ABSTRACT
OBJECTIVE: To investigate the lipopolysaccharide (LPS) antagonizing biological activity of densefruit pattany root-bark extract (DPR-2) in vitro. METHODS: The effect of DPR-2 in neutralizing LPS (0.1 microg/L) was detected by kinetic turbidimetric limulus test. The effect of different concentrations of DPR-2 (0,8.0,16.0,32.0,64.0 mg/L) on binding of FITC-conjugated LPS (FITC-LPS,100.0 microg/L) to murine RAW264.7 cells was analyzed with laser scanning confocal microscopy. The expression of TNF-alpha and IL-6 mRNA in RAW264.7 cells after exposure to LPS (100.0 microg/L) were determined by real-time RT-PCR. RESULTS: DPR-2 could neutralize LPS (P < 0.05 or P < 0.01), and inhibit the binding of FITC-LPS to RAW264.7 cells in a dose-dependent manner when the concentration of DPR-2 was above 16.0 mg/L. Furthermore, DPR-2 could markedly inhibit the expression of TNF-alpha and IL-6 mRNA in LPS-stimulated murine RAW264.7 cells. CONCLUSION: DPR-2 exhibit an anti-LPS effect in vitro, which may be related to its capacity to neutralize LPS and inhibit binding of LPS for its receptors.
Subject(s)
Drugs, Chinese Herbal/pharmacology , Endotoxins/antagonists & inhibitors , Lipopolysaccharides/antagonists & inhibitors , Monocytes/drug effects , Animals , Cell Line , In Vitro Techniques , Limulus Test , Mice , Monocytes/metabolism , Plant Extracts/pharmacologyABSTRACT
OBJECTIVE: To investigate the expression and analysis of its activity of anti-bacterial peptide gloverin in COS-7 cells. METHODS: The appearance frequency of all genetic codes in the cDNA sequence from the same species of protein Attacin A was analyzed, and its cDNA sequence was synthesized by PCR overlapping extension method in conjunction with the designation of the known protein sequence of gloverin. The genes were inserted into pCDSI, an eukaryotic vector, after being identified correctly. As a result, the vector pBZHG was constructed. Thereafter, the liposome FuGENE( trade mark ) 6 was employed as the vector, and the COS-7 cells were transfected with liposome pBZHG and blank vector pCDSI. The normal cells were taken as the control. The supernatant was collected for the detection of its bactericidal activity after 72 PBHs. RESULTS: The gloverin cDNA sequence designed artificially was expressed in COS-7 cells. The supernatant of the cells transfected by pBZHG exhibited bactericidal activity to E. coli J5 when compared with that from normal cells and in cells transfected with blank vectors. CONCLUSION: The designed cDNA sequence of gloverin was proved to be genuine, and it provided the basis for future study of its antibiotic and anti-endotoxin activities.
