Ke Xinlong, Cai Huajing, Luo Fangla, Zheng Xing, Hu Qian, Zhou Youfa, Wang Yongjie, Zhang Xiangnan, Chen Yeru, Chen Gang
Department of Anesthesiology, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Zhejiang, Hangzhou, China.
School of Pharmacy, Hangzhou Normal University, Zhejiang, Hangzhou, China.
CNS Neurosci Ther. 2025 Apr;31(4):e70368. doi: 10.1111/cns.70368.
Trigeminal neuropathic pain (TNP) is a debilitating condition characterized by chronic facial pain, yet its underlying mechanisms remain incompletely understood. Transient Receptor Potential Canonical 4 (TRPC4) has been reported to promote the development of abnormal pain or pain hypersensitivity in neuropathic pain. However, the specific contribution of TRPC4 to TNP pathogenesis remains unclear.
This study aimed to investigate the role of TRPC4 in a mouse model of trigeminal neuropathic pain induced by chronic constriction of the unilateral infraorbital nerve (CION).
Adult male/female mice were subjected to either CION surgery or sham surgery. Behavioral assays were conducted to assess facial pain-like responses over a 28-day period. TRPC4 distribution in the trigeminal ganglion (TG) was evaluated using Immunofluorescence. TRPC4 inhibitor ML204 and agonist Englerin A were employed to evaluate the impact of TRPC4 on facial pain-like behaviors. A TRPC4-overexpressing HEK293 cell model was conducted via plasmid transfection. To assess the function of TRPC4, we employed cellular calcium imaging technology to investigate the effects of modulating TRPC4 function by analyzing dynamic changes in intracellular calcium ion concentrations in primary trigeminal ganglion neurons and HEK293 cells. Trpc4 shRNA was used to specifically knock down TRPC4 in the trigeminal ganglion. Western blot analysis was used to assess the activation of ERK, P38, and ATF2 signaling pathways.
Mice subjected to CION exhibited persistent facial pain-like behaviors and a significant increase in TRPC4 expression in TG neurons. Trpc4 shRNA or pharmacological inhibition with ML204 attenuated CION-induced pain behaviors, while activation of TRPC4 with Englerin A induced pain-like responses in naive mice. Calcium imaging revealed that both Englerin A and TRPC4 overexpression elevated intracellular Ca² levels in TG neurons and HEK293 cells. This Ca² influx triggered the activation of ERK and P38, leading to enhanced ATF2 activation. Downregulation of TRPC4 in the TG reduced ERK/P38 phosphorylation and ATF2 expression and activation.
This study provides the first evidence that TRPC4 plays a critical role in CION-induced trigeminal neuropathic pain by promoting the activation of the downstream transcription factor ATF2 via the Ca²-ERK/P38 pathway.
三叉神经病理性疼痛（TNP）是一种以慢性面部疼痛为特征的使人衰弱的疾病，但其潜在机制仍未完全明确。据报道，瞬时受体电位香草酸亚型4（TRPC4）可促进神经性疼痛中异常疼痛或疼痛超敏反应的发展。然而，TRPC4在TNP发病机制中的具体作用仍不清楚。
本研究旨在探讨TRPC4在单侧眶下神经慢性缩窄（CION）诱导的三叉神经病理性疼痛小鼠模型中的作用。
成年雄性/雌性小鼠接受CION手术或假手术。进行行为学检测以评估28天内的面部疼痛样反应。采用免疫荧光法评估TRPC4在三叉神经节（TG）中的分布。使用TRPC4抑制剂ML204和激动剂Englerin A来评估TRPC4对面部疼痛样行为的影响。通过质粒转染构建TRPC4过表达的HEK293细胞模型。为评估TRPC4的功能，我们采用细胞钙成像技术，通过分析原代三叉神经节神经元和HEK293细胞内钙离子浓度的动态变化来研究调节TRPC4功能的效果。Trpc4 shRNA用于特异性敲低三叉神经节中的TRPC4。采用蛋白质免疫印迹分析来评估ERK、P38和ATF2信号通路的激活情况。
接受CION手术的小鼠表现出持续的面部疼痛样行为，且TG神经元中TRPC4表达显著增加。Trpc4 shRNA或用ML204进行药物抑制可减轻CION诱导的疼痛行为，而用Englerin A激活TRPC4可在未处理的小鼠中诱导出疼痛样反应。钙成像显示，Englerin A和TRPC4过表达均提高了TG神经元和HEK293细胞内的Ca²⁺水平。这种Ca²⁺内流触发了ERK和P38的激活，导致ATF2激活增强。三叉神经节中TRPC4的下调降低了ERK/P38磷酸化以及ATF2的表达和激活。
本研究提供了首个证据，即TRPC4通过Ca²⁺-ERK/P38途径促进下游转录因子ATF2的激活，在CION诱导的三叉神经病理性疼痛中起关键作用。
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