TRPM2/CREB通路在缺血/再灌注导致神经元损伤中的作用

The transient receptor potential melastatin-related 2 (TRPM2) were proved as a critical role in the delayed neuron damage after ischemia/reperfusion（I/P）, but their mechanism are still unknown; It’s well known, CaMKIV/CREB signalling reveal a critical role in neuroprotection after I/P. Our recent result shows that inhibition of TRPM2 activity are positively related to the expression of pCaMKIV in OGD-R. The hypothesis：CaMKIV/CREB probably is one of the mechanism of TRPM2 effect. The proposal start to explicit the effect of TRPM2 in neuron damage and their time-dynamic after I/P; secondly to detect the influence of TRPM2 over-expression on pCaMKIV and CREB by unsing TRPM2 transfection and CaMKIV- TRPM2 transgenic mice; furthermore, we will observe the neuron damage and CREB downstream apoptosis factors after I/P by inhibiting CaMKIV/ CREB expression using KN-62、KG-501 and CaMKIV/ CREB knockout mice. Taken together, CaMKIV/CREB signalling probably participate in the neuron damage mediated by TRPM2 after I/P, this study might provide valueable information for exploring targeted neuroprotectiv therapy.

TRPM2近期被证实在脑缺血/再灌注（I/P）的迟发性神经元损伤中起重要作用，但机制不清；已知CaMKIV/CREB是I/P后神经保护的重要调节通路。近期工作提示: 体外OGD-R模型中，抑制TRPM2活性与pCaMKIV蛋白表达正相关，由此推测CaMKIV/CREB可能是TRPM2作用调节机制之一。本项目拟建立体内外模型明确TRPM2对I/P后神经元凋亡的作用和时程；其次采用TRPM2转染和制备CaMKIV-TRPM2转基因小鼠，检测TRPM2高表达对pCaMKIV和pCREB的影响；进而采用KN-62、KG-501以及CaMKIV、CREB基因敲除小鼠，抑制CaMKIV/CREB表达，观察其对I/P后神经元损伤和CREB下游凋亡因子的影响，阐明CaMKIV/CREB通路可能参与了TRPM2脑缺血神经元损伤的调节，本项目将为探索脑缺血后新型靶向经保护治疗提供理论基础

背景：TRPM2近期被证实在脑缺血/再灌注（I/P）的迟发性神经元损伤中起重要作用，但机制不清；已知CaMKIV/CREB是I/P后神经保护的重要调节通路。.方法：本研究通过建立体内外模型明确TRPM2对I/P后神经元凋亡的作用和时程；其次采用TRPM2转染和制备TRPM2-/-转基因小鼠，检测TRPM2高表达对pCaMKIV和pCREB的影响；进而采用KN-62、KG-501以及注射AAV9-shRNA-CaMKIV、AAV9-shRNA-CREB，抑制CaMKIV/CREB表达，观察其对I/P后神经元损伤及CREB下游凋亡因子的影响。.结果：1. TRPM2在OGD6h/R3h时表达量最高，其下游凋亡因子bax，c-fos，cleaved caspase3也相应增高（P＜0.05）；2. TRPM2高表达组细胞活力显著下降，细胞凋亡比例增加，下游凋亡因子表达量明显升高，pCaMKIV/pCREB表达量显著高于对照组（P＜0.05）；3. 抑制CaMKIV/CREB表达后，TRPM2表达无增加，较未处理组细胞活力上升，细胞凋亡比例下降，下游凋亡因子表达量降低（P＜0.05）。.结论：TRPM2通过CaMKIV/CREB通路促进脑缺血后神经元损伤。.本项目为探索脑缺血后新型靶向神经保护治疗提供了理论基础和实验基础。