Wnt5a/Ryk信号参与骨癌痛的外周机制

随着全球癌症发病率的不断增高和癌症的慢性化趋势，治疗癌性疼痛的医疗需求及相关经济负担也日益增加。骨癌痛是由原发性骨肿瘤或肿瘤继发骨转移引起的临床最为常见的癌性疼痛类型。骨癌痛及其伴随的高钙血症、病理性骨折、易感染等情况不但严重影响患者的生活质量，而且给家庭和社会带来沉重的心理、经济和财政负担。目前临床上用于治疗癌性疼痛的药物包括世界卫生组织推荐的阿片类药物等对中、重度疼痛的治疗效果欠佳，无法达到充分缓解疼痛的目的，且大都存在安全性、成瘾性和药物耐受等方面的问题及严重的不良反应和其他副作用。因此，阐明癌性疼痛的发生发展机制，探索有效的防治措施是非常紧迫的科研和医疗任务。解决这一难题具有重要的学术和医疗价值，在挽救生命、维护人类健康等方面有着重大而深远的社会意义。

癌性疼痛发生发展的主要原因包括如下四个方面：1）肿瘤源性的分泌物介导的伤害性感受；2）骨质溶解导致的疼痛；3）肿瘤导致的神经损伤；4）神经系统的敏化与重构。这些改变使得外周神经末梢、初级感觉神经元的胞体和中枢突的兴奋性异常增高，脊髓以及更高级中枢信息传递的突触可塑性长期增强，从而导致外周痛觉感受器敏化和痛觉中枢敏化。但是，癌性疼痛发生发展的具体细胞和分子机制仍然很不清楚。

本文聚焦于研究骨癌痛发生和发展的外周神经机制，研究内容主要包括以下三个方面：1）通过转录组测序技术阐明骨癌痛发生发展不同阶段背根节中转录组学改变的特点，并比较不同慢性痛模型（炎性痛、神经病理性痛和骨癌痛）之间转录组差异改变的异同；2）初步阐明Wnt 信号家族β-catenin非依赖性信号通路在疼痛的初级传入感觉神经元（背根神经节）和外周神经末梢中的活动状况，及其和骨癌痛发生发展的相互关系；3）探索Wnt5a/Ryk信号在外周水平调控初级感觉神经元胞内钙活动及外周伤害性感受的离子通道机制。

本文采用RAN-seq技术研究了骨癌痛发生发展过程中背根节的转录组改变，并探讨了以离子通道和细胞因子改变为代表的神经病理性和炎症性特征在骨癌痛发生发展不同阶段的动态变化。研究结果显示骨癌引起背根节中数千个基因的表达发生改变，这些基因主要涉及免疫过程、炎症反应和细胞因子的胞内信号转导，且这些改变与骨癌痛的发展高度同步。其中，离子通道基因的改变有助于骨癌痛早期发生，而细胞因子信号通路相关基因的改变则有助于骨癌痛的维持。本文还比较了不同疼痛模型（炎性痛、神经病理性痛和骨癌痛）之间背根节中的转录组改变的差异。研究结果显示不同模型之间共享的基因很少，表明不同形式的疼痛发病机理有所不同。这部分的研究结论强调了细胞因子相关通路及免疫与炎症因素在骨癌痛发展中扮演的重要角色，以及不同疼痛模型之间发病机制的特殊性。此外，本文进一步研究了以Wnt5a/Ryk信号为代表的Wnt非经典通路在骨癌痛发展和维持中的作用和细胞及分子机制。结果显示外周Wnt5a/Ryk信号在骨癌痛的发展中被激活；其对大鼠背根节神经元的胞内钙活动和疼痛行为有显著的调控作用；抑制Wnt5a/Ryk信号可以显著缓解骨癌引起的疼痛行为。这部分的研究结论强调了外周Wnt5a/Ryk信号在热痛觉过敏和骨癌痛的发展中的重要作用。在机制研究中，结果显示外周Wnt5a/Ryk信号调控背根节中TRPV1的表达水平和JNK2/3的磷酸化水平；Wnt5a/Ryk/JNK信号通路调控TRPV1依赖的背根节神经元胞内钙活动和外周伤害性感受。本文提出了Wnt5a/Ryk信号通过调控外周神经系统中TRPV1离子通道的表达和活性从而调控背根节神经元中的中、小细胞胞内钙活动和大鼠热痛敏行为的观点，并初步探讨了背根节中JNK的磷酸化在介导Wnt5a/Ryk信号和调节TRPV1依赖的背根节神经元胞内钙活动中的作用。这些成果为后续研究骨癌痛的外周机制提供了理论和实验基础，也为临床治疗骨癌痛提供了新的潜在分子靶标。

