| With the rapid development of nanotechnology,nanotechnology has become one of the most promising technologies in the 21 st century.Nanoparticles have been widely used in the fields of physics,chemistry,biology and consumer goods.Fe3O4 nanoparticles(Fe3O4 NPs)have excellent properties,such as low toxicity,good biocompatibility and biodegradability,and it can be modified in a variety of ways.Therefore,Fe3O4 NPs have great application potential in the medical field.Fe3O4 NPs are mainly used in drug delivery,gene delivery,biosensor,magnetic resonance imaging,contrast agent,hyperthermia,magnetic field assisted radiotherapy and tissue engineering.Despite the remarkable advances in nanomedicine,relatively little is known about the specific interactions of NPs with the biological environment.It has been reported that NPs will interact with proteins when NPs enter the biological environment.Proteins in the environment prefer to combine on the surface of NPs,thus forming protein corona(PC).Current results suggest that PC formed on the surface of NPs will not only affect the biological response of NPs,including biological distribution,clearance,and potential toxicity,but also change the composition of PC with disease status and specific disease conditions.Based on previous work,this study systematically studied the applicability of Fe3O4 NPs in rat serum and plasmodium system,and established nanoproteomics strategy based on Fe3O4 NPs-PC to carry out research on disease and drug action targets.The main results are as follows:(1)Using stable FeCl3 iron powder as precursor,Fe3O4 NPs with uniform morphology and average particle size of 80-100 nm were successfully synthesized by solvothermal method.(2)Using rat serum as carrier,a nano-proteomics strategy based on Fe3O4 NPs-PC was finally established.Firstly,the amount of Fe3O4 NPs in rat serum was determined by BCA method,and the experimental process was optimized and determined.We then used the Ultimate 3000 RSLC Nano system in conjunction with Orbitrap Fusion Lumos Tribrid mass spectrometer to isolate and detect protein levels in Fe3O4 NPs treated and normal treated serum.It was found that after Fe3O4 NPs treatment the number of proteins in serum of rats increased significantly,and the proteins almost covered all regions of the standard database proteins.More interestingly,Fe3O4 NPs treatment had significantly more low protein abundance than normal treatment,while high protein abundance was significantly reduced.Meanwhile,the repeatability of Fe3O4 NPs treated samples was remarkably improved.We have basically established a nanoproteomics strategy based on Fe3O4 NPs-PC,which has the potential to be used to discover new therapeutic targets for diseases in serum.(3)Based on the nanoproteomics strategy of Fe3O4 NPs-PC,the molecular target detection of rheumatoid arthritis and the anti-rheumatoid arthritis mechanism of methotrexate(MTX)were studied using bovine type II collagen induced rheumatoid arthritis model(CIA).First of all,the pharmacology experiments have clearly showed the MTX has significant efficacy of rheumatoid arthritis.After treated with MTX for 4 weeks,the weight of CIA rats trended normally,and joint swelling of CIA rats improved significantly.In addition,the arthritis index of CIA rats and the expression of inflammatory factors obviously decreased,and the bone erosion and damage to joint structure of CIA rats reduced.Additionally,the Ultimate 3000 RSLC nano system and Orbitrap Fusion Lumos Tribrid mass spectrometer were adopted to measure serum protein levels in different groups of rats based on the Fe3O4 NPs-PC nanoproteomics strategy.We found that the expression levels of 67.72%CIA-induced up-regulated proteins were significantly decreased,while the expression levels of 41.81%CIAdownregulated proteins were significantly increased after 4 weeks of administration of MTX.It indicated that MTX could reverse most of the abnormal expression proteins of rheumatoid arthritis to nearly normal expression levels.MTX could significantly inhibit the regulation of actin and supramolecular fibers,coagulation,hemostasis,coagulation,platelet activation and other pathways induced by rheumatoid arthritis.In addition,MTX could significantly improve the humoral immune response caused by rheumatoid arthritis,the activation pathway of complement,the activation pathway of classical complement,the humoral immune response mediated by circulating immunoglobulin and other pathways down-regulation,thus playing an anti-rheumatoid arthritis role.(4)Using the Fe3O4 NPs-PC nanoproteomics strategy as the research object,a more comprehensive study on the mechanism of action of artesunate(ART)against malaria was carried out.Plasmodium lysates treated with Fe3O4 NPs and normal treatment were successively analyzed by the Ultimate 3000 RSLC Nano system combined with Orbitrap Fusion Lumos Tribrid mass spectrometer.After Fe3O4 NPs treatment,the number of proteins detected in plasmodium lysates increased and the fluctuation degree of samples decreased,indicating that Fe3O4 NPs-PC nanoproteomics strategy was also suitable for Plasmodium falciparum system,and it was a supplement to normal treatment methods.Combined the results from two strategies,we found that ART could significantly reverse the changes of protein spectrum induced by Plasmodium falciparum and improve the biological process of Plasmodium falciparum induction,thus playing a significant antimalarial role.The biological processes which ART regulated were as follows:small molecule metabolic process,catabolic process,carboxylic acid metabolic process,regulation of response to stimulus,organic substance catabolic process,regulation of metabolic process,regulation of macromolecule metabolic process,regulation of nitrogen compound metabolic process,regulation of cellular metabolic process.The combination of these two research strategies can help us fully explore the mechanism of ART and provide new ideas for better solving the problem of artemisinin drug resistance. |
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