GPER/β-alanine Positive Interaction in the Dorsal Root Ganglion Uncovers Potential Mechanisms: Mediating Continuous Neuronal Sensitization and Neuroinflammation Responses in Neuropathic Pain

Background: The pathogenesis of neuropathic pain and the reasons for the prolonged unhealing are still unknown. Increasing evidence suggests that oestrogen sex differences play a role in pain sensitivity, but few studies focused on the role of oestrogen receptor which maybe an important molecular component contributing to peripheral pain transduction. We aimed to investigate the impact of ooestrogen receptors in nociceptive neuronal response in the dorsal root ganglion (DRG) and spinal dorsal horn using a spared nerve injury (SNI) rat model of chronic pain. Methods: We used a class of oestrogen receptors antagonists and agonists intrathecal (i.t.) administrated to male rats with SNI or normal rats to identify the main receptor. Moreover, we applied genes identified through genomic metabolic analysis to determine the key metabolism point and elucidate potential mechanisms mediating continuous neuronal sensitisation and neuroinflammation responses in neuropathic pain. The excitability of DRG neurons was detected using the patch clamp technique. Primary culture was used to extract microglia and DRG neurons, and siRNA transfection was used to silence receptor protein expression. Immunofluorescence, Western blotting, qPCR and behavioral testing were used to assess the expressions, cellular distributions, and actions of main receptor and its related signaling molecules. Results: Increasing the expression and function of G protein-coupled oestrogen receptor (GPER), but not oestrogen receptor-α (ERα) and oestrogen receptor-β (ERβ), in the DRG neuron and microglia, but not the dorsal spinal cord, contributed to SNI-induced neuronal sensitisation. Inhibiting GPER expression in the DRG alleviated SNI-induced pain behaviours and neuroinflammation by downregulating iNOS, IL-1β and IL-6 expression as well as restoring GABAα2 expression simultaneously. Additionally, the positive interaction between GPER and β-alanine, β-alanine accumulation enhances pain sensation and promotes chronic pain development. Conclusion: GPER activation in the DRG causes a positive interaction of β-alanine with iNOS, IL-1β and IL-6 expression and represses GABAα2 involved in post-SNI neuropathic pain development. Blocking GPER and eliminating β-alanine in the DRG neuron and microglia may prevent neuropathic pain development.背景：神经病理性疼痛的发病机制及其迁延不愈的原因至今尚未明确。越来越多的证据表明，雌激素相关的性别差异在疼痛敏感性中发挥作用，但鲜有研究聚焦于雌激素受体的功能——其可能是参与外周疼痛传导的重要分子组分。本研究旨在采用慢性神经病理性疼痛的保留性神经损伤（spared nerve injury, SNI）大鼠模型，探究雌激素受体对背根神经节（dorsal root ganglion, DRG）及脊髓背角伤害性神经元反应的影响。 方法：本研究采用鞘内（intrathecal, i.t.）给药的一类雌激素受体拮抗剂与激动剂，分别对保留性神经损伤（SNI）模型雄性大鼠及正常雄性大鼠进行干预，以明确发挥主要作用的雌激素受体亚型。此外，本研究通过基因组代谢分析筛选得到差异基因，以此确定关键代谢节点，并阐明介导神经病理性疼痛中持续神经元敏化与神经炎症反应的潜在机制。采用膜片钳技术检测背根神经节神经元的兴奋性。通过原代培养提取小胶质细胞与背根神经节神经元，并采用小干扰RNA（small interfering RNA, siRNA）转染技术沉默受体蛋白的表达。采用免疫荧光、蛋白质印迹（Western blotting）、实时荧光定量聚合酶链反应（quantitative real-time PCR, qPCR）以及行为学实验，对主要受体及其相关信号分子的表达、细胞定位与功能进行评估。 结果：在背根神经节神经元与小胶质细胞（而非脊髓背角）中，G蛋白偶联雌激素受体（G protein-coupled oestrogen receptor, GPER）而非雌激素受体α（oestrogen receptor-α, ERα）与雌激素受体β（oestrogen receptor-β, ERβ）的表达与功能增强，可促进SNI诱导的神经元敏化。在背根神经节中抑制GPER的表达，可通过下调诱导型一氧化氮合酶（inducible nitric oxide synthase, iNOS）、白细胞介素1β（interleukin-1β, IL-1β）与白细胞介素6（interleukin-6, IL-6）的表达，同时恢复γ-氨基丁酸A型受体α2亚基（γ-aminobutyric acid type A receptor α2 subunit, GABAα2）的表达，从而缓解SNI诱导的疼痛行为与神经炎症反应。此外，GPER与β-丙氨酸（β-alanine）之间存在正向相互作用，β-丙氨酸的积累可增强疼痛感知并促进慢性疼痛的发生发展。 结论：背根神经节中GPER的激活可促使β-丙氨酸与iNOS、IL-1β及IL-6的表达产生正向相互作用，并抑制参与SNI后神经病理性疼痛发生的GABAα2的表达。在背根神经节神经元与小胶质细胞中阻断GPER并清除β-丙氨酸，或可阻止神经病理性疼痛的发生发展。