Autophagy Plays an Important Role in Anti-inflammatory Mechanisms Stimulated by Alpha7 Nicotinic Acetylcholine Receptor.
Autophagy Plays an Important Role in Anti-inflammatory Mechanisms Stimulated by Alpha7 Nicotinic Acetylcholine Receptor.
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自噬在 Alpha7 烟碱乙酰胆碱受体刺激的抗炎机制中发挥重要作用
DOI：
10.3389/fimmu.2017.00553
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发表时间：
2017
影响因子：
7.3
通讯作者：
Liu C
中科院分区：
文献类型：
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作者：
Shao BZ;Ke P;Xu ZQ;Wei W;Cheng MH;Han BZ;Chen XW;Su DF;Liu C
α 7烟碱乙酰胆碱受体（α 7 nAChR）已被报道可减轻神经炎症。本研究旨在探讨自噬在α 7 nAChR介导的神经炎症抑制中的作用及其机制。实验性自身免疫性脑脊髓炎（EAE）小鼠和脂多糖刺激的BV 2小胶质细胞分别用作体内和体外神经炎症模型。采用神经功能评分评价EAE的严重程度。免疫印迹法检测自噬相关蛋白Beclin 1、LC 3-II/I、p62/SQSTM 1。用透射电镜观察自噬体的形态，用mRFP-GFP-LC 3串联荧光质粒检测自噬通量。实时荧光定量PCR检测IL-6、IL-1β、IL-18和肿瘤坏死因子-α（TNF-α）的mRNA水平。我们分别用3-甲基腺嘌呤（3-MA）和自噬相关基因5小干扰RNA（Atg 5 siRNA）在体内和体外阻断自噬。PNU 282987激活α 7 nAChR可减轻EAE小鼠的严重程度和脊髓炎性浸润。PNU 282987处理还增强了EAE小鼠脊髓和脾脏中单核细胞/小胶质细胞自噬（Beclin 1、LC 3-II/I比率、p62/SQSTM 1、CD 45或CD 68阳性细胞与LC 3的共定位）。PNU 282987对EAE小鼠的有益作用被自噬抑制剂3-MA部分消除。在体外，PNU 282987处理增加自噬并促进自噬通量。通过Atg 5 siRNA或巴弗洛霉素A1阻断自噬减弱了PNU 282987对IL-6、IL-1β、IL-18和TNF-α mRNA的抑制作用。我们的研究结果首次表明，激活α 7 nAChR增强单核细胞/小胶质细胞自噬，从而抑制神经炎症，从而在EAE中发挥预防作用。
Alpha7 nicotinic acetylcholine receptor (α7nAChR) has been reported to alleviate neuroinflammation. Here, we aimed to determine the role of autophagy in α7nAChR-mediated inhibition of neuroinflammation and its underlying mechanism. Experimental autoimmune encephalomyelitis (EAE) mice and lipopolysaccharide-stimulated BV2 microglia were used as in vivo and in vitro models of neuroinflammation, respectively. The severity of EAE was evaluated with neurological scoring. Autophagy-related proteins (Beclin 1, LC3-II/I, p62/SQSTM1) were detected by immunoblot. Autophagosomes were observed using transmission electron microscopy and tandem fluorescent mRFP-GFP-LC3 plasmid was applied to test autophagy flux. The mRNA levels of interleukin-6 (IL-6), IL-1β, IL-18, and tumor necrosis factor-α (TNF-α) were detected by real-time PCR. We used 3-methyladenine (3-MA) and autophagy-related gene 5 small interfering RNA (Atg5 siRNA) to block autophagy in vivo and in vitro, respectively. Activating α7nAChR with PNU282987 ameliorates EAE severity and spinal inflammatory infiltration in EAE mice. PNU282987 treatment also enhanced monocyte/microglia autophagy (Beclin 1, LC3-II/I ratio, p62/SQSTM1, colocalization of CD45- or CD68-positive cells with LC3) both in spinal cord and spleen from EAE mice. The beneficial effects of PNU282987 on EAE mice were partly abolished by 3-MA, an autophagy inhibitor. In vitro, PNU282987 treatment increased autophagy and promoted autophagy flux. Blockade of autophagy by Atg5 siRNA or bafilomycin A1 attenuated the inhibitory effect of PNU282987 on IL-6, IL-1β, IL-18, and TNF-α mRNA. Our results demonstrate for the first time that activating α7nAChR enhances monocyte/microglia autophagy, which suppresses neuroinflammation and thus plays an alleviative role in EAE.