Our results show that Tb4 restored the levels of Cu/Zn-SOD and catalase close to normal physiological level even under oxidative stress and thus scavenging the extra H2O2-induced ROS from the cellular system. One of the traditional hallmarks of ROS initiated cell death is mitochondrial CP-358774 cost dysfunction and energy depletion. This is manifested by opening of the mitochondrial permeability transition pore, the collapse of the mitochondrial membrane potential and a concomitant drop in ATP production. These events lead to cascade of cell destruction and apoptosis. Increased production of ROS in the failing heart leads to mitochondrial permeability transition, which causes loss of mitochondrial membrane potential, swelling of mitochondrial matrix, release of apoptotic signaling molecules, such as cytochrome c, from the inter-membrane space, and irreversible injury to the mitochondria. Increased ROS in our Nutlin-3 system led to a decrease in the Dym as evident by the staining with MitoTracker Red. This loss of Dym was prevented by pretreatment of Tb4. At this point, it is beyond our scope to investigate how Tb4 modulate mitochondrial membrane potential under oxidative stress and, therefore, warranted further investigation. Tb4 was extremely effective in reducing intracellular ROS in H2O2 treated cardiac fibroblasts. Tb4 acts via upregulation of selected antioxidant genes like Cu/Zn-SOD and catalase. In fact, the intracellular ROS level was markedly reduced when the cells were pretreated with Tb4 at least 2 h prior to H2O2 exposure, suggesting that it might activate the key molecules that play an important role in the enzymatic antioxidant defense system. Another particularly relevant protein that loses function upon oxidation is Mn-SOD; its loss of function would further compromise antioxidant capacity and lead to further oxidative stress. Both Mn-SOD and Cu/Zn-SOD have been reported to play a crucial role in protecting the cardiac cells from oxidative damage by scavenging ROS. In our experimental system, we found that Tb4 upregulated the expression levels of Cu/Zn-SOD and not Mn-SOD in cardiac fibroblast thus affording cardiac protection which is in contrast to the previous report by Ho et al. This could be probably due to the different cell type used in the study. Catalase, which was directly responsible for H2O2 clearance, was upregulated by Tb4 both at protein and gene level in the presence of H2O2, indicating that Tb4 preferentially targets catalase which enables effecting scavenging of the H2O2 from the system. The mechanism of Cu/Zn-SOD and catalase upregulation by Tb4 is currently unknown but, a transcription factor mediator activity has been postulated. Tb4 has been reported to translocate into the nucleus by an active transport mechanism or possibly through its cluster of positively charged amino acid residues but the exact function is still obscure. Alternatively, it might be the similar event like nuclear localization of actin where it is postulated that it might involve in chromatin remodeling, mRNA processing and transport.