ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, July 2006, p. 2420–2427
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Vol. 50, No. 7
The Antimalarial Artemisinin Synergizes with Antibiotics To
Protect against Lethal Live Escherichia coli Challenge by
Decreasing Proinflammatory Cytokine Release
Jun Wang,1Hong Zhou,1* Jiang Zheng,2* Juan Cheng,1Wei Liu,1Guofu Ding,1Liangxi Wang,1
Ping Luo,1Yongling Lu,1Hongwei Cao,2Shuangjiang Yu,1Bin Li,1and Lezhi Zhang1
Department of Pharmacology, College of Medicine, The Third Military Medical University, Chongqing 400038,
People’s Republic of China,1and Medical Research Center, Southwestern Hospital,
The Third Military Medical University, Chongqing 400038, People’s Republic of China2
Received 16 August 2005 /Returned for modification 29 September 2005/Accepted 14 April 2006
In the present study artemisinin (ART) was found to have potent anti-inflammatory effects in animal models
of sepsis induced by CpG-containing oligodeoxy-nucleotides (CpG ODN), lipopolysaccharide (LPS), heat-
killed Escherichia coli 35218 or live E. coli. Furthermore, we found that ART protected mice from a lethal
challenge by CpG ODN, LPS, or heat-killed E. coli in a dose-dependent manner and that the protection was
related to a reduction in serum tumor necrosis factor alpha (TNF-?). More significantly, the administration
of ART together with ampicillin or unasyn (a complex of ampicillin and sulbactam) decreased mortality from
100 to 66.7% or 33.3%, respectively, in mice subjected to a lethal live E. coli challenge. Together with the
observation that ART alone does not inhibit bacterial growth, this result suggests that ART protection is
achieved as a result of its anti-inflammatory activity rather than an antimicrobial effect. In RAW264.7 cells,
pretreatment with ART potently inhibited TNF-? and interleukin-6 release induced by CpG ODN, LPS, or
heat-killed E. coli in a dose- and time-dependent manner. Experiments utilizing affinity sensor technology
revealed no direct binding of ART with CpG ODN or LPS. Flow cytometry further showed that ART did not
alter binding of CpG ODN to cell surfaces or the internalization of CpG ODN. In addition, upregulated levels
of TLR9 and TLR4 mRNA were not attenuated by ART treatment. ART treatment did, however, block the
NF-?B activation induced by CpG ODN, LPS, or heat-killed E. coli. These findings provide compelling evidence
that ART may be an important potential drug for sepsis treatment.
Sepsis is a potentially lethal condition that results from a
harmful or damaging host response to infection (1, 3, 8, 11).
Sepsis is triggered by bacteria and bacterial components, such
as bacterial DNA (bDNA) and lipopolysaccharide (LPS) (26,
28, 29). Delivery of CpG-containing oligodeoxy-nucleotides
(CpG ODN) can trigger sepsis by mimicking the immunostim-
ulatory effects of bDNA and therefore has provided a useful
animal model of the sepsis condition (21, 23, 26).
Recent surveys conducted in the United States and in
Europe have indicated that approximately 2 to 11% of all
hospital and intensive care unit admissions can be attributed to
severe sepsis. Despite improvements in supportive care and the
increased availability of effective antibacterial agents, hospital
mortality rates from severe sepsis and septic shock (50 to 60%)
have not improved over recent decades (1, 28). 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 (28). Thus, it is important to
investigate additional inflammatory antagonist-based treat-
ments with the aim of developing a clinically effective antisep-
Antimalarial drugs such as chloroquine (CQ) and artemisi-
nin (ART) are promising candidates for sepsis treatment. CQ
has been demonstrated to protect mice from CpG ODN and
LPS challenges in vivo via a mechanism that involves a reduc-
tion of proinflammatory cytokine release (14, 18). The protec-
tive effects afforded by CQ may be tightly related to interrupt-
ing endosome maturation (14, 18). The antimalarial drug ART
inhibits the endocytosis of macromolecular tracers by up to
85% in Plasmodium falciparum (15) and may suppress tumor
necrosis factor alpha (TNF-?) and interleukin-6 (IL-6) release
by inhibiting endocytosis of CpG ODN. ART has traditionally
been used to treat malaria (20, 22). It is the active ingredient in
the Chinese herb sweet wormwood, and its derivatives include
dihydroartemisinin, artesunate, artemether, and arteethe. ART
has many applications in addition to treatment of severe malaria,
including use as an antitumor agent (22).
In the present study we examined whether ART provides
protection against animal models of sepsis. Specifically, we
investigated the effects of ART on CpG ODN, LPS, and heat-
killed Escherichia coli- or live E. coli-challenged mice in vitro
and in vivo and further examined the possible molecular mech-
anisms involved in ART inhibition of proinflammatory cyto-
* Corresponding authors. Mailing address for H. Zhou: Department of
Pharmacology, College of Medicine, The Third Military Medical Univer-
sity, Gaotanyan Street 30, Shapingba District, Chongqing 400038, Peo-
ple’s Republic of China. Phone: 86-23-6875-2266. Fax: 86-23-6875-2266.
E-mail: email@example.com. Mailing address for J. Zheng:
Medical Research Center, Southwestern Hospital, The Third Military
Medical University, Gaotanyan Street 30, Shapingba District, Chongqing
400038, People’s Republic of China. Phone: 86-23-6875-4435. Fax: 86-23-
6875-4435. E-mail: firstname.lastname@example.org.
This study was supported by grant 30572365 from the National
Natural Science Foundation of China and a grant from Sci-Tech of
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