Yong Huang1, Jing Liu, Li-zhen Wang, Wei-yu Zhang, Xing-zu Zhu. 1. 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.
Abstract
AIM: 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. CONCLUSION: 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.
AIM: 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. CONCLUSION: 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.
Authors: Jens Neumann; Steven Sauerzweig; Raik Rönicke; Frank Gunzer; Klaus Dinkel; Oliver Ullrich; Matthias Gunzer; Klaus G Reymann Journal: J Neurosci Date: 2008-06-04 Impact factor: 6.167