基于GABAB受体介导的信号通路探讨疏肝安神法调节失眠大鼠睡眠时相的分子机制

Based on many years of clinical observation, it is currently accepted that liver depression and qi stagnation are the main cause of insomnia. Combined with the theory of traditional Chinese medicine on treatment of insomnia from the liver, Shugan Anshen method is considered as the primary method for prevention and treatment of insomnia. Our previous studies have showed that Song-Yu-An-Shen-Fang, the herbal compound of prescription for Shugan Anshen method, had a reliable curative effect on insomnia, and the underlying mechanism may be relevant to the regulation of GABA level and GABAB receptor in hypothalamus. On the basis of early researches, we propose that the treatment of daily oral gavage with Song-Yu-An-Shen-Fang for a period of 7 days will modulate the sleep stage of insomnia model rat and improve the concentration of GABA in hypothalamus. The electroencephalogram（EEG）and electromyogram（EMG）signals are recorded by the sleep/wake analytical system before and after treatment to verify the effect of the Shugan Anshen method on states of sleep-waking cycle in rats. After the rats are sacrificed, the ventrolateral preoptic area (VLPO) in hypothalamus is immediately separated. Real-time polymerase chain reaction is performed to determine GABAB receptor, AMP, PKA and CREB mRNA expression. Western-blot is performed to measured the level of protein expression.In vitro, cerebral cortex nerve cells were intervented by serum containing Song-Yu-An-Shen-Fang, through actibating or blocking GABAB receptors, AMP, PKA and CREB phosphorylation was measured by western-blot. The present study will explore the mechanism of cAMP/PKA/CREB signal pathway in insomnia rats treated by Shugan Anshen method, and provide the reliable experimental envidence for the clinical therapy of insomnia.

课题组多年临床观察发现肝气郁结是失眠的主要原因，结合失眠从肝论治中医理论，提出失眠多肝郁，疏肝安神法是防治失眠的主要方法。前期研究证实以疏肝安神法为组方的松郁安神方治疗失眠疗效确切，且与调节下丘脑GABA含量及GABAB受体mRNA表达有关。本课题用松郁安神方干预肝郁失眠大鼠，通过记录大鼠脑电和肌电，观察大鼠睡眠时相变化。选取大鼠下丘脑VLPO系统，通过检测GABA含量、GABAB受体mRNA及蛋白表达以及GABAB受体介导的下游信号通路关键蛋白AMP、PKA和CREB mRNA及蛋白磷酸化，探讨疏肝安神法对cAMP-PKA-CREB通路的调节。体外实验采用松郁安神方含药血清干预大脑皮层神经细胞，通过观察激活或阻断GABAB受体对AMP、PKA和CREB蛋白磷酸化的影响，阐明松郁安神方调节cAMP-PKA-CREB信号通路的分子机制，为疏肝安神法治疗失眠的作用靶点和分子机制提供实验依据。

失眠多肝郁，疏肝安神法是防治失眠的主要方法。为了阐明疏肝安神法治疗失眠的作用机制，本研究从睡眠时相、内源性睡眠物质、神经递质、作用受体及其介导的信号通路方面，从体内和体外实验研究疏肝安神法治疗失眠的作用机制，以期为临床治疗失眠提供新靶点。结果显示：（1）通过24小时连续记录EEG/EMG信号，观察松郁安神方对PCPA致失眠大鼠睡眠时相（觉醒期W、非快速眼动睡眠期NREM、快速眼动睡眠期REM）的影响，结果发现PCPA可以明显降低大鼠白天时段NREM和REM睡眠总量，增加觉醒量，而给予松郁安神方后，大鼠白天时段NREM和REM睡眠总量均明显增加，觉醒量减少。实验结果表明，松郁安神方可明显改善PCPA致失眠大鼠睡眠质量，其主要通过增加白天时段NREM和REM睡眠总量改善睡眠。（2）PCPA致失眠后，大鼠下丘脑PGD2含量明显降低，给予松郁安神方治疗后，PGD2含量明显增加，表明松郁安神方可调节失眠大鼠下丘脑PGD2含量，从内源性睡眠物质变化进一步证实了松郁安神方改善睡眠的作用。（3）松郁安神方能明显降低PCPA致失眠大鼠下丘脑Glu含量及Glu/GABA比例，推测其治疗失眠的机制可能是通过调节Glu和GABA的含量来减轻脑内兴奋性神经毒性，起到了保护脑神经元的作用。（4）松郁安神方能增加PCPA诱导的大鼠下丘脑GABAB mRNA 及蛋白表达的减少。提示松郁安神方可能通过增加GABAB受体表达达到改善睡眠的作用。（5）松郁安神方能明显增加大鼠下丘脑CREB mRNA 及 CREB蛋白 磷酸化水平，增加PCPA致失眠大鼠下丘脑cAMP含量，提示松郁安神方可能通过增加GABAB受体表达，激活 cAMP-PKA-CREB 信号通路而发挥镇静安神作用。（6）细胞实验发现，松郁安神方可增加GABAB受体的表达，而给予GABAB受体抑制剂CPG5426可明显降低GABAB、PKA和CREB蛋白磷酸化水平。以上结果提示以疏肝安神法为组方的松郁安神方可改善失眠大鼠的睡眠，其作用机制可能通过调节大鼠下丘脑中GABA含量，激活GABAB受体及其下游cAMP-PKA-CREB信号通路起作用。该研究为疏肝安神法治疗失眠的作用靶点和分子机制提供实验依据，为松郁安神方的临床应用提供了理论支持，更好地发挥中医药治疗失眠的优势。