear, Middle / Q000166:cytology*; D004847:Epithelial Cells / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D007004:Hypoglycemic Agents / Q000494:pharmacology*; D066298:In Vitro Techniques; D007249:Inflammation; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008687:Metformin / Q000494:pharmacology*; D010033:Otitis Media / Q000378:metabolism*; D018384:Oxidative Stress / Q000187:drug effects; D012333:RNA, Messenger / Q000187:drug effects* / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014409:Tumor Necrosis Factor-alpha / Q000187:drug effects / Q000378:metabolism
D016207:Cytokines; D007004:Hypoglycemic Agents; D008070:Lipopolysaccharides; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D014409:Tumor Necrosis Factor-alpha; D008687:Metformin; D051546:Cyclooxygenase 2
Human middle ear epitheilial cells; LPS; Metformin; Otitis media