Efficacy of the nucleotide-binding oligomerzation domain 1 inhibitor Nodinhibit-1 on corneal alkali burns in rats

Abstract

Aim: To evaluate the therapeutic effect of Nodinhibit-1 on alkali-burn-induced corneal neovascularization (CNV) and inflammation. The nucleotide-binding oligomerzation domain 1 (NOD1) is a potent angiogenic gene.

Methods: The alkali-burned rat corneas (32 right eyes) were treated with eye drops containing Nodinhibit-1 or phosphate buffered solution (PBS, PH 7.4) only, four times per day. CNV and inflammation were monitored using slit lamp microscopy, and the area of CNV was measured by formula. Vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF) was determined by Western blot analysis. The TUNEL assay was used to assess the corneal apoptosis cells.

Results: Alkali-burn-induced progressive CNV and inflammation in the cornea. After treatment for 7d and 14d, there were statistically significant differences in the CNV areas and inflammatory index on that between two group(P<0.05, respectively). Epithelial defect quantification showed a significant difference between the two groups at days 4 and 7 after the alkali burns (P<0.05). The apoptotic cells on days 1, 4, and 7 between the two groups showed significant differences at all time points (P<0.05, respectively). Compared to that in control group, the protein level of VEGF expression was significantly reduced whereas the PEDF expression was increase in the Nodinhibit-1 groups on day 14 (P<0.05, respectively).

Conclusion: Topical application of 10.0 µg/mL Nodinhibit-1 may have potential effect for the alkali burn-induced CNV and inflammation. The effect of Nodinhibit-1 on CNV may be by regulation the equilibrium of VEGF and PEDF in the wounded cornea.

Keywords: Nodinhibit-1; alkali burn; cornea; inflammation; neovascularization.

Figure 1. Nodinhibit-1 inhibits CNV and promotes corneal epithelial wound healing after alkali burns

A: On day 14 after the injury, there was only slight new blood vessel formation in the limbal areas in the Nodinhibit-1 treatment group, and the corneas remained transparent on day 14. In contrast, new blood vessels reached the central corneas in the control group, and there was a remarkable decrease in corneal transparency; B: CNV area in the control group increased from day 1 to day 7, and there was a mild decrease on day 14 after the alkali burns. In contrast, corneas treated with Nodinhibit-1 showed only a mild increase in CNV area on day 7. There was a significant difference between the two groups on days 7, 14 (P<0.05); C: The inflammatory index of the ocular surface declined from day 1 to day 14 in both groups. However, it was significantly lower in eyes treated with Nodinhibit-1 on days 7, 14 (P<0.05, respectively).

Figure 2. Nodinhibit-1 promotes corneal epithelial wound healing after alkali burns

A: Fluorescein staining showed central corneal epithelial defects at day 1 (D1) after the alkali burns, and there was gradual decrease of the epithelial defect areas at day 4 (D4) and day 7 (D7) in both groups. The epithelial defects were completely healed on day 7, if the corneas were treated with Nodinhibit-1, while the corneas treated with PBS did not heal. Images at different time points are sequential pictures from the same rat eye treated with either PBS or Nodinhibit-1; B: Epithelial defect quantification showed a significant difference between the two groups at days 4 and 7 after the alkali burns (P<0.05).^cP<0.05 vs control.

Figure 3. Nodinhibit-1 reduces alkali burn-induced apoptosis of corneal cells

A: A TUNEL assay showed only sporadic apoptotic cells in the corneal epithelia at day 7, in contrast, in the PBS group, there were many apoptotic cells in the epithelia and endothelia (B); C: There were no apoptotic cells present in normal rat corneas; D: A statistical analysis of the apoptotic cells on days 1, 4, and 7 between the two groups showed significant differences at all time points (P <0.05). ^cP <0.05 vs control.

Figure 4. Effect of Nodinhibit-1 on VEGF and PEDF expression after corneal alkali burns

A: Western blot analysis results showed that VEGF was expressed at high levels at day 14 (D14) after alkali burns in the control group. However, in the Nodinhibit-1 treatment group, there was a dramatic down regulation of VEGF on D14. PEDF was decreased on day 14 after alkali burns, whereas it was restored after Nodinhibit-1 treatment; B: Densitometry of protein expression showed significant differences between the control group and the Nodinhibit-1 treatment group in VEGF and PEDF on day 14 (P<0.05). ^cP <0.05 vs control.