The effect of danshen on tau phosphorylation: a possible treatment strategy for Alzheimer's disease.

Hung Shieh-Jung Fanny.Thesis submitted in: August 2002.Thesis (M.Phil.)--Chinese University of Hong Kong, 2004.Includes bibliographical references (leaves 97-109).Abstracts in English and Chinese.Acknowledgements --- p.iAbstract --- p.ii摘要 --- p.ivContent --- p.viList of Abbreviations --- p.xiiiList of Figure --- p.xvList of Tables --- p.xixChapter Chapter 1 --- Introduction --- p.1Chapter 1.1 --- Alzheimer's Disease (AD) --- p.1Chapter 1.1.1 --- Clinical features --- p.2Chapter 1.2 --- Histopathological studies of AD --- p.2Chapter 1.2.1 --- Neuritic plaques --- p.2Chapter 1.2.2 --- Neurofibrillary tangles (NFT) --- p.4Chapter 1.2.3 --- Tau --- p.5Chapter 1.3 --- Kinases and Alzheimer's Disease --- p.7Chapter 1.4 --- Free radical damage --- p.8Chapter 1.5 --- Available treatment for AD --- p.7Chapter 1.6 --- A Chinese medicinal material 一 Danshen （（Salviae miltiorrhizcie) --- p.11Chapter 1.6.1 --- Chemical constituents --- p.11Chapter 1.6.1.1 --- Lipophilic Compounds of Danshen --- p.12Chapter 1.6.1.2 --- Water-soluble Compounds of Danshen --- p.17Chapter 1.6.2 --- Pharmacological usage --- p.20Chapter 1.6.2.1 --- Action on Coronary system --- p.20Chapter 1.6.2.2 --- Bacteriostatic action --- p.21Chapter 1.6.2.3 --- Actions on the immune system --- p.21Chapter 1.6.3 --- Biological activity on brain --- p.22Chapter 1.7 --- Objectives and scope of the project --- p.23Chapter Chapter 2 --- General Materials and Method --- p.24Chapter 2.1 --- Recombinant DNA techniques --- p.24Chapter 2.1.1 --- Preparation of E. coli strain DH-5a competent cells --- p.24Chapter 2.1.2 --- Transformation of plasmid DNA into competent cells --- p.25Chapter 2.1.3 --- Preparation of plasmid DNA using QIAGEN Plasmid Maxipreps kit --- p.25Chapter 2.1.4 --- Phenol/ choroform extraction of DNA --- p.26Chapter 2.1.5 --- Spectrophotometric quantitation of the amount and purity of DNA --- p.27Chapter 2.2 --- Drugs preparation --- p.27Chapter 2.2.1 --- Preparation of aqueous extracts of Traditional Chinese Medicine (TCM) --- p.27Chapter 2.2.2 --- Preparation of ethanol extracts of Traditional Chinese Medicine (TCM) --- p.27Chapter 2.3 --- "3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium (MTT) assay " --- p.28Chapter 2.4 --- Analysis of proteins from culture cells --- p.28Chapter 2.4.1 --- Extraction of total proteins from culture cells --- p.28Chapter 2.4.2 --- Quantitation of protein by Bradford method --- p.29Chapter 2.4.3 --- Protein separation by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) --- p.29Chapter 2.4.4 --- Western blot analysis --- p.31Chapter 2.5 --- Reagents and buffers --- p.32Chapter 2.5.1 --- Reagents for competent cell preparation --- p.32Chapter 2.5.2 --- Reagents provided by QIAGEN Plasmid Maxipreps kit --- p.33Chapter 2.5.3 --- Reagents for SDS-PAGE --- p.34Chapter 2.5.4 --- Reagents and buffers for Western Blotting --- p.35Chapter 2.5.5 --- Cell lines --- p.36Chapter 2.5.6 --- Antibodies --- p.37Chapter 2.5.7 --- Plasmids --- p.37Chapter 2.5.8 --- Other Chemicals --- p.38Chapter Chapter 3 --- The effect of Danshen on GSK-3 induced hyperposphorylation of tau in Cos7 cellsChapter 3.1 --- Introduction --- p.39Chapter 3.1.1 --- Glycogen synthase kinase-3 (GSK-3) --- p.39Chapter 3.1.2 --- Structure