星形胶质细胞α7 烟碱型乙酰胆碱受体的抗炎作用是通过抑制 NF-κB 通路和激活 Nrf2 通路来介导的。

BACKGROUND: α7 nicotinic acetylcholine receptors (nAChRs) are widely distributed throughout the central nervous system and are reported to have neuroprotective properties. α7 nAChRs are expressed on astrocytes, which are key regulators of neuroinflammation and oxidative stress in several neurodegenerative diseases. However, the anti-inflammatory and antioxidant properties of astroglial α7 nAChRs are not well studied. Therefore, we evaluated the role of astroglial α7 nAChR activation in neuroinflammation. METHODS: Anti-inflammatory and antioxidant effects of α7 nAChR activation were evaluated in an in vitro mouse model of neuroinflammation using lipopolysaccharide (LPS) in primary astrocyte cultures. α7 nAChR anti-inflammatory effects on the NF-κB pathway were evaluated using ELISA, gene expression analysis, immunofluorescence, and western blotting. Antioxidant effect of α7 nAChR activation on expression profiles of canonical Nrf2 target genes was examined by quantitative PCR and western blotting. The role of the Nrf2 pathway in α7 nAChR-mediated anti-inflammatory response was evaluated using Nrf2 knockout astrocytes. Brain ex vivo NF-κB luciferase signals were evaluated after treatment with an α7 nAChR agonist in lipopolysaccharide (LPS)-injected NF-κB luciferase reporter mouse model. RESULTS: Astrocytes treated with the α7 nAChR partial agonist (GTS21) showed significantly reduced LPS-mediated secretion of inflammatory cytokines and this effect was reversed by the α7 nAChR antagonist methyllycaconitine (MLA) and by knockdown of α7 nAChR expression with a short hairpin RNA. Further, α7 nAChR activation blocked LPS-mediated NF-κB nuclear translocation indicating that the observed anti-inflammatory effect may be mediated through inhibition of the NF-κB pathway. Treatment with GTS21 also upregulated canonical Nrf2 antioxidant genes and proteins suggesting antioxidant properties of α7 nAChR in astrocytes. Using an astrocyte conditioned media approach, we demonstrated reduction in neuronal apoptosis when astrocytes were pretreated with GTS21. Finally, in an in vivo neuroinflammation model using LPS in NF-κB luciferase reporter mice, we demonstrated reduction in LPS-induced NF-κB activity and pro-inflammatory cytokines with GTS21 treatment in brain tissue. CONCLUSION: Our results suggest that activating astroglial α7 nAChRs may have a role in neuroprotection by decreasing inflammation and oxidative stress, and therefore could have therapeutic implication for disease modifying treatments of neurodegenerative diseases.

背景：α7 烟碱型乙酰胆碱受体（nAChRs）广泛分布于中枢神经系统，据报道具有神经保护作用。α7 nAChRs 表达于星形胶质细胞上，星形胶质细胞是几种神经退行性疾病中神经炎症和氧化应激的关键调节因子。然而，星形胶质细胞 α7 nAChRs 的抗炎和抗氧化特性尚未得到充分研究。因此，我们评估了星形胶质细胞 α7 nAChR 激活在神经炎症中的作用。

方法：我们使用脂多糖（LPS）在原代星形胶质细胞培养物中建立体外小鼠神经炎症模型，评估 α7 nAChR 激活的抗炎和抗氧化作用。使用 ELISA、基因表达分析、免疫荧光和 Western blot 评估 α7 nAChR 对 NF-κB 途径的抗炎作用。通过定量 PCR 和 Western blot 检查 α7 nAChR 激活对经典 Nrf2 靶基因表达谱的抗氧化作用。使用 Nrf2 基因敲除星形胶质细胞评估 Nrf2 通路在 α7 nAChR 介导的抗炎反应中的作用。使用 α7 nAChR 激动剂在脂多糖（LPS）注射的 NF-κB 荧光素酶报告小鼠模型中处理后，评估脑离体 NF-κB 荧光素酶信号。

结果：用 α7 nAChR 部分激动剂（GTS21）处理的星形胶质细胞显示 LPS 介导的炎症细胞因子分泌明显减少，这种作用被 α7 nAChR 拮抗剂甲基六氢吡啶（MLA）和短发夹 RNA 下调 α7 nAChR 表达所逆转。此外，α7 nAChR 激活阻断 LPS 介导的 NF-κB 核易位，表明观察到的抗炎作用可能是通过抑制 NF-κB 途径介导的。用 GTS21 处理还上调了经典的 Nrf2 抗氧化基因和蛋白，表明星形胶质细胞中 α7 nAChR 具有抗氧化作用。通过星形胶质细胞条件培养基方法，我们证明了当星形胶质细胞用 GTS21 预处理时，神经元凋亡减少。最后，在 LPS 在 NF-κB 荧光素酶报告小鼠中的体内神经炎症模型中，我们证明了 GTS21 治疗可降低脑内 LPS 诱导的 NF-κB 活性和促炎细胞因子。

结论：我们的结果表明，激活星形胶质细胞 α7 nAChR 可能通过减少炎症和氧化应激在神经保护中发挥作用，因此可能对神经退行性疾病的疾病修饰治疗具有治疗意义。