Cell culture, virus and treatment

The HCECs (ATCC, USA) were cultured in Hams F-12 Dulbecco's modified Eagle's medium (DMEM/F12; Gibco, USA) with 10% fetal bovine serum (FBS; Gibco, USA) and 1% penicillin/streptomycin solution (NCM Biotech, China). These were maintained at 37 ˚C with 5% CO2 and 95% humidity environment. To ensure continued growth and prevent overcrowding, the cells were transferred to new culture vessels or plates twice a week. The HSV-1 F strain was provided by the Center for Public Health Research, Nanjing University Medical School. Plaque assays on Vero cells were utilized to propagate HSV-1 F strain, and assess its titers.

Once the HCECs reached 60–80% confluence, they were divided into various groups for a 24-h culture. The normal control (NC) group received no treatment. The virus group was inoculated with the HSV-1 at different multiplicities of infection (MOI). N-acetyl-L-cysteine (NAC; MCE, China) was dissolved in sterile phosphate-buffered saline (PBS) to concentrations of 5 and 10 mM for treatment. Short hairpin RNA against JAG1 (sh-JAG1) was transfected into cells. For N-(N-[3,5-difuorophenacetyl]−1-alanyl)-S-phenylglycine t-butyl ester (DAPT) and rapamycin (RAPA) treatment, 10 mM of DAPT (Selleck, China) and 200 nM of RAPA (Selleck, China) were dissolved using 0.1% dimethyl sulfoxide (DMSO; Sigma, China).

Cell transfection

The HCECs were cultured using 6-well plates and transfected at 60–80% confluence via liposomal transfection method. Four sh-JAG1 constructs and a negative control (sh-NC) from GenePharma (GenePharma, China) were transfected with Lipofectamine 3000 (Thermo Fisher Scientific, China) following the manufacturer's protocols. The specific shRNA sequences are showed in Table 1.

Table 1 Oligonucleotide sequencesAnimals

Males of six-week-old male BALB/c mice with normal eye development were obtained from Cyagen Biosciences (Cyagen Biosciences, China). They were housed in a pathogen-free environment providing unrestricted food and water, adhering to a 12-h day/night schedule (lights on at 08:00 and off at 20:00). The experiments had the approval of Institutional Animal Care and Use Committee of Jinling Hospital, and complied with Association for Research in Vision and Ophthalmology guidelines for animal use.

Herpes simplex keratitis mouse model and treatment

The mice were allowed to adjust to the environment for 7 days before setting up animal model. Corneal infection was induced under general anesthesia by intraperitoneally administering 1% sodium pentobarbital (80 mg/kg; Shanghai Reagent Company, China). Under a dissection microscope (Carl Zeiss, Germany), a 30-gauge needle was used to scratch the corneal epithelium horizontally and vertically five times in the right eye. Then, 10 μL of the HSV-1 (107 PFU/mL) was inoculated into right eyes, while the mock group received 10 μL of DMEM. A gentle eyelid massage was performed to improve contact with the viral solution or DMEM.

The mice (n = 70) were randomly divided into an NC group (n = 12) and an HSV-1 infected group (n = 58). The infected mice were then randomly allocated to the HSK group (n = 28) or other groups with different treatments. Mice in the NAC group (n = 6) were treated with NAC eye drops (0.3%, 5 μL/eye, three times daily [TID]) for 3 continuous days. Mice in the DAPT (n = 6) and RAPA (n = 6) groups received DAPT eye drops (0.1%, 5 μL/eye, TID) and RAPA eye drops (0.5%, 5 μL/eye, TID), respectively, for 3 continuous days. To avoid solvent interference, PBS and 0.1% DMSO were used as solvent control groups (n = 12). HSK severity was observed and recorded at 1, 3, 5, and 7 days post infection (dpi) using a dissection microscope (Carl Zeiss, Germany). At 1, 3, 5, and 7 dpi, mice were euthanized to harvest the normal uninfected and HSV-1-infected corneas for hematoxylin and eosin (H&E) staining and immunofluorescence co-location.

High-throughput RNA sequencing (RNA-Seq)

TRIzol reagent (Vazyme Biotech, China) was utilized to isolate the total RNA from HCECs with or without infection by HSV-1 for 24 h post-infection (hpi). Then, microspectrophotometer (Nano-500; MIULAB, China) was used to measure RNA concentration and purity. Library construction and sequencing were carried out by LC Bio Technology (LC Bio Technology, China). Differential expression analysis was conducted using DESeq to find differentially expressed genes (DEGs) whose fold Change exceeding 2 and false discovery rate (FDR) less than 0.001.

Quantitative real-time polymerase chain reaction (RT-qPCR)

TRIzol reagent (Vazyme Biotech, China) was utilized to isolate the total RNA as described previously. One microgram of total RNA was converted into cDNA utilizing the Evo M-MLV RT Mix Kit (Accurate Biology, China). To determine mRNA expression levels, RT-qPCR was carried out using AceQ qPCR SYBR Green Master Mix (Vazyme Biotech, China) on an ABI 7500 Real-Time PCR System (Thermo Fisher, USA). Results were quantified employing the standard 2−ΔΔCt method, using GAPDH serving as a reference gene. The sequences of primers used are provided in Table 2.

