NAD+通过抑制TRPM2通道拮抗胆红素听觉中枢超兴奋作用机制

Hearing loss is one of the most serious sequelae of bilirubin encephalopathy. However, the effective medication is deficiency. The key mechanism of bilirubin-induced hyperexcitation in cochlear nucleus neurons is that free bilirubin can lead to calcium overload. Transient potential receptor melastatin-2 (TRPM2), a Ca2+-permeable cation channel, is mainly activated by intracellular adenosine diphosphate ribose (ADPR) in response to oxidative stress. Excess Ca2+ influx induced by TRPM2 is likely contribute to bilirubin-induced hyperexcitation. NAD+ is a ubiquitous molecule with multifaceted biological functions in various biological processes in human body. Previous study confirmed NAD+ can effectively inhibit bilirubin-induced enhancement of excitatory neurotransmission in cochlear nucleus. Pilot experiments show bilirubin can promote neuronal excitability through TRPM2 and NAD+ can both reverse and prevent increase of calcium channel current induced by bilirubin. As a result, we come up with the hypothesis that NAD+ antagonizes the effect of bilirubin-induced hyperexcitation by inhibiting ADPR-induced TRPM2. Here, we verify the hypothesis by further confirming the exist of ADPR-induced TRPM2 in cochlear nucleus, exploring its effect in bilirubin-induced hyperexcitation and its role in neuroprotection of NAD+. The study can lend support to NAD+ as a promising endogenous agent for the treatment associated with hearing loss caused by bilirubin encephalopathy of newborn.

听觉损伤是胆红素脑病的严重后遗症，尚缺乏安全有效的治疗药物。胆红素引起听觉核团神经元钙超载是其超兴奋的关键环节。高胆红素血症使新生儿处于氧化应激的状态，该状态下胞内生成的ADPR能激活TRPM2通道引起钙离子大量内流，本课题组推测这可能是胆红素超兴奋的重要机制之一。NAD+是人体的关键辅酶，申请人前期研究证实NAD+能有效抑制胆红素引起的突触传递增强。预试验结果显示胆红素能通过TRPM2通道增加神经元兴奋性，而NAD+能有效拮抗胆红素引起的钙电流增加。因此本研究提出如下假说：NAD+能通过抑制ADPR激活的TRPM2通道拮抗胆红素超兴奋，发挥神经保护作用。本研究拟通过明确ADPR激活的TRPM2通道在耳蜗核神经元上的表达，观察其在胆红素超兴奋中的作用，探索其在NAD+拮抗听觉神经元胆红素诱导的超兴奋机制中的作用。本项研究有望对内源性药物治疗胆红素脑病听觉障碍的研究提供有效的理论依据。

听觉障碍是新生儿胆红素脑病的严重后遗症之一，但具体机制不明，因此临床缺乏针对机制的有效治疗药物。高胆红素血症使新生儿处于氧化应激的状态，该状态下胞内生成的生腺苷二磷酸-核糖（ADPR）能激活瞬时受体电位M2型（TRPM2）通道引起钙离子大量内流，造成听觉核团神经元钙超载，这是胆红素超兴奋的关键环节。烟酰胺腺嘌呤二核苷酸（NAD+）是人体的关键辅酶，对多种因素引起的神经元损伤具有保护作用。既往研究证实NAD+能有效抑制胆红素引起的突触传递增强，而胆红素能通过电压门控性钙通道（VGCC）促进大量钙离子内流造成钙超载，突触传递、神经递质释放由VGCC开放引起钙内流决定，钙离子作为第二信使，能进一步促进胆红素能通过电压门控性钠通道（VGSC）提高神经元细胞膜兴奋性，提示NAD在听觉神经元胆红素诱导的超兴奋机制中具有拮抗作用。本研究项目通过电生理膜片钳技术、钙成像技术和免疫荧光技术，发现ADPR激活的TRPM2通道存在于耳蜗核神经元以及全脑。通过观察TRPM2通道阻滞剂本身对神经元自发放电情况的影响研究了ADPR激活的TRPM2通道在听觉神经元胆红素超兴奋中的作用，发现TRPM2阻滞剂可以拮抗胆红素所致放电频率的增加，证实TRPM2通道参与了神经元电活动的产生和发放过程，并且证实胆红素能促进TRPM2电流，产生兴奋毒性。研究了NAD+对胆红素引起的耳蜗核神经元超兴奋的作用，细胞外加入内原性辅酶NAD+对耳蜗核神经元胆红素超兴奋具有抑制和预防作用，NAD+能通过VGCC有效抑制胞内钙超载，推测NAD+能通过抑制ADPR激活的TRPM2通道拮抗胆红素超兴奋，发挥神经保护作用。NAD+可作为安全有效的内源性药物用于治疗和预防胆红素脑病。