| ObjectiveEphBs receptors and ephrinBs ligands are present in the adult brain and peripheral tissue and play a critical role in modulating multiple aspects of physiology and pathophysiology. Recent studies have showed that activation of spinal ephrinBs/EphBs signaling induces hyperalgesia through a MAPKs-mediated mechanism. In the present study, we investigated whether PI3K, as the downstream effectors, participate in modulation of peripheral sensitization related to ephrinBs/EphBs signaling.Methods①The study is to investigate whether intrathecal injection of ephrinBl-Fc induced a time-and dose-dependent hyperalgesia. Intrathecal injected various doses of ephrinB1-Fc (0.02,0.1,0.5μg in10μl saline), paw withdrawal latency to the radiant heat and paw withdrawal threshold to mechanical stimuli were recorded at0.5h,1h,2h,4h,8h,24h after i.t. ephrinB1-Fc; To investigate whether intrathecal injection of ephrinB1-Fc induced a dose-dependent spinal Fos protein expression, intrathecal injected various doses of ephrinB1-Fc (0.02,0.1,0.5μg), Spinal Fos protein expression was assayed at2h after the last injection.②To investigate whether ephrinB1-Fc could activate PI3K and AKT in a time-dependent manner, the expression of PI3K-p110γ and p-AKT in the spinal were assayed at10min,30min,1h,4h and8h after i.t. ephrinB1-Fc (0.5μg/10μl) by western blot; To investigate whether ephrinB1-Fc could activate PI3K and AKT in a dose-dependent manner, the expression of PI3K-p110y and p-AKT in the spinal were assayed at30min after i.t. various doses of ephrinB1-Fc (0.02,0.1,0.5μg/10μl) by western blot; To explore the role of PI3K in AKT activation by ephrinBl-Fc, we intrathecal injected PI3K inhibitor wortmannin (0.4μg/10μl) or LY294002(5μg/10μl) at30min before ephrinB1-Fc (0.5μg/10μl) injection, and then tested the expression of p-AKT in the spinal at30min after ephrinB1-Fc injection by western blot.③To study the effect of PI3K inhibitors on the pain behavior induced by i.t.ephrinBl-Fc, PI3K inhibitors wortmannin (0.016,0.08,0.4μg/10μl) or LY294002(0.2,1,5μg/10μl) were injected at30min before or after ephrinB1-Fc injection. Paw withdrawal latency to the radiant heat and paw withdrawal threshold to mechanical stimuli were recorded at0.5h,1h,2h,4h,8h,24h and48h in the pre-treatment experiment and at0.5h,1h,2h,4h,8h in the post-treatment after i.t. ephrinB1-Fc (0.5μg/10μl); To study the effect of PI3K inhibitors on the Fos protein expression induced by i.t. ephrinB1-Fc. PI3K inhibitor wortmannin (0.4μg/10μl) or LY294002(5μg/10μl) was intrathecal injected at30min before or after i.t. ephrinBl-Fc (0.5μg/10μl). Spinal Fos protein expression was assayed at2h after the last injection.④To investigate whether EphBs receptors involve in activation of spinal PI3K induced by formalin, EphBl-Fc (0.5μg) was injected at30min before intraplantarly injection of formalin. The time spent licking and biting the injected paw and the number of flinching paw at0～5min and15～30min after formalin injection were observed. The expression of spinal PI3K, p-AKT and spinal Fos protein were assayed at30min and2h after the injection of formalin respectively.⑤To investigate whether EphBs receptors involves in activation of spinal PI3K induced by neuropathic pain, EphBl-Fc (0.5μg) was consecutive intrathecal injected1h before CCI and day1, day3or intrathecal injection of a single dose of EphBl-Fc on the fifth day after CCI, Paw withdrawal latency to the radiant heat and paw withdrawal threshold to mechanical stimuli were recorded at1d,3d,5d,7d in consecutive injection group or1d,3d,5d (0.5h,1h,2h,4h,8h after injection),6d,7d in single injection group after CCI. The expression of spinal PI3K, p-AKT and spinal Fos protein were assayed on5d in consecutive injection group and on2h after injection on5d in single injection group respectively.⑥To study whether peripheral PI3K pathway contributes to ERK activation induced by ephrinBl-Fc, PI3K inhibitors wortmannin (0.4μg/10μl) or LY294002(5μg/10μl) was intrathecally injected at30min before i.t. ephrinB1-Fc (0.5μg/10μl).The expression of spinal p-AKT was assayed at30min after i.t. ephrinBl-Fc.Results①Intrathecal injection of ephrinB1-Fc induced a dose-dependent thermal hyperalgesia and mechanical allodynia in mice, which lasted at least up to24h and returned to baseline level on48h after injection of ephrinB1-Fc. Intrathecal injection of ephrinBl-Fc induced a dose-dependent Spinal Fos Expression.②Intrathecal injection of ephrinB1-Fc (0.5μl/10μl) increased the expression of spinal PI3K-p110γ, reaching maximal levels at30min and returning to baseline level at8h after injection. The same injection caused an increase of spinal p-AKT, already detectable at10min after injection, which reached its peak at1h and lasted at least up to8h after injection; Intraplantar injection of ephrinB1-Fc (0.02,0.1,0.5μg/10μl) produced a dose-dependent increase of spinal PI3K-p110y and p-AKT expression. Pre-treatment with wortmannin or LY294002prevented the increase of p-AKT expression induced by i.t. ephrinBl-Fc.③Pre-or post-treatment with PI3K inhibitors wortmannin or LY294002partly prevented or reversed thermal and mechanical hyperalgesia induced by i.t. ephrinBl-Fc in a dose-dependent manner; Pre-or post-treatment with PI3K inhibitors wortmannin or LY294002prevented or reversed the spinal Fos expression induced by i.t. ephrinBl-Fc.④Pre-treatment with EphBl-Fc decreased the time spent licking and biting the injected paw, the number of flinching paw, spinal Fos protein and PI3K, p-AKT expression induced by formalin.⑤Pre-or post-treatment with EphB1-Fc partly prevented or reversed thermal and mechanical hyperalgesia, spinal Fos protein and PI3K, p-AKT expression induced by CCI.⑥Both PI3K inhibitors wortmannin and LY294002prevented the activation of ERK induced by i.t.ephrinB1-Fc.ConclusionPI3K was involved in pain behavior induced by activation of spinal ephrinBs/EphBs signaling in mice, which was partly mediated by activation of ERK signaling. |
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