The Molecular Mechanism Of AWRK6 Neutralizing Endotoxin

Antimicrobial peptides(AMPs)are low molecular weight peptides which produced by organism itself.AMPs have the activity against bacteria,viruses,protists and fungi.AMPs also significantly have the inhibiting effect on some inflammatory reactions such as sepsis.Their bactericidal effect depends on changing the permeability of cytomembrane,as well as the target inside bacteria.They can take part in the metabolic activity of bacteria,in which way,they can inhibit and kill bacteria.When AMPs play the role of damaging bacteria,the bacteria won't release the endotoxin,without the bacterial resistance as well.Some studies have shown that AMPs could blocking LPS-stimulated inflammation by neutralizing endotoxin.Dybowskin-2CDYa is a kind of antibacterial peptide which is found in frog skin,it is reformed and named by our laboratory as AWRK6(SWVGKHGK KFGLKKHKKH).The molecular weight of AWRK6 is 2130.5.It is a cationic antibacterial peptide with ?-helix.The previous studies have shown that AWRK6 has significant activity of killing bacteria,neutralizing endotoxin in vitro or in vivo,as well as blocking LPS-stimulated inflammatory cytolines,such as TNF-? and IL-8.Moreover,it is safe for RAW264.7.These advantages and abilities make AWRK6 a novel peptide which may be used for neutralizing endotoxin and a new type of therapy against sepsis and septic shock.In order to study the mechanism of AWRK6 neutralizing endotoxin,the effect of AWRK6 blocking the binding of LPS and LBP is measured,as well as the mRNA expression level and protein expression level of some possible site.The effect of blocking the binding of LPS and LBP was studied by Enzyme-linked immunosorbent assay(ELISA),in order to explore the preliminary mechanism of AWRK6 neutralizing endotoxin.By using Real Time PCR(FQ-PCR),the mRNA expression level of TLR4,JNK and I?B were measures.For further study,the protein expression level of TLR4 and I?B was tested by western blotting.The result of ELISA showed that AWRK6 had a statistically significant activity of blocking the combination of LPS with LBP,further more,there was a activity of inhibiting combinated LPS-LBP.The result of FQ-PCR showed that AWRK6 could inhibit the change of TLR4,JNK and I?B mRNA expression level caused by LPS in vitro as well as vivo.Meanwhile,AWRK6 could make the mRNA expression level of these factors more closing to normal.The study of western blotting showed thatAWRK6 could inhibit the change of TLR4 and I?B m RNA expression level caused by LPS in RAW264.7 and make it close to the normal level.The research above aimed to study the mechanism of AWRK6 neutralizing endotoxin,which would provide us experimental foundation and theoretical basis of developing AWRK6 as a novel medicine to neutralize LPS.