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標題: | 介白素-1β 於初代培養大鼠胚胎皮質神經元中透過 IL-1R1–MyD88 訊息傳遞路徑增強神經傳導 Interleukin-1β Enhances Neurotransmission via IL-1R1– MyD88 Signaling Pathway in Primary Cultured Rat Embryonic Cortical Neurons |
作者: | 洪茂庭 Mao-Ting Hung |
指導教授: | 潘建源 Chien-Yuan Pan |
關鍵字: | 興奮性突觸後電流,介白素-1β,胞內鈣庫,骨髓分化初級反應蛋白 88,神經發炎,神經傳遞,電壓依賴型鈉離子通道, excitatory postsynaptic currents,interleukin-1β,intracellular Ca2+ stores,myeloid differentiation primary response 88,neuroinflammation,neurotransmission,voltage-gated Na+ channels, |
出版年 : | 2023 |
學位: | 碩士 |
摘要: | 介白素-1β (IL-1β) 是免疫反應的發炎前期細胞激素 (proinflammatory cytokines) 之一,且為許多神經疾病及退化的指標。IL-1β 的典型訊息路徑是與其受體蛋白 IL-1R1 結合,活化下游骨髓分化初級反應蛋白 88 (MyD88),進而透過轉錄因子 NF-κB,啟動發炎相關基因的表現。因此多數關於 IL-1β 的研究,多著重於長期處理對細胞活性的影響,但對於 IL-1β 對神經傳導的立即影響及機制則了解較少。在本篇研究中,我們利用全細胞膜片鉗技術及鈣離子螢光影像,紀錄初代培養大鼠胚胎皮質神經元受 IL-1β 刺激時的電訊號與胞內鈣離子濃度 ([Ca2+]i) 的變化。實驗結果顯示 IL-1β (10 ng/mL) 顯著地增加了自發性興奮性突觸後電流 (EPSCs) 及動作電位的頻率。將細胞預處理 IL-1R1 阻斷劑,IL-1RA (1 ng/mL) 及 MyD88 抑制劑,ST2825 (10 μM),則抑制了此頻率的增加。然而 IL-1β 處理並不影響神經細胞的靜止態維持電流 (holding current) 及靜止膜電位 (resting membrane potential);且對麩氨酸 (glutamate) 誘導的電流,亦無顯著影響。紀錄電流-電壓關係,IL-1β 可些微增加向內鈉離子電流,但不改變向外鉀離子電流。IL-1β 對電壓依賴型鈉離子通道 (Nav) 的失活態電壓 (inactivation voltage) 及從失活態恢復到活性態的時間 (recovery time) 也沒有影響。透過鈣離子螢光染劑 fura-2 紀錄 [Ca2+]i 的變化,我們發現 IL-1β濃度在 0.5-1μg/mL之間,皆可顯著地增加 [Ca2+]。 ST2825(10μM) 及 thapsigargin(3μM,使內質網鈣離子耗盡) 阻斷了此 [Ca2+]i 的增加。APV (100 μM) 與 DNQX (1 μM) 分別為 N-甲基-D-天門冬胺酸受體(NMDARs) 及 α-氨基-3-羥基-5-甲基-4-異惡唑丙酸受體 (AMPARs) 拮抗劑,處理細胞後,可抑制 IL-1β 引起的 [Ca2+]i 增加。此外,thapsigargin (3 μM) 的預處理同樣也抑制了 IL-1β 所誘導的 EPSCs 頻率上升。綜合這些結果我們認為 IL-1β 透過 IL-1R1–MyD88 訊息傳遞路徑,由胞內鈣庫釋放鈣離子,在不影響細胞膜離子通道活性狀況下,導致神經遞質釋放;透過神經突觸傳導,在神經之間正向回饋提升神經興奮性,增加 EPSCs 及動作電位的頻率。因此在神經發炎初期,尚未引起神經細胞程序性死亡機制時,IL-1β 濃度的上升,會使神經細胞更加容易興奮,進而影響神經傳導活性。 Interleukin-1β (IL-1β) is a proinflammatory cytokine and a hallmark of many neurologic disorders, including neurodegeneration. The canonical signaling pathway of IL-1β is to activate myeloid differentiation primary response 88 (MyD88) after the binding of IL-1β to its receptor (IL-1R1), activating NF-κB to transcript inflammatory-related genes. Most studies focused on the long-term effects of IL-1β on cellular activities. However, the immediate influence of IL-1β on neurotransmission is unclear. In this study, we applied the whole-cell patch clamp technique to record the electric activities and Ca2+ fluorescence imaging to monitor the changes in the intracellular Ca2+ concentration ([Ca2+]i) from the primary cultured rat embryonic cortical neurons under IL-1β treatment. Our results show that IL-1β (10 ng/mL) significantly increases the frequency of excitatory postsynaptic currents (EPSCs) and action potentials firing. Pretreating the cultured neurons with IL-1RA (1 ng/mL), an IL-1R1 antagonist, and ST2825 (10 μM), a MyD88 inhibitor, suppressed these increments in electric activities. However, IL-1β treatment did not affect the holding current, resting membrane potential, and glutamate-evoked currents. The current-voltage curve shows that IL-1β slightly and significantly increased the inward Na+ currents when depolarized to -20 mV from a holding potential of -70 mV, but did not change the outward K+ currents. The inactivation voltage and recovery time of the voltage-gated Na+ channels showed no difference after IL-1β treatment. We then loaded the cultured neurons with fura-2, a Ca2+-sensitive fluorescence dye, to monitor the changes in [Ca2+]i. IL-1β at 0.5-1 μg/mL could significantly elevate the [Ca2+]i. ST2825 (10 μM) and thapsigargin (3 μM, to deplete intracellular Ca2+ ER stores) blocked this elevation; blocking synaptic transmission by the N-methyl-D-aspartate receptors (NMDARs) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) antagonists, APV (100 μM) and DNQX (1 μM), respectively, also suppress this [Ca2+]i increment. Moreover, pretreating the cultured neurons with thapsigargin (3 μM) inhibited the enhancement effects of IL-1β on EPSCs frequency. These results demonstrate that IL-1β facilitates the excitability of neurons by releasing Ca2+ from ER Ca2+ stores via IL-1R1–MyD88 pathway without affecting the ion channels on the cell membrane. This elevation of [Ca2+]i triggers neurotransmitter release and enhances neuron excitability resulting in positive feedback among the cultured neurons through synaptic transmission. Therefore, at the early stage of neuroinflammation, before the activation of programmed cell death, the increase in the concentration of IL-1b will enhance the neuron excitability to regulate the excitability of neuronal circuits. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/88365 |
DOI: | 10.6342/NTU202301985 |
全文授權: | 同意授權(限校園內公開) |
顯示於系所單位: | 生命科學系 |
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