| Phosvitin (PV) is the one of the key phosphoprotein in egg yolk, where50%of amino acidsare composed of serine that has been mostly phosphorylated. Due to the special structure of PV,both PV and Phosvitin Phosphopeptides (PPPs) have strong abilities to bind metal. Accordingto the former research, PPPs has a better anti-oxidative and oxidative stress capacity. In ourresearch, PV was firstly extracted and purified, and then the properties of stability, oxidationresistance, antibacterial, anti-inflammatory of PV and PPPs are investigated respectively,especially focusing on the anti-inflammatory mechanism of PPPs. Moreover, a new kind ofsynthesized phosphopeptide named [Ser (PO3)](PP-2or PP-2for short) was subject toanti-inflammatory and mechanism research, as well as the cell signaling pathways of PP-2.This study will lay a foundation for the development and clinical application of more and morenew anti-inflammatory products with no adverse effects.In order to optimize the extraction of phosvitin, the technology model was established basedon the quadratic general rotation union test design. Genetic algorithm was used to obtain theoptimal parameters for extraction processing, and the quadratic regression wasobtained,y=121.51.86z1.4295z0.0135zz+2.79z2+0.00785z2121212, and the optimalparameters were as bellow: ratio of n-hexane and ethonal3.4/1(V/V), dosage of the organicsolvent148mL, respectively, and the purity of phosvitin reached81.88%. The SDS-PAGE wasused to determine the Molecule weight of phosvtin. The results were obtained as follows:12%separating gel was the helpful for the results, and the target band is clear and dark, whichindicated a high contents of the target protein, however, there are still two obvious bands forthe mixed protein with a M.W. ranging from66.2kD to97.4kD, which indicated that the targetprotein needs to be purified in the future.The properties of thermo stability and emulsification and stability of phosvitin (PV) andphosvitin phosphopeptides (PPPs) as well as the influence of Pulsed Electric Field (PEF) onPV and PPPs were investigated respectively, in addition, the antioxidative ability, DPPHclearing capabilities, reductive ability and chelating capabilities were determined. The results indicated that, both PV and PPPs have good thermo stabilities, emulsification and stabilities.Based on the conditions of100℃and30min, the absorption of PV had not changedsignificantly, by contrast, the absorption of PPPs also had not changed much under theconditions of100℃and30min. PV had better emulsifying properties compared with PPPs, aswell as a better stability. After the samples were treated by PEF, the secondary structure of PVwas not obviously modified, which indicated that PEF can't have significant influence on thesecondary structure of PV. Moreover, PV and PPPs have good bioactivities of clearing DPPHfree radicals, reductive ability and metallic ion chelating ability. Consequently, both PV andPPPs have good anti-oxidative abilities, in particular, PPPs was superior to PV in the ability ofchelating metallic ion.Three types of cells (Caco-2, HT-29and RAW264.7)were induced by TNF-α and LPS togenerate inflammation. Biological methods of ELISA, RT-PCR were employed to study theanti-inflammatory effects of PPPs, as well as its mechanism. The impact of PPPs oninflammation induced by tumor necrosis factor (TNF-α) and lipopolysaccharide (LPS) wasinvestigated, using intestinal epithelial cells (HT-29Caco-2) and macrophage RAW264.7asthe model, In addition, the impact of PPPs on the gene expression of pro-inflammatorycytokines (IL-8, IL-12, IL-6, MCP-1, TNF-α, IL-1β, iNOS in) were investigated.Cytotoxicity experiments indicated that PPPs had no significant impact on three kinds of cellsinduced by TNF-α or LPS, regardless of PPPs with low concentration or high concentration(0.01μg/mL-1000μg/mL), all three cell toxicity. The results of the induction time show thatthe optimal induction time for TNF-α is4h, and the optimal induction time for LPS is6h. Inthe model of Caco-2and HT-29cells induced by TNF-α, the contents of IL-8in the resultsindicated that PPPs could significantly reduce the secretion of IL-8. In the model ofLPS-induced Caco-2and HT-29, the contents of IL-8in the results indicated that PPPs had nosignificant effect on the inhibition of IL-8secretion, but it can inhibit the secretion of IL-8tosome extent. In the LPS-induced RAW264.7cell