Luteolin exhibits anti-inflammatory effects by blocking the activity of heat shock protein 90 in macrophages Dan Chen a , Aijing Bi a , Xiaoliang Dong a , Yi Jiang b , Bing Rui a , Jinjiao Liu a , Zhimin Yin b,⇑ , Lan Luo a,⇑ a State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing 210097, China b Jiangsu Province Key Laboratory for Molecular and Medicine Biotechnology, College of Life Science, Nanjing Normal University, Nanjing 210046, China article info Article history: Received 16 November 2013 Available online 7 December 2013 Keywords: Luteolin Macrophage Heat shock protein 90 Lipopolysaccharide High mobility group B-1 c-Jun abstract Septic diseases represent the prevalent complications in intensive care units. Luteolin, a plant flavonoid, has potent anti-inflammatory properties; however, the molecular mechanism beneath luteolin mediated immune modulation remains unclear. Here in vitro investigations showed that luteolin dose-dependently inhibited LPS-triggered secretion and relocation of high mobility group B-1 (HMGB1) and LPS-induced production of tumor necrosis factor alpha (TNF-a) and nitric oxide (NO) in macrophages. The mechanism analysis demonstrated that luteolin reduced the release of HMGB1 through destabilizing c-Jun and sup- pressed HMGB1-induced aggravation of inflammatory cascade through reducing Akt protein level. As an inhibitor of Hsp90, luteolin destabilized Hsp90 client protein c-Jun and Akt. In vivo investigations showed that luteolin effectively protected mice from lipopolysaccharide (LPS)-induced lethality. In conclusion, the present study suggested that luteolin may act as a potential therapeutic reagent for treating septic diseases. Ó 2013 Elsevier Inc. All rights reserved. 1. Introduction Endotoxic shock that occurs in overwhelming inflammatory re- sponses to bacterial infections are generally caused by lipopolysac- charide (LPS), an outer membrane component of gram-negative bacteria [1]. LPS acts on many different cell types inducing the expression of cytokines and adhesion molecules that contribute to the inflammatory response [2]. Tumor necrosis factor-a (TNF- a) is an important pro-inflammatory cytokine [3]; but as TNF-a is released early in systemic inflammatory responses, its acute kinetics only provides an extremely narrow therapeutic window for administration of antagonists. Most anti-TNF agents failed to show efficacy in clinical trials of sepsis [4]. High mobility group B-1 (HMGB1), originally identified as a highly conserved DNA-binding factor in the nucleus, translocates from the nucleus to the cytosol and then exocytose [5]. When HMGB1 releases from macrophage cells in response to infectious agents [6,7], it can serve as a cytokine contributed to high lethality in sepsis [8,9]. Unlike TNF-a and IL-1b, HMGB-1 is secreted late after injection of LPS in mice, starting only after 8 h and remaining detectable up to 48 h thereafter, so it has been implicated as a late mediator of sepsis. Secreted HMGB1 binds toll-like receptor 2/4 (TLR2/4) and the receptor for advanced glycation end products (RAGE) [10], and activates a series of signaling components includ- ing mitogen-activated protein kinases (MAPKs) and Akt, which play an important role in inflammation [11]. HMGB1-neutralizing anti- body may prevent organ damage in animal sepsis models [12]. Luteolin, a plant flavones, exhibits anticancer [13] and anti- inflammatory [14] properties. Luteolin reduced LPS-induced lethality [15] and suppressed LPS-stimulated the release of NO and interleukin-6 (IL-6) in RAW 264.7 cells [16]. Up till now the anti-inflammatory effects of luteolin and related mechanisms re- main elusive. Our previous investigation demonstrated that luteo- lin could bind to Heat shock protein 90 (Hsp90), a molecular chaperone, to block the activity of Hsp90 [13]. In this study, we provide evidence that luteolin prevented production and release of HMGB1, and treatment of mice with luteolin after LPS challenge reduced the mortality of LPS-induced endotoxin shock. 2. Materials and methods 2.1. Reagents Luteolin was purchased from Sigma and dissolved in ethyl alcohol at 10 mM stock solutions. LPS (from Escherichia coli 0006-291X/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.bbrc.2013.11.122 Abbreviations: HMGB1, high mobility group B-1; RAGE, receptor for advanced glycation end products. ⇑ Corresponding authors. Fax: +86 25 83686657. E-mail addresses: yinzhimin@njnu.edu.cn (Z. Yin), lanluo@nju.edu.cn (L. Luo). Biochemical and Biophysical Research Communications 443 (2014) 326–332 Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc