加兰他敏调节IL-1β/IL-1RA比率改善炎症微环境的机制探讨

doi:10.5246/jcps.2022.10.067

中国药学(英文版) ›› 2022, Vol. 31 ›› Issue (10): 773-781.DOI: 10.5246/jcps.2022.10.067

加兰他敏调节IL-1β/IL-1RA比率改善炎症微环境的机制探讨

康静婷, 纪超^*()

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:2022-05-21 修回日期:2022-06-24 接受日期:2022-08-31 出版日期:2022-10-31 发布日期:2022-10-31
通讯作者: 纪超
作者简介:
+ Tel.: +86-10-65105032, E-mail: jichao@ibms.pumc.edu.cn
基金资助:
National Natural Science Foundation of China (Grant No. 81100801); and Peking Union Medical College Small-Scale Characteristic School Education Project.

The mechanism of galanthamine regulating IL-1β/IL-1RA ratio to ameliorate inflammatory microenvironment

Jingting Kang, Chao Ji^*()

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:2022-05-21 Revised:2022-06-24 Accepted:2022-08-31 Online:2022-10-31 Published:2022-10-31
Contact: Chao Ji

摘要/Abstract

摘要：

本研究拟探讨阿尔茨海默氏病(AD)的经典治疗药物加兰他敏调节IL-1β/IL-1RA比率改善炎症微环境的作用机制。研究首先通过建立海马神经元细胞系HT-22与小胶质细胞系BV-2的共培养体系, 评价加兰他敏对脂多糖(LPS)诱导的炎症相关因子表达的影响。随后MTT法观察加兰他敏对神经元的保护作用, ELISA和qPCR方法分别检测加兰他敏对白细胞介素1β(IL-1β)和IL-1受体拮抗剂(IL-1RA)的蛋白与mRNA表达的影响。预处理α7亚型的N胆碱受体(α7 nAChR)阻断剂α-银环蛇毒(α-bun)、M胆碱受体(mAChR)阻断剂阿托品(Atr)、PI3K抑制剂LY294002、IKKβ抑制剂SC514或MEK抑制剂PD98059, 检测IL-1β和IL-1RA表达的变化。结果显示, 加兰他敏显著抑制LPS诱导的IL-1β和IL-1RA的表达增加, 维持IL-1β/IL-1RA的比率稳定。α-bun阻断了加兰他敏对IL-1β和IL-1RA的调节作用。分别阻断PI3K和NF-κB通路, 显著抑制加兰他敏对IL-1β表达的调节; 分别阻断PI3K和MEK通路, 显著抑制加兰他敏对IL-1RA表达的调节作用。本研究证明, 加兰他敏通过α7 nAChR影响PI3K/NF-κB与PI3K/MAPK通路, 调节IL-1亚家族炎症活性发挥神经保护作用, 为AD治疗提供新的思路。

关键词: 加兰他敏, 白细胞介素1β, IL-1受体拮抗剂, 脂多糖, 炎症反应

Abstract:

In the present study, we aimed to evaluate the anti-inflammatory mechanism of galanthamine, a classic therapeutic drug for Alzheimer’s disease (AD). The co-culture system of hippocampal nerve cell line HT-22 and microglial cell line BV-2 was established to observe the effect of galanthamine on the expressions of inflammatory factors induced by lipopolysaccharide (LPS). MTT method was used to observe the protective effect of galanthamine on neurons. ELISA and qPCR methods were used to detect the expressions of interleukin-1β (IL-1β) and IL-1 receptor antagonist (IL-1RA) at the protein and mRNA levels, respectively. IL-1β and IL-1RA were evaluated by the ELISA method after pretreating with galanthamine and α7 nAChR blocker α-bungarotoxin (α-bun), mAChR blocker atropine (Atr), PI3K inhibitor LY294002, IKKβ inhibitor SC514, or MEK inhibitor PD98059, respectively. The results showed that galanthamine significantly inhibited LPS-induced increased IL-1β and IL-1RA expressions and maintained the ratio of IL-1β/IL-1RA. α-Bun could block the regulatory effect of galanthamine on IL-1β and IL-1RA. As PI3K and NF-κB pathways were blocked, the regulatory effect of galanthamine on the IL-1β expression was significantly inhibited. Blocking PI3K and MEK pathways could significantly inhibit the regulation of galanthamine on IL-1RA expression. In summary, galanthamine regulated the inflammatory activity of the IL-1 subfamily to play an anti-inflammatory role mediated by α7 nAChR and PI3K/NF-κB/MAPK pathways, which probably provided a new strategy for AD treatment.

