NR2B-CREB-miR212/132-CRTC1-CREB信号通路在疼痛调节中的作用：离体和在体实验
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The Role of NR2B-CREB-miR212/132-CRTC1-CREB Signal Network in Pain Regulation In Vitro and In Vivo
背景与目的
慢性疼痛蚕食身心，使人变得衰弱，威胁着人类健康，而其分子机制仍未明了。已有的研究证实cAMP反应元件结合蛋白（CREB）在痛觉调节中起重要作用；而CREB转录共激活因子1（CRTC1）及微小miRRNA（212/132）在突触可塑性中同样也很重要。然而，在疼痛状态下，这些因素之间的相互作用知之甚少。我们进行这项离体实验的目的主要是为了确定CREB、CRTC1及mir212 / 132之间的串扰。此外，在体实验中，我们还探讨了给予CREB相关的腺病毒载体、CRTC1相关的腺病毒载体和绑定mir212 / 132的核酸（LNA）后，慢性缩窄性损伤（CCI）小鼠痛觉过敏的变化。
方 法
培养小鼠胚胎脊髓原代神经元，在培养的神经元中加入外源性谷氨酸以模拟体内疼痛过程。实时定量聚合酶链反应测定NR2B，crtc1，CREB和mir212 / 132在mRNA水平的变化；Western blot检测p-NR2B，p-CREB，CRTC1在蛋白质水平的表达情况。通过Von Frey纤维丝测痛仪研究CCI小鼠模型机械性痛觉过敏。鞘内注射CREB-miR（腺病毒载体干扰CREB基因），CREB-AD（腺病毒载体过表达CREB基因）；CRTC1-miR（腺病毒载体干扰CRTC1基因），CRTC1-AD（腺病毒载体过表达 CRTC1基因）及mir212 / 132 LNA 。
结 果
离体实验中，100 μmol/L谷氨酸诱导p-CREB and miR212/132-LNA表达。CRTC1蛋白被CREB-miR 和 miR212/132-LNA所下调。CRTC1 mRNA被CREB-AD上调而被CREB-miR 和 miR212-LNA所下调。P-CREB 被 CRTC1-AD所上调而被miR212/132所下调。CREB mRNA 可被CRTC1-AD上调而被CRTC1-miR下调。MiR212/132 被 CRTC1-AD 和 CREB-AD上调，被CREB-miR下调。在体实验中，CRTC1-miR，CREB-miR和 miR212/132-LNA不同程度上增加拨抓痛阈。
结 论
NR2B-CREB-miR212/132-CRTC1-CREB信号通路在疼痛的调节中起着重要作用。此信号通路中任何分子途径的干预都能使疼痛减轻。
原始文献摘要
Address correspondence to Xiaoping Gu, PhD, MD, and Zhengliang Ma,PhD, MD, Department of Anesthesiology, Affiliated Drum Tower Hospital of Medical Department of Nanjing University, 321 Zhong Shan Rd, Nanjing, Jiangsu 210008, P.R. China. Address e-mail to xiaopinggu@nju.edu.cn; mazhengliang1964@nju.edu.cn. Copyright © 2017 International Anesthesia Research Society DOI: 10.1213/ANE.0000000000001880
BACKGROUND:
Chronic pain is a debilitating threat to human health, and its molecular mechanism remains undefined. Previous studies have illustrated a key role of cAMP response element-binding protein (CREB) in pain regulation; CREB-regulated transcription coactivator 1 (CRTC1) and microRNA212/132 (miR212/132) are also vital in synaptic plasticity. However, little is known about the interaction among these factors in pain condition. We conducted this experiment mainly to determine the crosstalk between CREB, CRTC1, and miR212/132 in vitro. Moreover, we explored the changes in hyperalgesia on chronic constrictive injury (CCI) mouse in vivo when given CREB-related adenovirus vectors, CRTC1-related adenovirus vectors, and miR212/132-locked nucleic acid (LNA).
METHODS:
We cultured primary neurons in the spinal cord of mouse embryos. Exogenous glutamate was added to cultured neurons to simulate in vivo pain process. Real-time quantitative polymerase chain reaction was used to determine changes of NR2B, CRTC1, CREB, and miR212/132 at the mRNA level; Western blot was used to detect p-NR2B, p-CREB, and CRTC1 at protein level. Von Frey cilia were used to study mechanical hyperalgesia in a murine model of CCI. CREB-miR (adenovirus vector interfering CREB gene), CREB-AD (adenovirus vector overexpressing CREB gene); CRTC1-miR (adenovirus vector interfering CRTC1 gene), CRTC1-AD (adenovirus vector overexpressing CRTC1 gene), and miR212/132-LNA were injected intrathecally.
RESULTS:
In vitro, 100 μmol/L glutamate induced p-CREB and miR212/132-LNA. CRTC1 protein was downregulated by CREB-miR and miR212/132-LNA. CRTC1 mRNA was upregulated by CREB-AD and downregulated by CREB-miR and miR212-LNA. P-CREB was upregulated by CRTC1-AD and downregulated by miR212/132. CREB mRNA was upregulated by CRTC1-AD and downregulated by CRTC1-miR. MiR212/132 was upregulated by CRTC1-AD and CREB-AD; downregulated by CREB-miR. In vivo, CRTC1-miR, CREB-miR, and miR212/132-LNA increased paw withdrawal mechanical threshold in various degrees.
CONCLUSIONS:
The NR2B-CREB-miR212/132-CRTC1-CREB signal network plays an important role in the regulation of pain. Intervening with any molecule in this signal network would reduce pain perception.
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