A cell-permeable peptide inhibitor of p55PIK signaling alleviates suture-induced corneal neovascularization and inflammation

Abstract

To prepare an ophthalmic solution with a cell-permeable TAT peptide (TAT-N24) as the main cell-permeable peptide inhibitor of p55PIK signaling and observe its therapeutic effect on suture-induced corneal neovascularization (CNV) in rats. Sprague-Dawley rats were used to establish a corneal suture (CS) model of CNV. The vehicle and 0.9% TAT-N24 ophthalmic solution was topically administered. CNV induction was assessed on the basis of the clinical performance of each group. Hematoxylin-eosin staining was used to observe pathological changes, and immunohistochemical staining and confocal immunofluorescence were used to determine the localization of factors associated with corneal tissue. The mRNA expression levels of hypoxia-inducible factor (HIF-1α), vascular endothelial growth factor (VEGF-A), nuclear transcription factor κB (NF-κB p65), tumor necrosis factor (TNF-α), interleukin-1β (IL-1β), and interleukin (IL)-6 were determined using real-time quantitative polymerase chain reaction. Western blotting was performed to detect the protein expression levels of HIF-1α and NF-κB p65. TAT-N24 slowed CNV production and reduced the expression of HIF-1α and inflammatory factors in CS models. The mRNA levels of HIF-1α, VEGF-A, NF-kB, TNF-α, IL-1β, and IL-6 significantly decreased. Moreover, the protein levels of HIF-1α and NF-κB p65 were significantly decreased. TAT-N24 can treat CNV and ocular inflammation by inhibiting the HIF-1α/NF-κB signaling pathway in CS. In the early treatment of corneal foreign body trauma, topical application of TAT-N24 can not only reduce the inflammatory response but also inhibit corneal neovascularization.

Keywords: Corneal neovascularization; Corneal suture; HIF-1α; Inflammation; NF-κB; P55PIK; TAT-N24.

Fig. 1

Topical application of TAT-N24 alleviated the clinical symptoms and scores of CS. (A) Representative digital microscope images showing changes in corneal morphology at two time points (days 3 and 7) for each group. (B) CNV area scores for each group on days 3 and 7 in each group (****p < 0.0001 vs. the normal group; ^####p < 0.0001 vs. the CS+ Vehicle group). (C) CNV grade scores for each group on days 0, 3, and 7 in each group (***p < 0.001, ****p < 0.0001 vs. the normal group; ^##p < 0.01, ^####p < 0.0001 vs. the CS+ Vehicle group). Data are plotted as means ± SD.

Fig. 2

TAT-N24 alleviates the pathological morphology of the CS model. Figure a, b, c, d, e, f: Representative H&E-stained images (scale bar: 200×) showing corneal tissue at two time points (days 3 and 7) for each group.

Fig. 3

Figure a, b, c: Representative images of immunohistochemical staining. Localization of HIF-1α in the corneal tissues of each group on days 3 and 7. HIF-1α was mainly expressed in the corneal epithelium and stroma.

Fig. 4

Representative merged images of HIF-1α co-localized with NF-κB p65 immunofluorescence in each group of corneas on days 3 and 7. DAPI (blue) was used to stain the nuclei; CY3 (red)-labeled NF-κB p65; FITC (green)-labeled HIF-1α. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5

Approximate expression of HIF-1α and NF-κB p65 in the corneal tissue on days 3 and 7. We used the average fluorescence intensity by immunofluorescence to indicate the fluorescence signal level of (A) HIF-1α (B) NF-κB p65. Data are normalized to normal (of normal). Data are plotted as means ± SD. (****p < 0.0001 vs. the normal group; ^####p < 0.0001 vs. the CS+ Vehicle group). Data are plotted as means ± SD.

Fig. 6

On days 3 and 7, TAT-N24 reduces the mRNA levels of hypoxia-inducible signals and inflammatory cytokines in CS. Gene expression levels of (A) HIF-1α, (B) NF-kBp65, (C) VEGF-A, (D) TNF-α, (E) IL-1β, and (F) IL-6 mRNA were determined by RT-qPCR. GAPDH was used as an internal standard for comparisons with normal. Data are plotted as means ± SD. (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. the normal group; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001, ^####p < 0.0001 vs. the CS+ Vehicle group; NS means no significance).

Fig. 7

TAT-N24 reduced the expression levels of HIF-1α/NF-κB pathway-related proteins in the CS corneal tissue. The relative protein expression levels of (A) HIF-1α, (B) NF-kB p65 were determined by Western blotting. Data are normalized to β-actin. (****p < 0.0001 vs. the normal group; ^####p < 0.0001 vs. the CS+ Vehicle group). Data are plotted as means ± SD.