Table 2 Primers used for qRT-PCRWestern blotting (WB)

The pretreated HCECs were lysed using radioimmunoprecipitation buffer (Beyotime, China) including protease and phosphatase inhibitors (Beyotime, China). Denatured proteins in equal amounts were loaded onto 8–12% polyacrylamide gel (Beyotime, China). After being sorted by molecular weights, the target proteins were transferred onto polyvinylidene fluoride membranes (Merck, Germany), and blocked using 5% skim milk, followed by overnight incubation with primary antibodies. Then, they were probed with horseradish peroxidase-conjugated secondary antibodies, and the UltraSignal ECL Western Blotting Detection Reagent (4A Biotech Co., Ltd., China) was applied to visualize protein bands. Detection of protein bands was performed utilizing a Gel imaging system, and quantification was conducted through ImageJ software (National Institutes of Health, USA). All antibodies used for WB and subsequent immunostaining are detailed in Table 3.

Flow cytometry analysis

Flow cytometry analysis (fluorescence-activated cell sorting [FACS]) was applied to assess apoptosis and intracellular ROS levels in the pretreated HCECs. Cells were trypsinized, harvested by washing, and centrifuged using cold PBS for further processing.

We used an Annexin V-fluorescein isothiocyanate (FITC)/ propidium iodide (PI) apoptosis detection kit (Vazyme Biotech, China) to evaluate cell apoptosis. In brief, the harvested HCECs were resuspended and incubated with a binding solution containing Annexin V-FITC/PI for 15 min. FITC/PI fluorescence was accessed using a BriCyte E6 flow cytometer (Mindray, China). We used FlowJo V10.0 software (FlowJo, USA) to analyze data, with the proportion of apoptotic cells determined based on quadrant statistics for the early and late apoptotic regions relative to the total cell population.

We assessed intracellular ROS levels with a Reactive Oxygen Species assay kit (Beyotime, China). The pre-treated HCECs were incubated in 2’,7’-dichlorodi-hydrofluorescein diacetate (DCFH-DA) probe (1:1000 dilution) for half an hour at 37 ˚C. DCFH-DA, a non-fluorescent ester, can readily enter cells and is rapidly oxidized to DCF due to ROS. Changes in DCF fluorescence, indicative of intracellular ROS levels, were assayed and analyzed using a BriCyte E6 flow cytometer and FlowJo V10.0.

Hematoxylin and eosin (H&E) staining

Eyeballs of different groups were fixed overnight using 4% paraformaldehyde at 4 ˚C. Sections were then dehydrated through graded ethanol solutions and embedded in paraffin. Paraffin blocks were cut into 4 µm-thick sections with a rotary microtome, followed by H&E staining. We use light microscope to observe and record the results.

Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining

We assessed cell apoptosis by TUNEL staining with a TMR TUNEL Cell Apoptosis Detection Kit (Servicebio, China). Staining was visualized and recorded using a ZEISS LSM 980 with AiryScan2 (Zeiss, Germany).

Immunofluorescence staining and confocal imaging

HCECs from different treatment groups were fixed with paraformaldehyde. Moreover, paraffin sections of the mouse corneal tissues were deparaffinized and underwent heat-induced antigen retrieval. Both the pre-treated cells and tissue slices were permeabilized and blocked with 5% bovine serum albumin and 0.5% Triton X-100 (Solarbio, China), followed by overnight incubation using corresponding primary antibodies. Subsequently, samples were incubated with the corresponding fluorescent secondary antibodies, and an antifluorescence quenching agent containing 4,6-diamidino-2-phenylindole (DAPI) (Beyotime, China) was applied. Images were visualized and captured using a ZEISS LSM 980 with AiryScan2 (Zeiss, Germany).

Transmission electron microscopy (TEM)

The morphology of HCECs after different treatments was examined utilizing TEM. Briefly, pre-treated cells were digested and fixed in an electron microscopy fixative (Electron Microscopy Sciences, USA). HCECs were then rinsed and centrifuged with PBS (pH 7.4) for three times. Following dehydration in graded ethanol solutions, the samples were embedded, and stained with uranyl acetate and lead citrate. We visualized and capture HCECs ultrastructure using a TEM (Hitachi, Japan).

Statistical analysis

We analyzed and visualized the data through GraphPad Prism 8.0 software (GraphPad, USA). When comparing two-sample data, we used Student's t-test. To compare multi-sample data, we first conducted tests for homogeneity of variance on the data. When the data satisfied the assumptions of homogeneity of variance, we utilized one-way analysis of variance with Bonferroni’s post hoc test. When the data did not meet these assumptions, the Kruskal–Wallis rank test was utilized, with the Dunn method applied for post hoc test. All experiments were independently repeated at least three times, and all data were presented as the mean ± standard deviation. P < 0.05 was considered to have statistical significance in all analyses.