Abstract
Trichostatin A (TSA) has been shown to prevent fibrosis in vitro and in vivo. The present study aimed at investigating the role of reactive oxygen species (ROS) scavenging by TSA on transforming growth factor-β (TGF-β)-induced myofibroblast differentiation of corneal fibroblasts in vitro. Human immortalized corneal fibroblasts were treated with TGF-β in the presence of TSA, the NAD(P)H oxidase inhibitor diphenyleneiodonium (DPI), the antioxidant N-acetyl-cysteine (NAC), the NF-E2–related factor 2-antioxidant response element (Nrf2-ARE) activator sulforaphane, or small interfering RNA. Myofibroblast differentiation was assessed by α-smooth muscle actin (α-SMA) expression, F-actin bundle formation, and collagen gel contraction. ROS, H2O2, intracellular glutathione (GSH) level, cellular total antioxidant capacity, and the activation of Nrf2-ARE signaling were determined with various assays. Treatment with TSA and the Nrf2-ARE activator resulted in increased inhibition of the TGF-β–induced myofibroblast differentiation as compared with treatment with DPI or NAC. Furthermore, TSA also decreased cellular ROS and H2O2 accumulation induced by TGF-β, whereas it elevated intracellular GSH level and cellular total antioxidant capacity. In addition, TSA induced Nrf2 nuclear translocation and up-regulated the expression of Nrf2-ARE downstream antioxidant genes, whereas Nrf2 knockdown by RNA interference blocked the inhibition of TSA on myofibroblast differentiation. In conclusion, this study provides the first evidence implicating that TSA inhibits TGF-β–induced ROS accumulation and myofibroblast differentiation via enhanced Nrf2-ARE signaling.
Footnotes
The research was supported by National Natural Science Foundation of China [Grants 81170816, 81170815]; and the National Basic Research Program of China [Grant 2012CB722409]. Qingjun Zhou is supported in part by the Taishan Scholar Program [20081148] and Shandong Provincial Excellent Innovation Team Program. Patrik Danielson was supported in part by the Cronqvist Foundation [Grants SLS-120651, 249071], Stiftelsen Kronprinsessan Margaretas Arbetsnämnd för Synskadade [Grants 30:2010, 26:2012], and Ögonfonden.
- Received July 8, 2012.
- Accepted January 2, 2013.
- Copyright © 2013 by The American Society for Pharmacology and Experimental Therapeutics
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