In total, 40 male ICR mice (age, 12 weeks; weight 30±2 g) were chosen to build CoNV model, and were purchased from Nanjing Qinglongshan Experimental Animal Center. The mice were raised under standard conditions (temperature, 22±2°C; humidity, 50±5%) with a controlled 12-h light/dark cycle and had free access to water and standard laboratory chow. The mice were anesthetized by intraperitoneal injection of chloral hydrate at a dose of 350 mg/kg. No signs of peritonitis were observed during the experiment. Corneal alkali-burn was performed by applying a 2.5 mm diameter filter paper soaked with 1 mol/l NaOH on the corneal center for 25 sec. After the filter paper was removed, the eye was rinsed with the sterilized saline for 1 min. An eyedrop of SKLB1002 (0.05 mg/ml) or sodium carboxymethyl cellulose (CMC-Na; 0.5%) was used on the surface of cornea 3 times each day. Corneal neovascularization was observed using a slit lamp. The animals were euthanized by cervical dislocation.

Hematoxylin and eosin (H&E) staining was performed to detect the histopathological change of cornea. The mice were treated with SKLB1002, CMC-Na (0.5%), or PBS 3 times per day. The eyeballs were collected and fixed in 4% paraformaldehyde (Beyotime Institute of Biotechnology) at 4°C for 24 h. Then, the eyeballs were dehydrated by immersion in a series of increased concentrations of alcohol, embedded in paraffin wax and sectioned at 5-µm thickness. The paraffin sections were deparaffinized by xylene for 30 min and rehydrated by an alcohol gradient. After washing with ddH₂O, the sections were stained with hematoxylin at room temperature for 10 min. Then, the sections were washed with ddH₂O and stained with eosin at room temperature for 1 min. After washing with ddH₂O, the sections were dehydrated using alcohol and mounted using resinene. The images were captured using a light microscope at magnification, ×10.

Human umbilical vein endothelial cells (HUVECs) were obtained from Lonza Group, Ltd. (cc-2159) and cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) at 37°C with 5% CO₂ in a humidified atmosphere.

MTT assay was performed to detect cell viability. HUVECs were seeded onto 96-well plate (3,000-4,000/well) and treated with different concentrations of SKLB1002 for 48 h. HUVECs were also treated with p38 inhibitor SB203580 (10 µM; cat. no. S1863; Beyotime Institute of Biotechnology), ERK inhibitor U0126 (10 µM; cat. no. S1901; Beyotime Institute of Biotechnology) or JNK inhibitor SP600125 (10 µM; cat. no. S1876; Beyotime Institute of Biotechnology) for 1 h, and incubated with VEGF (10 ng/ml), VEGF + SKLB1002 or left untreated at 37°C for 48 h. Then, MTT (5 mg/ml, Beyotime Institute of Biotechnology) was added at 37°C for 4 h. After removing the medium, the crystals were dissolved with isopropanol and determined at 570 nm wavelength using a microplate reader.

Ki67 staining was performed to determine cell proliferation. In brief, HUVECs were seeded onto 24-well plate. Following the required treatment, they were washed with PBS buffer, fixed in 4% paraformaldehyde (Beyotime Institute of Biotechnology) at room temperature for 15 min and blocked in 5% BSA (Biofroxx GmbH) at 37°C for 0.5 h. Ki67 antibody (1:200; Abcam; cat. no. ab16667) was added to each well overnight at 4°C. The plate was placed at room temperature to rewarm for 1 h and incubated with the secondary antibody for 3 h. The nuclei were stained with DAPI (1:1,000; Biosharp) at room temperature for 10 min. The plate was imaged using a fluorescence microscope.

