Effects Of β-amyloid Peptide (25-35) And Tea Catechins On Human Erythrocyte: A Preliminary Study

β-amyloid peptide 25-35 (Aβ_25-35) is a fragment ofβ-amyloid peptide39～40, which is the main components of the neurofibrillar tangles in Alzheimer's disease(AD). The amino acid sequence of Aβ_25-35 is GSNKGAIIGLM. It has been well-known that Aβ_25-35 can induce human erythrocyte hemolysis, although the underlying mechanism is kept unclear. Tea catechins could inhibit the neurotoxicity ofβ-amyloid peptidel-40/42. To deeply understand the neurotoxic mechanism of amyloid peptide, by using human erythrocyte as an in vitro model, this study explores the cytotoxic events of Aβ_25-35 in the absence and presence of several tea catechins, including release dynamics of hemoglobin and potassium, alterations of membrane proteins, glutathione levels and cellular morphology. In addition, the effects of tea catechins on hemolysis and Aβfibrillation were also investigated.Results: Hemolysis of erythrocyte induced by Aβ_25-35 did not involve in permeably acrossing membrane of cytosolic solutes. Among the catechins tested, (-) epicatechin-3-gallate (ECG) and (-) epigallocatechin-3-gallate (EGCG) show opposite actions on the hemolysis induced by Aβ_25-35. This phenomenon is possibly a result of the distinctive fibrillar morphology of Aβ_25-35 formed in the presence of EGCG and ECG.Methods and Conclusions:Part One. The hemolytic mechanism of Aβ_25-351. Time course of hemoglobin and K⁺ releasing induced by Aβ_25-35.Visible light spectrophotometer and atomic absorb spectrophotometer were adopted to monitor the release of hemoglobin and K⁺ from erythrocytes incubated with Aβ_25-35. There was no difference between the time courses of hemoglobin and K⁺ releases, i. e. the hemolysis of the erythrocyte induced by Aβ_25-35 is not involved in membrane permeability.2. Glutathion level of erythrocyte in the presence of Aβ_25-35Ellman method was used to determine content of erythrocyte GSH. The result showed that GSH level in erythrocyte decreased upon addition of Aβ_25-35, with a dose-dependent manner in the range of concentration lower than 20μg/zg/mL of Aβ_25-35. The maximum GSH consumption was 17%. This indicates that the oxidization damage by Aβ_25-35 is cytosolic controllable.3. Aβ_25-35 had no influence on the membrane protein pattern of erythrocyteGhost membrane was separated from the hemolysate of erythrocyte treated with Aβ_25-35, followed with SDS-PAGE to detected alteration of membrane protein. The result showed there was no protein aggregation produced in the ghost membrane.4. Morphological change of erythrocyte upon treating by Aβ_25-35Compared to the control sample, the treatment of erythrocytes with Aβ_25-35 resulted in the formation of stomatocytes and global vesicles with various sizes, as visualized under electron microscope. The global vesicles were possibly the outcomes of protuberance release and resealing and/or fusing of membrane pieces and debris.Part Two. Effects of tea catechins on hemolysis induced by Aβ_25-35Five tea catechins have been tested. Among them EGCG and ECG affected Aβ_25-35-induced hemolysis obviously. EGCG strongly inhibited hemolysis, in contrast to an opposite action of ECG which promoted hemolysis. Aβ_25-35 underwent conformation change and aggregation in the presence of EGCG and ECG.1. The antioxidative properties of EGCG and ECGMany reports evidenced the antioxidant activity of EGCG and ECG in biological system was determined for their polyphenol structure. In this experiment, EGCG and ECG similarly reduced the production of MDA, a marker of lipid peroxidation of erythrocyte membrane triggered by t-butylhydroperoxide (t-BHP). This fact indicated that hemolysis induced by Aβ_25-35 did not involve in oxidation passway.2. The hemolytic effect of mixture of Aβ_25-35 EGCG and ECGBoth the patches of tea catechin-pretreated erythrocyte and tea catechin-pretreated Aβ_25-35 showed same hemolytic behaviors. That is, hemolysis was inhibited with EGCG treated samples and in the case of ECG, hemolysis was promoted.3. The conformational events of Aβ_25-35 treated with EGCG and ECGThe formation of protofibrils withβ-sheet structure is a key characteristic of amyloid peptide with cytotoxicity. The treatment of Aβ_25-35 with EGCG and ECG respectively resulted in different fibrillar shapes. As showed under transmission electron microscope, fibrous structure was found in the control sample, big amorphous aggregates in the EGCG treated patch and small amorphous aggregation like protofibrils upon ECG treatment. This result is consistent with what have accepted that protofibrils are much toxic than mature fibrils and amorphous aggregates.