Inhibitory effects of S100A4 gene silencing on alkali burn-induced corneal neovascularization: an in vivo study

Abstract

Objective: The purpose of this study is to explore the inhibitory effects of S100A4 gene silencing on alkali burn-induced corneal neovascularization (CNV) in rabbit models.

Methods: Sixty-five rabbits were used to establish alkali-induced CNV models. After the operation, rabbits were given daily antibiotic eye drops and an eye ointment to prevent infection. The models were assigned to either an S100A4 siRNA or an empty vector group. Thirty rabbits were selected as the normal control group. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to detect the mRNA expression of S100A4, vascular endothelial growth factor (VEGF), and tumor necrosis factor-α (TNF-α) in corneal tissues. Immunohistochemistry was used to detect the protein expression of VEGF in corneal tissues, and an enzyme-linked immunosorbent (ELISA) assay was used to detect the protein expression of VEGF and TNF-α in the aqueous humor.

Results: The qRT-PCR results showed that S100A4 mRNA expression was lower in the S100A4 siRNA group than in the empty vector group at 1, 3, 7, 14, and 28 days after an alkali burn. When compared with the empty vector group, the expression of VEGF and TNF-α mRNA was downregulated in the S100A4 siRNA group. The immunohistochemistry results revealed that VEGF protein expression was downregulated in the S100A4 siRNA group when compared to the empty vector group at 1, 3, 7, 14, and 28 days after an alkali burn. The ELISA results suggest that VEGF and TNF-α protein expression is downregulated in the S100A4 siRNA group in comparison to the empty vector group at 1, 3, 7, 14, and 28 days after an alkali burn.

Conclusions: These findings indicate that S100A4 gene silencing can inhibit alkali burn-induced CNV in rabbits.

Figure 1

Growth of CNV after alkali burns at each time point (× 400). Notes: The dotted line represents the edge of CNV; CNV, corneal neovascularization.

Figure 2

HE staining in corneal sections after alkali burn (× 40). Note: HE, haematoxylin eosin.

Figure 3

The mRNA expressions of S100A4, VEGF, and TNF-α after alkali burns at each time point for each group. Notes: mRNA, microRNA; VEGF, vascular endothelial growth factor; TNF, tumor necrosis factor; * and ** refer to p<0.05 and p<0.01, respectively when compared with the normal control group; ^# and ^## refer to p<0.05 and p<0.01, respectively when compared with the empty vector group.

Figure 4

The protein expression of VEGF in the corneal tissues of each group by immunohistochemical detection (× 200) Notes: VEGF, vascular endothelial growth factor; the arrow indicates the brown particles as a positive signal.

Figure 5

VEGF protein expression after alkali burn in the aqueous humor at different time points. Note: μg /l refers to protein concentration; * and ** refer to p<0.05 and p<0.01 when compared with the normal control group, respectively; # and ## refer to p<0.05 and p<0.01 when compared with the empty vector group, respectively. VEGF, vascular endothelial growth factor.

Figure 6

TNF-α protein expression at different time points in the aqueous humor after alkali burn. Notes: μg /l refers to protein concentration; * and ** refer to p<0.05 and p<0.01 when compared with the normal control group, respectively; # and ## refer to p<0.05 and p<0.01 when compared with the empty vector group, respectively. TNF, tumor necrosis factor.