Thymosin Beta 4 Prevents Oxidative Stress by Targeting Antioxidant and Anti-Apoptotic Genes in Cardiac Fibroblasts

Rationale: Thymosin beta-4 (T beta 4) is a ubiquitous protein with diverse functions relating to cell proliferation and differentiation that promotes wound healing and modulates inflammatory responses. The effecter molecules targeted by T beta 4 for cardiac protection remains unknown. The purpose of this study is to determine the molecules targeted by T beta 4 that mediate cardio-protection under oxidative stress. Methods: Rat neonatal fibroblasts cells were exposed to hydrogen peroxide (H2O2) in presence and absence of T beta 4 and expression of antioxidant, apoptotic and pro-fibrotic genes was evaluated by quantitative real-time PCR and western blotting. Reactive oxygen species (ROS) levels were estimated by DCF-DA using fluorescent microscopy and fluorimetry. Selected antioxidant and antiapoptotic genes were silenced by siRNA transfections in cardiac fibroblasts and the effect of T beta 4 on H2O2-induced profibrotic events was evaluated. Results: Pre-treatment with T beta 4 resulted in reduction of the intracellular ROS levels induced by H2O2 in the cardiac fibroblasts. This was associated with an increased expression of antioxidant enzymes Cu/Zn superoxide dismutase (SOD) and catalase and reduction of Bax/Bcl(2) ratio. T beta 4 treatment reduced the expression of pro-fibrotic genes [connective tissue growth factor (CTGF), collagen type-1 (Col-I) and collagen type-3 (Col-III)] in the cardiac fibroblasts. Silencing of Cu/Zn-SOD and catalase gene triggered apoptotic cell death in the cardiac fibroblasts, which was prevented by treatment with T beta 4. Conclusion: This is the first report that exhibits the targeted molecules modulated by T beta 4 under oxidative stress utilizing the cardiac fibroblasts. T beta 4 treatment prevented the profibrotic gene expression in the in vitro settings. Our findings indicate that T beta 4 selectively targets and upregulates catalase, Cu/Zn-SOD and Bcl(2), thereby, preventing H2O2-induced profibrotic changes in the myocardium. Further studies are warranted to elucidate the signaling pathways involved in the cardio-protection afforded by T beta 4.