of GSK-3 --- p.39Chapter 3.1.3 --- The importance of GSK-3 in AD --- p.39Chapter 3.2 --- Materials and Methods --- p.41Chapter 3.2.1 --- Transfection of Gsk-3 and tau into Cos7 monkey kidney cells --- p.43Chapter 3.2.2 --- Extraction of total proteins from culture cells --- p.44Chapter 3.2.3 --- Quantitation of protein by Bradford method --- p.44Chapter 3.2.4 --- Protein separation by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) --- p.44Chapter 3.2.5 --- Western blot analysis --- p.44Chapter 3.3 --- Results --- p.45Chapter 3.3.1 --- Toxicity test on Cos7 cells --- p.45Chapter 3.3.2 --- The effect of ethanol extract of Danshen on GSK-3 β induced tau phosphorylation --- p.45Chapter 3.3.3 --- The effect of aqueous extract of Danshen on GSK-3 β induced tau phosphorylation --- p.48Chapter 3.3.4 --- The effect of Protocatechualdehyde on GSK-3β induced tau phosphorylation --- p.48Chapter 3.3.5 --- The effect of Salvianolic acid B on GSK-3β induced tau phosphorylation --- p.49Chapter 3.4 --- Discussion --- p.60Chapter Chapter 4 --- Cdk5 induced hyperposphorylation of tau in CHO cellsChapter 4.1 --- Introduction --- p.63Chapter 4.1.1 --- Cyclin dependent kinase 5 (Cdk5) --- p.63Chapter 4.1.2 --- Structure of Cdk5 --- p.63Chapter 4.1.3 --- Neurological functions of Cdk5 --- p.64Chapter 4.2 --- Materials and Methods --- p.66Chapter 4.2.1 --- Transfection of p35 and tau into CHO cells --- p.66Chapter 4.2.2 --- Extraction of total proteins from culture cells --- p.67Chapter 4.2.3 --- Quantitation of protein by Bradford method --- p.67Chapter 4.2.4 --- Protein separation by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) --- p.67Chapter 4.2.5 --- Western blot analysis --- p.67Chapter 4.3 --- Results --- p.68Chapter 4.3.1 --- Toxicity test on CHO cells --- p.68Chapter 4.3.2 --- Tau transfection in Cdk5/p35 and TauON3R transiently transfected in CHO cells --- p.68Chapter 4.3.3 --- Effect of roscovitine treatment on the transiently tau and p35 transfection in CHO cells --- p.74Chapter 4.3.4 --- "Effects of aqueous active components of Danshen, PCAH and SAB on the transiently tau and p35 transfection in CHO cells " --- p.74Chapter 4.4 --- Discussion --- p.79Chapter Chapter 5 --- Antioxidant effect of Danshen and its active components on lipid peroxidationChapter 5.1 --- Introduction --- p.81Chapter 5.1.1 --- Red-blood-cell hemolysis model --- p.82Chapter 5.2 --- Materials and methods --- p.84Chapter 5.2.1 --- Red-blood-cell hemolysis model --- p.84Chapter 5.2.2 --- Materials --- p.85Chapter 5.2.2.1 --- Animals --- p.85Chapter 5.2.2.2 --- Chemicals --- p.85Chapter 5.3 --- Results --- p.86Chapter 5.3.1 --- Aqueous and ethanol extracts of Danshen --- p.86Chapter 5.3.2 --- Active components ´ؤ Protocatechualdehyde and Salvianolic acid B --- p.87Chapter 5.4 --- Discussion --- p.91Chapter Chapter 6 --- General discussion and OutlookChapter 6.1 --- General discussion --- p.93Chapter 6.2 --- Proposed study in the future --- p.95Chapter 6.2.1 --- In vitro kinase assay using gamma32 P ATP and substrate with or without TCM --- p.95Chapter 6.2.2 --- Use of neuroblastoma cells (SHSY-5Y) to study the effect of Danshen and its active components on tau phosphorylation --- p.95Chapter 6.2.3 --- Thiobarbituric acid-reacting substances (TBARS) assay --- p.96Chapter 6.2.4 --- In vitro phosphatase kinase assay --- p.9