Targeting CpG DNA to screen and isolate anti-sepsis fraction and monomers from traditional Chinese herbs using affinity biosensor technology Xin Liu a, 1 , Juan Cheng b, 1 ,2 , Xinchuang Zheng a , Yiguo Chen a , Chong Wu b,2 , Bin Li b,2 , Jianfeng Fu a , Hongwei Cao a , Yongling Lu a , Jun Li b,2 , Jiang Zheng a, ⁎, Hong Zhou b, ⁎ a Medical Research Center, Southwestern Hospital, The Third Military Medical University, Chongqing 400038, PR China b Department of Pharmacology, College of Pharmaceutical, The Third Military Medical University, Chongqing 400038, PR China abstract article info Article history: Received 4 August 2007 Received in revised form 24 March 2009 Accepted 26 March 2009 Keyword: CpG DNA Sepsis Affinity biosensor Traditional Chinese herbs Radix et Rhizoma Rhei Rhein Bacterial DNA/CpG DNA is recognized as a key molecule during the pathogenesis of sepsis. Therefore, preventing CpG DNA from binding to its receptor is considered as the most promising strategy. In the present experiments, Radix et Rhizoma Rhei had the highest CpG DNA-binding ability among the seventy-eight traditional Chinese herbs. After the isolation of silica gel chromatography and high performance liquid chromatography (HPLC) and evaluation with affinity biosensor, the active fraction was confirmed and named Fraction D. It was found that in vitro, Fraction D bound to both CpG DNA and lipid A with high affinity, and strongly inhibited LPS- and CpG DNA-induced TNF-α release from RAW264.7 cells in a dose-dependent manner. Furthermore, Fraction D reduced the expression of TLR9 mRNA up-regulated by CpG DNA. In vivo, Fraction D protected mice challenged with lethal heat-killed E. coli. Using HPLC method, two monomers with high affinity for CpG DNA were isolated and identified as rhein and emodin. Rhein could significantly reduce CpG DNA- and LPS-induced TNF-α release, but emodin only reduced CpG DNA-induced TNF-α release. Rhein in combination with emodin could play synergistic inhibitory effect on both CpG DNA and LPS-induced TNF- α release, which contributed to the bioactivity of Fraction D. In conclusion, we successfully established the platform to screen anti-CpG DNA components of traditional Chinese herbs using affinity biosensor technology, got active Fraction D from Radix et Rhizoma Rhei and determined rhein and emodin as the main bioactive ingredients in Fraction D. © 2009 Elsevier B.V. All rights reserved. 1. Introduction Sepsis is a generalized inflammatory response to an infection. Severe sepsis results in multi-organ dysfunction, septic shock and death. Recent surveys conducted in the U.S. and in Europe have indicated that approximately 2%–11% of all hospital and intensive care unit admissions can be attributed to severe sepsis. Despite improve- ments in supportive care and the increased availability of effective antibacterial agents, hospital mortality rates from severe sepsis and septic shock (50%–60%) have not improved over recent decades [1,2]. Unfortunately, many experimental inflammatory antagonist-based therapies have failed in sepsis trials, and currently there is only one adjuvant therapy in clinical use, e.g. activated protein C, which targets the coagulation system [3]. However, APC is only recommended in patients at high risk of death (septic shock, sepsis-induced acute respiratory distress syndrome, Acute Physiology and Chronic Health Evaluation II score of ≥25, and sepsis-induced multi-organ failure) without bleeding risk [4,5]. Thus it is important to investigate additional inflammatory antagonist-based treatments with the aim of developing a clinically effective anti-sepsis drug. Sepsis is triggered by the presence of invasive bacteria and bacterial components, such as bacterial DNA (bDNA) and lipopoly- saccharide (LPS [endotoxin]) [6,7]. Synthetic oligodeoxynucleotides containing CpG motifs (CpG DNA) mimic the activity of bDNA. Excessive bDNA and CpG DNA-driven immune activation may induce hyperinflammatory responses, even systemic inflammatory response syndrome (SIRS) and sepsis [7]. It is therefore necessary to develop treatments, which can be used to balance the activity of CpG DNA and reduce the release of cytokines induced by CpG DNA exposure. Previously, some neutralizing or suppressive CpG ODNs (CpG-N ODNs) were developed to inhibit bioactivities of bDNA [8,9]. In our lab, chloroquine, an acidic inhibitor of endosome, was discovered to inhibit proinflammatory cytokines release induced by bDNA in vitro and in vivo [10]. International Immunopharmacology 9 (2009) 1021–1031 ⁎ Corresponding authors. Zheng is to be contacted at Medical Research Center, Southwestern Hospital, The Third Military Medical University, Gaotanyan Street 30, Shapingba District, 400038, Chongqing 400038, PR China. Tel.: +86 23 68754435; fax: +86 23 65460584. Zhou, Department of Pharmacology, College of Medicine, The Third Military Medical University, Gaotanyan Street 30, Shapingba District, Chongqing 400038, PR China. Tel./fax: +86 23 68752266. E-mail addresses: zhengj@mail.tmmu.com.cn (J. Zheng), zhouh64@mail.tmmu.com.cn (H. Zhou). 1 They equally contributed to this work. 2 Tel./fax: +86 23 68752266. 1567-5769/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.intimp.2009.03.023 Contents lists available at ScienceDirect International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp