牛磺酸通过激活SD大鼠AVCN神经元抑制性GABA-R和/或glycine-R拮抗胆红素兴奋毒性

Auditory nuclei is highly sensitive to bilirubin and vulnerable to bilirubin-induced neurotoxicity. It has been proposed that excitotoxicity plays a crucial role in bilirubin-induced toxicity, which causes excess influx of Na+, Ca2+, and water into the cell, inducing cell swelling and triggering cell death. It has been demonstrated that glutamate, a major excitatory neurotransmitter in the central nervous system, contributes to bilirubin-induced excitotoxicity. Despite intensive investigation of hyperbilirubinemia, an effective treatment for bilirubin-induced neuronal injury is lacking. The current method used to treat neonatal hyperbilirubinemia is based on the total serum bilirubin level. Nevertheless, the free bilirubin concentration is the main criterion for the severity of neonatal hyperbilirubinemia, because free bilirubin can easily pass the blood-brain barrier and exert its toxic effects. Taurine is one of the most abundant free amino acids in the brain and has many physiological functions. Taurine protects neurons from excitotoxicity, as it can also freely pass through the blood-brain barrier, creating conditions that do not favor bilirubin-induced neurotoxicity. The neuroprotective functions of taurine to prevent bilirubin-induced apoptosis and reduce oxidative stress are based on its role inhibiting the NMDA receptor and voltage-gated calcium channels. It is well known that taurine conjugates with bile acid play an important role in accelerating bilirubin excretion. However, the electrophysiological effect of taurine on neuronal excitability and hyperexcitation needs to clarification. The results of the present study add a new line of electrophysiological evidence for a powerful neuronal protective action of taurine against bilirubin-induced excitotoxicity. Considering that excitotoxicity is the main mechanism involved in bilirubin-induced neurological disorders, we believe that activating inhibitory neurotransmitter receptors can suppress increased excitability. Taurine is considered an inhibitory neurotransmitter and produces its inhibitory effects by activating GABAARs and/or GlyRs. The type of inhibitory receptor activated by taurine depend on the brain region and the concentration of taurine. We demonstrated previously that taurine activates both inhibitory GABAARs and GlyRs in AVCN neurons. In present study, patch clamp, cell culture, Western blot and confocal laser scanning microscopy were used to investigate the mechanisms underlying the protective effect of taurine. These data has been proposed taurine as a potential therapy for bilirubin-induced neurotoxicity.

听力障碍是新生儿胆红素脑病的常见后遗症，目前缺乏有效的治疗药物。胆红素诱发听觉核团神经元产生兴奋毒性是造成听觉损失的主要原因。牛磺酸是哺乳动物大脑内含量最多的氨基酸之一，既往研究证实牛磺酸具有神经保护功能，它能通过激活抑制性GABA受体和/或甘氨酸受体降低神经元兴奋性。我们的预实验结果提示牛磺酸能够激活大鼠腹前侧耳蜗核神经元中抑制性GABA受体和甘氨酸受体，为此我们提出如下假说：牛磺酸可能通过激活以上两种抑制性氨基酸受体拮抗胆红素引起的腹前侧耳蜗核神经元兴奋毒性。本项目拟通过膜片钳、细胞培养等技术明确牛磺酸是否能够抑制胆红素引起的耳蜗核神经元超兴奋及兴奋毒性，并进一步探索抑制性GABA和甘氨酸受体在牛磺酸拮抗胆红素神经毒性机制中的作用。该项研究有望为胆红素脑病患儿听力损失的治疗提供新的方向。

项目背景：.听力障碍是新生儿胆红素脑病的常见后遗症，但目前缺乏有效的治疗药物。胆红素的兴奋毒性是其造成听觉损失的主要原因，既往研究证实牛磺酸具有神经保护功能，它能通过激活抑制性GABA受体和/或甘氨酸受体降低神经元兴奋性。.主要研究内容：.1、明确牛磺酸在SD大鼠腹前侧耳蜗核神经元激活何种抑制性氨基酸受体；.2、明确牛磺酸是否通过激活SD大鼠腹前侧耳蜗核的抑制性GABA受体和/或甘氨酸受体降低胆红素所致神经元兴奋毒性。.重要结果：.1、胆红素能显著增加神经元动作电位的发放，产生兴奋毒性；牛磺酸能拮抗胆红素引起的兴奋毒性；.2、牛磺酸在不同核团中激活的抑制性氨基酸受体也不一样，我们经研究发现牛磺酸在大鼠腹前侧耳蜗核神经元中，能够同时激活抑制性GABA受体和甘氨酸受体；.3、GABA受体阻滞剂和甘氨酸受体阻滞剂能阻断牛磺酸对胆红素兴奋毒性的拮抗作用。.关键数据：.1、在电流钳记录模式下，胆红素导致腹前侧耳蜗核神经元动作电位大量发放，胆红素组动作电位发放数量为对照组的848.0 ± 223.2%（P＜0.01），冲洗掉胆红素后，动作电位发放频率仍高于对照组，为对照组的623.3 ± 290.9%，但是组间无统计学差异（P＞0.05），证实胆红素会导致腹前侧耳蜗核神经元超兴奋，这种兴奋效应即使在冲洗掉胆红素之后的短时间内仍然存在；.2、联合灌流胆红素和牛磺酸，神经元动作电位的发放数量较单独灌流胆红素组减少（P＜0.01），冲洗掉牛磺酸后，动作电位发放频率下降，但是仍高于单独灌流胆红素组（P＜0.05），证实胆红素能减轻胆红素引起的腹前侧耳蜗核神经元超兴奋；.3、低浓度、高浓度牛磺酸诱发的电流，都被GABAA-R阻滞剂部分阻断，联合加入glycine-R阻滞剂之后几乎完全被阻断，说明被阻断的电流其实是牛磺酸激活GABA-R和glycine-R诱发的，证实不同浓度牛磺酸均能激活GABA-R和glycine-R；.4、牛磺酸联合胆红素培养能减轻胆红素引起的SD大鼠腹前侧耳蜗核神经元活性减弱、突触长度缩短、凋亡指数增加及胞内Ca2+超载；GABAA-R阻滞剂和glycine-R阻滞剂能阻断牛磺酸的上述保护作用。.科学意义：本研究通过研究抑制性GABA和甘氨酸受体在牛磺酸拮抗胆红素神经毒性机制中的作用，有望为胆红素脑病患儿听力损失的治疗提供新的方向。