This dissertation, "A mechanistic study of the inhibitory effect of magnesium tanshinoate B on stress-activated protein kinase in ischaemia/reperfusion" by Ka Wai, Au-Yeung, 歐陽嘉慧, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Abstract Abstract of thesis entitled A MECHANISTIC STUDY OF THE INHIBITORY EFFECT OF MAGNESIUM TANSHINOATE B ON STRESS-ACTIVATED PROTEIN KINASE IN ISCHAEMIA/REPERFUSION submitted by Au-Yeung Ka Wai for the degree of Doctor of Philosophy at the University of Hong Kong in August 2003 Heart disease is the leading cause of death worldwide. c-Jun NH -terminal kinase (JNK) is known to be involved in the induction of apoptosis caused by ischaemia/reperfusion. Previous results have demonstrated that magnesium tanshinoate B (MTB) (isolated from Radix Salviae miltiorrhizae or Danshen) protects the heart against apoptosis by directly inhibiting JNK and preventing nuclear translocation. However, there may be further underlying mechanisms of actions of MTB. The aim of this study was to elucidate the mechanisms by which MTB affected the regulation of JNK signaling in ischaemia/reperfusion. JNK isoforms may have distinct roles in individual cell types, and are dependent on specific stimulus. The Langendorff isolated rat heart model and simulated ischaemia/reperfusion in rat heart cells were used to study the effect of MTB on the expression of JNK isoforms and to determine whether it was isoform- specific. The scaffold proteins, in particular JIP-1 and JSAP-1, can explain partly the specificity and the regulation of JNK cascade. The expressions of these two scaffold proteins were investigated. Lipoproteins have been shown to activate JNK in various cell types, including smooth muscle cells and endothelial cells, and trigger apoptosis and eventual plaque instability. It is therefore likely that MTB can protect the heart from lipoprotein-induced apoptosis mediated by the JNK pathway. Human umbilical vein endothelial cells (HUVECs) were employed to study the effect of MTB on lipoproteins-induced apoptosis. Various kinds of lipoproteins including very-low- density lipoprotein (VLDL), low-density lipoprotein (LDL) and high-density lipoprotein (HDL) were isolated from normal subjects by sequential ultracentrifugation. cDNA microarray was used to study the gene expression pattern following ischaemia/reperfusion. MTB did not show isoform-specificity towards its inhibition on JNK isoforms. It inhibited JNK1 and JNK2, as well as the upstream kinases of JNK. The results also indicated that JIP-1 and JSAP-1 were differentially expressed in ischaemia/ reperfusion, and that MTB could reverse the changes in their expression. Next, the effect of MTB on lipoprotein-induced JNK activation was studied. JNK activities increased markedly in HUVECs treated with both native and oxidized forms of VLDL and LDL and such elevation was significantly inhibited by MTB. Two apoptotic parameters: level of released cytochrome c and caspase 3 activity were also studied to corroborate the effect of MTB on lipoprotein-induced apoptosis. Both native and oxidized forms of VLDL and LDL induced the release of high level of cytochrome c from mitochondria into cell culture medium time-dependently. The caspase 3 activity was closely related to the level of cytochrome c. All lipoproteins except HDL increased caspase 3 activity in a time-dependent manner. Lastly, MTB also modulated alterations in expression of some genes after ischaemia/reperfusion. In conclusion, the present study demonstrates (1) that the inh