Department of Anesthesiology, Xiangya Hospital, Central South University, Changsha, China.
PLoS One. 2012;7(7):e42068. doi: 10.1371/journal.pone.0042068. Epub 2012 Jul 31.
Morphine tolerance is a common drawback of chronic morphine exposure, hindering use of this drug. Studies have shown that PKCã may play a key role in the development of morphine tolerance, although the mechanisms are not fully known.
METHODOLOGY/PRINCIPAL FINDINGS: In a rat model of morphine tolerance, PKCã knockdown in the spinal cord was successfully carried out using RNA interference (RNAi) with lentiviral vector-mediated short hairpin RNA of PKCã (LV-shPKCã). Spinal cords (L4-L5) were obtained surgically from morphine-tolerant (MT) rats with and without PKCã knockdown, for comparative proteomic analysis. Total proteins from the spinal cords (L4-L5) were extracted and separated using two-dimensional gel electrophoresis (2DGE); 2D gel images were analyzed with PDQuest software. Seven differential gel-spots were observed with increased spot volume, and 18 spots observed with decreased spot volume. Among these, 13 differentially expressed proteins (DEPs) were identified with matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS), comparing between MT rats with and without PKCã knockdown. The DEPs identified have roles in the cytoskeleton, as neurotrophic factors, in oxidative stress, in ion metabolism, in cell signaling, and as chaperones. Three DEPs (GFAP, FSCN and GDNF) were validated with Western blot analysis, confirming the DEP data. Furthermore, using immunohistochemical analysis, we reveal for the first time that FSCN is involved in the development of morphine tolerance.
CONCLUSIONS/SIGNIFICANCE: These data cast light on the proteins associated with the PKCã activity during morphine tolerance, and hence may contribute to clarification of the mechanisms by which PKCã influences MT.
吗啡耐受是慢性吗啡暴露的常见缺点，阻碍了这种药物的使用。研究表明，PKCã可能在吗啡耐受的发展中起关键作用，尽管其机制尚不完全清楚。
方法/主要发现：在吗啡耐受大鼠模型中，成功地使用慢病毒载体介导的 PKCã短发夹 RNA（LV-shPKCã）进行了脊髓中的 PKCã 敲低。从吗啡耐受（MT）大鼠和 PKCã 敲低大鼠的脊髓（L4-L5）获得手术样本，进行比较蛋白质组学分析。使用二维凝胶电泳（2DGE）提取和分离脊髓（L4-L5）中的总蛋白；使用 PDQuest 软件分析 2D 凝胶图像。观察到 7 个差异表达斑点，其斑点体积增加，18 个斑点体积减少。其中，通过基质辅助激光解吸/电离飞行时间质谱（MALDI-TOF MS）在 MT 大鼠和 PKCã 敲低大鼠之间比较，鉴定了 13 个差异表达蛋白（DEP）。鉴定的 DEP 具有细胞骨架、神经营养因子、氧化应激、离子代谢、细胞信号转导和伴侣的作用。使用 Western blot 分析验证了 3 个 DEP（GFAP、FSCN 和 GDNF），证实了 DEP 数据。此外，通过免疫组织化学分析，我们首次揭示 FSCN 参与吗啡耐受的发展。
结论/意义：这些数据揭示了与吗啡耐受期间 PKCã 活性相关的蛋白质，从而可能有助于阐明 PKCã 影响 MT 的机制。
