1. HEWL Interacts With Oleic Acid Micells And Decreases Oleic Acid Cytotoxicity 2.Effect Of S100A9-Mediated NF-?B(p65) Nuclear Dynamic Changes On The Biological Effects Of SH-SY5Y Cell Line

Objective: In this experiment,oleic acid(OA)micelles and hen egg white lysozyme(HEWL)were used as mimic molecules to analyze the aggregation mechanism of amyloid protein,and to compare the biological effects of different concentrations of OA micelles and OA-HEWL aggregates on SH-SY5Y cells and neural stem cells(NSCs),to preliminary study the effect of OA micelles on amyloid plaque formation and further to improve the understanding of the pathogenesis of Alzheimer's disease(AD).Method: In order to maintain the conformation of OA micelles,all samples were continuously shaken at 800 rpm,pH 2.3 and 57 °C.The amyloid fibrosis process was characterized by atomic force microscopy(AFM),ThT binding and CD spectroscopy.AFM is an important experimental method to study the dynamic changes of proteins during aggregation.The morphology of amyloid oligomers and fibrils formed under different conditions can be observed and at the same time the height of protein fibers were measured.The kinetics of protein fibrosis was monitored by ThT binding method;CD spectroscopy was employed to analyze the convertion of protein secondary structure during protein fibrosis.The effects of different concentrations of OA micelles and OA-HEWL aggregates on the activity of SH-SY5Y cells and NSCs were measured by WST-1 method and fluorescence microscope real time imaging.Result: Compared with the control group,the AFM results showed that the OA100 group formed a large number of circular oligomers,which were further combined to form a fibrous structure with a fiber height of up to 60 nm.The ThT fluorescent dye can specifically bind to the ?-sheetcontaining protein,and the ThT fluorescence intensity of the protein sample was measured by FluoroMax-2 spectrophotometer,the results showed that the OA100 group had the highest ThT fluorescence intensity before 50 h,however,the growth rate is slower.After 50 h,the HEWL group had the fastest growth rate,and the ThT fluorescence intensity continues to increase,which was higher than that of the OA10 group and the OA100 group,while the OA100 group had the lowest fluorescence intensity.The results of CD spectroscopy showed that at 10 h,the curve obtained by HEWL group,OA10 group and OA100 group had the lowest negative peak at 208 nm,which is similar to the characteristic peak of ?-helical structure.And at 7d,there is a lowest negative peak at 217 nm,it is consistent with the characteristic peak of the ?-sheet structure.The results of WST-1 method and fluorescence microscopy showed that OA had significant proliferation inhibition effect on SH-SY5Y and NSCs at OA concentration of 140 ?M(P<0.01),and when OA was co-incubated with HEWL,the toxicity of OA-HEWL to cells was significantly decreased.Conclusion: Co-incubation of OA micelles and HEWL promoted the formation of amyloid protein and significantly reduced the toxic effects of OA on SH-SY5Y cells and NSCs.Objective: By extracting,purifying S100A9 protein and constructing a lentiviral vector that contain nuclear factor kappa B(NF-?B)p65 gene and transfecting SH-SY5Y cells in vitro to observe the biological effects of S100A9-mediated dynamic changes of NF-?B(p65)nuclear translocation on SH-SY5Y cell line.Methods: Successfully constructed prokaryotic plasmid p ET28a-S100A9 in previous experimentswas induced by using IPTG.Extracted and purified protein was identified by SDS-PAGE and Western blotting(WB).Protein was isolated and purified by a nickel affinity column.Protein powder was obtained by dialysis,concentration and lyophilization.Target gene RELA(p65)was obtained by PCR.After successful identification by agarose gel electrophoresis,reconstructed lentiviral vector was transfected into 293 T cells.SH-SY5Y cells were infected with optimal MOI,and SH-SY5Y stably transformed strain was screened.Fluorescence microscopy and flow cytometry were used to observe viral transfection efficiency.The effect of S100A9 protein on the cell viability of SH-SY5Y stably transformed strain was detected by CCK-8 method.Matlab analysis software was used to analyze effect of S100A9 protein on the expression of p65 in SH-SY5Y stably transfected cells.Results: SDS-PAGE and WB results demonstrated successful acquisition of high purity proteins.Agarose gel electrophoresis results confirmed that a lentiviral vector overexpressing the p65 gene was successfully constructed.Results of CCK-8 showed that S100A9 had significant proliferation inhibition effect on SH-SY5Y cells(P<0.05),and inhibition of p65-GFP group was not stronger compared to the other two groups.There was no significant difference between control and blank group.Results of fluorescence microscopy and flow cytometry showed that the transfection rate of the SH-SY5Y stable transformed strain was about 90%.Matlab(R2017a)analysis showed that S100A9 protein can activate NF-?B signaling pathway and cause p65 to translocate into the nucleus.Conclusion: S100A9 promote cell viability inhibiton of SH-SY5Y by activating the NF-?B singaling pathway.