乙酰胆碱通过调节IL-1β/IL-1RA平衡改善炎症微环境的机制探讨

doi:10.5246/jcps.2023.04.023

中国药学(英文版) ›› 2023, Vol. 32 ›› Issue (4): 260-267.DOI: 10.5246/jcps.2023.04.023

乙酰胆碱通过调节IL-1β/IL-1RA平衡改善炎症微环境的机制探讨

马宁¹^,², 纪超¹^,³^,^*()

1. 中国医学科学院基础医学研究所&北京协和医学院基础学院, 药理学系, 北京 100005
2. 中国人民解放军总医院第七医学中心儿科研究所, 北京 100700
3. 中国医学科学院基础医学研究所&北京协和医学院基础学院, 基础医学国家级实验教学示范中心(北京协和医学院), 北京 100005

收稿日期:2022-11-23 修回日期:2022-12-26 接受日期:2023-01-14 出版日期:2023-04-29 发布日期:2023-04-29
通讯作者: 纪超
作者简介:
+ Tel.: +86-10-65105021, E-mail: jichao@ibms.pumc.edu.cn
基金资助:
National Natural Science Foundation of China (Grant No. 81100801); Peking Union Medical College Small-Scale Characteristic School Education Project.

Acetylcholine ameliorates inflammatory microenvironment via regulating the balance of IL-1β/IL-1RA

Ning Ma¹^,², Chao Ji¹^,³^,^*()

1 Department of Pharmacology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & School of Basic Medicine, Peking Union Medical College, Beijing 100005, China
2 Institute of Pediatrics, the Seventh Medical Center of PLA General Hospital, Beijing 100700, China
3 National Demonstration Center for Experimental Basic Medical Education (Peking Union Medical College), Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & School of Basic Medicine, Peking Union Medical College, Beijing 100005, China

Received:2022-11-23 Revised:2022-12-26 Accepted:2023-01-14 Online:2023-04-29 Published:2023-04-29
Contact: Chao Ji

摘要/Abstract

摘要：

本研究旨在探讨乙酰胆碱的抗炎机制, 为阿尔茨海默病(Alzheimer's disease, AD)炎性病理机制的验证和治疗药物的筛选提供进一步依据。研究首先建立Aβ_25?35诱导小胶质细胞系BV-2细胞炎性损伤的体外模型, 观察氯化乙酰胆碱(ACh)抑制炎症反应的作用。于有或无α7 nAChR的阻断剂(α-银环蛇毒)存在的情况下, 采用ELISA方法评价ACh作用后IL-1β和IL-1RA的表达及二者比率, 并分别利用Western blot和EMSA方法测定MAPK(JNK, p38和ERK)及NF-κB通路相关分子的表达及磷酸化水平。结果显示, ACh能够显著降低IL-1β/IL-1RA比率, 对抗Aβ_25?35诱导的IL-1系统炎症活性变化, 维持IL-1亚家族平衡。预先给予α7 nAChR阻断剂能够阻断ACh对IL-1β和IL-1RA蛋白表达的抑制作用。同时, α7 nAChR阻断剂能够阻断ACh对下游ERK1/2 MAPK蛋白的磷酸化水平和NF-κB的活化的调节作用。总之, 乙酰胆碱能够抑制Aβ诱导的BV-2细胞中IL-1亚家族的炎症活性、维持该亚家族的平衡稳定, 该作用由α7 nAChR介导, 有ERK1/2 MAPK和NF-κB通路的参与。该研究为发现探索AD发病的关键机制提供依据, 同时也为寻找AD治疗的新靶点提供参考。

关键词: 乙酰胆碱, IL-1β, IL-1受体拮抗剂, β-淀粉样蛋白, 阿尔茨海默病

Abstract:

