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Objective To evaluate the role of spinal CX3C chemokine receptor 1 (CX3CR1) in inflammatory pain and the relationship with calmodulin (CaM)-calmodulin-dependent protein kinaseⅡ(CaMKⅡ) signaling pathways in mice.Methods Ninety-six pathogen-free healthy male C57BL6 mice,weighing 25-27 g,were divided into 3 groups using a random number table:control group (group C,n=30),inflammatory pain group (group IP,n=36) and CX3CR1 antagonist group (group CA,n=30).Inflammatory pain was induced by injecting complete Freund′s adjuvant (CFA) 50 μl into the plantar surface of right hind paws in IP and CA groups,while the equal volume of normal saline was given instead in group C.In group CA,CX3CR1 antagonist (diluted to 1 μg/5 μl in phosphate buffer solution) was intrathecally injected at 1 h before CFA injection.The thermal paw withdrawal latency (TWL) was measured at 30 min before CFA injection (T0) and 30 min,1 h,2 h and 4 h after CFA injection (T2-4).The animals were then sacrificed,and the spinal cord was removed for determination of the expression of phosphorylated CaMKⅡ (p-CaMKⅡ),phosphorylated cyclic adenosine monophosphate response element-binding protein (p-CREB) and c-fos (by Western blot) and expression of CaMKⅡ,CREB and c-fos mRNA (using real-time polymerase chain reaction).Immunofluorescence was used to determine that p-CAMKⅡ was expressed in microglia.Results Compared with group C,the TWL was significantly shortened at T2-4,and the expression of p-CaMKⅡ,p-CREB and c-fos protein and mRNA was up-regulated at T1-4 in IP and CA groups (P<0.05).Compared with group IP,the TWL was significantly prolonged at T2-4,and the expression of p-CaMKⅡ,p-CREB and c-fos protein and mRNA was down-regulated at T1-4 in group CA (P<0.05).p-CaMKⅡ was co-expressed with the microglial specific biomarker.Conclusion CX3CR1 is involved in the development and maintenance of inflammatory pain through activating CaM-CaMKⅡsignaling pathways in mice.
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Objective To investigate the role of CaM/CaMK-Ⅱ signaling pathways in inflammatory pain in mice.Methods Sixty male C57BL6 mice,weighing 25-27 g,were randomly divided into 3 groups (n =20):control group (group C),complete freunds adjuvant (CFA) group (group F) and KN-93+CFA group (group KF).Saline 50 μl were injected into the right side of the claw in group C.CFA 50 μl were injected into the right claw foot for the preparation of inflammatory pain models in group F.KN-93 45 nmol was injected i.c.v.30 min before CFA injection in group KF.The thermal withdrawal latency (TWL) were measured 30 min before injection,1 h and 4 h after injection.The protein expressions of CaMK-Ⅱ,c-fos and CREB in the spinal cord were measured at above time by Western blot.Results Compared with group C,TWL were lower in groups F and KF 1 h and 4 h after injection (P＜0.05).Compared with groups F,TWL in group KF were higher 1 h and 4 h after injection (P＜0.05).Compared with group C,the protein expressions of p-CaMK-Ⅱ,p-CREB,e-fos and mRNA expression of CaMK-Ⅱ,CREB,c-fos were higher in group F and KF 1 h and 4 h after injection (P＜0.05).Compared with group F,the protein expression of p-CaMK-Ⅱ,p-CREB,c-fos and mRNA expressions of CaMK-Ⅱ,CREB,c-fos in group KF were lower 1 h and 4 h after injection (P＜0.05).Conclusion CaM/CaMK-Ⅱ signaling pathways involved in inflammatory pain in mice.
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Objective To investigate the role of calcineurin(CaN)in inflammatory pain in rats.Methods Seventy-five male Harlan-Sprague-Dawley rats,weighting of 200-300 g were randomly divided into 3 groups (n=25): group control (group C),group CFA (complete Freunds adjuvant) (group F) and group CaN+CFA (group NF).100 μl CFA were injected on the right hind claw preparaing for inflammatory pain models in groups F and NF,100 μl saline were injected on the right hind claw in group C.CaN 10 U was intracerebroventricular injected 1 d before CFA injection in group NF.Paw withdrawal thermal latency (PWTL) were measured in 30 min prior to (T0),0.5 h (T1),1 h (T2),2 h (T3) and 4 h (T4) after injection.The expression of CaN and nuclear factor kappa B (NF-κB),IL-1β,TNF-α and IL-10 in spinal cord were measured at each time point.Results The PWTL was significantly shorter at T2-T4 in group F,at T3,T4 in group NF than that at T0and in group C (P<0.05);The PWTL at T2-T4 in group NF was significantly longer than that in group F (P<0.05).CaN protein expression in spinal cord at T1-T4 in group F,at T2-T4 in group NF was significantly lower than that of T0 and in the group C,NF-κB p65 protein expression was significantly higher than that of T0 and in the group C (P<0.05).CaN gene and IL-10 protein content at T2-T4 in groups F and NF were significantly lower than that of group C and at T0,NF-κB gene and IL-1β,TNF-α protein content was significantly higher than that of group C and at T0 (P<0.05).CaN protein and CaN gene expression,IL-10 protein content in spinal cord tissue at T1-T4in group NF was significantly higher than that of group F,NF-κB p65 protein and NF-κB gene expression and contents of IL-1β,TNF-α protein were significantly lower than that of group F (P<0.05).Conclusion CaN adjusts pro-inflammatory and anti-inflammatory cytokines by reducing NF-κB and inhibiting the process of inflammatory pain in rats.
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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.
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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.
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Objective To evaluate the role of autophagy in HL-1 cardiomyocyte injury induced by lipopolysaccharide( LPS).Methods Primary cultured HL-1 cardiomyocytes were randomly divided into 4 groups ( n =15each): normal control group( group C),LPS group,rapamycin( a autophagy inducer) group( group R) and 3-MA(a autophagy inhibitor) group.In group C cardiomyocytes were cultured continuously for 24 h.In group LPS cardiomyocytes were incubated with LPS (final concentration 1 μg/ml) for 24 h.In groups R and 3-MA,rapamycin and 3-MA was given 48 h before LPS (final concentration 1 μg/ml) incubation with final concentration of 0.2 μg/ml and 10 mmol/L respectively.The lipidated microtubule-associated protein 1 light chain 3 Ⅱ (LC3 Ⅱ ) expression,mitochondrial membrane potential,autophagosome number and optical density of mitochondria were determined,and ultrastructure of mitochondria was observed at 4 h of LPS incubation.Apoptosis rate and Caspase-3 activity were determined at 24 h of LPS incubation.Results LPS significantly increased LC3 Ⅱ expression,autophagosome number,optical density of mitochondria,apoptosis rate and Caspase-3 activity,decreased mitochondrial membrane potential in group LPS as compared with group C ( P ＜ 0.05).The LC3 Ⅱ expression,autophagosome number and mitochondrial membrane potential were higher,optical density of mitochondria,apoptosis rate and Caspase-3 activity lower in group R than in group LPS( P ＜ 0.05).The LC3 Ⅱ expression,autophagosome number and mitochondrial membrane potential were lower,optical density of mitochondria,apoptosis rate and Caspase-3 activity higher in group 3-MA than in group LPS (P ＜ 0.05).Mitochondrial histopathologic injury was reduced in group R and aggravated in group 3-MA as compared with group LPS.Conclusion Autophagy can reduce LPS-induced HL-1 cardiomyocyte injury by improving mitochondrial function and inhibiting apoptosis.