硫化氢供体的光学探针调节ATP敏感性钾通道治疗癫痫的分子机制

As the high incidence of epilepsy,and low cure rate,early exogenous low concentration of H2S can effectively inhibit seizures, high concentrations increased their attacks, however, mechanism of the treatment of epilepsy by low concentration of H2S is still not clear.In this study, we firstly synthesize the hydrogen sulfide donor probe of BODIPY structure, detect the safety of the probe by CCK-8, apoptosis, laser confocal microscopy . At cell level, we culture the hippocampal neurons without magnesium to establish epilepsy cell model then to analyze the cell cycle changes and trace the metabolic status of optical probe,to determine the expression of KATP channel mRNA and protein by Western blot and RT-qPCR , and use the patch clamp to record the membrane KATP channel current and action potential changes in section of brain neuron. Then to investigate the molecular mechanism of hydrogen sulfide donor probe restrain intermittent firing of hippocampal neurons. At the animal level, we establish the epilepsy animal models,provide hydrogen sulfide exogenously by intracerebroventricular injection of hydrogen sulfide probe, then screen the optimal concentration of hydrogen sulfide in treatment of epilepsy through the behavior observation, EEG and hippocampal pathology detection method et al , further, analyzed in detail the molecular biological indexes of brain tissue slices,.and investigate the mechanism of low concentration of H2S in the regulation of KATP channels, then provide the theoretical basis for the treatment of epilepsy.

癫痫发病率高，治愈率低，早期外源性给予低浓度H2S可抑制癫痫发作，高浓度则加剧其发作，然而低浓度H2S治疗癫痫的作用机制尚不清楚。本项目拟合成自身发光的BODIPY结构硫化氢供体探针，通过cck-8、细胞凋亡、激光共聚焦等检测探针的安全性，在细胞水平，建立海马神经元无镁细胞外液癫痫细胞模型，分析细胞周期变化，示踪光学探针在细胞的代谢情况，Western blot和RT-qPCR检测KATP通道mRNA和蛋白的表达，膜片钳记录神经元膜KATP通道电流及动作电位变化，探讨硫化氢供体探针对海马神经元间歇性放电抑制的分子作用机制；在动物水平，建立动物癫痫模型，侧脑室注射硫化氢探针外源性补充硫化氢，通过行为学观察、脑电及海马病理检测等方法筛选硫化氢治疗癫痫的最适浓度，并详细分析脑组织切片的上述分子生物学指标；深入探讨体内低浓度H2S调节KATP通道的分子作用机制，为癫痫的治疗提供理论基础。

本项目合成了新型H2S供体探针（专利号：201910164930.3），开展了如下研究：①采用扫描电镜、红外吸收光谱及核磁共振氢谱分别对新型H2S供体探针进行表征;②分别在细胞和动物实验筛选新型H2S供体探针在细胞和动物实验的最大安全有效浓度和作用时间；③分别在细胞和动物水平探讨了新型H2S供体探针对H2S/CBS酶的影响，验证新型H2S供体探针在细胞和动物组织中释放H2S的情况；④分别在细胞和动物水平探讨了新型H2S供体探针对癫痫模型的作用及机制。我们的实验结果表明：①新型H2S供体探针具有细胞成像能力，不同激发波长，发不同颜色的光；②细胞实验H2S供体探针最大安全有效浓度为400μM，作用时间为12h,动物实验H2S供体探针最大安全有效浓度为500μM,作用时间为4h；③最大安全有效浓度的新型H2S供体探针可释放H2S；④新型H2S供体探针对海马神经元有保护作用，可抑制大鼠癫痫发作，其对神经元的保护作用及对癫痫的抑制作用与KATP通道有关。这些结果将为癫痫防治新靶点提供实验依据，为进一步揭示癫痫的发病机制提供新思路。