Inhibition Of Flavonoids On Lysozyme Amyloid Fibrillation Process

Protein aggregation will lead to changes in protein conformation and loss of protein of normal biological function. The fibers gathered to form the deposition in cells. This leads to cellular dysfunction and pathological changes in the tissues and organs eventually which causes a variety of diseases, such as Alzheimer’s disease,type II diabetes, Parkinson’s disease, Alzheimer’s disease and other. Studies the inhibitory effect of drug molecules on the protein amyloid fibrosis and the prevention and treatment of the disease as well as the development of new drugs are significant.Studies have indicated that natural phenolic compounds have the potential to inhibit the protein aggregation. Flavonoids are natural polyphenols which widely exist in nature. But study of inhibition effect of flavonoids on protein aggregation focused on catechol flavonoids at present. Lysozyme is the first protein of markers of disease,which is known to spherical protein classical model of forming amyloid. Therefore,this study selected 18 kinds of non-catechin flavonoids of different structures and lysozyme as a model in vitro. Through investigation condition of lysozyme aggregation, a research model of lysozyme aggregation was established. Based on the condition, we used 8-Anilion-1-naphthalenesulfonic acid(ANS), Thioflavine T,(ThT),Congo red probe technology, Cirular Dichrosin(CD), transmission electron microscopy(TEM) studying inhibitory effect flavonoids on exposure of hydrophobic region and increasing of β sheet in the process of lysozyme aggregation. Through molecular docking technology, the mechanism of inhibitory effect of flavonoids on lysozyme aggregation was investigated preliminarily. The results provided the beneficial reference and basis for we exploring the flavonoids inhibitor screening and molecular mechanism. Specific content is as follows:1. Construction and characterization of the self-aggregation process of lysozyme.We investigated the effects of different pH, temperature, concentration of organic solvent on lysozyme aggregation using ThT as a fluorescent probe and determined the experimental model lysozyme self-aggregation. The results showed that 50 μMlysozyme in pH 2.0 glycine-HCl buffer solution containing 0.5% DMSO and 65℃incubited 16 days that can reach self-aggregation forming amyloid fibril and growth curve is S-shaped. The growth process is a dynamic process of nucleation—fibers extend—mature fibers. Under certain conditions, we detected exposure of hydrophobic region and changes of β sheet in the process of lysozyme aggregation through ANS fluorescent probe method, Congo red UV probe method, CD spectra and TEM. The results showed that secondary structure of lysozyme had obvious change that α-helical structure gradually transformed β sheet structure and exposure of hydrophobic regions aggravated and uniform solution gradually grew long silky fibers with the intensification of the process of lysozyme aggregation.2. Effects of flavonoids on the process of lysozyme self-aggregationUnder the same experimental conditions, flavonoids of different concentration ratio(1:1, 1:0.5 1:0.2) were added lysozyme samples incubated together. The effect of flavonoids on lysozyme aggregation was detected by ThT, ANS fluorescence method,Congo red absorption method and CD, transmission electron microscopy. The results showed that ThT fluorescence intensity, ANS fluorescence intensity and Congo red absorption intensity of lysozyme solution containing flavonoids compared with mature fibrils decreased at different degree and degree of decreasing and was positively correlated with flavonoids concentration. CD spectra show that, absorption of β sheet structure decreased gradually, absorption of α-helical structure increased gradually in process of lysozyme aggregation after the addition of flavonoids. TEM morphology observation showed that lysozyme fibrils were shorter and taperer after the addition of flavonoids. Puerarin, naringenin, sophoricoside, quercetin, silymarin,naringin, chrysin, baicalin, baicalein and luteolin in 18 kinds of flavonoids showed strong inhibition on lysozyme aggregation. At 1:1 of concentration ratio, the inhibition rate on exposure of hydrophobic region and increasing of β sheet of lysozyme aggregation reached above 75% which showed the stronger inhibition.3.Study of interaction of flavonoids and lysozyme through molecular docking methodThe experimental results showed that inhibitory effect of flavonoids on lysozyme aggregation occurred mainly the early stage in the formation of lysozyme fibrosis,which meaned that inhibitory effect of flavonoids on lysozyme aggregation mainly owed to the interaction of flavonoids and lysozyme monomer. So we selected three-dimensional structure of 2LYZ as a model protein and investigated molecular docking of lysozyme and flavonoid molecules with Autodock 4.0 software. Molecular docking study results showed that Hydrogen bonding and van der Waals force were formed between the amino acid residues of lysozyme active site and flavonoids;Hydrophobic interaction was formed between hydrophobic amino acids residues of lysozyme hydrophobic cavity and flavonoids; The π-π stacking effect may be formed between lysozyme aromatic amino acid residues and aromatic ring of flavonoids when their aromatic ring were parallel. These forces can maintain the stability of the conformation of lysozyme and inhibited the aggregation of lysozyme.