Date of Award

1-2020

Degree Type

Thesis

Degree Name

Master of Science in Biomedical Sciences

First Advisor

Qian Chen, PhD

Second Advisor

Susan Hingley, PhD

Third Advisor

Mei Xu, PhD

Abstract

Oxidative stress (e.g., increased hydrogen peroxide [H2O2] levels) and dicarbonyl stress (e.g. increased methylglyoxal [MG]) serve as initiators for the pathogenesis of cardiovascular diseases and diabetic complications. Increased H2O2-derived free radicals and MG can be highly reactive and attack cellular components resulting in cell damage and even cell death. Normally, endogenous antioxidants and glyoxalase degrade H2O2 and MG, respectively. Additionally, autophagy facilitates the break-down of damaged cellular components allowing the components to be recycled by the cells. Researchers are still unclear about whether autophagy exacerbates cell damage or protects cells from higher doses of H2O2 and MG. This study investigated cell damage and autophagy changes when rat cardiac H9c2 myoblasts were treated with H2O2 or MG. Moreover, autophagy enhancing or inhibiting drugs were tested to evaluate their effects on H2O2- or MG-induced cell damage. We found that H2O2 (100 - 600 μM, n=4) dose-dependently decreased cell viability as determined by measuring live cell dehydrogenase activity. H2O2 (600 μM, n=4) decreased cell viability to 30 ± 5% of the control. Similarly, MG (400 - 1200 μM, n=5) decreased cell viability in a dose-dependent manner. MG (1200 μM, n=5) decreased cell viability to 30 ± 6% of the control. These results were further confirmed with the use of calcein staining, which contained calcein-AM for labeling live cells. By using Cyto ID autophagy detection dye, we found that H9c2 cells exhibited higher autophagy after incubation of H2O2 (100 - 600 μM, n=2) or MG (400 - 1200 μM, n=2), for 1 and 4 hours, and 1 and 24 hours, respectively. Intracellular ROS was also detected, and the data suggested that ROS levels increased as the dose of H2O2 and MG increased. Experimentation using autophagy regulatory drugs revealed that trehalose (500 μM - 100 mM, n=4), an autophagy enhancer, increased cell viability at 24 hours for cells exposed to H2O2 (600 μM), and increased cell viability at 1, 4, and 24 hours for cells exposed to MG (1200 μM) compared to the positive controls (600 M H2O2 or 1200 M MG). Trehalose (100 mM, n=4) increased cell viability by 78  31% in MG-treated cells when compared to cells treated with MG (1200 M) alone at one-hour post treatment. Trehalose also decreased intracellular ROS levels. Rapamycin (100 nM – 20 μM), another autophagy enhancer, reduced intracellular ROS levels in cells exposed to H2O2 and cells exposed to MG, but generally did not increase cell viability for cells exposed to 600 M H2O2 or 1200 M MG. 3-methyladenine (3-MA), an autophagy inhibitor, increased intracellular ROS levels. 3-MA (50 – 10,000 μM) provided no increase in cell viability compared to H2O2 (600 μM), but for MG at 1 and 4 hours, 3-MA did increase cell viability. Our data suggests that trehalose has potential to provide protection against H2O2 or MG-induced damage, while rapamycin does not. 3-MA seems to provide protection against MG, but not H2O2 .

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