Hydrogen peroxide-mediated oxidative stress and collagen synthesis in cardiac fibroblasts: Blockade by tanshinone IIA

Ethnopharmacological relevance: We have recently reported that tanshinone IIA attenuated cardiac fibrosis in two-kidney, two-clip renovascular hypertensive rats via inhibiting NAD(P)H oxidase. However, little is known about the cellular and molecular mechanisms of tanshinone IIA mediated anti-fibrotic effects in cardiac fibroblasts after H2O2 stimulation. The present study was performed to investigate whether H2O2 may increase collagen synthesis in cardiac fibroblasts by affecting the expression and activity of NAD(P)H oxidase and whether the effects of H2O2 on cardiac fibroblasts can be blocked by treatment of tanshinone IIA. Materials and methods: Cardiac fibroblasts were treated with H2O2 (100 mu mol/L) in the presence or absence of tanshinone IIA (1 mu mol/L), NAD(P)H oxidase inhibitors diphenyleneiodonium (10 mu mol/L), siRNA-p47phox, siRNA-Nox2 and siRNA-Nox4. Collagen synthesis was measured by [H-3]proline incorporation, O-2(-) production were determined by flow cytometry and DHE fluorescence microscopy. NAD(P)H oxidase activity was measured by lucigenin-enhanced chemiluminescence. Results: H2O2 induced the activity of NAD(P)H oxidase, O-2(-) production, collagen synthesis and fibronectin expression in cardiac fibroblasts, and DPI abolished this induction. Exposure of adult rat cardiac fibroblasts to H2O2 had time-dependent increase in the expression of p47phox, Nox2 and Nox4 oxidases. In addition, tanshinone IIA significantly inhibited H2O2-induced collagen synthesis via attenuation of O-2(-) generation and NAD(P)H oxidase activity. Moreover, siRNA-mediated knockdown of p47phox, Nox2 and Nox4 inhibited H2O2-induced NADPH oxidase activity. H2O2-induced collagen synthesis and fibronectin expression were also inhibited by p47phox, Nox2 and Nox4 knock down. Conclusions: Our data show that NAD(P)H oxidase plays a significant role in regulating collagen synthesis in H2O2-stimulated cardiac fibroblasts. Inhibition of NAD(P)H oxidase with tanshinone IIA completely blocked the H2O2-stimulated collagen production, which will raise the experimental basis for using tanshinone IIA to cardiac fibrosis in clinic. (C) 2012 Elsevier Ireland Ltd. All rights reserved.