Department of Pharmacology, Second Military Medical University, Shanghai, China.
Naval Convalescent Zone of Hangzhou Sanatorium, Nanjing Military Command, Hangzhou, China.
CNS Neurosci Ther. 2017 Nov;23(11):875-884. doi: 10.1111/cns.12758. Epub 2017 Sep 21.
To evaluate whether activating α7 nicotinic acetylcholine receptor (α7nAChR) could inhibit the NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome through regulation of β-arrestin-1 in monocyte/macrophage system, thus contributing to the control of neuroinflammation.
The protein levels of NLRP3, caspase-1 (Casp-1) p20 and proCasp-1, interleukin-1β (IL-1β) p17 and proIL-1β, IL-18 and proIL-18 were measured using Western blotting. The mRNA levels of Casp-1 and IL-1β were detected by real-time PCR (RT-PCR). The colocalization and interaction of NLRP3 protein and β-arrestin-1 were measured by immunofluorescence staining and immunoprecipitation.
The expression of β-arrestin-1 was significantly increased and colocalized with CD45-positive cells in spinal cord of experimental auto-immune encephalomyelitis (EAE) mice when compared with the sham mice, which was attenuated by pretreatment with PNU282987, a specific α7nAChR agonist. PNU282987 also significantly inhibited the activation of NLRP3 inflammasome and thus decreased the production of IL-1β and IL-18 both in lipopolysaccharide (LPS)/ATP-stimulated BV2 microglia in vitro and spinal cord from EAE mice in vivo, while inverse effects were observed in α7nAChR knockout mice. Furthermore, overexpression of β-arrestin-1 attenuated the inhibitory effect of PNU282987 on NLRP3 inflammasome activation in LPS/ATP-stimulated BV2 microglia. PNU282987 inhibited the interaction between β-arrestin-1 and NLRP3 protein in vitro.
The present study demonstrates that activating α7nAChR can lead to NLRP3 inflammasome inhibition via regulation of β-arrestin-1 in monocyte/microglia system.
评估激活α7 烟碱型乙酰胆碱受体 (α7nAChR) 是否能够通过调节单核细胞/巨噬细胞系统中的β-arrestin-1 抑制 NOD 样受体家族，富含吡啶结构域的 3 (NLRP3) 炎性小体，从而有助于控制神经炎症。
使用 Western blot 法测定 NLRP3、半胱氨酸蛋白酶-1（Caspase-1）p20 和 proCasp-1、白细胞介素-1β（IL-1β）p17 和 proIL-1β、IL-18 和 proIL-18 的蛋白水平。通过实时 PCR（RT-PCR）检测 Caspase-1 和 IL-1β 的 mRNA 水平。通过免疫荧光染色和免疫沉淀测定 NLRP3 蛋白与β-arrestin-1 的共定位和相互作用。
与假手术组相比，实验性自身免疫性脑脊髓炎 (EAE) 小鼠脊髓中β-arrestin-1 的表达明显增加，并与 CD45 阳性细胞共定位，而 PNU282987（一种特异性α7nAChR 激动剂）预处理可减弱这种共定位。PNU282987 还显著抑制 NLRP3 炎性小体的激活，从而减少脂多糖 (LPS)/三磷酸腺苷 (ATP) 体外刺激的 BV2 小胶质细胞和体内 EAE 小鼠脊髓中 IL-1β 和 IL-18 的产生，而在α7nAChR 敲除小鼠中则观察到相反的效果。此外，β-arrestin-1 的过表达减弱了 PNU282987 在 LPS/ATP 刺激的 BV2 小胶质细胞中对 NLRP3 炎性小体激活的抑制作用。PNU282987 抑制了体外β-arrestin-1 与 NLRP3 蛋白之间的相互作用。
本研究表明，激活α7nAChR 可通过调节单核细胞/小胶质细胞系统中的β-arrestin-1 抑制 NLRP3 炎性小体。
