Substance P influenced gelatinolytic activity via reactive oxygen species in human pulp cells.
OBJECTIVE
To investigate the effects of substance P (SP) on gelatinolytic activity of matrix metalloproteinases (MMPs) in human pulp cells.


METHODS
Human dental pulp cells were isolated and cultured. Subconfluent cells, between the third and sixth passages, were maintained under serum deprivation for 18 h followed by the treatment of varying doses of SP (1 pmol L(-1), 100 pmol L(-1), 10 nmol L(-1), 1 micromol L(-1) and 100 micromol L(-1)). Conditioned media were then subjected to gelatin zymography using 8% sodium dodecyl sulphate polyacrylamide gel electrophoresis minigels containing 1.5 g L(-1) gelatin. The effect of SP on intracellular reactive oxygen species (ROS) was also examined by confocal microscopy. ROS scavenger N-Acetyl-L-cysteine (NAC, 5 mmol L(-1)) was utilized to evaluate the roles of ROS pathway in mediating the impact of SP on cellular gelatinolytic activity. Data were analysed using analysis of variance with Bonferroni correction for multiple comparisons or an unpaired Student's t-test.


RESULTS
Substance P, at levels above 1 micromol L(-1), remarkably enhanced MMP-2 activity reflected by the band migrating at 66 kDa (P < 0.05). A gelatinolytic band at approximately 44 kDa appeared to be intensified in a SP dose-dependent manner. In addition, it was demonstrated that SP could induce ROS production in pulp cells and ROS scavenger NAC was further found to significantly reduce MMP-2 activity (P < 0.05), as well as other bands of gelatinolytic proteinases.


CONCLUSIONS
Substance P can influence gelatinolytic activity in human pulp cells via ROS pathway.
Wang
F-M
FM
State Key Laboratory of Oral Diseases, Sichuan University, Chengdu, China.
Hu
T
T
Cheng
R
R
Tan
H
H
Zhou
X-D
XD
D000111:Acetylcysteine / Q000494:pharmacology; D002478:Cells, Cultured; D017077:Culture Media, Conditioned; D003782:Dental Pulp / Q000187:drug effects* / Q000201:enzymology; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D016166:Free Radical Scavengers / Q000494:pharmacology; D018093:Gelatinases / Q000008:administration & dosage / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D006801:Humans; D020778:Matrix Metalloproteinase 2 / Q000187:drug effects; D061965:Matrix Metalloproteinase Inhibitors; D018613:Microscopy, Confocal; D017382:Reactive Oxygen Species / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D013373:Substance P / Q000008:administration & dosage / Q000494:pharmacology*
D017077:Culture Media, Conditioned; D016166:Free Radical Scavengers; D061965:Matrix Metalloproteinase Inhibitors; D017382:Reactive Oxygen Species; D013373:Substance P; D018093:Gelatinases; C522361:MMP2 protein, human; D020778:Matrix Metalloproteinase 2; D000111:Acetylcysteine