In the present study, we aimed to explore the anti-inflammatory mechanism of acetylcholine (ACh) and to provide further evidence for the investigation of the inflammatory pathogenesis of Alzheimer’s disease (AD) and its therapeutic drugs. The in vitro model of Aβ_25?35-induced inflammatory injury in microglial cell line BV-2 cells was established to observe the anti-inflammatory effect of ACh chloride. In the presence or absence of α7 nAChR blocker (α-bungarotoxin), the expressions of IL-1β and IL-1RA and their ratio after ACh treatment were evaluated by ELISA. The expression and phosphorylation levels of MAPK (JNK, p38, and ERK1/2) and NF-κB pathway molecules were determined by Western blot analysis or EMSA, respectively. The results showed that ACh could significantly reduce the ratio of IL-1β/IL-1RA, antagonize the inflammatory activity of the IL-1 system induced by Aβ_25?35, and restore the viability of BV-2 cells. Pretreatment with α7 nAChR blocker could block the inhibitory effect of ACh on the IL-1β/IL-1RA ratio. Meanwhile, α7 nAChR blocker could block the phosphorylation level of ERK1/2 MAPK protein and NF-κB activation downstream. Our study suggested that ACh could regulate IL-1 system inflammatory response induced by Aβ_25?35 in BV-2 cells and maintain IL-1 subfamily balance, which was mediated by α7 nAChR and involved ERK1/2 MAPK and NF-κB pathways. This study provided a basis for exploring AD inflammatory pathogenesis and also a reference for discovering new targets for AD treatment.

Key words: Acetylcholine, Interleukin-1β, IL-1 receptor antagonist, β-Amyloid protein, Alzheimer's disease

Supporting:

马宁, 纪超. 乙酰胆碱通过调节IL-1β/IL-1RA平衡改善炎症微环境的机制探讨[J]. 中国药学(英文版), 2023, 32(4): 260-267.

Ning Ma, Chao Ji. Acetylcholine ameliorates inflammatory microenvironment via regulating the balance of IL-1β/IL-1RA[J]. Journal of Chinese Pharmaceutical Sciences, 2023, 32(4): 260-267.

图/表 6

Figure 1. The effect of ACh on the viability of BV-2 cells by the MTT method. The cells were incubated with ACh of indicated concentrations for 1 h and then stimulated with Aβ25?35 10 μM for another 24 h. n = 6, *P < 0.01 vs. control group; #P < 0.05 and ##P < 0.01 vs. Aβ group.

Figure 2. The effects of ACh on IL-1β (A), IL-1RA (B), and IL-1β/IL-1RA ratio (C) by ELISA. The cells were incubated with ACh 10 or 40 μM for 1 h and then stimulated with 10 μM Aβ25?35 for another 24 h. n = 6, *P < 0.05 and **P < 0.01 vs. control group; #P < 0.05 and ##P < 0.01 vs. Aβ group.

Figure 3. α-Bun restores the inhibitory effect of ACh on IL-1β (A) and IL-1RA (B) expressions stimulated by 10 μM Aβ25?35, and the ratio of IL-1β/IL-1RA was also displayed here (C). n = 6, *P < 0.05 vs. control group; #P < 0.05, ##P < 0.01 vs. Aβ group; &P < 0.05, &&P < 0.01 vs. ACh group.

Figure 4. α-Bun restores the effect of ACh on MAPK protein phosphorylation level stimulated by 10 μM Aβ25?35 by Western blotting analysis. n = 6, *P < 0.05 vs. control group; #P < 0.05 vs. Aβ group; &P < 0.05 vs. ACh group.

Figure 5. α-Bun restores the inhibitive effect of ACh on NF-κB p65 protein activation stimulated by 10 μM Aβ25?35 by the EMSA.

Figure 6. The underlying mechanism of ACh regulating IL-1β/IL-1RA ratio to play an anti-inflammatory role.

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乙酰胆碱通过调节IL-1β/IL-1RA平衡改善炎症微环境的机制探讨

Acetylcholine ameliorates inflammatory microenvironment via regulating the balance of IL-1β/IL-1RA

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