Pterostilbene Attenuates Fructose-Induced Myocardial Fibrosis by Inhibiting ROS-Driven Pitx2c/miR-15b Pathway

Abstract

Excessive fructose consumption induces oxidative stress and myocardial fibrosis. Antioxidant compound pterostilbene has cardioprotective effect in experimental animals. This study is aimed at investigating how fructose drove fibrotic responses via oxidative stress in cardiomyocytes and explored the attenuation mechanisms of pterostilbene. We observed fructose-induced myocardial hypertrophy and fibrosis with ROS overproduction in rats. Paired-like homeodomain 2 (Pitx2c) increase, microRNA-15b (miR-15b) low expression, and p53 phosphorylation (p-p53) upregulation, as well as activation of transforming growth factor-β1 (TGF-β1)/drosophila mothers against DPP homolog (Smads) signaling and connective tissue growth factor (CTGF) induction, were also detected in fructose-fed rat hearts and fructose-exposed rat myocardial cell line H9c2 cells. The results from p53 siRNA or TGF-β1 siRNA transfection showed that TGF-β1-induced upregulation of CTGF expression and p-p53 activated TGF-β1/Smads signaling in fructose-exposed H9c2 cells. Of note, Pitx2c negatively modulated miR-15b expression via binding to the upstream of the miR-15b genetic loci by chromatin immunoprecipitation and transfection analysis with pEX1-Pitx2c plasmid and Pitx2c siRNA, respectively. In H9c2 cells pretreated with ROS scavenger N-acetylcysteine, or transfected with miR-15b mimic and inhibitor, fructose-induced cardiac ROS overload could drive Pitx2c-mediated miR-15b low expression, then cause p-p53-activated TGF-β1/Smads signaling and CTGF induction in myocardial fibrosis. We also found that pterostilbene significantly improved myocardial hypertrophy and fibrosis in fructose-fed rats and fructose-exposed H9c2 cells. Pterostilbene reduced cardiac ROS to block Pitx2c-mediated miR-15b low expression and p-p53-dependent TGF-β1/Smads signaling activation and CTGF induction in high fructose-induced myocardial fibrosis. These results firstly demonstrated that the ROS-driven Pitx2c/miR-15b pathway was required for p-p53-dependent TGF-β1/Smads signaling activation in fructose-induced myocardial fibrosis. Pterostilbene protected against high fructose-induced myocardial fibrosis through the inhibition of Pitx2c/miR-15b pathway to suppress p-p53-activated TGF-β1/Smads signaling, warranting the consideration of Pitx2c/miR-15b pathway as a therapeutic target in myocardial fibrosis.