With the increasing incidence of cancer and the trend of chronic cancer worldwide, the medical needs and related financial burdens for the treatment of cancer pain are also increasing. Bone Cancer Pain (BCP) in the clinic is the most common type of cancer pain caused by primary bone sarcomas or secondary bone metastases from non-bone cancer. BCP and its accompanying hypercalcemia, pathological fractures, susceptibility to infection not only seriously affect patients’ life quality, but also bring heavy psychological, economic, and financial burdens to the family and society. The currently available clinical medication for the treatment of cancer pain is not enough effective in the treatment of moderate and severe pain, while most of the drugs have safety, addiction, and drug tolerance problems, e.g., serious adverse reactions, and other side effects. Therefore, scientific research and medical tasks including clarifying the mechanism of generation and development of cancer pain and exploring effective approaches for prevention and treatment are urgently needed. Solving these problems has important academic and medical value as well as great social significance in saving lives and maintaining human health.

The hypotheses of previous studies explaining the mechanism of the generation and development of cancer pain mainly include: 1) nociception mediated by tumor-derived secretions; 2) pain induced by osteolysis; 3) nerve damage caused by tumor growth, and 4) sensitization and reconstruction of the nervous system. These changes induce the increased abnormal excitability of peripheral nerve endings, cell bodies, and central processes of primary sensory neurons, and the long-term potentiation of the synaptic plasticity of the spinal cord and higher brain centers involved in pain information transmission, which leads to the sensitization of peripheral nociceptors and central sensitization. However, the cellular and molecular mechanisms of the generation and development of cancer pain remain unclear. This research focuses on understanding the peripheral mechanisms involved in the development of BCP. The main findings include threefold. First, we have clarified the characteristics of transcriptomic changes in the dorsal root ganglion (DRG) at different stages of development of bone cancer pain and compared the similarity and uniqueness of transcriptome differences among different chronic pain models including inflammatory pain, neuropathic pain, and BCP. Second, we have demonstrated the critical role of Wnt β-catenin-independent signaling within the dorsal root ganglion (DRG) including its peripheral terminals, and its contribution to the development of BCP. Third, we have demonstrated the regulatory effect of Wnt5a/Ryk/JNK signaling on intracellular calcium activity.

We employed the RNA-seq technology to clarify the total transcriptomic changes at different stages of development of BCP and explored the dynamic changes of neuropathological features represented by ion channels and the inflammatory features represented by cytokines in BCP. Our results show that bone cancer cause alterations in the expression of thousands of genes in the DRG. These genes are mainly involved in the immune process, inflammatory response, and intracellular signal transduction of cytokines, and these alterations are highly synchronized to the development of BCP. Among them, the alterations of ion channel genes contribute to the early occurrence of BCP and the alterations of genes of cytokine signaling pathway contribute to the maintenance of BCP. We also compared the differences in transcriptome alterations in the DRG between different pain models. The results show that there are few genes shared between different pain models, indicating that the pathogeneses of different forms of pain are different. The conclusions of this part of the research emphasize the important role of cytokine-related pathways and immune and inflammatory factors in the development of BCP, as well as the uniqueness of the pathogeneses of different pain models. In addition, we have further studied the role and cellular and molecular mechanisms of Wnt non-canonical pathways represented by Wnt5a/Ryk signaling in the development and maintenance of BCP. We found that the peripheral Wnt5a/Ryk signal is activated during the development of BCP, which has a significant regulatory effect on the intracellular calcium activity of rat DRG neurons and pain behavior. Inhibition of the Wnt5a/Ryk signal can significantly alleviate painful behavior caused by bone cancer. The conclusions of this part of the study emphasize the important role of peripheral Wnt5a/Ryk signaling in the development of thermal hyperalgesia and BCP. In the mechanism study, we found that the peripheral Wnt5a/Ryk signaling regulates the expression level of TRPV1 and the phosphorylation level of JNK2/3 in the DRG, and Wnt5a/Ryk/JNK signaling pathway regulates the TRPV1-dependent intracellular calcium activity of small and medium DRG neurons and peripheral nociception. We proposed that Wnt5a/Ryk signal regulates the expression and activity of TRPV1 ion channels in the peripheral nervous system to trigger the intracellular calcium activity in DRG neurons and thermal hyperalgesia in rats. In addition, we have preliminary explored the role of JNK phosphorylation in DRG in mediating Wnt5a/Ryk signaling and regulating TRPV1-dependent intracellular calcium activity of DRG neurons. These findings provide a theoretical and experimental basis for the follow-up study of the peripheral mechanism of BCP and also provide new potential molecular targets for the clinical treatment of BCP.

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