ABSTRACT
Objective To determine whether p38 mitogen-activated protein kinase (p38MAPK) signaling pathway is involved in cerebral fractalkine-induced hyperalgesia in mice.Methods Two hundred and twenty-five male Kunming mice weighing 30-40 g were randomly divided into 4 groups:group control ( group C,n =55 ) ;group fractalkine (group F,n =60); group anti-CX3CR1 + fractalkine (group CF,n =55) and group SB203580 (p38MAPK inhibitor) + fractalkine (group SF,n =55).Fractalkine 100 ng was injected into cerebral lateral ventricle (i.c.v.) in groups F,CF and SF.Anti-CX3CR1 1 μg and SB203580 1 μg were injected i.c.v.at 1 h before fractalkine injection in groups CF and SF respectively.Paw withdrawal latency to a thermal nociceptive stimulus (PWL) was measured at 30 min before the drugs were injected into cerebral lateral ventricle and 30,60,120 and 240 min after fractalkine injection.Five animals were sacrificed after PWL measurement at each time point and their brains were removed for determination of phosphorylated p38MAPK protein expression (by Western blot analysis).Five animals were sacrificed at 30 min before the drugs were injected into cerebral lateral ventricle and 6,12 and 24 h after fractalkine injection for determination of IL-1β and TNF-α contents in the brain (by ELISA) in all the 4 groups.In group F 5 animals were sacrificed at 4 h after fractalkine injection for determination of action of fractalkine on microglia or astrocyte (by immunofluorescence).Results Fractalkine i.c.v.injection significantly reduced PWL and increased phosphorylated 38MAPK,IL-1β and TNF-α levels in group F as compared with group C.Pretreatment with anti-CX3CR1 or SB203580 significantly decreased fractalkine-induced hyperalgesia and phosphorylated-p38MAPK,IL-1β and TNF-α levels in groups CF and SF as compared with group F.Fractalkine was localized at microglia.Conclusion p38MAPK signal transduction pathway is involved in cerebral fractalkine-induced hyperalgesia in mice.
ABSTRACT
Objective To evaluate the role of inositol triphosphate receptor (IP3 R) in the fractalkine-induced activation of p38MAPK signaling pathway in BV-2 microglial cells.Methods BV-2 microglial cells were seeded in 3.5 cm diameter dishes (5 ml/dish),50 ml culture flasks (8 ml/flask) or 24-well plates (1 ml/hole) with a density of 1 × 105/ml and randomly divided into 5 groups (n =25 each) ∶ control group (group C),fractalkinegroup (group F),CX3C chemokine receptor 1 (CX3CR1) antibody anti-CX3CR1 + fractalkine group (group CF),IP3R antagonist 2-APB + fractalkine group (group AF) and p38 mitogen-activated protease (p38MAPK) inhibitor SB203580 + fractalkine group (group SF).Fractalkine 10 nmol/L was added to the culture medium in groups F,CF,AF and SF.The anti-CX3CR1 15 μmol/L,2-APB 50 μmol/L and SB203580 10 μmol/L were added to the culture medium in groups CF,AF and SF,respectively,1 h before addition of fractalkine.The cells were then cultured for 24 h.The intracellular Ca2+ concentration ([Ca2+]i) was measured during the 10 min incubation with fractalkine.The phosphorylation of p38MAPK was measured at 0,30,60,120 and 240 min of incubation with fractalkine.The concentrations of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in theculture medium were determined at 24 h of incubation with fractalkine.Results Compared with group C,[Ca2+]i,and the phosphorylation of p38MAPK and concentrations of IL-1β and TNF-α were significantly increased in groups F,CF,AF and SF (P ＜ 0.05).[Ca2+]i was significant lower in groups AF and CF and phosphorylation of p38MAPK and concentrations of IL-1β and TNF-α were significantly lower in groups CF,AF and SF than in group F (P ＜ 0.05).Conclusion IP3 R is involve in the fractalkine-induced activation of p38MAPK signaling pathway in BV-2 microglial cells.