Effects Of Two Polyphenols On Aβ42 Aggregation And Cytotoxicity And Studies On Aβ1-16 Toxicity And Inflammatory Response

ObjectiveAlzheimer's disease (AD) is a neurodegenerative disorder with insidious onset and progression of cognitive decline. AD has achieved more and more researchers' attention because of the increasing of old people. Histopathologically, AD is characterized by extracellular "senile plaques" containing amyloidβ-peptide (AP) fibrils and intracellular "neurofibrillary tangles" containing hyperphosphorylated tau protein. AP42 is a peptide containing 42 amino acids, which is physiologically soluble and non-toxic. Aβcan induce neuron apoptosis and loss of synapse. influence the balance of Ca2+, and induce the production of reactive oxygen species (ROS). Aggregation of Aβmonomer into multimeric aggregates has been strongly associated with the neurodegenerative pathology and a cascade of harmful event related to AD. There are soluble monomers, oligomers and insoluble fibrils in AD brains, and soluble Aβoligomers are more toxic in vitro and in vivo than fibrils, and may represent the primary pathological species.Finding molecules to intervene Aβaggregation and toxicity provides an optimum selection for therapeutic target. Recnetly, moderate wine consumption is receiving increasing attention in AD research. Some reports suggest that red wine intake may protect against AD and attenuate AD-type cognitive deterioration and amyloid neuropathology. Red wine contains a broad range of polyphenols that may interact with some peptides and proteins and have wide-ranging properties on preventing and treating various diseases including neuroprotective effects both in vivo and in vitro. Polyphenols have potent anti-amyloidogenic activities. Polyphenols extracted from grape seeds were able to inhibit Aβaggregation, reduced Aβproduction and protected against Aβneurotoxicity in vitro. In present study, the effects of two polyphenols resveratrol and ellagic acid on Aβ42 aggregation and cytotoxicity were elucidated. Moreover, we reported the toxicity and inflammatory response of Aβ1-16.Experimental methods1. Effects of resveratrol on Aβ42 aggregation and cytotoxicity(1) The effects of resveratrol at different concentrations on AP42 aggregation by ThT-induced fluorescence detection(2) The effects of resveratrol on Aβ42 morphologies by TEM observation(3) The effects of resveratrol on AP42 secondary structure by CD measure-ments(4) The effects of resveratrol on AP42 oligomers formation by Elisa and dot-blot detection(5) Disaggregation of Aβ42 fibrils by resveratrol(6) Cytotoxicity of Aβ42 and fAβ42 co-incubated with resveratrol by MTT assay2. Effects of ellagic acid on Aβ42 aggregation and cytotoxicity(1) The effects of ellagic acid on Aβ42 aggregation processes by TEM observation(2) Measurement of AP42 oligomer levels by Elisa and western-blot(3) Conformational changes of Aβ42 by CD measurements(4) Cell viability measurement3. The toxicity and inflammatory responses of Aβ1-16(1) Measurement of Aβ1-16 toxicity by MTT assay (2) Inflammatory factors released by BV-2 glial cells stimulated by Aβ1-16(3) Morris water maze(4) ImmunohistochemistryResults1. Effects of resveratrol on Aβ42 aggregation and cytotoxicity(1) Resveratrol inhibited Aβ42 aggregation as detected by ThT, TEM and CD.(2) Resveratrol did not inhibit Aβ42 oligomer formation. Resveratrol main-tained a higher oligomer level over the test period after a short initial phase while Aβ42 alone showed decreasing signals with incubation proceeding after the oligomerization phase, indicating that some of Aβ42 oligomers in the samples of Aβ42 alone aggregated to fibrils and resveratrol could stabilize the formed Aβ42 oligomers.(3) Resveratrol disaggregated performed AP42 fibrils as shown by ThT, western-blot and TEM.(4) Resveratrol could effectively attenuate the cytotoxicity of Aβ42 towards SH-SY5Y cells.2. Effects of ellagic acid on Aβ42 aggregation and cytotoxicity(1) Ellagic acid accelerated Aβ42 aggregation into fibrils by TEM and western-blot detection.(2) Ellagic acid significantly reduced Aβ42 oligomers levels detected by Elisa.(3)The addition of ellagic acid acceleratedβ-sheet formation as shown by CD results.(4) Ellagic acid reduced Aβ42 cytotoxicity toward SH-SY5Y cells by MTT assay.3. The toxicity and inflammatory responses of Aβ1-16 (1) The morphologies and numbers of SH-SY5Y cells obviously changed after Aβ1-16 (30μM) added for 48 h.(2) The cell viability of SH-SY5Y cells were affected by Aβ1-16 in a dose dependent manner, but not for Aβ1-8.(3) The toxicity of Aβ1-16 decreased after incubation at different temperature (4℃,37℃)for 3dor7d.(4)Aβ1-16 stimulated the secretion of TNF-a, but not IL-1βreleased by BV-2 microglia cells, and 100μM Aβ1-16 significantly increased IL-4 levels.(5) Morris water maze:The latencies and crosses of Aβ1-16 group were no better than the other two groups, which indicated the memory deficiency of Aβ1-16 group.(6) The astrocytes were activated by Aβ1-16 as shown by the results of immunohistochemistry.Conclusions1. Resveratrol inhibited Aβ42 aggregation but did not prevent oligomer formation.2. Resveratrol could disaggregate performed fibrils.3. Resveratrol attenuated Aβ42 cytotoxicity.4. Ellagic acid promoted Aβ42 aggregation with less time of oligomer existence, and inhibited Aβ42 cytotoxicity.5. The above two polyphenols influenced AP42 aggregation in two different pathway, but both alleviated the cytotoxicity. The one reason might attribute to their antioxidant activity, and the other more important one might be the conformation changes of oligomers due to the interaction between the polyphenols and Aβ42.6. Aβ1-16 decreased the viability of SH-SY5Y cells in a dose dependent manner, and stimulated the secretion of TNF-a and IL-4 by BV-2 microglia cells. Injection of Aβ1-16 to hippocampus of C57 mouse led to memory deficiency.