Targeting CpG DNA to screen and isolate anti-sepsis fraction and monomers from traditional Chinese herbs using affinity biosensor technology.
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-alpha 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-alpha release, but emodin only reduced CpG DNA-induced TNF-alpha release. Rhein in combination with emodin could play synergistic inhibitory effect on both CpG DNA and LPS-induced TNF-alpha 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.
Liu
Xin
X
Medical Research Center, Southwestern Hospital, The Third Military Medical University, Chongqing, PR China.
Cheng
Juan
J
Zheng
Xinchuang
X
Chen
Yiguo
Y
Wu
Chong
C
Li
Bin
B
Fu
Jianfeng
J
Cao
Hongwei
H
Lu
Yongling
Y
Li
Jun
J
Zheng
Jiang
J
Zhou
Hong
H
D031841:Aconitum / Q000276:immunology*; D000818:Animals; D000880:Anthraquinones / Q000494:pharmacology; D015374:Biosensing Techniques; D002460:Cell Line; D005591:Chemical Fractionation; D002851:Chromatography, High Pressure Liquid; D018899:CpG Islands / Q000235:genetics; D004269:DNA, Bacterial / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D004353:Drug Evaluation, Preclinical; D004357:Drug Synergism; D004365:Drugs, Chinese Herbal / Q000494:pharmacology / Q000627:therapeutic use; D004642:Emodin / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000276:immunology*; D004927:Escherichia coli Infections / Q000188:drug therapy* / Q000235:genetics / Q000276:immunology; D005260:Female; D005786:Gene Expression Regulation / Q000187:drug effects / Q000276:immunology; D007113:Immunity, Innate / Q000187:drug effects; D008050:Lipid A / Q000276:immunology / Q000378:metabolism; D008264:Macrophages / Q000187:drug effects / Q000276:immunology / Q000378:metabolism* / Q000473:pathology; D051379:Mice; D008815:Mice, Inbred Strains; D008517:Phytotherapy; D011485:Protein Binding; D018805:Sepsis / Q000188:drug therapy* / Q000235:genetics / Q000276:immunology; D051217:Toll-Like Receptor 9 / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000276:immunology / Q000378:metabolism
D000880:Anthraquinones; D004269:DNA, Bacterial; D004365:Drugs, Chinese Herbal; D004791:Enzyme Inhibitors; D008050:Lipid A; D051217:Toll-Like Receptor 9; D014409:Tumor Necrosis Factor-alpha; D004642:Emodin; C020491:rhein