论文部分内容阅读
Objective Cerebral hypoxic preconditioning (HPC) could initiate intracellular signaling pathways to provide neuroprotection.Our previous studies have demonstrated that the activation of novel protein kinase Cε (nPKCε) was involved in the development of cerebral HPC.The present study was designed to indentify the nPKCε-interacting proteins and its possible signaling pathways in the brain of HPC mice.Methods Using our established "auto-hypoxia"-induced HPC and middle cerebral artery occlusion (MCAO)-induced focal cerebral ischemia mouse models, and the TTC staining, Nissl staining, TUNEL and Western blot were applied to examine the role ofnPKCε in HPC induced neuroprotection.Co-immunoprecipitation, 2-dimensional electrophoresis (2DE)and matrix-assisted laser desorption/ionization time of flight mass spectrometer (MALDI-TOF MS) were used to identify the nPKCε signaling molecules.We also examined the HPC and MCAO induced change of nPKCεbinding with one of the interacting proteins, 14-3-3γ.Results HPC could reduce MCAO-induced cerebral infarct volume, edema ratio, cell loss and cell apoptosis in the peri-infarct region, and inhibit the decrease of nPKCεmembrane translocation in peri-infarct region of ischemic cortex.However, nPKCε inhibitor εV1-2 could block this HPC-induced neuroprotection.In total, 15 nPKCε-interacting proteins were successfully identified by MALDITOF MS in the cytosolic fraction of both normoxic control and HPC mouse brain, including 3 up and 1 downregulated proteins more than 1.5 times in brain of HPC mice.Among the 24 identified nPKCε-interacting proteins in particulate fraction of both normoxic control and HPC mouse brain, 5 up-and 2 down-regulated proteins were observed during HPC.In addition, we found the levels of nPKCε-interacting 14-3-3γ protein expressions increased significantly both in cytosolic and particulate fractions of cerebral cortex of HPC mice, and HPC could increase the binding of nPKCε and 14-3-3γ both in the ischemic core and peri-infarct region of ischemic cortex.Conclusion A total of 39 nPKCε-interacting proteins were identified in the the brain of HPC mice;especially, nPKCε-14-3-3γsignaling molecules are involved in the protective effect of HPC.