title: Hydrogen peroxide-mediated oxidative stress and collagen synthesis in cardiac fibroblasts: blockade by tanshinone IIA.
abstract: BACKGROUND
  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 H(2)O(2) stimulation. The present study was performed to investigate whether H(2)O(2) may increase collagen synthesis in cardiac fibroblasts by affecting the expression and activity of NAD(P)H oxidase and whether the effects of H(2)O(2) on cardiac fibroblasts can be blocked by treatment of tanshinone IIA.
  
  
  METHODS
  Cardiac fibroblasts were treated with H(2)O(2) (100 μmol/L) in the presence or absence of tanshinone IIA (1 μmol/L), NAD(P)H oxidase inhibitors diphenyleneiodonium (10 μmol/L), siRNA-p47phox, siRNA-Nox2 and siRNA-Nox4. Collagen synthesis was measured by [(3)H]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
  H(2)O(2) 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 H(2)O(2) had time-dependent increase in the expression of p47phox, Nox2 and Nox4 oxidases. In addition, tanshinone IIA significantly inhibited H(2)O(2)-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 H(2)O(2)-induced NADPH oxidase activity. H(2)O(2)-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 H(2)O(2)-stimulated cardiac fibroblasts. Inhibition of NAD(P)H oxidase with tanshinone IIA completely blocked the H(2)O(2)-stimulated collagen production, which will raise the experimental basis for using tanshinone IIA to cardiac fibrosis in clinic.
authors.affiliation: Shenzhen Institute for Drug Control, Shenzhen, China.
authors.forename: Ping
authors.initials: P
authors.lastname: Wang
authors.forename: Sigui
authors.initials: S
authors.lastname: Zhou
authors.forename: Lipeng
authors.initials: L
authors.lastname: Xu
authors.forename: Yao
authors.initials: Y
authors.lastname: Lu
authors.forename: Xi
authors.initials: X
authors.lastname: Yuan
authors.forename: Huijie
authors.initials: H
authors.lastname: Zhang
authors.forename: Ruifang
authors.initials: R
authors.lastname: Li
authors.forename: Jian
authors.initials: J
authors.lastname: Fang
authors.forename: Peiqing
authors.initials: P
authors.lastname: Liu
mesh_terms: D045784:Abietanes / Q000494:pharmacology*; D000818:Animals; D002316:Cardiotonic Agents / Q000494:pharmacology*; D003094:Collagen / Q000096:biosynthesis*; D004247:DNA / Q000096:biosynthesis; D004347:Drug Interactions; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D005353:Fibronectins / Q000096:biosynthesis; D015870:Gene Expression / Q000187:drug effects; D055785:Gene Knockdown Techniques / Q000379:methods; D006321:Heart / Q000187:drug effects; D006861:Hydrogen Peroxide / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D008297:Male; D008562:Membrane Glycoproteins / Q000096:biosynthesis; D000074662:NADPH Oxidase 2; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000096:biosynthesis / Q000378:metabolism; D018384:Oxidative Stress / Q000187:drug effects* / Q000502:physiology; D061251:Primary Cell Culture / Q000379:methods; D034741:RNA, Small Interfering / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species / Q000378:metabolism
chemical_list: D045784:Abietanes; D002316:Cardiotonic Agents; D004791:Enzyme Inhibitors; D005353:Fibronectins; D008562:Membrane Glycoproteins; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; C021751:tanshinone; D003094:Collagen; D004247:DNA; D006861:Hydrogen Peroxide; C500593:Cybb protein, rat; D000074662:NADPH Oxidase 2; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; C498911:Nox4 protein, rat; C060505:neutrophil cytosolic factor 1