Date of Award
12-27-2021
Degree Type
Thesis
Degree Name
MPhil in Biological and Biomedical Sciences
First Advisor
Dr. Satwat Hashmi
Second Advisor
Dr. Azhar Hussain
Third Advisor
Dr. Fazal Arain
Department
Biological and Biomedical Sciences
Abstract
Coronary heart disease, caused by narrowing of coronary vasculature, is a leading cause of death in Pakistan. As a consequence of reduction in blood supply to the heart, ischemia reperfusion (IR) injury to cardiomyocytes ensues. Myocardial IR injury is a result of initial ischemic insult followed by further damage due to reactive oxygen species (ROS) release during sudden outburst of oxygen during reperfusion. This study focuses on the role of two pathways, nuclear factor (erythroid-derived 2)-like factor 2 (Nrf2) and Wingless-related integration site (Wnt) pathway, that are activated as a result of insult to the myocardium that otherwise remain quiescent. Nrf2 has been shown to be regulated by canonical Wnt pathway in the same fashion as its downstream target ß catenin in multiple studies. However, this link has apparently not been studied in the heart. Pyrvinium Pamoate, an inhibitor of Wnt was used to study the effect of Wnt inhibition on its downstream target, ß catenin and whether inhibition of Nrf2 accompanies inhibition of ß catenin. Male BALB/c mice (n=32) were divided into four groups, IR group pretreated with Pyrvinium Pamoate and its sham operated group, untreated mice that underwent IR surgery and its sham operated group (n=8 in each group). A murine model of IR injury was established whereby left anterior descending artery (LAD) was surgically ligated for 30mins followed by reperfusion. Since Wnt pathway peaks at 7 days, samples were collected a week after surgery for tissue processing (H&E staining, Masson’s trichrome staining, immunohistochemistry) and protein analysis (Western blot, ELISA, total antioxidant capacity assay). Mice with IR injury exhibited an increased infarct size (IR: 19.5±6.3%, Sham: 3.8±1.9%) and upregulation of Nrf2 (IR: 68.73±0.81%, Sham: 60.17±3.47%) and ß catenin (IR: 73.35±3.36%, Sham: 53.58±18.55%) compared to sham. Group pre-treated with Pyrvinium Pamoate displayed Wnt inhibition confirmed by reduction in expression of ß catenin (IR: 73.34±3.36% Treated IR: 47.42±1.63%). Contrary to our hypothesis, Nrf2 did not decrease in the same fashion as ß catenin. There was upregulation of Nrf2 (IR: 68.73±0.81%, Treated IR: 77.09±0.92%). Destruction of ß catenin resulted in a reduction in total antioxidant capacity (IR: 52.16±27.08µM, Treated IR: 21.89±1.74µM) suggestive of an increase in oxidative stress; followed by activation of Akt (IR: 6.15±1.93%, Treated IR: 10.75±0.23%). Akt activation resulted in downregulation of GSK-3b. This was confirmed by upregulation of inactivated GSK-3: p-GSK-3 (IR: 8.22±0.82%, Treated IR: 15.84±1.46%). Upregulation of Nrf2 and Akt, and inactivation of GSK-3b suggests Nrf2 is regulated by Akt/GSK-3b/Nrf2 pathway when Wnt is inhibited. Furthermore, reduction of apoptosis by Akt and decrease in oxidative stress by Nrf2 most likely resulted in a decrease in myocardial IR injury in Pyrvinium Pamoate treated group which then resulted in reduced infarct size (IR: 18.9±3.7%, Treated IR: 8.9 ± 2.6%). By establishing this link, future strategies can be devised to minimize cellular insult due to oxidative stress in IR injury by targeting Nrf2 pathway through modulation of Wnt protein.
First Page
1
Last Page
91
Recommended Citation
Zuberi, S. K.
(2021). Role of NRF2 and WNT pathway in regulating oxidative stress after myocardial ischemia reperfusion injury. , 1-91.
Available at:
https://ecommons.aku.edu/etd_pk_mc_mphil-bbs/20