2-Hydroxycircumdatin C通过抑制TLR4/NF-κB/MAPK和JAK2/STAT3通路对脂多糖诱导BV2小胶质细胞发挥抗炎作用

doi:10.5246/jcps.2022.04.021

中国药学(英文版) ›› 2022, Vol. 31 ›› Issue (4): 239-249.DOI: 10.5246/jcps.2022.04.021

• 【研究论文】 • 下一篇

2-Hydroxycircumdatin C通过抑制TLR4/NF-κB/MAPK和JAK2/STAT3通路对脂多糖诱导BV2小胶质细胞发挥抗炎作用

张婵娟¹^,², 胡立坤², 张贺¹, 漆心怡¹, 黄健¹, 刘东²^,^*()

1. 北京大学医学部药学院化学生物学系, 北京 100191
2. 北京大学医学部药学院天然药物及仿生药物国家重点实验室, 北京 100191

收稿日期:2021-12-14 修回日期:2022-01-16 接受日期:2022-01-29 出版日期:2022-04-30 发布日期:2022-04-30
通讯作者: 刘东
作者简介:
+ Tel.: +86-10-82805771, E-mail: liudong_1982@126.com
基金资助:
National Key Research and Development Program of China (Grant No. YFC0310900), National Natural Science Foundation of China (Grant No. 81991525, 21861142006, 81872793, 81630089).

2-Hydroxycircumdatin C inhibits LPS-induced BV2 cells inflammatory response via down-regulation of TLR4-mediated NF-κB/MAPK and JAK2/STAT3 pathways

Chanjuan Zhang¹^,², Likun Hu², He Zhang¹, Xinyi Qi¹, Jian Huang¹, Dong Liu²^,^*()

1 Department of Chemical Biology, School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing 100191, China
2 State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing 100191, China

Received:2021-12-14 Revised:2022-01-16 Accepted:2022-01-29 Online:2022-04-30 Published:2022-04-30
Contact: Dong Liu

摘要/Abstract

摘要：

2-Hydroxycircumdatin C (2-HCC)是从珊瑚附生真菌赭曲霉(Aspergillus ochraceus LZDX-32-15)中分离出来的一种喹唑啉苯并二氮杂?生物碱。本文研究了2-HCC抑制小胶质细胞炎症反应的作用机制及其对神经元细胞的保护作用。在LPS诱导的小鼠小胶质BV2细胞炎症反应中, 2-HCC抑制LPS诱导的BV2细胞产生的肿瘤坏死因子(TNF-α)和白细胞介素(IL-1β)的分泌, 以及诱导型一氧化氮合酶(iNOS)和环氧氧酶2(COX-2)的表达。此外, 通过原代小胶质细胞及原代神经元细胞共培养实验发现, 2-HCC能够抑制LPS诱导的共培养体系中原代小胶质细胞炎性因子的过度表达和释放, 进而起到保护原代神经元细胞的作用。进一步研究发现, 2-HCC通过抑制LPS诱导的TLR4/NF-κB/MAPK和JAK2/STAT3信号通路发挥抗炎作用。综上所述, 2-HCC能够通过下调BV2细胞中TLR4/NF-κB/MAPK和JAK2/STAT3信号传导通路, 抑制炎症反应从而发挥神经保护作用。

关键词: 2-Hydroxycircumdatin C, 小胶质细胞, 神经炎症, NF-κB, MAPK, JAK/STAT

Abstract:

2-Hydroxycircumdatin C (2-HCC) is a circumdatin-type alkaloid isolated from a coral-associated fungus Aspergillus ochraceus LZDX-32-15. In the present study, we aimed to assess the neuroprotective effects of 2-HCC on the microglia-mediated inflammatory response as well as underlining molecular mechanisms. 2-HCC could significantly down-regulate the overproduction of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) induced by lipopolysaccharide (LPS) both in BV2 cells and primary microglial cells without affecting cell viability. In addition, 2-HCC exerted obvious neuroprotective effects against inflammatory injury in neurons when cocultured with LPS-induced microglia. Mechanism investigation indicated that the anti-inflammatory effect of 2-HCC involved the inhibition of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) and alleviation of the LPS-induced TLR4-NF-κB/MAPK pathway. Furthermore, 2-HCC treatment attenuated LPS-induced activation of the JAK2/STAT3 pathway. In conclusion, these results indicated that the anti-inflammatory and neuroprotective properties of 2-HCC, at least partially, depended upon TLR4-NF-κB/MAPK and JAK2/STAT3 signaling pathways.

