| Protein is an important component of organism cells and tissues.Every kind of protein usually has its own specific natural conformation to perform its particular physiological functions.However,in some abnormal environments,the structure of protein will be changed,and protein further misfolds to form insoluble fibrils,which can accumulate into amyloid deposits or intracellular endosomes,resulting in metabolic disorders.Pathological studies have shown that about 50 amyloid proteins are closely related to specific human diseases,such as Alzheimer’s disease,Parkinson’s disease and type 2 diabetes.These diseases have brought great harm to human health and social development.Delaying or inhibiting the fibrotic aggregation of amyloid protein has become a potential therapeutic strategy for amyloidosis.At the same time,the fibrotic aggregation property of protein affects the production,storage and transportation process of protein drugs,causing tremendous risk to their safety use,which is also an urgent problem to be solved in biomedical field.In this paper,we selected insulin as the model protein to investigate the protein aggregation using two kinds of inhibitors.The modern biophysical techniques and molecular modeling methods were performed to study the effects of inhibitors on insulin aggregation,including identifying the aggregation kinetics,aggregation morphology and cytotoxicity induced by insulin fibrils,and analyzing the interaction mechanism between insulin and inhibitors,and the corresponding inhibitory mechanism.In short,the relevant research results could provide theoretical reference and application guidance for the treatment of amyloidosis and the storage and clinical use of biological agents(such as insulin).The main findings of this paper are as follows:1)Two kinds of morin-modified gold nanoparticles with three different sizes were synthesized by two different methods(conventional reduction method using trisodium citrate and in-situ reduction method using morin),and then their inhibitory effect on insulin aggregation was studied.It was found that both kinds of gold nanoparticles had great inhibitory effect.Th T fluorescence experiments showed that inhibitors could prolong the nucleation phase of aggregation process and reduce the fluorescence intensity in the stationary phase.CD results showed that inhibitors could reduce the β-sheet content of insulin fibrils and retain a certain degree of α-helix structure.TEM and AFM images showed that the addition of inhibitors could make insulin form shorter and thinner fibrils,even amorphous aggregates.Cytotoxicity experiments showed that inhibitors could reduce the cytotoxicity induced by insulin aggregation.At the same time,it was also found that the inhibitory effect of two types of gold nanoparticles were concentration-dependent manner,and closely related to the size of nanoparticle and preparation method.The gold nanoparticles obtained by the in-situ reduction method had better inhibitory effect than that of conventional reduction method.In addition,the inhibitory effect of morin-modified gold nanoparticles were compared with that of pure gold nanoparticles and morin,from which it could be obtained that morin-modified gold nanoparticles had a synergistic advantage.2)The inhibitory effect of three kinds of crown ethers with different structures(unmodified,hydroxymethyl modified and benzene modified crown ethers)on insulin aggregation were studied.It was found that the crown ether modified by benzene had the best inhibitory effect.It significantly reduced the Th T fluorescence intensity and β-sheet content of insulin fibrils,and then induced insulin to form more shorter fibrils.In addition,the inhibitory effect of three types of crown ethers were also concentrationdependent manner. |
