The Mechanism Against Gram-negative Bacteria And The Effect On Lipopolysaccharide-incduced Inflammatory Responses Of Chensinin-1b,an Antimicrobial Peptide Analogue From Chinese Brown Frog,Rana Chensinensis

Lipopolysaccharide(LPS)is the major structural component of the cell wall of gram-negative bacteria.Besides the protect activity from antibiotics,it is also the trigger of inflammatory response.Excessive and uncontrolled inflammation leads to the organ or tissue damage from SIRS or sepsis,and even causes death of the organism.At present,there are no drugs which can effectively neutralize LPS in clinical.Antimicrobial peptides with alternative sources are proved to be an important component of the innate immune system of all organisms,they have broad-spectrum antimicrobial activity,no toxicity to mammals and less acquired resistance.Some of antimicrobial peptides have the properties of neutralizing LPS and immunoregulatory functions.For these reasons,antimicrobial peptides are surely the most promising candidate of anti-inflammatory drugs.Chensinin-1,a natural antimicrobial peptide containing 18 amino acid residues,was isolated and purified from the skin secretions of Chinese brown frog,Rana chensinensis.Chensinin-1 is a cationic peptide with low molecular weight,which shows a potential anti-bacterial activity against gram-positive bacteria.In previous study,chensinin-lb was designed by replacing Gly residues with Trp residues,and by rearranging the amino acid sequence to improve its amphipathy.Chensinin-1b exhibited a higher antimicrobial activity against both gram-positive and gram-negative bacteria than its parent peptide,chensinin-1.In this study,the reason which caused the selectivity differences between chensinin-1 and chensinin-1b was investigated firstly.We performed NPN assay to study the ability of chensinin-1b to permeate the outer membrane of gram-negative bacteria.Chensinin-1b showed a better permeabilization ability than chensinin-1.Both peptides had higher depolarizing ability and antibacterial activity against spheroplasts of E.coli.Chensinin-1 displayed an obvious decrease in the antimicrobial ability due to its weak interaction ability with LPS.The mechanism of action between the antimicrobial peptides and LPS was analyzed by various biophysical methods.ITC and Zeta potential experiments were used to evaluate the binding activity of the peptides to LPS.ITC experiments showed that the process of the binding of chensinin-1b with LPS was an exothermic process at the beginning,then an endothermic process,which meant the binding and inserting of peptide to LPS was driven by electrostatic and hydrophobic interaction force separately.In addition,the results of zeta potential measurements were consistent with the results of the ITC analysis.Chensinin-lb showed a higher binding affinity than chensinin-1.DLS and FITC-LPS disaggregation experiments showed that after binding of antimicrobial peptides with LPS,LPS aggregates were disaggregated.the polymerization state of LPS molecules became smaller particle size,due to the penetration of antimicrobial peptides.Chensinin-lb could completely eliminate the LPS aggregates,which is more effective than chensinin-1.The Trp fluorescence spectroscopy showed a blue shift with an increased fluorescence intensity,which indicated that the Trp fluorescence could not been quenched by the quenchers.IR spectroscopy displayed a drastic change in the PO2-band shape and intensities after the addition of chensinin-1b,indicating the penetration and stuctrual change in LPS.The result of LAL assay proved that chensinin-lb can neutralise LPS effectively.LPS is the trigger of inflammatory pathway,and this thesis also explored the inhibitory effect of chensinin-1b on the model of LPS-induced inflammation.The results showed the antimicrobial peptides can inhibit the release of LPS-induced inflammatory cytokines like TNF-?,IL-6,IL-1? and NO.Chensinin-lb showed potent anti-inflammatory effects.The confocal laser scanning microscope experiments showed that chensinin-lb can enter RAW264.7 cells in a short time,and indicated an anti-inflammatory interaction was taken place inside the cells.Therefore,we performed a pretreatment experiment of chensinin-lb to investigate the anti-inflammatory effect within RAW264.7 cells.The result revealed that the pretreatment of chensinin-lb also inhibited the release of TNF-a and IL-6,which value was about 80%compared with that of normal treatment group,these results indicated the anti-inflannatory action was main happened inside the cells,instead of neutralization of LPS outside of the cells.Western blot experiment proved the overexpression of the cytosolic protein NOD1/NOD2 induced by LPS was inhibited by chensinin-1b.NOD1 and NOD2 activated the downstream signaling pathways,NF-?B and MAPK.Experiments showed that chensinin-1b decreased the mRNA expression of NF-?B mediated by NOD1/NOD2 while also inhibiting the phosphorylation ERK/JNK/p38 in the MAPK pathway.The mechanisms of internalizing action of chensinin-1b were also studied,and the results indicated it didn't exhibit the depolarization of cell membrane and affect the permeability of membrane.By low temperature and endocytosis inhibitors treatment,the results revealed that chensinin-1b relied on Na+/H+ ion channels mediated endocytic pathways to enter RAW264.7 cells.In conclusion,chensinin-1b had the ability of inhibiting on LPS-induced cytokines and neutralising LPS.The main anti-inflammatory mechanisms of chensinin-1b was downregulated expression of NOD1/NOD2,blocking their downstream NF-?B and MAPK pathways by entering into cells through Na+/H+ ion channels mediated endocytic pathways.