Peptide Amyloid Interaction Between The Fibers And The Cell Membrane

Amyloid fibrillation is a causal factor of various human diseases,for example,the Alzheimer's Disease is caused byβ-amyloid deposits.It is known that amyloid firbril is cytotoxic,and may lead to hemolysis.The mechanism is still remained debate and unclear.In order to understand the mechanism of amyloid fibrils' cytotoxicity,erythrocyte is used as an in vitro membrane model to investigate the interaction between erythrocyte membrane and insulin fibril,as well as Aβ_25-35,a fragment of Aβ.Hemolysis,morphological and membrane cytoskeleton changes,and membrane fluidity were investigated.The effect ofγ-radiation on the Aβ_25-35-induced cytotoxicity has been also studied.The results showed that both insulin fibrils and Aβ_25-35 caused injury to erythrocyte, but through different pathways.Under the condition used herein,γ-radiation showed to increase the fluidity of erythrocyte membrane and to protect erythrocytes from the Aβ_25-35-induced hemolysis. Method and result:Ⅰ.The cytotoxicity of insulin fibril.1.The effect of insulin fibril on erythrocyte osmotic fragilityErythrocyte treated with insulin fibrils was dissolved in buffers of different osmotic pressure. Hemolytic rates were determined spectrometrically.According to the osmotic fragility curve,it was found that insulin fibrils increased the membrane fragility of erythrocyte.2.Kinetics of sulfhydryl exposure during insulin fibrillation and its relationship to cytotoxicity of the fibrils.The hemolytic rates and sulfhydryl exposure were investigated during insulin fibrillation.The data showed that the hemolytic effect of the fibrils increased after 3-hour incubation and decreased after 7.5-hour incubation.Insulin exposed its sulfhydryl groups during the incubation.However, sulfhydryl exposing was independent of the fibril-induced hemolysis.Ⅱ.The cytotoxicity ofβamyloid fibril.1.Hemolysis effect of Aβ_25-35Erythrocytes were incubated with Aβ_25-35 for different times and the hemolytic rates were determined at 540 nm.The data showed that hemolysis increased with time in 40 minutes and decreased upon further incubation.Moreover,Aβ_25-35 induced hemolysis in a dose-dependent manner.2.The effect of Aβ_25-35 on erythrocyte morphology and skeletal organization Treating erythrocytes with Aβ_25-35 resulted in morphological alteration and aggregation of the cells.Erythrocyte ghosts were treated with Aβ_25-35 and then observed under atomic force microscope.Compared with the control sample,the Aβ_25-35-treated ghosts showed irregular skeletal meshwork,indicating the spectrin distribution was changed.This fact can be an explanation for the morphological alternations induced by Aβ_25-35.3.Effect of Aβ_25-35 on erythrocyte membrane fluidityDPH was used as a fluorescent dye to probe the membrane fluidity.Erythrocytes were incubated with DPH for 30 min at room temperature prior to the anisotropy was detected.The membrane fluidity of erythrocyte treated with Aβ_25-35 has no difference from that of control sample.Ⅲ.The effect ofγradiation on Aβ_25-35 mediated erythrocyte injuryTwo doses(50 Gy,100 Gy) ofγ-ray irradiation were used to investigate their effects on the Aβ_25-35-induced cytotoxicity.1.Aβ_25-35-induced hemolysisBoth the two doses ofγradiation decreased significantly the Aβ_25-35-induced hemolysis.2.Morphological change of erythrocyteAfterγ-ray irradiation,erythrocytes were treated with Aβ_25-35.The results indicatedγradiation protected the cells from the morphological alternation by Aβ_25-35.3.Osmotic fragility of erythrocyteErythrocytes irradiated byγ-rays were suspended in buffers with different osmotic pressures and hemolysis was detected at 540 nm.The curve of osmotic fragility of erythrocyte was drawn. The results showed that erythrocyte irradiated byγ-ray had lower osmotic fragility.4.Membrane fluidity of erythrocyteErythrocytes irradiated withγ-rays were treated with Aβ_25-35.Membrane fluidity was detected using DPH as a fluorescent probe.Erythrocytes irradiated byγ-rays showed higher lipid fluidity than that of the control sample;in contrast to the Aβ-treated ceils which no change was observed.ConclusionsBoth insulin fibrils and Aβ_25-35 caused injury to erythrocytes,leading to hemolysis, morphological changes and other membrane disruptions.The hemolytic effect of amyloid fibrils increased with incubation time,indicating the cytotoxicity of amyloid fibrils depended on the conformational transitions of native peptides.The fact that further incubation decreased the cytotoxicity of Aβ_25-35 suggested that Aβ_25-35 formed amyloid fibirls faster than insulin did. Sulfhydryl exposure was observed during insulin fibrillation.Cell aggregation rather than hemolysis increased upon the sulfhydryi groups exposed,suggesting the exposed sulfhydryl could trigger the thiol-disulfide exchange between membrane skeletal proteins.As a consequence cell membrane fusion and morphological change can be triggered.Aβ_25-35 led to disrupting the membrane skeleton organization,but not the fluidity of lipid bilayers,implicating the interaction between Aβ_25-35 and cytoskeleton is a causal event of the erythrocyte injury.In this experiment,γray irradiation could protect erythrocyte against Aβ_25-35 mediated membrane injury and increase membrane fluidity.