Hsp27的翻译后调控在短暂全脑缺血后低氧后处理神经保护中的分子机制

Our previous data indicated that hypoxic postconditioning (HPC) ameliorates transient global cerebral ischemia-induced hippocampal CA1 neuronal death in adult rats. However, the possible molecular mechanisms for neuroprotection of this kind are largely unknown. Heat shock proteins 27 (Hsp27) has been demonstrated to exert neuroprotective effects against cerebral ischemic injury. However, the precise mechanism underlying this protection is currently unknown. In our preliminary studies, significant increases in the expression of Hsp27 protein were observed in CA1 subregion after 10 min of transient global cerebral ischemia (tGCI) with HPC in adult rats, but apparent difference was not observed regarding to Hsp27 mRNA. The results suggest that the difference in the expression of Hsp27 protein between HPC and tGCI groups did not result from the up-regulating transcription of Hsp27. Whether post-translational regulation plays a key role in the expression of Hsp27 by HPC and which molecular mechanism is involved remain unknown. To investigate whether the overexpression of Hsp27 induced by HPC is capable of protecting the brain from tGCI, recombinant lentivirus encoding Hsp27 cDNA was constucted by molecular biological techniques. To further our understanding of the molecular mechanisms of overexpression of Hsp27 induced by HPC, we will determine whether phosphorylated Hsp27 which is regulated by p38MAPK/MK2 signaling pathway is involved in the neuroprotection of HPC against tGCI. Also, in order to test whether there was a difference in protein degradation in tGCI rats with or without HPC, Hsp27 protein degradation rate will be determined by immunoblotting. Meanwhile, to understand whether the degradation of Hsp27 is mediated by the proteasome degradation pathway, rats will be treated by protease inhibitors and the expression of Hsp27 will be measured. To further dissect the alterations of the autophagic-lysosomal degradation pathway in tGCI rats with or without HPC, several autophagy-related genes will be monitored. More insights will be obtained on the neuroprotective molecular mechanisms of Hsp27 in cerebral ischemia from the study, and it will be supported that Hsp27, as a new therapeutic target, is very important in the neuroprotection against cerebral ischemia.

我们近期发表的研究显示，低氧后处理对大鼠短暂全脑缺血后CA1区神经元起保护作用，但其确切机制仍不清。研究表明Hsp27在脑缺血后的神经保护中起着极为关键的作用。我们在预实验中发现，低氧后处理使脑缺血后CA1区Hsp27蛋白表达增加，而mRNA则无变化，提示低氧后处理不是通过转录水平来调控Hsp27的表达。由此我们提出低氧后处理可能通过翻译后的调节，影响Hsp27蛋白的表达，发挥神经保护作用。本研究拟采用大鼠短暂全脑缺血低氧后处理模型，通过免疫印迹、荧光定量PCR、慢病毒载体构建等技术，阐明低氧后处理通过对Hsp27翻译后水平的调控，即一方面通过抑制缺血后CA1区自噬-溶酶体途径的激活，减少Hsp27蛋白降解，维持Hsp27的过表达，另一方面通过激活MK2通路，使Hsp27磷酸化水平增加，产生神经保护作用。研究成果对阐明脑缺血耐受的机制和确立以Hsp27为药物治疗的"新靶点",具有重要意义。

我们近期发表的研究显示，低氧后处理对大鼠短暂全脑缺血后CA1区神经元起保护作用，但其确切机制仍不清。研究表明Hsp27在脑缺血后的神经保护中起着极为关键的作用。本研究在前期研究基础上，阐明低氧后处理通过对Hsp27翻译后水平的调控。我们的结果发现（1）低氧后处理使脑缺血后CA1区Hsp27蛋白表达增加，而mRNA则无变化，提示低氧后处理不是通过转录水平来调控Hsp27的表达。（2）腹腔注射放线菌酮抑制剂蛋白合成后，低氧后处理组CA1区Hsp27蛋白水平比缺血组明显增加，蛋白降解减少；缺血后24 h侧脑室注射溶酶体抑制剂亮肽素后CA1区Hsp27蛋白水平明显增加，提示缺血后Hsp27蛋白可能通过溶酶体途径降解。（3）电镜结果表明，缺血后CA1区神经元内自噬体生成明显增加，Western blot结果显示缺血后CA1区LC3II蛋白表达明显增多，提示缺血后自噬激活。缺血前通过侧脑室注射3-MA后，神经元死亡减少，Hsp27蛋白增加；低氧后处理前通过侧脑室注射雷帕霉素后，神经元死亡增加，Hsp27蛋白减少。（4）与缺血组比较，低氧后处理增加缺血后CA1区p-MK2和p-Hsp27的表达，低氧后处理前通过侧脑室注射SB203580后，p-MK2和p-Hsp27的表达明显减少。低氧后处理通过对Hsp27翻译后水平的调控，即一方面通过抑制缺血后CA1区自噬-溶酶体途径的激活，减少Hsp27蛋白降解，维持Hsp27的过表达，另一方面通过激活MK2通路，使Hsp27磷酸化水平增加，对成年Wistar 大鼠产生神经保护作用。研究成果对阐明脑缺血耐受的机制和确立以Hsp27为药物治疗的"新靶点",具有重要意义。