After the required treatment, the cells were cultured with EdU medium (50 µM, 300 µl/well; Guangzhou RiboBio Co., Ltd.) at 37°C for 4 h. Cells were fixed with 4% paraformaldehyde (Beyotime Institute of Biotechnology) at room temperature for 30 min and incubated with 0.5% Triton X-100 for 10 min. After washing with PBS for three times, cells were incubated with Apollo Dye solution (Guangzhou RiboBio Co., Ltd.; cat. no. C10310) at 37°C for 1 h in the dark. Then, the cells were incubated with 0.5% Triton X-100 at room temperature for 15 min. Cell nuclei were stained by Hoechst 33342 at room temperature for 30 min in the dark. The images were captured using a fluorescent microscope at magnification, ×40.

HUVECs (5×10⁵ cells/well in 6-well plates) were incubated with or without SKLB1002 at 37°C for 24 h. They were harvested using a 0.05% trypsin solution, washed twice with PBS and centrifuged at 1,200 × g at 4°C for 5 min. Then, cell suspension was stained with fluorescein isothiocyanate-labeled Annexin V (BD Pharmingen; Becton, Dickinson and Company) and counterstained with propidium iodide (PI; BD Pharmingen; Becton, Dickinson and Company) at room temperature for 10 min in the dark. Finally, the percentage of apoptotic HUVECs were determined using a flow cytometer (cytoFLEX; Beckman Coulter, Inc.) and analyzed using CytExpert 2.3 (Beckman Coulter, Inc.).

HUVECs were seeded onto a 6-well plate. After they reached >90% confluence, a 10 µl pipette tip was used to make a straight line in the middle of confluent monolayer. The floating cell debris was washed with PBS buffer and the injured cell monolayers were cultured in serum-free medium. The injured area was observed and the images captured at different time points (0, 24 and 48 h).

HUVECs were seeded onto the upper Transwell inserts (Corning, Inc.) at 2×10⁴/well and allowed for migrating through the hole for 10 h. Meanwhile, serum-free media was added into the upper insert and the complete media (10% FBS in DMEM) was put into the lower chamber as the chemoattractant. These non-migrated cells were removed by cotton swabs. The migrated cells were fixed with methyl alcohol at room temperature for 15 min and stained with 0.5% crystal violet solution at room temperature for 30 min. Finally, the stained cells were counted under a light microscope and images were captured at magnification, ×20.

The tube formation assay was performed to detect the angiogenic ability of HUVECs. The 24-well plate was frozen in advance, thawed Matrigel (BD Biosciences; Becton, Dickinson and Company) was added onto the bottom of wells and incubated at 37°C for 1 h to solidify. After the required treatment, HUVECs were cultured onto these wells at 1×10⁵/well. The tube formation was observed by a light microscope and the tube length was calculated using ImageJ 1.52p software (National Institutes of Health).

After the required treatment, HUVECs were collected and lysed in RIPA lysis buffer (Beyotime Institute of Biotechnology). Following centrifugation at 10,000 × g at 4°C for 15 min, the supernatant was collected and the concentration of protein was determined using a BCA Protein Assay kit (Thermo Fisher Scientific, Inc.). The extracted protein (30 µg/lane) was separated on 10% SDS-PAGE and transferred onto the PVDF membranes (EMD Millipore). After blocking with 5% non-fat milk at room temperature for 0.5 h, the membranes were incubated with ERK1/2 (1:1,000, 9102), p-ERK1/2 (1:1,000; 4370), JNK (1:1,000; 9252), p-JNK (1:1,000; 9251), p38 (1:1,000; 9212), p-p38 (1:1,000; 9215), or GAPDH (1:1,000; 2118; all from Cell Signaling Technology, Inc.) overnight at 4°C. After washing with TBST (containing 0.05% Tween) for 3 times, the PVDF membranes were incubated with the HRP-conjugated secondary antibody (1:1,000; Beyotime Institute of Biotechnology) at room temperature for 3 h. The results of blots were visualized using an ECL detection system (Nanjing KeyGen Biotech Co., Ltd.) and the densitometry was measured using ImageJ 1.52p software (National Institutes of Health).

All experiments were repeated at least 3 times. All quantitative data were presented as mean ± standard error of the mean. Statistical significance was calculated using unpaired or paired Student's t test or one-way ANOVA followed by Bonferroni post hoc test. P<0.05 was considered to indicate a statistically significant difference.