Key words: Galanthamine, Interleukin-1β, IL-1 receptor antagonist, Lipopolysaccharide, Inflammatory response

Supporting:

康静婷, 纪超. 加兰他敏调节IL-1β/IL-1RA比率改善炎症微环境的机制探讨[J]. 中国药学(英文版), 2022, 31(10): 773-781.

Jingting Kang, Chao Ji. The mechanism of galanthamine regulating IL-1β/IL-1RA ratio to ameliorate inflammatory microenvironment[J]. Journal of Chinese Pharmaceutical Sciences, 2022, 31(10): 773-781.

图/表 8

Table 1. Sequences of primers.

Figure 1. The effects of galanthamine on the viability of HT-22 (A) and BV-2 (B) cells by MTT method. The cells were incubated with galanthamine of indicated concentrations for 1 h and then stimulated with 1 μg/mL LPS for another 24 h. n = 3, **P < 0.01 vs. control group; #P < 0.05 and ##P < 0.01 vs. LPS group.

Figure 2. The effects of galanthamine on IL-1β (A), IL-1RA (B), and IL-1β/IL-1RA (C) ratio by ELISA. The cells were incubated with 10 or 40 μM of galanthamine for 1 h and then stimulated with 1 μg/mL LPS for another 24 h. n = 3, *P < 0.01 vs. control group; #P < 0.05 and ##P < 0.01 vs. LPS group.

Figure 3. Effects of galanthamine on the mRNA expressions of IL-1β (A) and IL-1RA (B) by qPCR analysis. The cells were incubated with 10 μM of galanthamine for 1 h and then stimulated with 1 μg/mL LPS for another 24 h. n = 3, *P < 0.01 vs. control group; #P < 0.05, ##P < 0.01 vs. LPS group.

Figure 4. α-Bungarotoxin (α-bun) 10 nM, but not atrophine (Atr) 10 nM, restores the inhibitory effect of 10 μM galanthamine on IL-1β (A) and IL-1RA (B) expressions stimulated by 1 μg/mL LPS, and the ratio of IL-1β/IL-1RA was also displayed here (C). n = 3, *P < 0.05, **P < 0.01 vs. vehicle-treated culture; #P < 0.05 vs. LPS group; ΔP < 0.05 vs. galanthamine group.

Figure 5. The effects of PI3K inhibitor LY294002 25 μM (A), IKKβ inhibitor SC514 20 μM (B), and MEK inhibitor PD98059 25 μM (C), respectively, on IL-1β expression in the co-culture system treated with LPS and galanthamine. n = 3, *P < 0.05, **P < 0.01 vs. control group; #P < 0.05 vs. LPS group.

Figure 6. The effects of PI3K inhibitor LY294002 25 μM (A), IKKβ inhibitor SC514 20 μM (B), and MEK inhibitor PD98059 25 μM (C), respectively, on IL-1RA expression in the co-culture system treated with LPS and galanthamine. n = 3, *P < 0.01 vs. control group; #P < 0.05 vs. LPS group.

Figure 7. The mechanism of galanthamine regulating IL-1β/IL-1RA ratio to play anti-inflammatory effect.

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加兰他敏调节IL-1β/IL-1RA比率改善炎症微环境的机制探讨

The mechanism of galanthamine regulating IL-1β/IL-1RA ratio to ameliorate inflammatory microenvironment

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