Diaporisoindole B通过抑制MyD88/NF-κB/MAPKs通路对LPS诱导的RAW 264.7巨噬细胞发挥抗炎作用

doi:10.5246/jcps.2021.08.055

摘要/Abstract

摘要：

Diaporisoindole B(DPB)是从红树内生真菌Diaporthe sp. SYSU-HQ3的次级代谢产物中分离得到的一种异吲哚类衍生物。前期研究表明DPB可抑制LPS诱导的RAW 264.7巨噬细胞产生NO, 说明其具有潜在的抗炎活性。本文进一步研究了DPB对LPS诱导的RAW 264.7巨噬细胞的抗炎活性及可能的作用机制。DPB(3.125、6.25、12.5和25 μM)可显著性抑制LPS引起的RAW 264.7小鼠巨噬细胞中PGE2的水平, 及iNOS和COX-2的表达, 并呈剂量依赖性。DPB也可显著抑制LPS引起的RAW 264.7小鼠巨噬细胞中细胞因子TNF-α, IL-1β和IL-6的表达。此外, 我们还研究了DPB发挥抗炎作用的机制。DPB可通过下调上游MyD88 蛋白水平抑制NF-κB信号通路的激活。此外, DPB还能够抑制MAPKs三个亚型(包括ERK 1/2、JNK和p38)的磷酸化。因此, DPB可能通过下调RAW 264.7巨噬细胞中MyD88蛋白的水平抑制NF-κB和MAPKs的表达, 从而达到抗炎的作用。

关键词: Diaporisoindole B, 抗炎作用, RAW 264.7巨噬细胞, NF-κB, MAPKs, MyD88

Abstract:

Diaporisoindole B (DPB), an isoprenylisoindole alkaloid isolated from the mangrove endophytic fungus Diaporthe sp. SYSU-HQ3, has been proved to inhibit the production of nitric oxide (NO) in lipopolysaccharide (LPS)-challenged RAW 264.7 mouse macrophages, showing potent anti-inflammatory effects. In this study, we further investigated the anti-inflammatory effects of DPB and explored the possible mechanisms in LPS-challenged RAW 264.7 mouse macrophages. The results showed that DPB (3.125, 6.2, 12.5 and 25 μM) could significantly reduce LPS-induced levels of PGE2, and inhibit the expressions of iNOS and COX-2 in a dose-dependent manner. In addition, DPB also inhibited LPS-induced production of inflammatory cytokines, including TNF-α, IL-1β, IL-6. Moreover, we further investigated signal transduction mechanisms by which DPB exerted anti-inflammatory effects. DPB could affect LPS-mediated nuclear factor kappa B (NF-κB) signaling pathway activation via down-regulating the upstream myeloid differentiation protein 88 (MyD88) at the protein level. Additionally, DPB also strongly inhibited the phosphorylation of mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinase (ERK) 1/2, c-Jun N-terminal kinase (JNK) and p38. Therefore, DPB might exert anti-inflammatory effects by suppressing NF-κB activation and MAPKs pathways via down-regulating MyD88 in RAW 264.7 cells.

Key words: Diaporisoindole B, Anti-in?ammation, RAW 264.7 macrophage cells, NF-κB, MAPKs, MyD88

Supporting:

刘红菊, 李静, 谢慧仪, 王玲玲, 张志珍, 闫冲. Diaporisoindole B通过抑制MyD88/NF-κB/MAPKs通路对LPS诱导的RAW 264.7巨噬细胞发挥抗炎作用[J]. 中国药学(英文版), 2021, 30(8): 675-685.

Hongju Liu, Jing Li, Huiyi Xie, Lingling Wang, Zhizhen Zhang, Chong Yan. Anti-inﬂammatory effects of diaporisoindole B in LPS-stimulated RAW 264.7 macrophage cells via MyD88 activated NF-κB and MAPKs pathways[J]. Journal of Chinese Pharmaceutical Sciences, 2021, 30(8): 675-685.

图/表 7

Figure 1. Chemical structure of diaporisoindole B.

Figure 2. Inhibitory effects of DPB on LPS-induced iNOS mRNA (A) and protein levels (B) in RAW 264.7 cells. Data are means ± SD of three independent experiments; #P < 0.05 compared with the negative group, *P < 0.05, **P < 0.01 and ***P < 0.001 compared with the LPS-treated group.

Figure 3. Inhibitory effects of DPB on LPS-induced PGE2 and COX-2 levels in RAW264.7 cells. (A) DPB inhibited LPS-induced PGE2 in RAW264.7 cells. DPB inhibited LPS-induced COX-2 expression at the mRNA (B) and protein (C) levels in RAW 264.7 cells. Data are means ± SD of three independent experiments; #P < 0.05 compared with the negative group, *P < 0.05, **P < 0.01 and ***P < 0.001 compared with the LPS-treated group.

Figure 4. Inhibitory effects of DPB on LPS-induced pro-inflammatory cytokines in RAW 264.7 cells. DPB inhibited LPS-induced pro-inflammatory cytokines TNF-α (A), IL-1β (B) and IL-6 (C) in RAW 264.7 cells. DPB inhibited LPS-induced pro-inflammatory cytokines TNF-α (D), IL-1β (E) and IL-6 (F) at the mRNA level in RAW 264.7 cells. Data are means ± SD of three independent experiments; #P < 0.05 compared with the negative group, *P < 0.05, **P < 0.01 and ***P < 0.001 compared with the LPS-treated group.

Figure 5. Effects of DPB on LPS-induced IκB-α and NF-κB p65 phosphorylation in RAW 264.7 cells. (A) DPB inhibited LPS-induced phosphorylation of IκB-α. (B) DPB inhibited LPS-induced phosphorylation of NF-κB p65. Quantitative data of IκB-α, p-IκB-α, NF-κB p65 and p-NF-κB p65 were analyzed using Image J software. Data are means ± SD of three independent experiments; #P < 0.05 compared with the negative group, *P < 0.05, **P < 0.01 and ***P < 0.001 compared with the LPS-treated group.

Figure 6. Effects of DPB on LPS-induced phosphorylation of ERK (A), JNK (B), p38 (C) in RAW 264.7 cells. Data are means ± SD of three independent experiments; #P < 0.05 compared with the negative group, *P < 0.05, **P < 0.01 and ***P < 0.001 compared with the LPS-treated group.

Figure 7. Effects of DPB on LPS-induced upregulation of MyD88 protein in RAW 264.7 cells. Data are means ± SD of three independent experiments; #P < 0.05 compared with the negative group, *P < 0.05, **P < 0.01 and ***P < 0.001 compared with the LPS-treated group.

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