Abstract
RationaleThymosin beta-4 (Tβ4) 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 Tβ4 for cardiac protection remains unknown. The purpose of this study is to determine the molecules targeted by Tβ4 that mediate cardio-protection under oxidative stress.MethodsRat neonatal fibroblasts cells were exposed to hydrogen peroxide (H2O2) in presence and absence of Tβ4 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 Tβ4 on H2O2-induced profibrotic events was evaluated.ResultsPre-treatment with Tβ4 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/Bcl2 ratio. Tβ4 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 Tβ4.ConclusionThis is the first report that exhibits the targeted molecules modulated by Tβ4 under oxidative stress utilizing the cardiac fibroblasts. Tβ4 treatment prevented the profibrotic gene expression in the in vitro settings. Our findings indicate that Tβ4 selectively targets and upregulates catalase, Cu/Zn-SOD and Bcl2, 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 Tβ4.
Highlights
Reactive oxygen species (ROS) plays an important role in regulating a variety of cellular functions, including gene expression, cell growth and cell death and has been implicated as one of the major contributors of cardiac damage in various cardiac pathologies [1,2,3]
Primary antibody for Mn-superoxide dismutase (SOD) was purchased from Upstate/Millipore (Billerica, MA, USA); Cu/ Zn-SOD from Assay Designs Inc., (Ann Arbor, myocardial infarction (MI), USA); catalase from Santa Cruz Biotechnology (Santa Cruz, CA, USA); collagen type I and collagen type III from Rockland Immunochemicals, (Gilbertsville, PA, USA); connective tissue growth factor (CTGF) from Abcam Inc. (Cambridge, MA, USA), Bax, Bcl2, caspase-3 and GAPDH were purchased from Cell Signaling Technology (Beverly, MA, USA)
The optimal concentration of H2O2 was determined from MTT experiment and standardized dose of 100 mM H2O2 which was used throughout the study
Summary
Reactive oxygen species (ROS) plays an important role in regulating a variety of cellular functions, including gene expression, cell growth and cell death and has been implicated as one of the major contributors of cardiac damage in various cardiac pathologies [1,2,3]. It has been suggested that an increased level of oxidative stress heart is primarily due to the functional uncoupling of the respiratory chain caused by inactivation of complex I in the mitochondria [10] or due to impaired antioxidant capacity, such as reduced activity of Cu/Zn superoxide dismutase (Cu/Zn-SOD) and catalase [6], or stimulation of enzymatic sources, including xanthine oxidase, cyclo-oxygenase, nitric oxide synthase, and nonphagocytic NAD(P)H oxidases [11]. Considerable attempts have been made in the recent decades to discover an ‘‘ideal’’ cardio-protective agent, which can abrogate the oxidative damage and maladaptive changes in the heart. In this pursuit, thymosin b4 (Tb4) emerged as a powerful candidate
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