Identification of differentially expressed proteins in the spinal cord of neuropathic pain models with PKCgamma silence by proteomic analysis

Abstract In order to elucidate the mechanisms that PKCγ regulates neuropathic pain (NP), and detect proteins that are associated with the function of PKCγ in NP, we exploited a chronic constriction injury (CCI)-induced neuropathic pain rat (CCI-NP rat) model in which PKCγ knockdown in the spinal cord was successfully carried out with stable RNA interference (RNAi). The spinal cords (L4–L5) were surgically obtained from CCI-NP rats with and without PKCγ knockdown, for comparative proteomic analysis. The total proteins from the spinal cords (L4–L5) were extracted and were separated with two-dimensional gel electrophoresis (2DGE). 2D gel images were analyzed with PDQuest software. Nineteen differential gel-spots were identified with spot-volume increased and 17 spots with spot-volume decreased. Among them, eighteen differentially expressed proteins (DEPs) were identified with matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) between CCI-NP rats with and without PKCγ knockout. Those DEPs are involved in transmission and modulation of noxious information; cellular homeostasis and metabolism; antioxidant proteins, heat shock proteins and chaperones; membrane receptor trafficking; and cytoskeleton. Three DEPs (SNAP-25, TERA and AR) were validated with Western blot analysis, and confirmed the DEP data. Further study showed that AR-selective inhibitor epalrestat totally turned over the upregulated expression of AR in CCI-NP rats. Those DEP data are extensively associated with the function of PKCγ that regulates NP, and would contribute to the clarification of the mechanisms of PKCγ in NP.