Neuroprotective effects of cyclooxygenase-2 inhibitor celecoxib against toxicity of LPS-stimulated macrophages toward motor neurons.
OBJECTIVE
To establish an in vitro injured motor neuronal model and investigate the neuroprotective effects and possible mechanism of celecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor, on this model.


METHODS
After macrophages were stimulated with lipopolysaccharide (LPS)+interferon-gamma (IFN-gamma) in the presence or absence of celecoxib for 24 h, the cell-free supernatant of LPS-stimulated macrophages was transferred to the culture of NSC34 cells. Viability of NSC34 cells was assessed by MTT assay after a further 24 h and 72 h incubation. After macrophages were stimulated by LPS+IFN-gamma for 12 h or 24 h, the release of prostaglandin E2 (PGE2), nitric oxide (NO), reactive oxygen species (ROS), tumor necrosis factor alpha (TNF-alpha) and interleukin-1beta (IL-1beta) from macrophages was measured by radioimmunoassay, Griess assay, fluorescence assay and enzyme-linked immunosorbent assay, respectively. The mRNA levels of COX-2, inducible nitric oxide synthase (iNOS), TNF-alpha and IL-1beta in macrophages were determined by reverse transcription-polymerase chain reaction after macrophages were stimulated for 6 h and 12 h.


RESULTS
The supernatant of LPS-stimulated mouse macrophages induced the death of NSC34 cells and celecoxib protected the NSC34 cells against this toxicity. The LPS-induced increases in the release of PGE2, NO, TNF-alpha and IL-1beta from macrophages were attenuated by pre-treatment with celecoxib. However, celecoxib showed no effect on the ROS levels upregulated by LPS+IFN-gamma in the macrophage supernatant. The mRNA levels of COX-2, iNOS, TNF-alpha and IL-1beta were increased in LPS-activated macrophages and, except COX-2, reduced by pre-treatment with celecoxib.


CONCLUSIONS
An in vitro injured motor neuronal model was established by using the toxicity of LPS-stimulated mouse macrophages toward motor neuronal NSC34 cells. In this model, celecoxib exerted neuroprotective effects on motor neurons via an inhibition of the neurotoxic secretions from activated macrophages.
Huang
Yong
Y
Department of Pharmacology, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Graduate School of Chinese Academy of Sciences, Shanghai 201203, China.
Liu
Jing
J
Wang
Li-zhen
LZ
Zhang
Wei-yu
WY
Zhu
Xing-zu
XZ
D000818:Animals; D000068579:Celecoxib; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D018920:Coculture Techniques; D051546:Cyclooxygenase 2 / Q000235:genetics; D052246:Cyclooxygenase 2 Inhibitors / Q000494:pharmacology*; D015232:Dinoprostone / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005260:Female; D007371:Interferon-gamma / Q000494:pharmacology; D007375:Interleukin-1 / Q000235:genetics; D008070:Lipopolysaccharides / Q000633:toxicity*; D008262:Macrophage Activation / Q000187:drug effects; D017737:Macrophages, Peritoneal / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D009046:Motor Neurons / Q000166:cytology / Q000187:drug effects*; D018696:Neuroprotective Agents / Q000494:pharmacology; D009569:Nitric Oxide / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000235:genetics; D011720:Pyrazoles / Q000494:pharmacology*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011863:Radioimmunoassay; D017382:Reactive Oxygen Species / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013449:Sulfonamides / Q000494:pharmacology*; D013997:Time Factors; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics
D052246:Cyclooxygenase 2 Inhibitors; D007375:Interleukin-1; D008070:Lipopolysaccharides; D018696:Neuroprotective Agents; D011720:Pyrazoles; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D013449:Sulfonamides; D014409:Tumor Necrosis Factor-alpha; D009569:Nitric Oxide; D007371:Interferon-gamma; D052247:Nitric Oxide Synthase Type II; D051546:Cyclooxygenase 2; D000068579:Celecoxib; D015232:Dinoprostone