科研之友
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摘要
Objective To investigate molecular mechanisms mediating anti-fibrotic effect of SAHA in the canine cornea using an in vitro model. We hypothesized that SAHA attenuates corneal fibrosis by modulating Smad-dependent and, to a lesser extent, Smad-independent signaling pathways activated by TGF-beta 1, as well as matrix metalloproteinase (MMP) activity. Methods Cultured canine corneal fibroblasts (CCF) were incubated in the presence/absence of TGF-beta 1 (5 ng/mL) and SAHA (2.5 mu M) for 24 h. Western blot analysis was used to quantify non-phosphorylated and phosphorylated isoforms of Smad2/3, p38 MAP kinase (MAPK), ERK1/2, and JNK1. Real-time PCR and zymography were utilized to quantify MMP1, MMP2, MMP8, and MMP9 mRNA expressions and MMP2 and MMP9 protein activities, respectively. Results TGF-beta 1 treatment caused a significant increase in phospho-Smad2/3 and phospho-p38 MAPK. SAHA treatment reduced TGF-beta 1-induced phosphorylation of Smad2/3 but not of p38 MAPK. TGF-beta 1 did not modulate the phosphorylation of ERK1/2 or JNK1. SAHA caused a significant reduction in phospho-ERK1/2 expression regardless of concurrent TGF-beta 1 treatment. Neither SAHA alone nor in combination with TGF-beta 1 altered phospho-JNK1 expression. TGF-beta 1 significantly increased MMP1 and MMP9 mRNA expressions but did not alter MMP2 mRNA. SAHA treatment attenuated TGF-beta 1-induced MMP9 mRNA expression while significantly enhancing TGF-beta 1-induced MMP1 mRNA expression. Zymography detected reduced expression of MMP2 and MMP9 proteins in untreated control CCF. TGF beta 1 treatment did not alter their expression, but SAHA treatment +/- TGF-beta 1 significantly increased MMP2 and MMP9 protein expressions. Conclusions The corneal anti-fibrotic effects of SAHA involve multiple mechanisms including modulation of canonical and non-canonical components of TGF-beta 1 intracellular signaling and MMP activity.
- 出版日期2016-11
