Neuroinflammation and ER-stress are key mechanisms of acute bilirubin toxicity and hearing loss in a mouse model.
Neuroinflammation and ER-stress are key mechanisms of acute bilirubin toxicity and hearing loss in a mouse model.
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DOI：
10.1371/journal.pone.0201022
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发表时间：
2018
期刊：
影响因子：
3.7
通讯作者：
Forsythe ID
中科院分区：
文献类型：
--
作者：
Schiavon E;Smalley JL;Newton S;Greig NH;Forsythe ID
高胆红素血症（黄疸）是由血液中未结合胆红素水平升高引起的。严重时，包括小脑和听觉脑干在内的易感脑区受损，导致神经系统后遗症，如共济失调、听力损失和核黄疸。胆红素发挥其毒性作用的机制迄今尚未完全了解。在这项研究中，我们研究了胆红素引起神经毒性导致听力损失的急性机制。我们开发了一种新的小鼠模型，该模型表现出在人类胆红素诱导的神经功能障碍（BIND）综合征中观察到的神经学特征，我们用行为评分和听觉脑干反应（ABR）进行了评估。在将胆红素应用于体外培养细胞的初始实验的指导下，我们在胆红素暴露后对小鼠脑组织（小脑和听觉脑干）进行了全基因组基因表达测量，以获得对受影响的生化过程的机械见解，并使用免疫印迹进一步研究。然后，我们比较了胆红素诱导的基因变化，细菌脂多糖（LPS），一个很好的特点诱导神经炎症，以评估它们之间的相似程度。最后，我们研究了炎症的遗传扰动以及已知和新型抗炎药物可以保护听力免受胆红素诱导的毒性的程度。体外结果表明，胆红素诱导的基因表达的变化与内质网（ER）应激和未折叠蛋白反应（UPR）的激活一致。这些基因变化与已知的内质网应激诱导剂毒胡萝卜素的基因表达特征相似。它还诱导与炎症和NF-κB活化相关的基因表达变化。体内模型显示出行为障碍和听觉阈值升高。全基因组基因表达分析证实炎症是听觉通路中胆红素神经毒性的关键机制，并且通过暴露于细菌脂多糖（LPS）（一种充分表征的神经炎症诱导剂）诱导了共同的基因表达标志。有趣的是，在该模型中，胆红素对听觉系统的损害比LPS更严重，但与我们的假设一致，即神经炎症是胆红素毒性的主要部分，听力损失通过干扰炎症反应而得到保护。这是使用脂质运载蛋白-2（LCN 2）-null小鼠进行遗传学研究的，脂质运载蛋白-2（LCN 2）是一种炎症细胞因子，响应胆红素而高度上调。最后，我们测试了已知和新型抗炎化合物（干扰NF-κB和TNFα信号传导），并证明了对胆红素毒性的听觉系统保护作用。我们已经开发了一种新的，可逆的，黄疸模型，显示运动障碍和听力损失符合人类症状。我们使用该模型来建立ER应激和炎症作为胆红素毒性的主要贡献者。由于这种新型模型中毒性的快速和可逆发作，它代表了筛选治疗化合物的系统。我们已经通过遗传靶向炎症和抗炎小分子来证明这一点，这些小分子提供了对胆红素毒性的保护。这也表明抗炎药可能对高胆红素血症具有治疗作用。
Hyperbilirubinemia (jaundice) is caused by raised levels of unconjugated bilirubin in the blood. When severe, susceptible brain regions including the cerebellum and auditory brainstem are damaged causing neurological sequelae such as ataxia, hearing loss and kernicterus. The mechanism(s) by which bilirubin exerts its toxic effect have not been completely understood to date. In this study we investigated the acute mechanisms by which bilirubin causes the neurotoxicity that contributes to hearing loss. We developed a novel mouse model that exhibits the neurological features seen in human Bilirubin-Induced Neurological Dysfunction (BIND) syndrome that we assessed with a behavioural score and auditory brainstem responses (ABR). Guided by initial experiments applying bilirubin to cultured cells in vitro, we performed whole genome gene expression measurements on mouse brain tissue (cerebellum and auditory brainstem) following bilirubin exposure to gain mechanistic insights into biochemical processes affected, and investigated further using immunoblotting. We then compared the gene changes induced by bilirubin to bacterial lipopolysaccharide (LPS), a well characterized inducer of neuroinflammation, to assess the degree of similarity between them. Finally, we examined the extent to which genetic perturbation of inflammation and both known and novel anti-inflammatory drugs could protect hearing from bilirubin-induced toxicity. The in vitro results indicated that bilirubin induces changes in gene expression consistent with endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR). These gene changes were similar to the gene expression signature of thapsigargin–a known ER stress inducer. It also induced gene expression changes associated with inflammation and NF-κB activation. The in vivo model showed behavioural impairment and a raised auditory threshold. Whole genome gene expression analysis confirmed inflammation as a key mechanism of bilirubin neurotoxicity in the auditory pathway and shared gene expression hallmarks induced by exposure to bacterial lipopolysaccharide (LPS) a well-characterized inducer of neuroinflammation. Interestingly, bilirubin caused more severe damage to the auditory system than LPS in this model, but consistent with our hypothesis of neuroinflammation being a primary part of bilirubin toxicity, the hearing loss was protected by perturbing the inflammatory response. This was carried out genetically using lipocalin-2 (LCN2)-null mice, which is an inflammatory cytokine highly upregulated in response to bilirubin. Finally, we tested known and novel anti-inflammatory compounds (interfering with NF-κB and TNFα signalling), and also demonstrated protection of the auditory system from bilirubin toxicity. We have developed a novel, reversible, model for jaundice that shows movement impairment and auditory loss consistent with human symptoms. We used this model to establish ER-stress and inflammation as major contributors to bilirubin toxicity. Because of the rapid and reversible onset of toxicity in this novel model it represents a system to screen therapeutic compounds. We have demonstrated this by targeting inflammation genetically and with anti-inflammatory small molecules that offered protection against bilirubin toxicity. This also suggests that anti-inflammatory drugs could be of therapeutic use in hyperbilirubinemia.
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影响因子：
6.7
作者：
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DOI：
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影响因子：
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影响因子：
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作者：
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通讯作者：
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