TRPV4在颞叶癫痫发病中的作用及其分子机制研究

Neuronal synchronized hyperexcitability and synaptic remodeling are important mechanisms accounting for temporal lobe epilepsy (TLE) seizure and spontaneous seizures in the chronic TLE. Transient receptor potential vanilloid 4 (TRPV4) plays an important role in the modulation of cell excitability, vascular tone and angiogenesis. Based on our previous study that TRPV4 protein of hippocampus markedly increased in the process of TLE and blockage of TRPV4 significantly prolonged the latency of seizure and inhibited the epileptiform discharge in hippocampal slice of a rat model of TLE, we propose that activation of TRPV4 is involved in the pathogenesis of TLE through neuronal-vascular multi-target mechanisms. We will firstly study the changes of TRPV4 protein expression in the process of TLE and the effect of TRPV4 blockage on the epiepsy behavior and eletroencephalogram in the rat/mouse model of TLE; secondly explore whether activation of TRPV4 is involved in the pathogenesis of TLE through (1) increasing the intrinsic excitability of neurons and enhancing the communication between neurons and that between neurons and gliocytes to facilitate the neuronal synchronized hyperexcitability; (2) leading to the synaptic remodeling in the hippocampal dentate gyrus and the cerebral vessels remodeling in the epileptogenic zone by modulating MAPK/ERK and PI3K-Akt signaling pathways; (3) leading to abnormal cerebralvascular function by modulating the release of endothelium-derived relaxing factors, calcium-activated potassium channels and/or neurovascular coupling. The results are expected to provide a new target for the clinic treatment of TLE.

神经元同步化过度兴奋和突触重塑是导致颞叶癫痫（TLE）发作和慢性期反复自发发作的重要机制。TRPV4对细胞兴奋性、血管舒缩和血管生成起重要调节作用。基于TLE大鼠海马的TRPV4蛋白表达增加，阻断TRPV4能延长癫痫发作的潜伏期、抑制TLE大鼠海马脑片痫性放电的预实验结果，我们提出“激活TRPV4通过神经-血管多靶点联合机制介导TLE的发生发展”。本课题首先研究TRPV4在TLE模型鼠中的表达，阻断TRPV4对癫痫发作行为、脑电图的影响；并阐明激活TRPV4是否通过（1）提高神经元自身的兴奋性、增强神经元之间及神经元-胶质细胞之间的兴奋传递等易化神经元同步化过度兴奋；（2）调节MAPK/ERK和PI3K-Akt等信号通路导致海马齿回突触和病灶区脑血管重塑；（3）调节内皮源性舒张因子的释放、钙激活钾通道、神经血管偶联等改变局部脑血流量等机制介导TLE的发病过程，为临床防治TLE提供新的靶点。

神经元同步化过度兴奋和突触重塑是导致颞叶癫痫（TLE）发作和慢性期反复自发发作的重要机制。本课题以TRPV4受体为靶点，主要研究TRPV4受体在TLE发病过程中的作用及分子机制。研究结果如下：（1）TLE小鼠在造模成功后的3h至30d海马组织TRPV4受体蛋白水平升高；预先给予TRPV4受体阻断剂HC-067047能降低TLE的造模成功率、延长痫性发作的潜伏期；HC-067047能抑制TLE小鼠海马脑片的痫性放电。（2）TRPV4受体激动剂GSK1016790A抑制海马神经元延迟整流钾电流（IK），CaMKII阻断剂能阻断上述作用；HC-067047能增加TLE小鼠海马组织Kv1.2和Kv2.1的表达。（3）GSK1016790A能抑制海马神经元GABAA受体介导的电流，AMPK或PKC阻断剂或PI3K激动剂能阻断上述作用。（4）GSK1016790A能增强甘氨酸激活的电流，PKC或CaMKII阻断剂能阻断上述作用；GSK1016790A能提高海马组织GlyR α2，α3和β亚基的表达。（5）GSK1016790A能增加海马组织Cx43的蛋白水平，HC-067047能降低TLE小鼠海马组织Cx43的蛋白水平。（6）HC-067047能阻断TLE小鼠海马脑区胶质细胞活化，降低NLRP3炎性小体及炎性细胞因子的蛋白水平，抑制痫后神经元损伤；GSK1016790A促进胶质细胞活化，提高NLRP3炎性小体和炎性细胞因子的表达。（7）GSK1016790A能增加海马齿回1-day-old BrdU+和Ki67+数目，增加CDK2、CDK6、cyclin E1、cyclin A2及p-Rb的表达；ERK或p38MAPK的阻断剂能阻断GSK1016790A的上述作用。（8）GSK1016790A能降低海马齿回DCX+及突起数目，AMPK阻断剂或PI3K激动剂能阻断上述作用；GSK1016790A能增加p-AMPK的表达、降低p-Akt、p-mTOR及p-p70S6K的表达。（9）GSK0106790A对基底动脉的舒缩活动具有双相调节作用，高剂量使其收缩，低剂量使其舒张；GSK1016790A可以增加内皮细胞p-ERK1/2、 CDK2和cyclin E1的表达，促进血管内皮细胞增殖。因此，TRPV4受体过度激活后可通过提高神经元兴奋性、减弱抑制性神经递质系统的功能，促进胶质细胞之