Study Of An Active Compound Antagonizing Lipopoly-saccharide Isolated From Densefruit Pittany Root-bark

Introduction Sepsis is a complex clinical syndrome that results from a harmful host response to infection, which may result in septic shock, multiple organ failure (MOF) and ultimately death. A recent epidemiological study from North America found that the incidence was approximately 3.0 cases per 1,000 population, which translated into an annual burden of approximately 750,000 cases. The overall mortality is approximately 30%, rising to 40% in the elderly and is 50% or greater in patients with the most severe syndrome, septic shock. The most common cause of sepsis is an exposure to the structural component of the Gram-negative bacterial outer membrane, lipopolysaccharide (LPS, known also as endotoxin).LPS is thought to be the main toxic element that induces pro-inflammatory cytokine production after interaction with CD14 and toll-like receptor 4 (TLR4). Pro-inflammatory cytokines such as TNF-α, IL-1 and IL-6 are induced by LPS, which may damage cells and lead to organ injuries. Despite improved care, the hospital mortality rate from severe sepsis and septic shock has not improved significantly over recent decades. Thus, it is important to investigate new anti-LPS drugs to identify a relevant anti-sepsis drug.The use of traditional Chinese medicines has a long history in sepsis therapy. A lot of herbs have been shown to possess potent anti-LPS and anti-inflammatory properties. However, herbs possess a large number of complex constituents and it is extremely difficult to identify which is the anti-LPS component. In the present study, we undertook to confirm the presence of anti-LPS component(s) in a panel of 60 traditional Chinese herbs, using affinity biosensor technology. The herb, densefruit pittany root-bark, was selected from this panel, and components were then isolated that possessed the highest lipid A-binding ability. Finally, the active compound isolated from densefruit pittany root-bark was examined for its anti-LPS activity both in vitro and in vivo.Objective To obtain the active compound from traditional Chinese herbs that can antagonize LPS and investigate its associated mechanism.Methods 1. Lipid A was immobilized on the optical biosensor's hydrophobic cuvette and employed to monitor the interactions of the components from Chinese herbs with lipid A. Sixty aqueous extractions of traditional Chinese medicine were determined the exact capacity for binding to lipid A using the affinity biosensor technology. The aqueous extraction from densefruit pittany root-bark, which possessed higher binding activity to lipid A, were selected for further study. Briefly, the fraction obtained was subjected to macroporous adsorptive resins, n-butanol solvent extraction, polyamide laminar analysis, and the fraction with the highest binding activity to lipid A was further purified by high performance liquid chromatography (HPLC). Then the neutralization of HPLC-purified products DPR1~4 for LPS (0.1 ng/ml) was detected by kinetic turbidimetric limulus test. 2. The Kd value for the lipid A of active compound, DPR-2, was measured by biosensor, and the activity of DPR-2 (0.5, 1, 2 and 4μg/ml) neutralizing LPS (0.1 ng/ml) was detected by kinetic turbidimetric limulus test. Then the binding of FITC-conjugated LPS (100 ng/ml) to murine RAW264.7 cells was analyzed by laser scanning confocal microscopy (LSM) and the influence of DPR-2 (8, 16, 32 and 64μg/ml) on TLR4 expression in RAW264.7 cells simulated with LPS (100ng/ml) was detected by immunocytochemical staining. Meanwhile, the expressions of TLR4, TNF-αand IL-6 mRNA and the release of cytokines were detected with real-time RT-PCR and ELISA, respectively. 3. In vivo, different dosage of LPS (2.5, 5, 10, 15 and 20mg/kg body wt) was injected into BALB/c mice by tail vein to prepare the murine model of sepsis and endotoxemia murine model, and the effects of DPR-2 were observed. Then, in an endotoxemia murine model (12mg/kg body wt), blood samples were collected at different time (2, 6, 12, 24, 48 and 72 h) after inception of the experiment and assayed using the appropriate ELISA kits, and the TNF-αand IL-6 mRNA expressions in liver and lung were analysed by real-time RT-PCR. Meanwhile, pathological changes of hepatic tissue and lung tissue were observed.Results 1. Among the 60 Chinese herbs examined, four herbs were found to possess higher lipid A-binding activities (RU>300 arc seconds). Four chemical components DPR1~4 with different affinity for lipid A from densefruit pittany root-bark were obtained by above methods. The active compound DPR-2 was purified and confirmed as a compound with tetrasaccharide. 2. DPR-2 bound with high-affinity to lipid A with Kd value of 8.63μM and neutralized LPS in a dose-dependent manner. Furthermore, DPR-2 inhibited the binding of FITC-LPS to RAW264.7 cells in a dose-dependent manner and TLR4 expression of RAW264.7 cells induced by LPS. Moreover, DPR-2 could inhibit markedly the expressions of TNF-αand IL-6 mRNA and the release of cytokines in LPS-stimulated murine RAW264.7 cells. 3. DPR-2 was found to protect BALB/c mice from lethal challenge by LPS and decreased the plasma LPS level in endotoxemic mice. In the endotoxemia murine model, DPR-2 could potently suppress LPS-induced expression of TNF-αand IL-6 mRNA in hepatic tissue and lung tissue and significantly attenuate the release of cytokines in plasma. In addition, it could alleviate the pathological changes in liver and lung.Conclusion The active compound, DPR-2, was obtained from aqueous extraction of densefruit pittany root-bark using biosensor technology. DPR-2 was found to neutralize LPS and possess anti-LPS capacity in vitro and in vivo. This might be associated with its hight-affinity for lipid A.