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Title: The cardioprotective mechanisms of dietary flavonoids
Author: Daubney, J.
ISNI:       0000 0004 5920 7815
Awarding Body: Nottingham Trent University
Current Institution: Nottingham Trent University
Date of Award: 2015
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Mitotic rat embryonic cardiomyoblast-derived H9c2 cells are widely used as a model cardiomyocyte to study the protective mechanisms of dietary flavonoids, but they lack features of fully differentiated cardiomyocytes. Therefore, this present study aimed to investigate the cytoprotective and cytotoxic effects of the dietary flavonoids quercetin, kaempferol, myricetin and two major quercetin metabolites, quercetin-3-glucoronide and 3’-O-methylquercetin, on fully differentiated H9c2 cells for the first time. The cardiomyocyte-like phenotype of the differentiated H9c2 cells was confirmed by monitoring the expression of cardiac specific troponin T, as well as through the identification of other cardiac specific cytoskeletal markers using MALDI-TOF MS/MS. The cytoprotective effect of quercetin, kaempferol, myricetin, quercetin-3-glucoronide and 3’-O-methylquercetin against hypoxia and H2O2-induced cell death was assessed by monitoring MTT reduction and LDH release. Furthermore the effect of quercetin pre-treatment on ERK1/2, PKB, JNK and p38 MAPK phosphorylation was monitored using western blotting. It was shown that quercetin was the most potent flavonoid at inducing a protective effect, and 3’-O-methylquercetin the most potent metabolite. Using western blotting it was shown that this protective effect is most likely due to quercetin-mediated inhibition of ERK1/2, PKB, JNK and p38 MAPK. Specific inhibitors of these protein kinases did not modulate the observed cytoprotective effect, or cause significant protection alone. The cytotoxic effects of dietary flavonoids, particularly quercetin, was monitored with MTT reduction, LDH release, western blotting to monitor phosphorylation of ERK1/2, PKB, JNK and p38 MAPK and activation of caspase-3, and monitoring intracellular ROS generation with DCFDA assay. The cytotoxic effect of quercetin was shown to be linked to intracellular ROS generation, caspase-3 activation and phosphorylation of ERK1/2, PKB, JNK and p38 MAPK. MALDI-TOF MS for the first time identified several proteins associated with the flavonoid-mediated cytoprotective effect and flavonoid pre-treatment in differentiated H9c2 cells. Most were shown to be linked to the regulation of MAPK and PI3K cell signalling pathways. This present study for the first time demonstrates the cytoprotective and cytotoxic effects of flavonoids on differentiated H9c2 cells, and has identified novel proteins associated with the cytoprotective effect.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available