Endotoxin-neutralizing Activity Of Hen Egg Phosvitin

Phosvitin (Pv) is one of the major egg yolk proteins formed by the proteolyticprocessing of the large precursor molecule vitellogenin. Its amino acid composition isunique in that it has approximately55%serine residues, which are almost fullyphosphorylated. Thus Pv is an extremely highly phosphorylated protein, carrying avery high net negative charge in the central hydrophilic area surrounded by two smallhydrophobic areas at the N-and C-termini. Due to its peculiar physical and chemicalcharacteristics, Pv possesses numerous functional and biological properties, includingion chelating activity, powerful emulsifying properties, antioxidant activity,antithrombin action and antimicrobial activity. The aim of this study was toinvestigate the immune modulatory activity of chicken Pv.First, the antibacterial activity of chicken Pv was tested by colony-forming unit(CFU) assay. The results showed that Pv exerted a lethal effect on the Gram-negativebacterium Escherichia coli (E. coli) and the Gram-positive bacterium Staphylococcusaureus (S. aureus) under thermal stress at50oC, although it exhibited no significantbactericidal effect on the two bacteria at37°C. Then FITC-labeled Pv was incubatedwith E. coli and S. aureus to test the binding activity of chicken Pv to microbial cells.It was found that Pv was able to bind both E. coli and S. aureus. To better understandthe mechanisms of bacterial binding activity, an enzyme-linked immunosorbent assay(ELISA) was carried out to test what molecules on the microbial surfaces arerecognized by Pv. The results showed that Pv had a significantly stronger affinity tothe immobilized pathogen-associated molecular patterns (PAMPs) such aslipopolysaccharide (LPS), lipoteichoic acid (LTA) and peptidoglycan (PGN)compared to the control BSA. These data indicated that like fish Pv, chicken Pv is amultivalent pattern recognition receptor (PRR), capable of binding to LPS, LTA andPGN, and has an antimicrobial activity against E.coli and S.aureus in atemperature-dependent manner.Trauma, surgery, and burns can all result in a severe complications, sepsis, which is a systemic inflammatory response syndrome (SIRS) caused by bacterial infection.Sepsis is the major cause of mortality in patients in intensive care units (ICU),accounting for about one million deaths per year over the world. LPS, also calledendotoxin, is the major initiator of sepsis, which targets monocytes and macrophagesvia activating the cascade of inflammatory signal transduction pathway, resulting inthe production of large amounts of cytokines such as tumor necrosis factor-α (TNF-α),interleukin (IL), leukotriene (TL) and nitric oxide (NO) as well as other inflammatorymediators, and initiating an excessive autodestructive inflammation. Studies haveshown that many proteins and peptides with antibacterial activity also have immunemodulatory activity. They could bind to LPS and block its toxicity at the verybeginning and thus prevent the inflammatory cascade reaction. Usually these proteinsand peptides can inhibit the LPS-induced TNF-α production of macrophages, decreasethe mortality of septic mice and protect them from tissue damage and organ failure.Experiments in the first part of this study had shown that Pv had antibacterialactivity and LPS-bing capacity. We wonderd if Pv could block the LPS toxicity. In thesecond part, LPS detoxicating property of Pv was detected. TNF-α is considered as agood indicator of LPS toxicity and the sepsis syndrome. Thus, we cultured the murinemacrophages cell line RAW264.7and treated the cells with LPS followed by medium,or medium with Pv or PMB. The TNF-α concentration in the supernatants of differentgroups was tested using an ELISA kit. It was found that Pv could significantly inhibitLPS-induced TNF-α release from RAW264.7cells (P<0.05), suggesting the presenceof LPS detoxification in vitro. We next incubated RAW264.7cells and human redblood cells with Pv and examined the cytotoxicity of Pv on the two kind of cells viaMTT assay and homelysis assay, respectively. The results of which showed that Pvhad no cytotoxicity nor homelysis activity, indicating it could be used in vivo. Todetect whether Pv has protective effects against LPS in vivo, Kunming mice werestimulated with intraperitoneal (i.p.) injection of different dosage LPS to generate themurine endotoxin shock model. We chose the dose of50mg/kg to produce80-100%mortality in the mice. Groups of18mice were injected with50mg/kg LPS followedby injection of normal saline, Pv or PMB. In the challenge control group, mice began to die8h after the injection, and mortality was increasing between16h and24h, with50%of accumulated mortality at24h. Administration with Pv resulted in a prolongedsurvival of the mice compared to the challenge control group, and there was only1mouse died between16h and24h. In addition, Pv could improve the survival rate ofthe endotoxemia mice from22.22%to50%at the end of experiment, and exhibitedsignificantly increased survival rate between24h and64h (p<0.05). We further testedthe serum TNF-α concerntration in the mice received different administration, findingout a prominent reduction of TNF-α in Pv treated group compared to the challengecontrol group (p<0.01). These data together indicated that Pv can detoxicate LPS. AsPv is abundant in hen egg yolk, therefore, it can be used as adjunctive therapy inclinics to treat sepsis.