研究白介素-1β刺激提升初代培養大鼠胚胎皮質神經細胞胞內鈣離子濃度的訊息途徑

Characterization of the Signaling Cascade Underlying the IL-1β-induced Elevation of [Ca2+]i in Primary-cultured Rat Embryonic Cortical Neurons

高陵(Ling Kao)

指導教授 ： 潘建源

國立臺灣大學/生命科學院/生命科學系/碩士(2022年)

https://doi.org/10.6342/NTU202201817

摘要

白介素-1β (interleukin-1 β, IL-1β) 是一種促進發炎的細胞激素，在免疫和發炎反應中擔任重要角色。研究指出，在罹患慢性神經退化疾病患者的腦脊髓液中，相對較於一般人有較高濃度的IL-1β。然而，研究重心多在於IL-1β與細胞發炎機制間的交互作用，及對神經細胞存活的研究，而較少討論IL-1β對神經傳導的影響。鈣離子是神經傳導機制中重要的信息分子，調控神經細胞許多活性。在本研究中，利用鈣離子影像技術，研究IL-1β刺激並影響初代培養大鼠胚胎皮質神經細胞，細胞內鈣離子濃度 ([Ca2+]i)的變化。先將鈣離子螢光染劑Fura-2送入神經細胞中，再以不同濃度IL-1β刺激，觀測鈣離子螢光的變化ΔRatio。結果顯示ΔRatio會隨IL-1刺激濃度提高而增加，呈劑量依賴性曲線 (dose-dependent)，EC50為0.22±0.21 ng/ml；但相同濃度範圍下的IL-1α，卻無法引起鈣離子反應。使用IL-1β受體的抑制劑IL-1RA (1 ng/mL)，可顯著抑制ΔRatio變化，而IL-1受體訊息途徑的抑制劑，ST2825 (10 µM)，可幾乎完全抑制IL-1β引起的鈣離子反應。移除胞外鈣離子，或是去除胞內鈣庫中的鈣離子，也都可抑制IL-1β所引起的鈣離子反應。在AMPA受體抑制劑，DNQX (1 µM)、神經傳導物質釋放抑制劑，tetanus toxin (1 µM)、或是鈉離子通道抑制劑，tetrodotoxin (1 µM)處理下，IL-1β皆無法引起鈣離子反應。這些結果表明，在發炎反應過程中，IL-1β可能通過IL-1R誘導神經傳導物質的釋放，從而刺激突觸後神經元並升高[Ca2+]i，從而在神經迴路中產生正回饋。因此，控制 IL-1β不僅可以調節神經發炎，還可以調節突觸活動。

關鍵字

AMPA受體 ； 鈣離子恆定 ； IL-1β ； 胞內鈣庫 ； MyD88 ； 神經傳導

並列摘要

Interleukin-1β (IL-1β) is a pro-inflammatory cytokine that belongs to the IL-1 family and plays a crucial role in inflammatory responses. The cerebrospinal fluid of patients with chronic neurodegenerative diseases usually contains a higher level of IL-1. Most studies focus on the effects of IL-1β on neuron viability; it is not clear whether IL-1β affects the neurotransmission in the nervous system. In this study, we loaded the primary cultured rat embryonic cortical neurons with Fura-2, a Ca2+ sensitive fluorescence dye, and characterized how IL-1β affects the intracellular Ca2+ concentration ([Ca2+]i). The results showed that IL-1β but not IL-1α elevated the [Ca2+]i and the EC50 was 0.22 ± 0.21 ng/mL. IL-1RA (1 ng/mL), an antagonist of the IL-1 receptor, and ST2825 (10 µM), an inhibitor blocking the signaling pathway of IL1R, could significantly suppress this elevation. Removing extracellular Ca2+ and depletion of the intracellular Ca2+ stores inhibited IL-1 evoked Ca2+ responses significantly. In the presence of DNQX (1 µM), an inhibitor of AMPA receptors, tetanus toxin (1 µM), an inhibitor of neurotransmitter release, or tetrodotoxin (1 µM), a blocker of voltage-gated Na+ channels, IL-1β did not elicit a significant elevation in [Ca2+]i. These results suggest that, during the inflammatory response, IL-1β may induce the release of neurotransmitters via the IL-1R to stimulate the post-synaptic neurons and elevate the [Ca2+]i, resulting in positive feedback in neural circuits. Therefore, controlling the level of IL-1 does not only regulate neuroinflammation but synaptic activities as well.

並列關鍵字

AMPA receptor ； Ca2+ homeostasis ； interleukin-1β ； intracellular Ca2+ store ； MyD88 ； neurotransmission

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