CircMap1a调控L-苯丙氨酸代谢参与紫杉醇诱导慢性疼痛的作用及机制研究

Chronic pain caused by paclitaxel (Pacl) has been seriously affected the course of treatment and the quality of life in patients with cancer. However the underlying mechanism is not clear now. The metabolomics study showed that a variety of metabolites were involved in the development of chronic pain. Our preliminary experiments for the first time found that: intraperitoneal injection of paclitaxel increased the content of L-phenylalanine (L-Phe) in spinal dorsal horn, and the application of L-Phe can induce mechanical allodynia and enhance the dorsal horn NMDA receptor currents. However, the mechanism of paclitaxel induced up regulation of L-Phe was not clear. We also found that paclitaxel down-regulated the expression of circMap1a and phenylalanine hydroxylase (PAH, the rate-limiting enzyme of L-Phe). The prediction software further showed that there are potential binding sites between PAH mRNA with both miR-320 andmiR-125a, which can be “sponged” by circMap1a. Therefore, we hypothesized that paclitaxel might decrease the circMap1a expression in spinal dorsal horn, thereby enhancing the inhibition effect of miRNA on PAHmRNA, so that causing L-Phe accumulation to act on NMDA receptor, which eventually leads to central sensitization and chronic pain. This project will provide new evidence for elucidating the mechanism of chemotherapeutic drugs induced chronic pain and central sensitization.

紫杉醇所致的慢性疼痛严重影响了肿瘤患者的治疗进程及生活质量，其机制不清。组学研究显示多种代谢产物参与慢性疼痛的发生发展。我们预实验首次发现：腹腔注射紫杉醇上调脊髓背角L-苯丙氨酸（L-Phe）的含量，且应用L-Phe可诱导大鼠产生机械痛敏并增强背角NMDA受体电流，但紫杉醇诱导脊髓背角L-Phe上调的机制不清。预实验还发现：紫杉醇下调了脊髓背角circMap1a和L-Phe代谢限速酶-苯丙氨酸羟化酶（PAH）的表达。预测进一步显示：能够被circMap1a海绵吸附的miR-320和miR-125a与PAH mRNA间存在潜在的结合位点。因此，我们推测：紫杉醇可能通过下调脊髓背角circMap1a，减少对miRNA的吸附，增强其对PAH mRNA的抑制作用导致L-Phe堆积，堆积的L-Phe作用于NMDA受体，诱导中枢敏化介导慢性疼痛的发生。本项目将为阐明化疗药物诱导慢性疼痛提供全新的证据。

细胞代谢产物在一定程度上能够更加精准的反映机体内的变化，其含量异常改变与多种病理过程有着密切联系。我们的实验结果发现，化疗药紫杉醇诱导慢性疼痛的大鼠模型中，脊髓背角组织中代谢产物L-Phe及其代谢限速酶PAH的表达均出现异常。外源性补充过量的L-Phe能够引起正常大鼠产生机械性痛觉过敏，而在脊髓中激活或过表达PAH则能显著抑制紫杉醇诱导的中枢敏化及痛觉过敏。此外，高通量测序结果显示circMap1a的表达在慢性疼痛后出现明显的下调；通过数据库分析预测及分子实验验证，发现circMap1a能够海绵吸附miR-320，当circMap1a表达下降，游离的miR-320含量增加，其作用于PAH mRNA抑制其翻译，进而导致PAH的表达降低，引起L-Phe的代谢障碍并在脊髓内堆积，最终引起中枢敏化和慢性疼痛。以上结果从代谢及转录后调控的角度揭示了化疗药物诱导慢性疼痛的新机制，为慢性疼痛的治疗提供潜在治疗靶标。