| Antimicrobial peptides(AMPs)widely exist in organisms,play an important role in immune system to defense invasive pathogenic microorganisms.AMPs possess broad spectrum antimicrobial activities,against not only ordinary bacteria,fungi and viruses,but also many clinical antibiotic-resistance microorganisms.Additionally,AMPs also have many other biological activities,such as immune chemotaxis,histamine release,apoptosis,promoting wound healing,inducing angiogenesis and lymphocyte activation etc.In this thesis,we emploied a series of biological activity tests and some in vitro/vivo experiments for preliminary pharmaceutical study on a synthetic antimicrobial peptide ZY13.First,inhibition tests showed ZY13 had significant antimicrobial activities against Staphylococcus aureus,Escherichia coli,Bacillus subtilis and Candida albicans,including a large number of clinical isolates of antibiotic-resistance strains.Kinetic analysis showed it was lethiferous.At concentrations greater than 1× MIC,ZY13 could kill all of the tested Candida albicans strains within 30 min.Scanning Electron Microscopy results showed that ZY13 possibly caused microbial death via binding to the cell membrane of microorganisms,resulting in membrane perforation,and finally leading to the leakage of cell contents.Further experimental results showed that ZY13 kept good stability in serum and in different concentrations of salt ions without significant changes in the antibacterial activities.Animal model showed that ZY13 had highly significant therapeutic effects on the vaginitis in mice caused by the infection of Candida albicans.In summary,the broad-spectrum antimicrobial properties of ZY13 and its significant effects shown in animal models suggest that it is an excellent template or candidate for the development of antimicrobial drugs.Blood-sucking arthropods have developed effective mechanisms to overcome the immune responses of hosts to get blood meal successfully.Such arthropods produce wide sources of antihemostatic and immune suppressant compounds in their salivary glands.Numerous antihemostatic compounds have been identified from them,such as platelet aggregation inhibitors,vasodilators,anticoagulants,immunomodulatory peptides and antimicrobial peptides etc.These compounds are of great significance for us to understand the coevolutionary history between parasites and hosts and also provide new ideas of medicine design for us.Tsetse fly,a kind of bloodsucking insect,which belongs to Glossina,Glossinidae,Diptera,Insecta,is widespread in sub-Saharan Africa,and can cause sleeping sickness by carrying trypanosomes.Here,we tested the possible immunomodulatory activities of Gloss 2,a peptide in the salivary gland of tsetse fly,which was synthesised according to the sequence obtained from protein data banks and found the significant anti-inflammatory functions of this peptide.We verified that Gloss 2 could inhibit the secretion of cytokines in mice splenocytes,such as tumor necrosis factor secretion-a(TNF-?),interleukin-6(IL-6)and interferon-?(IFN-?)under the stimulation of LPS.We presumed that the inhibitory effect of Gloss 2 on inflammatory factors was closely related to the inhibitions of signal molecules in MAPKs pathway.Western Blot assay showed that Gloss 2 could inhibit the phosphorylation of ERK1/2,SAPK/JNK and p38 MAPK kinase produced by LPS-induced mice monocyte-macrophage cell line RAW 264.7,to regulate the secretion of the relative cytokines.We found it had no significant effects on the proliferation and cellular activity of mice splenocytes using MTT assay,indicating Gloss 2 has an immunomodulatory function independent of cytotoxicity.The above analyses confirmed immunomodulatory polypeptide Gloss 2 could inhibit the immune response by regulating the secretion of cytokines to help tsetse fly fullfill blood-sucking. |
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