Neuroprotective Effects Of Theaflavins On Alzheimer's Disease Model

Alzheimer's disease is a type of onset conceals the progressive development of the nervous system degenerative disease.In recent years,Alzheimer's disease is the most common one of dementia disease in worldwide.Unfortunately,despite decades of research,the etiology of AD is unknown,and many fundamental questions remain unanswered.In Alzheimer's disease,there are two primary histopathological features evident upon post-mortem examination of brain tissue amyloid plaques and neurofibrillary tangles.A vast amount of theories explained the mechanism of Alzheimer disease?AD?,such as“amyloid cascade hypothesis”,nerve cells apoptosis,oxidative stress,endoplasmic reticulum stress,mitochondrial dysfunction,inflammation,etc.In black tea,theaflavins monomer has been reported to have multi-purpose activity,especially these four monomers:theaflavins?TF1?,Theaflavin-3-gallate?TF2A?,Theaflavin-3'-gallate?TF2B?and Theaflavin-3,3'-gallate?TF3?.In our study,we systematically studied that theaflavins and found that it inhibited the generation of A?in vitro and in vivo.The inhibition includes inhibitory effect to A?₄₂ itself and age pigment products such as lipid peroxidation products and non-enzymatic glycosylation product which promote A?generation,regulate related A?metabolic pathways,and antiapoptotic and antioxidant protection on nerve cells both in vitro and in vivo.All in all,this study explored and showed that theaflavins have potential research and application value for the prevention and treatment of AD from different angles.1 Inhibition of theaflavins on protein carbonylation and aggregation in vitroCarbonyl stress is one of the core biochemical processes in biological aging.A protein carbonylation reaction system has been built in vitro with physiological conditions.Namely,MDA can react with BSA and generated lipofuscin?LF?,MGO can react with BSA and generated Advanced glycation end products?AGEs?,in addition,the model of A?₄₂aggregation.In order to study the effect of theaflavins on protein carbonylation and aggregation in vitro,different concentrations of theaflavins monomer intervened the above three reaction systems.The results as follow:?1?The model of carbonyl-ammonia crosslinking reaction system?BSA/MDA and BSA/MGO?in vitro has been built successfully.The best concentration of MDA or MGO for reaction was 4 mM or 20 mM,and for the optimized incubation of time is 2 d or 7 d,respectively.Moreover,the concentration of A?₄₂ was 30?M and the incubation time was 3d in the model of A?₄₂ aggregation.They were incubated under simulated physiological conditions?37?,120 r/min table,in PBS buffer PH 7.2?.?2?The experimental results of the BSA/MDA reaction treated with theaflavins showed that,there were some positive correlation between the production of?-sheet structure and the production of lipofuscin in the process of carbonyl-ammonia crosslinking reaction.We also found that theaflavins have inhibitory effect on carbonylation protein,in which they reduce the percentage of?-sheet structure in a concentration-dependent manner.?3?Coomassie blue and NBT staining of SDS-PAGE showed that theaflavins could directly bind BSA protein and generate quinones composition,reducing toxic oligomers rich in?-sheet structure produced in the fibrosis process of protein.These results were consistent with the results of Th T fluorescence detection.Imagine observations of electron microscope further illustrated that theaflavins directly changed the protein conformation and inhibit the formation of protein fibrosis and lipofuscin.?4?The results implied that theaflavins may play a role in inhibition of the formation of AGEs in a concentration-dependent manner by binding directly to the compound system of BSA/MGO.Compared with other two groups,the production of AGEs in the TF2B and TF3groups were lower at each concentration set up in this study,which indicated that the inhibitory effects of TF2B and TF3 on the formation of AGEs were better than those of TF1and TF2A.ThT fluorescence detection result showed that the four kinds of theaflavins monomer inhibits the production of?-sheet structure in the reaction system of BSA/MGO,which suggests that theaflavins can not only inhibit protein glycosylation but also reduce the level of protein aggregation.2 Neuroprotective effects of theaflavins against oxidative stress-induced injury in neural cellsOxidative stress can induce neuronal apoptosis via the production of superoxide and hydroxyl radicals.The occurrence and progression of AD are often associated with oxidative stress and apoptosis.In this study,we aimed to clarify whether theaflavins protect PC12cells from oxidative damage.A cell model of neural cells undergoing oxidative stress was created.The results as follow:?1?Three kinds of SH-SY5Y or PC12 cells damage models were established by exposing cells to 20?M A?₄₂,200?M H₂O₂ or 400?g/ml AGEs.Cell viability was monitored using the MTT assay and Hoechst 33258 staining,showing that theaflavins could enhance cell survival.?2?Intracellular reactive oxygen species?ROS?and MDA,antioxidant enzyme activities GSH-PX and CAT,which suggested that the neuroprotective effect of theaflavins against oxidative stress in neural cells is derived from suppression of oxidant enzyme activity.?3?Furthermore,RT-PCR and Western blot analyses indicated that theaflavins down regulated the ratio of pro-apoptosis/anti-apoptosis proteins Bax/Bcl-2,also downregulated the expression of Caspase-3 or the receptor of AGEs?RAGE?compared with oxidative damage group that had not been treated with theaflavins.This indicates that theaflavins possess anti-apoptotic properties,providing both cytoprotection and neuroprotection.3 Neuroprotective effect of theaflavins on SAMP8 miceOn the basis of above experiments in vitro,12 weeks senescence accelerated mouse-prone 8?SAMP8?were chosen to built animal model in vivo,the mice in drug-treated group were given intragastric administration of 10 mg·kg^-11 theaflavins once a day during the test?10 weeks?.The purpose is to examine whether theaflavins exerted their neuroprotective effects on SAMP8 mice.The results as follow:?1?A?₄₂ and AGEs in serum were measured by ELISA using a microplate reader.Both content were reduced significantly by theaflavins,which was consistent with the experimental results in vitro.?2?The cerebral cortex and hippocampus in mice were removed to detect hippocampal mitochondria,nucleus and organelles using transmission electron microscopy.The change of neuron in hippocampus was measured by HE staining,and the level of transcription or translation of Bax and Bcl-2 in the brain was evaluated by RT-PCR and Western blot.These results showed that theaflavins can exert protective effect on nerve cells by alleviating mitochondrial dysfunction,inhibiting nerve cell apoptosis.?3?In addition,the transcription or translation level correlated with A?metabolic pathways were measured by RT-PCR and Western blot.Results speculated that theaflavins in black tea extract maybe reduced the protein levels of BACE1,PS1,thereby reduced the activity of?and?secrete,and then the way of APP fragment into A?.Otherwise,the raising level of ADAM10 protein enhanced the non-amyloidogenic pathway of APP which was hydrolyzed into soluble APP by?secrete enzymes.What is more,the activity of NEP and IDE degrading A?were increased,indicating that theaflavins can suppress A?deposition by promoting A?fragment.