TAK1调控脊髓神经元-胶质细胞cross-talk参与吗啡耐受的机制研究

The mechanism of opioid tolerance remain elusive. It is an urgent task that finding a new and unique target and then improving the analgesic effects of opioid drugs. Recently, it has been demonstrated that TNFR, IL-1R and TLR-4 receptor and MAPK--NF-kB downstream signaling pathways play a crucial role in morphine tolerance. Strikingly, transforming growth factor β-activated kinase1 (TAK1), which belongs to MAP3K family, is a common gated molecular target of many inflammatory factor receptors and mediates MAPK--NF-kB signaling pathways. The lastest data of the applicant shows that, inhibition of spinal TAK1 can significantly enhance the analgesic effect of morphine and suppress morphine tolerance, while it also significantly suppressed morphine-induced spinal neuronal extra-excitation and neuroinflammation associated with glial cells activiation. Those findings suggest that TAK1 may play an important role in morphine tolerance. In addition, the data also shows that paeoniflorin probably is an inhibitor of TAK1. Paeoniflorin significantly inhibited morphine-induced increased protein level of TAK1, and significantly suppressed chronic morphine tolerance and morphine-induced neuroinflammation. However, the molecular mechanisms of TAK1 involved in morphine tolerance is still unclear, and the exact sites that paeoniflorin binding TAK1 is not known.This project aims to clarify the molecular mechanism of spinal TAK1 in morphine tolerance and find new drugs. The expected findings will not only help reveal the mechanism of morphine tolerance but also provide new targets for clinical drug development.

明确阿片药物的耐受机制，寻找新的靶点和药物改善阿片药物耐受是迫切任务。协同抑制TNFR，IL-1R和TLR-4及下游MAPK/NF-KB信号通路是改善吗啡耐受的良好策略。转化生长因子β活化激酶1（TAK1）隶属MAP3K，是介导多个炎症因子受体及MAPK-NF-KB信号通路活化的共同门控性分子靶点。申请者最新研究发现：抑制脊髓水平TAK1可显著增强吗啡镇痛效能，缓解吗啡耐受；显著抑制神经元敏化以及胶质细胞相关神经炎症。但TAK1参与吗啡耐受详细分子机制尙不清楚。预实验还提示：芍药苷可能是TAK1的抑制剂，其可显著抑制吗啡诱导TAK1升高，并可显著抑制吗啡耐受和吗啡所致神经炎症。但芍药苷与TAK1结合的关键性位点还需进一步筛选。本项目旨在阐明TAK1调控脊髓各类细胞功能及细胞间通讯的机制，并围绕芍药苷--TAK1，筛选抑制TAK1的小分子药物，研究成果为临床药物研发提供新靶标和先导化合物。

明确阿片药物的耐受机制，寻找新的靶点和药物改善阿片药物耐受是迫切任务。转化生长因子β活化激酶1（TAK1）是介导多个炎症因子受体及MAPK-NF-KB信号通路活化的共同门控性分子靶点。采用安全有效的药物靶向性干预TAK1或其上下游分子，协同抑制神经元、胶质细胞激活，是改善吗啡耐受的良好策略。我们发现吗啡处理可导致脊髓神经元及胶质细胞p-TAK1和TAK1水平的上升；抑制TAK1可协同抑制神经元敏化和小胶质细胞星形胶质细胞的活化、显著提升吗啡镇痛效果，显著延缓吗啡耐受。进一步探索发现，HMGB1作为体内DAMPs之一，是TLR4的重要配体，可由神经元释放进而促进神经病理性痛的发生。申请人利用细胞免疫荧光、活性胞工作站、组织免疫荧光双标、siRNA细胞转染、免疫印迹法等技术在体内外进行了详细的研究，结果发现：1）吗啡诱导神经元释放HMGB1，通过结合小胶质细胞上TLR4，促进神经炎症级联反应，最终导致吗啡耐受；2）吗啡诱发的HMGB1的释放是通过AMPK-HO-1信号通路所介导的。因此，AMPK-HMGB1-TLR4在介导神经元小胶质细胞的crosstalk中起到关键作用，未来有希望成为治疗吗啡耐受的潜在靶点。.其次，通过筛选和考察，小分子局麻药利多卡因成为候选药物。采用免疫印迹法、荧光双标、实时定量PCR技术验证利多卡因对于吗啡镇痛和吗啡耐受、神经元、小胶质细胞活化产物和分子指标的作用；利用荧光钙离子测定、免疫共定位、siRNA干扰等技术探索利多卡因与TAK1上游激酶AMPK以及SOCS1、SOCS3以及胞内Ca2+、cAMP、PKA的关系。结果发现发现：1）鞘内注射利多卡因显著延缓小鼠吗啡耐受、减少c-fos和CGRP表达、小胶质细胞活化及TNF-α、IL-1β释放；2）体内外数据表明利多卡因可直接激活AMPK，进而诱导脊髓小胶质细胞SOCS3蛋白水平的增高；3）利多卡因对小胶质细胞内Ca2+没有直接影响，而通过cAMP-PKA-CaMKKβ通路激活AMPK。因此，利多卡因可以通过AMPK-SOCS3依赖的方式反馈性抑制吗啡诱导的小胶质细胞激活，从而抑制神经炎症，缓解吗啡耐受。