| Objective: Sepsis is systemic inflammatory response syndrome (SIRS) induced by infection, which is a common complication in patients associated with extensive deep infection, severe traumas and major operation. Sepsis is the most frequent cause of the death to serious patients in hospital. Although great attentions have been paid to treatment, it still keeps a high mortality about 50%-60%. Sepsis is mainly triggered by Gram-positive bacteria or/and Gram-negative bacteria. Especially peptidoglycan (PGN), cytidine-phosphate-guanosine DNA (CpG DNA) and lipopolysaccharide (LPS /endotoxin) are recognized as the key molecules during the pathophysiology of sepsis, which at last lead to sepsis shock or even death. Therefore, it is considered as the most promising strategy to prevent all of the three key molecules from triggering sepsis.Many studys indicated that many traditional Chinese medicines (TCM) were natural antagonist of sepsis in vitro and in vivo, but a further study was obstructed on some potential anti-inflammatory TCM respecting the absence of an effective tracking approach. Therefore, targeting on PGN, CpG DNA and lipid A respectively, we screened anti-inflammatory TCM which possessed higher affinity with target. Then lycium bark was taken for object to extract effective components and prepared components were further investigated bioactivity of anti-PGN, anti-CpG DNA and anti-LPS in vitro and in vivo.Methods: (1) Dissoluble PGN and CpG DNA were immobilized onto the surface of biotin cuvette for establishing target. Another effective tracking approach was established by immobilizing Escherichia lipid A F583 onto the surface of Non-derivatised cuvett. The biosensor technology was applied to screen anti-inflammatory TCM targeting on three key molecules. (2) The active compositions were isolated by AB-8 macroreticular resin from lycium bark. After the activities of compositions were evaluated, the most effective compositions was confirmed. In vitro, the affinities of different concentrations composition E binding with PGN, CpG DNA and lipid A were measured separately. The effect of composition E on vigor of RAW264.7 cells were tested by MTT and CCK-8, and its inhibition on TNF-α, which was released from RAW264.7 cells induced by PGN, CpG DNA and LPS, was also tested by ELISA. In vivo, murine sepsis models were made by intravenously heat-killed E.coli and heat-killed S.aureus, then protection of composition E on mice sepsis model were observed.Results: (1) Each of the resonance peaks of immobilization is symmetrica singlet. The coating amount of PGN, CpG DNA and lipid A was 0.44 ng/mm2, 0.33 ng/mm2 and 1.19 ng/mm2 respectively. All of these datas were consistent with test standard given by company Thermo. The platform to screen the anti-inflamatory traditional Chinese medicine was established. Among the 114 traditional Chinese medicine, twenty-two ones possessed the higher binding activity to PGN, CpG DNA and lipid A, and seven ones possessed more content of active material. (2) The active composition E from lycium bark was confirmed as the best one. In vitro, composition E bound to PGN, CpG DNA and lipid A with high affinity, and had no effect on RAW264.7 cells, and strongly inhibited TNF-αrelease from RAW264.7 cells induced by PGN, CpG DNA and LPS in a dose-dependent manner. In vivo, composition E markedly protected mice from lethal challenge by heat-killed E. coli and heat-killed S. aureus.Conclusions: (1) Targeting on PGN, CpG DNA and lipid A, we successfully established the platform to screen anti-inflammatory traditional Chinese medicine using biosensor technology which is highly effective, objective and feasible. (2) The active composition E from lycium bark is confirmed as the most effective one. It possessed the higher binding activity to PGN, CpG DNA and lipid A. Test in vitro and in vivo showed that the active composition E had significant activity against three key molecules mediating sepsis.
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