Ghrelin inhibits interleukin-8 production induced by hydrogen peroxide in A549 cells via NF-kappaB pathway.
Ghrelin, a novel growth hormone-releasing peptide, can influence appetite and induce positive energy balances. Previous studies have reported that ghrelin ameliorated inflammatory responses of the heart, liver and pancreas. We examined whether ghrelin inhibits the proinflammatory cytokine interleukin-8 production induced by hydrogen peroxide (H2O2) in human lung epithelia cell line A549 and which mechanism is related with this effect of ghrelin. A549 cells were preincubated with vehicle or ghrelin (0.1 to 1000 ng/mL) in a concentration-dependent manner and then H2O2 (0 to 50 microM) was added. The interleukin-8 released by A549 in the medium was determined by ELISA, the mRNA expressions of interleukin-8 and ghrelin receptor were detected by RT-PCR. We also examined the phosphorylation of NF-kappaB/p65 protein and the degradation of inhibitory protein-kappaB (I-kappaB) in A549 by western blot analysis, the NF-kappaB DNA-binding activity by electrophoretic mobility shift assay, and then detected the generation of reactive oxygen species (ROS) in A549 by nitroblue tetrazolium reduction assay. Cells treated with H2O2 (50 microM) exhibited significantly higher interleukin-8 production and ghrelin receptor mRNA expression compared with cells treated with vehicle alone (P < 0.05). Ghrelin inhibited H2O2-induced interleukin-8 production by A549 at both mRNA and protein levels in a concentration-dependent manner (P < 0.05). Moreover, ghrelin attenuated H2O2-triggered NF-kappaB activation dependent on I-kappaB degradation dose-dependently in A549, but the intracellular ROS level after application of H2O2 was not affected by ghrelin (1000 ng/mL). Together, these results suggest that ghrelin inhibits H2O2-induced interleukin-8 production in A549 cells by targeting on NF-kappaB pathway, but not by directly scavenging intracellular ROS.
Hou
Yan
Y
Division of Pulmonary Diseases, State Key Laboratory of Biotherapy of China, West China Hospital of Sichuan University, Chengdu, Sichuan 610041, PR China.
An
Jing
J
Hu
Xiao-Rong
XR
Sun
Bei-Bei
BB
Lin
Jian
J
Xu
Dan
D
Wang
Tao
T
Wen
Fu-Qiang
FQ
D000894:Anti-Inflammatory Agents, Non-Steroidal; D000975:Antioxidants; D015153:Blotting, Western; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D024202:Electrophoretic Mobility Shift Assay; D004797:Enzyme-Linked Immunosorbent Assay; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism; D054439:Ghrelin / Q000494:pharmacology*; D006801:Humans; D006861:Hydrogen Peroxide / Q000037:antagonists & inhibitors* / Q000494:pharmacology*; D020672:I-kappa B Proteins / Q000096:biosynthesis; D007202:Indicators and Reagents; D016209:Interleukin-8 / Q000096:biosynthesis*; D016328:NF-kappa B / Q000502:physiology*; D016877:Oxidants / Q000494:pharmacology*; D018384:Oxidative Stress; D010766:Phosphorylation; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D017382:Reactive Oxygen Species / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects*; D051996:Transcription Factor RelA / Q000096:biosynthesis / Q000235:genetics
D000894:Anti-Inflammatory Agents, Non-Steroidal; D000975:Antioxidants; D054439:Ghrelin; D020672:I-kappa B Proteins; D007202:Indicators and Reagents; D016209:Interleukin-8; D016328:NF-kappa B; D016877:Oxidants; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D051996:Transcription Factor RelA; D006861:Hydrogen Peroxide