Department of Anesthesiology, Xiangya Hospital, Central South University, Changsha, Hunan 410008, China.
Brain Res. 2012 Feb 27;1440:34-46. doi: 10.1016/j.brainres.2011.12.046. Epub 2012 Jan 2.
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.
为了阐明蛋白激酶 Cγ（PKCγ）调控神经病理性疼痛（NP）的机制，并检测与 PKCγ 在 NP 中功能相关的蛋白，我们利用稳定 RNA 干扰（RNAi）成功地在脊髓中敲低 PKCγ，构建了慢性压迫损伤（CCI）诱导的 NP 大鼠（CCI-NP 大鼠）模型。从 CCI-NP 大鼠和 PKCγ 敲低的 CCI-NP 大鼠的脊髓（L4-L5）中获取脊髓组织，用于比较蛋白质组学分析。提取脊髓（L4-L5）的总蛋白，并用二维凝胶电泳（2DGE）分离。用 PDQuest 软件分析 2D 凝胶图像。通过体积增加识别出 19 个差异凝胶斑点，通过体积减少识别出 17 个斑点。其中，通过 CCI-NP 大鼠和 PKCγ 敲除大鼠之间的基质辅助激光解吸/电离飞行时间质谱（MALDI-TOF MS）鉴定了 18 个差异表达蛋白（DEPs）。这些 DEPs 涉及伤害性信息的传递和调节；细胞内稳态和代谢；抗氧化蛋白、热休克蛋白和伴侣；膜受体转运；和细胞骨架。用 Western blot 分析验证了 3 个 DEPs（SNAP-25、TERA 和 AR），并证实了 DEP 数据。进一步的研究表明，AR 选择性抑制剂依帕司他完全逆转了 CCI-NP 大鼠中 AR 的上调表达。这些 DEP 数据广泛与 PKCγ 调节 NP 的功能相关，将有助于阐明 PKCγ 在 NP 中的作用机制。
