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Author

Jiashu Xie

Document Type

Dissertation - University Access Only

Award Date

2014

Degree Name

Doctor of Philosophy (PhD)

Department

Pharmaceutical Sciences

First Advisor

Teresa M. Seefeldt

Abstract

Thiol redox state (TRS) refers to the balance between reduced thiols and their corresponding disulfides, and is mainly reflected by the ratio of GSH/GSSG. A decrease in GSH/GSSG, which reflects a state of thiol oxidative stress, and thiol modifications such as S-glutathionylation have been implicated in a variety of cardiovascular diseases. Research models that can selectively induce thiol oxidative stress are, therefore, valuable tools for studying the pathophysiology of these disease states and examining the impacts of pharmacological interventions on thiol pathways. In this project, the potential of 2-AAPA, a novel GR inhibitor, to be used as a pharmacological model of thiol oxidative stress was evaluated by determining the extent of thiol modifications induced in H9c2 rat cardiomyocytes and its impact on cellular functions. The extent of thiol oxidative stress produced by 2-AAPA was also compared to other models of oxidative stress including H2O2, diamide, BSO and BCNU. Results indicated that 2-AAPA effectively inhibited GR activity and decreased the GSH/GSSG ratio by causing a significant accumulation of GSSG. 2-AAPA also increased the formation of protein disulfides as well as protein S-glutathionylation. The alteration in TRS leads to loss of ΔΨm, release of cytochrome c and an increase in ROS production. Compared to other models, 2-AAPA is superior in selectively increasing GSSG and inducing thiol modification with lower cytotoxicity and more physiological relevance. 2-AAPA as a thiol oxidative stress model was then applied to study the cardioprotective effects of carvedilol and berberine in H9c2 cells. Both carvedilol and berberine were found to protect H9c2 cells against 2-AAPA induced cytotoxicity by increasing cell viability. However, the cells protection by carvedilol was associated with greater extent of thiol oxidative stress as carvedilol increased GSSG accumulation in 2- AAPA treated H9c2 cells. In contrast, berberine greatly decreased the GSSG accumulation as well as protein disulfides in 2-AAPA treated H9c2 cells. The decreased GSSG accumulation was found to associate with an increase in GR activity and GR mRNA expression. In conclusion, a model of thiol oxidative stress using a novel GR inhibitor, 2- AAPA, has been successfully developed and applied to study TRS related abnormal and normal biochemical processes in H9c2 rat cardiomyocytes.

Library of Congress Subject Headings

Oxidative stress
Heart cells
Thiols
Rats
Cardiovascular system -- Diseases -- Pathophysiology

Description

Includes bibliographical references (pages 123-134)

Format

application/pdf

Number of Pages

148

Publisher

South Dakota State University

Rights

In Copyright - Educational Use Permitted
http://rightsstatements.org/vocab/InC-EDU/1.0/

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