Figure 1.
TGF-β1 induced MAPK pathways phosphorylation in THSF cells.
THSF cells were incubated with TGF-β1 (3 ng/ml) for the times indicated. The total and phosphorylation of ERK (A), p38 (B) and JNK (C) MAPK were determined by using Western blot analysis. Data are representative of three independent experiments. *, P<0.05 vs. control cells without TGF-β1 stimulation.
Figure 2.
Inhibitory effect of PD98059, SB203580 or SP600125 on TGF-β1 induced MAPK pathways phosphorylation in THSF cells.
THSF cells were pretreated with ERK inhibitor (PD98059, 30 µM), p38 inhibitor (SB203580, 10 µM) or JNK inhibitor (SP600125, 30 µM) for 1 h, respectively. Then the cells were subsequently treated with TGF-β1 (3 ng/ml) for 15 min (A) or 30 min (B, C), followed by protein extraction and Western blot analysis for total and phosphorylated ERK1/2 (A), p38 (B), and JNK (C). Data are representative of three independent experiments. *, P<0.05 vs. control; #, P<0.05 vs. TGF-β1 group.
Figure 3.
SP600125 inhibited TGF-β1 induced CTGF expression and secretion in THSF cells.
THSF cells were pretreated with ERK inhibitor (PD98059, 30 µM), p38MAPK inhibitor (SB203580, 10 µM) or JNK inhibitor (SP600125, 30 µM) for 1 hour, respectively. Subsequently they were treated with TGF-β1 (3 ng/ml) for 24 hour. (A) CTGF mRNA expression levels were detected by real time PCR. (B) CTGF protein was measured in conditioned medium samples using ELISA and results were normalized for total protein concentration. Data are representative of tree independent experiments. *, P<0.05 vs. control; #, P<0.05 vs. TGF-β1 group.
Figure 4.
Effect of MAPK-specific inhibitors on expression of fibronectin and collagen I induced by TGF
-β1 in THSF cells. THSF cells were pretreated with ERK inhibitor (PD98059, 30 µM), p38MAPK inhibitor (SB203580, 10 µM) or JNK inhibitor (SP600125, 30 µM) for 1 hour, respectively. Subsequently they were treated with TGF-β1 (3 ng/ml) for 24 hour. Expression of fibronectin and collagen I protein was determined by Western blot analysis. (A) SB203580 or SP600125 significant inhibited TGF-β1 induced fibronectin expression. (B) SP600125 significant suppressed TGF-β1 induced collagen I expression. Data are representative of three independent experiments. *, P<0.05 vs. control; #, P<0.05 vs. TGF-β1 group.
Figure 5.
Evaluation of inhibitory effect of SP600125 on penetrating corneal wound induced JNK phosphorylation in Wistar rats.
(A) p-JNK was examined by immunofluorescence analysis. Five micrometer corneal sections were stained with antibodies to p-JNK (green) as well as with nuclear staining dye (blue). There was little expression of P-JNK in the normal mice cornea of normal rats. (B) Penetrating injury was made in the central cornea of Wistar rats, control group received daily subconjunctival injection of physiological saline. Positive p-JNK staining was markedly increased in the corneal stroma at 1 d after injury. (C) Subconjunctival injection of SP600125 notably inhibited JNK activation in the corneal stroma at 1 d after injury. n = 4 rat in each group, Bars: 40 µm.
Figure 6.
Evaluation of inhibitory effect of SP600125 on penetrating corneal wound induced CTGF expression in Wistar rats.
(A) Real time PCR was used to measure CTGF mRNA expression. The expression of CTGF mRNA was upregulated significantly in wounded corneas and reached a peak at 3 d after injury, subconjunctival injection of SP600125 significantly inhibited injury-induced CTGF mRNA expression. (B) Real time PCR was used to measure TGF-β1 mRNA expression. Subconjunctival injection of SP600125 did not influence the expression of TGF-β1. Data are representative of three independent experiments. *, P<0.05 vs. 0 d; #, P<0.05 vs. control group at the same time point. (C) immunofluorescence was used to measure CTGF protein expression. Five micrometer corneal sections were stained with antibodies to CTGF (red) as well as with nuclear staining dye (blue). There was dramatic expression of CTGF protein in the corneal stroma at 3 d after injury. Subconjunctival injection of SP600125 notably inhibited CTGF expression. n = 4 rat in each group, Bars: 40 µm.
Figure 7.
Evaluation of inhibitory effect of SP600125 on penetrating corneal wound induced corneal scarring in Wistar rats.
A penetrating corneal wound model was created with Wistar rats and inhibition of JNK activation by subconjunctival injection of SP600125 daily post-wounding. (A) HE stained histological sections showed that corneal epithelial healing was almost complete at 3 d in both groups. Subconjunctival injection of SP600125 after injury daily markedly improved the architecture of cornea and reduced scarring and did not have a significant impact on wound stroma healing at 14 d (B), 21 d (C). n = 4 rat in each group, Bars: 40 µm.