model, the contents of IL-8in the resultsindicated PPPs had no significant effect on the inhibition of TNF-α secretion, but to someextent, it could inhibit the secretion of TNF-α.RT-PCR technology was adopt in the evaluation of PPPs on regulating gene expression in HT-29cells induced by TNF-α and LPS, the results showed that the there were intact RNAwith good quality, PCR products with clear bands and the expected theoretical value. InTNF-α-induced HT-29cells, PPPs can significantly inhibit the gene expression of IL-8, ofMCP-1and IL-12. In LPS-induced HT-29cells, PPPs can significantly inhibit the geneexpression of IL-8and IL-12, although the PPPs couldn't significantly inhibit the geneexpression of TNF-α. In the LPS-induced RAW264.7cells, PPPs could significantly inhibitthe gene expression of TNF-alpha, IL-1β, IL-6and iNOS. Therefore, we concluded that PPPshad anti-inflammatory effect through the regulation of inflammatory factors in geneexpression, and thus played a role in suppression of inflammation.Based on the technology of ELISA, RT-PCR, Western blot and PCR-Array technology, theanti-inflammatory of PP-2and its mechanism were studied. The effect of PP-2on HT-29cellsand of Caco-2induced by TNF-α and LPS was investigated, and the results indicated that PP-2had not side effect on the three kinds of cells, even if the concentration of PP-2reached5mM.The results also indicated that PP-2could significantly inhibit the secretion of IL-8and keptdose-dependent relations. The RAW264.7cells induced by LPS were measured respectivelythrough the determination of secretion of TNF-α and cell-induced IL-8. And then the effect ofPPPs on regulating gene expression in HT-29cells induced by TNF-α and LPS and RAW264.7cells induced by LPS (including IL-8, IL-12, IL-6, MCP-1, TNF-α, IL-1β, iNOS) wereevaluated respectively. The results indicated that PP-2can inhibit the gene expression of IL-8,IL-12, MCP-1and TNF-α in the model of HT-29cells induced by TNF-α. By contrast, PP-2can significantly inhibit the gene expression of IL-8, IL-12, TLR-4, TNF-α and IL-6in themodel of HT-29cells induced by LPS, but no obvious effect on the gene expression of MCP-1although PP-2can have a certain effect on MCP-1compared with the POS group. In the modelof RAW264.7cells, PP-2could significantly inhibit the gene expression of IL-6, TNF-α andiNOS.Based on Western blot technology, the effect of PP-2on the phosphorylated gene expression ofJNK, ERK1/2, p38and IκB induced by TNF-α was studied respectively. The results showedthat PP-2could inhibit the phosphorylated gene expression of JNK, ERK1/2, p38and IκB. Inthe model of HT-29cells induced by LPS, PP-2can significantly inhibit the phosphorylated gene expression of ERK1/2, p38and IκB. Therefore, we concluded that the anti-inflammatoryfunctions of PP-2had something to do with two passways including NF-κB and MAPK.Employing PCR-Array, two pathway factors (NF-κB and MAPK) were investigated. Theresults indicated that, PP-2can inhibit most of the gene expression in the passway of NF-κBand MAPK in the model of HP-29cells induced by TNF-α. By contrast, PP-2can inhibit thegene expression in the passway of NF-κB and MAPK, and the down regulation rate was70%and56%, respectively. The results indicated that, the number of gene expression was differentwith the different induction reagent, and PP-2had less inhibition in the HT-29cells induced byLPS than that in the HT-29cells induced by TNF-α, which can be attributed to the highsensitivity of TNF-α. The above results were consistent with the ELISA results.PPPs derived from hen egg yolk have good stability, anti-oxidation, bacteriostasis, what ismore important, PPPs had anti-inflammatory effect, and PPPs can be transported into cancercells and macrophage in the intestinal tract, which can lead to the reduction of somepro-inflammatory factors, thus had the anti-inflammatory functions. As a new kind ofsynthesized peptides, PP-2had good anti-inflammatory functions, and the key mechanism isthat it can inhibit the gene expression of some pro-inflammatory factors (such as IL-8, IL-12,IL-6, MCP-1, TNF-α, IL-1β and iNOS) through inhibiting the passway of NF-κB and MAPK.In conclusion, PPPs and PP-2can be used as natural anti-inflammatory materials, which areexpected to lay a foundation for the development of new anti-inflammatory medicine orfunctional foods. |
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