PHLPP1调控Akt/Nrf2/ARE信号通路对脊髓损伤的影响及分子机制研究

The Akt/Nf-E2 related factor-2 (Nrf2)/ARE signaling pathway plays an essential role in maintaining cellular homeostasis on spinal cord injury (SCI). PH domain leucine-rich repeat protein phosphatase (PHLPP1) has been shown to block cell survival in severed neurons by dephosphorylating AKT. The activation of oxidative damage, neuroinflammation and mitochondrial dysfunction has been implicated in secondary pathomechanisms following SCI. These pathophysiological processes lead to cell death, thus, the specific mechanism of spinal cord injury is not clear now. In our previous work, we observed high levels of PHLPP1 in hippocampal neurons of both embryonic and adult rat hippocampus. Western blotting analysis demonstrated that hydrogen peroxide exposure significantly decreased Nrf2 levels of primary spinal cord neurons, but increased levels of PHLPP1. The expression of Nrf2 further reduce neurons after PHLPP1 transfection cell injury aggravated. We hypothesized that PHLPP1 can aggravate the SCI, which act as negative modulators of Akt/Nrf2/ARE pathways by inhibition of antioxidant gene expression in SCI.This project will study the effect of PHLPP1 on oxidative stress injury in primary cultured spinal cord neurons, identify the effective domain of PHLPP1 negatively regulates Akt/Nrf2/ARE signaling pathway, and clear PHLPP1 specific molecular mechanisms of negative regulation of this signaling pathway. The salutary effect of PHLPP1 removal on damage of SCI will be observed in acute Spinal Cord Injury Model. This work based on the neuron model and the experimental mouse model of SCI will shed light on the potential molecular mechanisms of PHLPP1 for and offer the theoretical basis for new pharmacological targets.

Akt/Nrf2/ARE信号通路在脊髓损伤后发挥重要的抗氧化作用，而PHLPP1可使Akt去磷酸化并加重神经损伤。前期研究发现：PHLPP1过表达可使过氧化氢处理后脊髓神经元的Nrf2表达明显降低。因此我们提出：PHLPP1负性调控Akt/Nrf2/ARE信号通路可加重脊髓损伤。本项目将观察基因沉默PHLPP1及PHLPP1过表达对体外培养永生化神经元氧化应激损伤后超微结构、增殖、凋亡、电生理及能量代谢的影响；分析PHLPP1通过大分子相互作用对Akt磷酸化水平的调节；检测处理PHLPP1后Akt/Nrf2/ARE信号通路及下游关键分子的表达；利用PHLPP1基因敲除小鼠构建脊髓损伤模型，验证干预PHLPP1后损伤脊髓组织学、能量代谢、电生理及细胞凋亡的变化。本研究的顺利实施将丰富脊髓损伤后内源性抗氧化通路激活理论，探索促进脊髓继发性损伤修复的新方法，为脊髓损伤治疗的基础研究提供实验依据。

目的：脊髓损伤（SCI）可使神经元细胞凋亡增加，导致脊髓不可逆转的功能障碍。在这项实验中，研究在PHLPP1基因沉默后对脊髓损伤和细胞凋亡的潜在调节的进展及影响。主要方法：用编码针对PHLPP1的小干扰RNA（siRNA）的重组腺病毒载体（AdsiPHLPP1）注射到脊髓局部及神经元转染，腺病毒在神经元中的稳定表达，显示建立体内外PHLPP1沉默模型成功。研究结果：结果显示沉默PHLPP1显著降低Nrf2表达水平。Western blot分析显示注射AdsiPHLPP1的小鼠，体内促凋亡因子（Bax和cleaved-caspase 3）的表达增加，神经营养因子（BDNF）和抗凋亡（Bcl-2）因子水平降低。注射AdsiPHLPP1的小鼠的运动功能比野生型（WT）小鼠恢复得更慢。此外，脊髓损伤后脊髓前角存活的运动神经元数量也减少。意义：PHLPP1的沉默可促进神经细胞凋亡并延缓运动功能恢复。PHLPP1可作为潜在治疗靶基因用于临床治疗脊髓损伤。