乳酸抗痫作用中GPR81+β-arrestin2/cAMP/KATP分子通路机制

Lactic acid is an important metabolic energy substrate in the neural network involved in the pathogenesis of epilepsy, and is an important metabolic signal molecules involved in the regulation of epileptic networks. In this study, we put forward the hypothesis can inhibit the electrical activity of epileptic networks by signal pathway of GPR81+ beta-arrestin2/cAMP /KATP. In this study, the pathway was demonstrated in two experimental conditions, in vivo and in vitro. In vivo, at the levels of the brain, in pilocarpine epileptic mouse model before and after Si-RNA- beta -arrestin2/ GPR81, we would detect EEG, morphology, GPR81 or beta-arrestin2, Kir6.1 and Kir6.2 mRNA and protein expression by an applications of EEG, immunohistochemistry, RT-PCR and Western blot before and after usage of three different doses of 2-DG, and cAMP inhibitor, and cAMP changes should be detected by ELISA. In vitro, at the levels of the cellular network, in neural network epilepsy model without magnesium before and after Si-RNA- beta -arrestin2/ GPR81, we would detect the KATP channel activity, GPR81 or beta -arrestin2, Kir6.1 and Kir6.2 mRNA and protein changes by an applications of patch clamp technique, RT-PCR and Western blot before and after usage of 2-DG, and KATP agonist and inhibitors; at the same time, an interaction between the clear GPR81, beta -arrestin2 should be investigated by an application of immune coprecipitation. In turn, lactic acid could regulated the epileptic networks through the GPR81+ beta -arrestin2/cAMP /KATP molecular pathway, which can inhibit the regulation of electrical activity in epileptic network, and these findings would provide a new perspective for the treatment of epilepsy.

乳酸作为癫痫发病过程中重要的代谢供能底物，同时，也作为重要的代谢信号分子参与痫性电活动调节。本研究提出乳酸受体分子GPR81介导的信号通路，即GPR81+β-arrestin2/cAMP /KATP分子通路对癫痫网络电活动有抑制调节作用。1）在匹罗卡品癫痫小鼠模型上采用脑电图、免疫组化、RT-PCR、Western-blot方法，论证GPR81与β-arrestin2结合后介导cAMP→KATP 通道发挥抗痫作用。2）在原代培养海马神经元细胞上，建立无镁诱导惊厥模型，采用膜片钳、RT-PCR、Western-blot方法，证实GPR81与β-arrestin2结合后介导cAMP→KATP 通道发挥抗痫作用。3）在原代培养海马神经元细胞上，应用免疫共沉淀方法明确GPR81、β-arrestin2之间存在相互作用。进而论证GPR81介导的分子通路，对癫痫发作有抑制作用，为抗痫治疗提供新视角。

癫痫是由多种病因引起神经元反复异常过度同步化放电导致的脑部疾病，然而，关于癫痫发作及终止的机制仍不十分清楚。此前有研究认为，脑部的酸性环境（如局部微环境的酸中毒）能阻止癫痫发作的进展。另外，癫痫发作时乳酸在脑内急剧升高，那么乳酸是否参与了癫痫发作或终止呢？为此，我们创造性地提出了乳酸受体GPR81介导的信号通路，即GPR81+β-arrestin2/cAMP/KATP分子通路对癫痫网络电活动有抑制调节作用。一方面，我们按照项目设计的实验路线，使用2-脱氧葡萄糖（一种影响乳酸生成的糖酵解抑制剂）干预匹鲁卡品诱导的C57小鼠急性癫痫模型，发现2-DG通过抑制代谢促进KATP通道的开放抑制小鼠的癫痫发作，进一步的研究表明mir-135b/Netrin-G1/KATP信号通路参与了2-DG的抗癫痫机制。另一方面，按照项目设计另一部分实验路线，我们直接使用GPR81的激动剂（3,5-DHBA）干预匹鲁卡品及海人酸诱导的C57小鼠急性癫痫模型，结果表明使用GPR81激动剂干预并不影响小鼠的癫痫发作；进一步的分子生物学实验也发现激活GPR81不影响KATP蛋白的mRNA和蛋白表达。此外，在本项目支撑下，我们在代谢控制癫痫和癫痫药物治疗等相关领域进行了一系列的探索并取得相应成果。我们系统阐述了代谢控制癫痫的理论基础、现状和可能途径，并指出通过调节代谢是开发癫痫治疗方法新的有前途的途径。另外，论证了停用抗癫痫药物的患者的复发率高于继续使用抗癫痫药物的患者，而单用不同抗癫痫药物（卡马西平、苯妥英钠、丙戊酸钠和苯巴比妥/普利米酮）治疗的无癫痫发作患者停药后癫痫复发率无显著差异。这些结果为癫痫相应细分领域提供了重要的指导和启示。