Key words: 2-Hydroxycircumdatin C, Neuroinflammation, Microglia, NF-κB, MAPK, JAK/STAT

Supporting:

张婵娟, 胡立坤, 张贺, 漆心怡, 黄健, 刘东. 2-Hydroxycircumdatin C通过抑制TLR4/NF-κB/MAPK和JAK2/STAT3通路对脂多糖诱导BV2小胶质细胞发挥抗炎作用[J]. 中国药学(英文版), 2022, 31(4): 239-249.

Chanjuan Zhang, Likun Hu, He Zhang, Xinyi Qi, Jian Huang, Dong Liu. 2-Hydroxycircumdatin C inhibits LPS-induced BV2 cells inflammatory response via down-regulation of TLR4-mediated NF-κB/MAPK and JAK2/STAT3 pathways[J]. Journal of Chinese Pharmaceutical Sciences, 2022, 31(4): 239-249.

图/表 6

Table 1. Primer sequences for real-time PCR.

Figure 1. Effects of 2-HCC on cell survivability of BV2 cells. (A) The structures of 2-HCC. (B) Cellular survivability was assessed using the MTT assay. The results shown are mean ± SD and represent three independent tests. N.S., no significant differences from the control cells.

Figure 2. 2-HCC down-regulates inflammatory cytokines in LPS-induced BV2 cells. (A) The production of TNF-α and IL-1β in culture media was measured by ELISA. (B) The expressions of TNF-α, IL-1β, iNOS, and COX-2 at the mRNA level were determined using RT-PCR. GAPDH served as a housekeeping gene. (C) The expressions of iNOS and COX-2 at the protein level were determined by Western blotting analysis. The data are represented as the mean ± SD from independent experiments performed in triplicate. #Compared with the control group, *compared with the LPS-treated group, #P < 0.05, ##P < 0.01, ###P < 0.001, **P < 0.01, ***P < 0.001.

Figure 3. 2-HCC prevents inflammation-induced neuronal death in microglial-neuronal co-culture. (A) Primary microglial cells were stimulated with 1 μg/mL LPS in the absence or presence of 2-HCC for measurement of NO, TNF-α, and IL-1β. (B) The apoptotic neuronal cells were determined by a TUNEL assay. (C) Crystal violet staining was performed to observe changes in morphology. Values represent the mean ± SD of three independent experiments. #Compared with the control group, *compared with the LPS-treated group, ##P < 0.01, *P < 0.05, **P < 0.01,***P < 0.001.

Figure 4. 2-HCC inhibits the expressions of TLR4 and MyD88 at the protein level in LPS-induced BV2 cells. Values represent the mean ± SD of three independent experiments. #Compared with the control group, *compared with the LPS-stimulated group, #P < 0.05, ##P < 0.01, *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5. 2-HCC inhibition of NF-κB activation and MAPK/STAT3 phosphorylation in LPS-induced BV2 cells. (A) Detection of the NF-κB p65 subunit translocation by immunocytochemistry. Green fluorescence represents the NF-κB p65 subunit, and blue fluorescence represents nuclear Hoechst staining. (B) NF-κB p65 levels in the nucleus and cytoplasm, the phosphorylated and total IKKβ and IκB proteins were determined by Western blotting analysis. Histone H3 and GAPDH were used as endogenous controls for nuclear and cytoplasmic proteins, respectively. (C) The phosphorylated and total ERK1/2-JNK1/2-p38 MAPKs were determined by Western blotting analysis. (D) The phosphorylated and total JAK2 and STAT3 proteins were determined by Western blotting analysis. Values represent the mean ± SD of three independent experiments. #Compared with the control group, *compared with the LPS-treated group, #P < 0.05, *P < 0.05, **P < 0.01, ***P < 0.001.

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2-Hydroxycircumdatin C通过抑制TLR4/NF-κB/MAPK和JAK2/STAT3通路对脂多糖诱导BV2小胶质细胞发挥抗炎作用

2-Hydroxycircumdatin C inhibits LPS-induced BV2 cells inflammatory response via down-regulation of TLR4-mediated NF-κB/MAPK and JAK2/STAT3 pathways

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