Corneal tissues were administered SKLB1002 (0.05 mg/ml), CMC-Na solution (0.5%), or PBS (Control) for 7 days. The histological change of the cornea was observed using H&E staining. Compared with the control group, no obvious morphologic change was observed after the administration of SKLB1002 or CMC-Na (Fig. 1A). MTT assay was performed to determine whether SKLB1002 had cytotoxicity in vitro. The result demonstrated that SKLB1002 had no obvious cytotoxicity on HUVECs ranging from 1 to 100 nm (Fig. 1B). Flow cytometry assays through Annexin V-FITC/PI double labeling revealed that compared with the control group, SKLB1002 administration did not lead to an increased percentage of apoptotic HUVECs (Fig. 1C). Collectively, these results show that SKLB1002 administration has no obvious cytotoxicity in vitro and tissue toxicity in vivo.

To determine whether SKLB1002 administration plays an inhibitory role in CoNV, a CoNV model was first constructed through alkali-burn injury and then the injured corneas were treated with SKLB1002 eyedrops (0.05 mg/ml) or CMC-Na solution (0.5%) 3 times per day. The results demonstrated that new corneal blood vessels appeared at 1 day and peaked at 7 days after alkali-burn injury. The image of the anterior segment was taken using a slit lamp at 7 days after alkali-burn injury. There was no neovascularization in normal cornea (Ctrl group). Furthermore, alkali-burn injury induced an increased number and length of pathological corneal blood vessels (Alkali group). Compared with Alkali group, the number and the length of new corneal blood vessels was significantly reduced after the administration of SKLB1002 (Alkali + SKLB1002 group), but not CMC-Na (Alkali + CMC-Na group; Fig. 2). Collectively, the above-mentioned results suggest that SKLB1002 administration can suppress CoNV in vivo.

To determine the effect of SKLB1002 administration on HUVEC viability, HUVECs were pre-treated with SKLB1002 and then treated with or without VEGF (10 ng/ml). VEGF treatment significantly increased the viability of HUVECs. However, pre-treatment with SKLB1002 (10 or 50 nM) significantly reduced VEGF-induced increase in HUVEC viability (Fig. 3A). EdU incorporation assay and Ki67 immunofluorescence staining demonstrated that pre-treatment with SKLB1002 significantly decreased the proliferation ability of HUVECs (Fig. 3B and C). Transwell migration assay and scratch wound healing assay demonstrated that pre-treatment of SKLB1002 significantly reduced the migration ability of HUVECs (Fig. 3D and E). Matrigel tube formation assay demonstrated that VEGF-mediated tube formation ability was interrupted after the administration of SKLB1002 (Fig. 3F).

VEGF and VEGFR-2 usually serve their roles through activation of MAPK signaling. Western blotting was performed to investigate whether SKLB1002 administration could affect the activation of the MAPK signaling pathway. VEGF treatment led to increased expression levels of phosphorylated ERK1/2, JNK and p38. The increased expression of these proteins was markedly interrupted after SKLB1002 administration (Fig. 4A). It was thus concluded that SKLB1002 played its anti-angiogenic role in endothelial cells through inactivation of MAPK signaling as demonstrated in Fig. 4B. To further verify whether SKLB1002 performed its anti-angiogenic role in endothelial cells through MAPK signaling, HUVECs were treated with p38 inhibitor (SB203580), ERK inhibitor (U0126), JNK inhibitor (SP600125), or SKLB1002 and then incubated with or without VEGF. The results demonstrated that SKLB1002 administration decreased the viability of HUVECs (Fig. 4C) and reduced the migration and tube formation ability of HUVECs (Fig. 4D and E), showing similar effects as the combined effects of SB203580, U0126 and SP600125 administration on endothelial angiogenic functions. These results provided additional evidence that SKLB1002 plays its anti-angiogenic role in endothelial cells through the inactivation of MAPK signaling.