Hwang Jung Seok, Kang Eun Sil, Han Sung Gu, Lim Dae-Seog, Paek Kyung Shin, Lee Chi-Ho, Seo Han Geuk
Department of Food Science and Biotechnology of Animal Products, Sanghuh College of Life Sciences, Konkuk University, Seoul, Korea.
Department of Biotechnology, CHA University, Seongnam, Korea.
PeerJ. 2018 Jan 3;6:e4208. doi: 10.7717/peerj.4208. eCollection 2018.
The release of high mobility group box 1 (HMGB1) induced by inflammatory signals acts as a cellular alarmin to trigger a chain of inflammatory responses. Although the inflammatory actions of HMGB1 are well studied, less is known about the therapeutic agents that can impede its release. This study investigated whether the isoflavonoid formononetin can modulate HMGB1 release in cellular inflammatory responses.
RAW264.7 murine macrophages were exposed to lipopolysaccharide (LPS) in the presence or absence of formononetin. The levels of HMGB1 release, sirtuin 1 (SIRT1) expression, and HMGB1 acetylation were analyzed by immunoblotting and real-time polymerase chain reaction. The effects of resveratrol and sirtinol, an activator and inhibitor of SIRT1, respectively, on LPS-induced HMGB1 release were also evaluated.
Formononetin modulated cellular inflammatory responses by suppressing the release of HMGB1 by macrophages exposed to LPS. In RAW264.7 cells, formononetin significantly attenuated LPS-induced release of HMGB1 into the extracellular environment, which was accompanied by a reduction in its translocation from the nucleus to the cytoplasm. In addition, formononetin significantly induced mRNA and protein expression of SIRT1 in a peroxisome proliferator-activated receptor δ (PPARδ)-dependent manner. These effects of formononetin were dramatically attenuated in cells treated with small interfering RNA (siRNA) against PPARδ or with GSK0660, a specific inhibitor of PPARδ, indicating that PPARδ is involved in formononetin-mediated SIRT1 expression. In line with these effects, formononetin-mediated inhibition of HMGB1 release in LPS-treated cells was reversed by treatment with SIRT1-targeting siRNA or sirtinol, a SIRT1 inhibitor. By contrast, resveratrol, a SIRT1 activator, further potentiated the inhibitory effect of formononetin on LPS-induced HMGB1 release, revealing a possible mechanism by which formononetin regulates HMGB1 release through SIRT1. Furthermore, modulation of SIRT1 expression by transfection of SIRT1- or PPARδ-targeting siRNA significantly counteracted the inhibitory effects of formononetin on LPS-induced HMGB1 acetylation, which was responsible for HMGB1 release.
This study shows for the first time that formononetin inhibits HMGB1 release by decreasing HMGB1 acetylation via upregulating SIRT1 in a PPARδ-dependent manner. Formononetin consequently exhibits anti-inflammatory activity. Identification of agents, such as formononetin, which can block HMGB1 release, may help to treat inflammation-related disorders.
炎症信号诱导的高迁移率族蛋白B1（HMGB1）释放作为一种细胞警报素，触发一系列炎症反应。尽管HMGB1的炎症作用已得到充分研究，但对于能够阻止其释放的治疗药物却知之甚少。本研究调查了异黄酮芒柄花黄素是否能在细胞炎症反应中调节HMGB1的释放。
将RAW264.7小鼠巨噬细胞在有或没有芒柄花黄素的情况下暴露于脂多糖（LPS）。通过免疫印迹和实时聚合酶链反应分析HMGB1释放水平、沉默调节蛋白1（SIRT1）表达和HMGB1乙酰化水平。还评估了白藜芦醇和SIRT1的激活剂和抑制剂sirtinol对LPS诱导的HMGB1释放的影响。
芒柄花黄素通过抑制暴露于LPS的巨噬细胞释放HMGB1来调节细胞炎症反应。在RAW264.7细胞中，芒柄花黄素显著减弱LPS诱导的HMGB1释放到细胞外环境中，同时伴随着其从细胞核向细胞质的转位减少。此外，芒柄花黄素以过氧化物酶体增殖物激活受体δ（PPARδ）依赖的方式显著诱导SIRT1的mRNA和蛋白表达。在用针对PPARδ的小干扰RNA（siRNA）或PPARδ的特异性抑制剂GSK0660处理的细胞中，芒柄花黄素的这些作用显著减弱，表明PPARδ参与芒柄花黄素介导的SIRT1表达。与这些作用一致，用靶向SIRT1的siRNA或SIRT1抑制剂sirtinol处理可逆转芒柄花黄素介导的对LPS处理细胞中HMGB1释放的抑制作用。相比之下，SIRT1激活剂白藜芦醇进一步增强了芒柄花黄素对LPS诱导的HMGB1释放的抑制作用，揭示了芒柄花黄素通过SIRT1调节HMGB1释放的可能机制。此外，通过转染靶向SIRT1或PPARδ的siRNA调节SIRT1表达，显著抵消了芒柄花黄素对LPS诱导的HMGB1乙酰化的抑制作用，而HMGB1乙酰化是导致HMGB1释放的原因。
本研究首次表明，芒柄花黄素通过以PPARδ依赖的方式上调SIRT1来降低HMGB1乙酰化，从而抑制HMGB1释放。因此，芒柄花黄素具有抗炎活性。鉴定能够阻断HMGB1释放的药物，如芒柄花黄素，可能有助于治疗炎症相关疾病。
