IL-33/SP 调控胆囊癌神经浸润促进癌性疼痛的机制研究

The underlying mechanisms of cancer pain remain elusive and methods of treating are very limited. Treating cancer pain is a major challenge for pain scientists and clinicians in the field. Gallbladder carcinoma patients are usually suffered severe pain, mainly induced by perineural invasion, however, the mechanism is unknown. Our previous studies indicated that, the expression of IL-33 and substance P (SP) in gallbladder carcinoma were significantly increased compared with the adjacent tissues. Moreover, a mutual promotion phenomenon was generated between IL-33 and SP. Our further experiments showed that SP promoted the cell proliferation, migration and invasion. IL-33 could increase and activate TAK1 in DRG. And TAK1 inhibitor reversed the effects of SP, as well as the allodynia caused by IL-33. Based on the above analysis, we hypothesis IL-33 and SP regulate the perineural invasion of gallbladder cancer cells in a positive feedback process, promote central sensitization, then participate in the induction and maintenance phase of cancer pain. Furthermore, the biological function of SP induced by IL-33 is mediated by TAK1. This study will use human samples, cells, and rodents to explore the interaction between IL-33 and SP, and their roles in the pathogenesis of cancer pain in gallbladder carcinoma, as well as the molecular mechanism. This study will provide new evidence that demonstrate, the critical roles of IL-33/SP in the development of cancer pain, thus open a newavenue of understanding mechanisms of cancer pain. Further, this study will find molecules that could effectively treat cancer pain in laboratory animals with potential in future-patients.

癌性疼痛的发生发展机制尚不明确，临床治疗效果有限，是极具挑战的理论和临床问题。临床上胆囊癌伴癌痛常见，主因可能是肿瘤细胞浸润神经，但具体机制不明。我们前期发现：与癌旁组织相比，胆囊癌组织IL-33与SP显著高表达，并相互促进表达；SP能够促进胆囊癌浸润神经；IL-33能够上调并激活背根神经元中TAK1，抑制TAK1可取消IL-33对SP的促进作用及其诱发的疼痛。据此，我们推测IL-33激活TAK1，上调SP，并形成正反馈，共同调控胆囊癌浸润神经，促进中枢敏化，参与癌性疼痛发生发展。本项目拟收集临床标本，应用胆囊癌/原代背根神经元共培养体系及癌痛模型，通过考察痛行为、神经元兴奋性、相关蛋白及钠离子通道电流的变化情况，阐明并验证TAK1在IL-33促进SP表达中的作用以及IL-33/SP在胆囊癌浸润神经纤维诱发维持癌性疼痛中的作用与相关机理，为肿瘤浸润神经导致癌性疼痛的防治提供新的策略和靶点。

临床中胆囊肿瘤诱发的癌性疼痛非常常见，但发生发展机制尚不明确，治疗方法和效果有 限，是极具挑战的理论和临床问题。本项目以前期研究及相关文献为基础，首先检测IL-33/SP 在人胆囊癌组织和癌旁组织中的表达情况，并分析其与胆囊癌临床病理特征、癌性疼痛程度的关系；分离培养背根神经节（DRG）神经元，利用膜片钳等方法考察干预IL-33/SP信号对DRG初级感觉神经元兴奋性的调控作用，并探索其中的分子机制，同时，体外培养胆囊癌细胞，利用免疫印迹、质粒转染、免疫荧光等实验手段，考察IL-33/SP在肿瘤增殖迁移、痛觉过敏等过程中的作用及其中可能机制；最后，利用裸鼠胆囊癌皮下移植瘤模型、慢性疼痛动物模型，进一步在体验证IL-33/SP促进胆囊癌肿瘤细胞浸润神经，增加神经元兴奋性，促进中枢敏化，进而引起并维持癌性疼痛的过程。我们的研究结果表明胆囊癌疼痛患者多伴有神经浸润；在胆囊癌疼痛状态下，与癌旁组织相比，胆囊癌组织IL-33与SP显著高表达，并相互促进表达，形成正反馈；SP能够促进胆囊癌浸润神经，抑制SP/NK-1R信号通路能够显著减少胆囊癌细胞增殖、迁移与侵袭，抑制神经浸润，并降低DRG神经元兴奋性，有效缓解痛觉过敏、异样性疼痛和自发性疼痛等慢性疼痛症状，且发现其中具体作用机制与AKT/NF-κB信号通路相关；IL-33能够上调并激活背根神经元中TAK1，抑制TAK1可取消IL-33对SP的促进作用及其诱发的疼痛，同时，IL-33能够上调SP和MMP-9的表达与释放，促进胆囊癌侵袭转移，浸润神经，促进中枢敏化，参与胆囊癌浸润神经诱发的痛觉过敏；慢性疼痛模型动物的脊髓和 DRG 水平 MMP-9 活力上调，抑制脊髓 MMP-9 能够有效缓解机械学超敏，抑制慢性疼痛维持期相关神经化学信号的改变以及髓鞘病变。本项目通过收集临床标本，应用细胞实验、多种慢性疼痛模型，考察痛行为、神经元兴奋性、相关蛋白及钠离子通道电流的变化情况，初步明确了 IL-33/SP 促进神经浸润诱发并维持癌性疼痛的分子机制及相关信号通路，为后续临床探寻防治癌痛提供新策略新靶点，为后续药物研发提供理论依据和实验研究基础。

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