MicroRNA124调控PRDX6在丙泊酚保护创伤性脑损伤的作用和机制

Traumatic brain injury (TBI) is defined as direct mechanical damage to the brain,which is a leading cause of mortality and morbidity in the population all over the world. As a difficult problem faced by the clinical treatment, the study for the treatment of TBI is increasingly interest. Previously, we have found that the The expressions of pro-inflammatory factors were significantly augmented but PRDX6 down-regulated after TBI, following propofol treatment, the expression of PRDX6 was significantly up-regulated and the pro-inflammatory factors was inhibited, furthermore, neurological severity scoring(NSS) improved also after treatment with propofol. These findings suggested that propofol was a neuro-protective agent in TBI treatment, the underlying mechanism was associated with inhibition of pro-inflammatory cytokines and protection of antioxidant function. In addition, by using bioinformatics it is possible that microRNA124 may regulate PRDX6. Based on above findings, this study is aiming to investigate the role of PRDX6 in the neuro-protection of propofol in TBI and the mechanism of microRNA124 regulation by using the technologies of gene cloning, RNA interference, immunocytochemistry, RT-PCR, Western Blot, Luciferce assay,site directed mutation.The results will contribute to elucidate the roles of PRDX6 in the neuroprotection of propofol in TBI and the mechanism of microRNA124 regualation, which has important scientific significance and clinical value. The findings may provide new intervention strategy for the treatment of TBI.

创伤性脑损伤是外伤引起的脑组织损伤，前期研究发现丙泊酚处理脑外伤后脑组织PRDX6上调，神经功能改善，提示丙泊酚脑保护机制可能与维持抗氧化功能有关。基因芯片分析显示脑损伤后microRNA124表达显著下降，丙泊酚处理后损伤脑组织microRNA124表达升高，生物信息学预测microRNA124的靶分子之一为PRDX6。本实验拟通过构建PRDX6过表达和低表达基因克隆HSV重组体和microRNA124的模拟物/抑制剂，研究PRDX6和microRNA124对体外新生大鼠大脑皮质神经元细胞和创伤性脑外伤大鼠的作用，以及microRNA124对PRDX6表达水平的影响，并用双荧光素酶报告基因表达系统检测microRNA124对PRDX6 表达水平的调控，最终确定PRDX6和microRNA-124在丙泊酚治疗创伤性脑损伤的作用和机制。

创伤性脑损伤是外伤引起的脑组织损伤，前期研究发现丙泊酚处理脑外伤后脑组织PRDX6上调，神经功能改善，提示丙泊酚脑保护机制可能与维持抗氧化功能有关。基因芯片分析显示脑损伤后microRNA124表达显著下降，丙泊酚处理后损伤脑组织microRNA124表达升高，生物信息学预测microRNA124的靶分子之一为PRDX6。为探讨PRDX6和microRNA-124在丙泊酚治疗创伤性脑损伤的作用和机制，本实验构建了PRDX6过表达和低表达基因克隆HSV重组体和microRNA124的模拟物/抑制剂，研究PRDX6和microRNA124对体外新生大鼠大脑皮质神经元细胞和创伤性脑外伤大鼠的作用。结果显示丙泊酚组和PRDX6过表达组TBI大鼠模型NSS评分与TBI组相比明显下降，说明丙泊酚和PRDX6过表达对大鼠神经功能恢复有促进作用，但叠加作用不明显；而丙泊酚联合PRDX6基因干扰组大鼠的神经功能评分增加，提示丙泊酚作用发挥需要PRDX6支持。体外实验中，在新生鼠大脑皮质神经元细胞培养模型中分别加入microRNA124 模拟物和抑制剂，结果显示在PC12，sy5y和神经元细胞损伤后加入miR-124模拟物，细胞损伤明显减轻；在sy5y 细胞培养模型中结果表明，与未加micRNA124的对照组相比，加入micRNA124抑制剂组PRDX6的蛋白水平明显上调，而加入micRNA124模拟物组的PRDX6水平明显减少。体内实验中，将microRNA124 模拟剂注入脑外伤模型后，大鼠神经行为学功能评分明显改善，神经元的凋亡明显减轻。本实验还通过用双荧光素酶报告基因表达系统检测microRNA124对PRDX6表达水平的调控，将PRDX6 3'-UTR 与microRNA124 可能结合的位点进行定点突变，再加入microRNA124 模拟物，观察microRNA124对PRDX6表达水平的影响。结果显示，miR-124能有效靶向PRDX6的 3'-UTR 并与之结合，从而影响荧光素酶报告基因表达活性。与对照组比，实验组荧光素酶报告基因表达活性明显降低。反之，PRDX6位点突变实验显示，miR-124加入未能影响荧光素酶报告基因活性，即miR-124不能与突变的PRDX6结合来改变荧光素酶报告基因表达活性，证明miR-124能特异靶向PRDX6，并发挥直接调控作用。