Study On Artemisinin To Prevent And Cure Bacterial Sepsis

Objective: Systemic inflammatory response syndrome (SIRS) is an organic reaction generally associated with an infection (sepsis) or any situation that involves intense tissue damage. SIRS is triggered by bacteria and bacterial components, such as bacterial DNA (bDNA) and lipopolysaccharide (LPS [endotoxin]). CpG-containing oligodeoxynucleotides (CpG DNA) can mimic the immunostimulatory effects of bDNA in triggering SIRS. Severe sepsis and septic shock are life threatening complications of infections and the most common cause of death in intensive care units. Recent US and European surveys have estimated that severe sepsis accounts for 2-11% of all admissions to hospital or intensive care units. Despite better supportive care, the hospital mortality from severe sepsis and septic shock (50% to 60%) has not changed significantly over the recent decades. Unfortunately, many experimental inflammatory antagonist-based therapies have failed in sepsis trials, and there are currently no effective anti-sepsis drugs in clinical use. Thus it is important to investigate additional inflammatory antagonist-based treatments in the hope of identifying a clinically relevant anti-sepsis drug. Artemisinin (ART) is traditionally used for treating malaria. ART is the active ingredient in artemisia annua L against malaria, and its derivatives include dihydroartemisinin, artesunate, artemether and arteethe. Besides the effect against severe malaria, ART has other uses, such as anti-tummor, anti-parasitic infections and regulate immune system, etc. ART was recently shown to decrease cytokine release from macrophages induced by LPS. However, it is not yet known whether ART is capable of reducing proinflammatory cytokines in vivo, nor has its effectiveness been tested in an animal sepsis model. With these considerations in mind, we undertook the current study to investigate ART on the CpG DNA, LPS and heat-killed E.coli or live E.coli challenged mice and the possible molecular mechanisms for its effects on proinflammatory cytokine release in vitro and in vivo. Methods: ①Murine RAW264.7 cells were cultivated and induced by CpG DNA, LPS or heat-killed E.coli to release TNF-αand IL-6; ②To establish murine sepsis models, then the protection of ART on the mouse was observed; ③Effect of ART on internalization of CpG DNA within RAW264.7 cells and CpG DNA binding to RAW264.7 cells were observed by using flow cytometry; ④Effect of ART on mRNA expression of TLR9 and TLR4 within RAW264.7 cells stimulated by CpG DNA, LPS and heat killed E.coli was observed using RT-PCR; ⑤Effect of ART on NF-κB activation within RAW264.7 cells stimulated by CpG DNA, LPS and heat killed E.coli were observed using electrophoretic mobility shift assay (EMSA). Results: In the present study, CpG DNA, LPS, and heat-killed E.coli were used to induce sepsis in animal models. We found that 50, 100, and 200 mg/kg of ART could protect mice from lethal challenge by CpG DNA, LPS, and heat-killed E.coli, and 200 mg/kg of ART could decrease serum TNF-αin mice injected with lethal doses of CpG DNA, LPS, and heat-killed E.coli. In addition, ART have a synergistic protective effect with ampicillin on mice challenge by active E.coli 35218. In vitro, treatment of murine macrophage RAW264.7 cells with ART potently inhibited the release of TNF-αand IL-6 induced by CpG DNA, LPS, and heat-killed E.coli in dose-and time-dependent manners. Furthermore, ART reduced the activation of NF-κB stimulated by CpG DNA, LPS, and heat-killed E.coli. However, ART didn't reduce the expression of TLR9 mRNA by RT-PCR. Flow cytometry revealed that ART had no influence on the binding of CpG DNA to the cell-surfce and the accumulation of CpG DNA within RAW264.7 cells, suggesting that the inhibitory effects of ART on CpG DNA -, LPS-, and heat-killed E.coli-induced cytokine release are unlikely due to its inhibitory effects on expression od TLR9 mRNA and accumulation of CpG DNA. Conclusions: Our results demonstrated that ART-mediated protection of lethal challenge by CpG DNA, LPS, and heat-killed E.coli was associated with the reduction of proinflammatory cytokine release. ART have a synergistic protective effect with antibiotics such as ampicillin on mice challenge by active E.coli 35218. ART is hopeful to prevent and treat bacterial sepsis in the future.