Six-to-eight-weeks-old female Sprague Dawley (SD) rats and Wistar rats were purchased from Guangzhou Southern Medical University Experimental Animal Technology Development Co., Ltd. (Guangzhou, Guangdong, China) To avoid the interference of sex hormones with immune responses, only female rats were used. The rats were housed in a specific pathogen free (SPF)-grade standardized feeding room at Zhongshan Ophthalmic Center, Yat-sen University. The experiments were approved by the Institutional Animal Care and Use Committee of Zhongshan Ophthalmic Center (protocol code O2021044).

Preoperatively, pentobarbital sodium (1%) was used as an anesthetic in intraperitoneal injection doses of 40–45 mg/kg. To establish allograft corneal transplantation models, the SD rats were used as donors, and the Wistar rats were used as recipients. Moreover, corneal autograft operations were performed on Wistar rats. Full-thickness corneal donors 3.5 mm in diameter were sutured to implant beds 3.0 mm in diameter using 10-0 nylon sutures (Alcon, Fort Worth, TX, United States) under a microscope.

Four experimental groups were formed. A normal group of Wistar rats did not undergo corneal transplantation and received no treatment. The Wistar rats in the autograft group underwent only autologous corneal transplantation and received no treatment. A control group of Wistar rats underwent corneal allograft surgery and received saline treatment. The resveratrol group consisted of Wistar rats undergoing corneal allograft surgery and receiving resveratrol treatment. Resveratrol solution (0.6 mL, 100 mg/kg, Sigma-Aldrich, St. Louis, MO, United States) or saline was injected intraperitoneally every day for 30 days postoperatively.

A human monocytic leukemia cell line (THP-1) was obtained from the cell bank of the Chinese Academy of Sciences in Shanghai. An RPMI-1640 culture medium (Gibco-BRL, Grand Island, NY, United States) with 10% FBS (Thermo Fisher Scientific, Waltham, MA, United States) and 1% penicillin-streptomycin (Sigma-Aldrich, St. Louis, MO, United States) was used. All cells were cultured in a cell incubator containing 5% carbon dioxide at 37°C. After cell line resuscitation, fourth-to-eighth-generation cells were used in the experiments. Adhesive THP-1 cells induced using phorbol 12-myristate-13-acetate (PMA; 100 ng/mL, P1585; Sigma-Aldrich, St. Louis, MO, United States) for 48 h were considered M0 macrophages. M0 macrophages were transformed into M1 macrophages using LPS treatment (1 μg/mL, from Escherichia coli O111:B4, Sigma-Aldrich, St. Louis, MO, United States) for 24 h.

The cells were divided into five groups. The control group consisted of untreated M0 macrophages. The cells in the LPS group were treated only with LPS. The cells in the resveratrol groups were treated with three resveratrol concentrations: 10, 25, and 50 μM.

The rat corneas in the autograft, control, and resveratrol groups were examined using slit-lamp microscopy every other day for 30 days postoperatively. The corneal allografts were assessed clinically using the Holland rejection scoring system (30, 31). Corneal graft clarity, edema, and neovascularization were used as indicators (Table 1). The three parameters were summed to obtain a rejection index (RI). An RI of ≥6 was considered to indicate rejection. Anterior segment photography was performed 7 and 14 days postoperatively to record the condition of the corneal grafts in the control and resveratrol groups. Anterior segment optical coherence tomography (AS-OCT) was performed 14 days postoperatively to measure central corneal thickness (CCT) in the control, resveratrol, and normal groups.

On the 14th postoperative day, the eyeballs of rats in the normal, control, and resveratrol groups were removed and placed in 4% paraformaldehyde (PFA; Biosharp, Hefei, Anhui, China) solution at 4°C overnight. After dehydration and paraffin embedding, sections approximately 4 μm thick were cut. After dewaxing and hydration, the sections were stained with hematoxylin and eosin (H&E; Servicebio, Wuhan, Hubei, China). Histological analysis was performed using an inverted microscope (Ts2FL; Nikon, Japan) and CapStudio software.

The eyes of rats in the normal, control, and resveratrol groups were removed and fixed with 4% PFA. The tissues were then embedded in paraffin wax and cut into 4 μm thick sections. The sections were stained with mouse anti-cluster of differentiation molecule 11b (CD11b; 1:100, sc-1186; Santa Cruz, CA, United States) and rabbit anti-inducible nitric oxide synthase (iNOS; 1:500, ab283655; Abcam, Cambridge, United Kingdom) primary antibodies at 4°C overnight. Subsequently, they were dyed with Goat anti-Mouse IgG (H + L) Highly Cross-Adsorbed Secondary Antibody (Alexa Fluor Plus 488, 1:200; Invitrogen, Carlsbad, CA, United States) and Goat anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody (Alexa Fluor Plus 594, 1:200; Invitrogen) for 1 h. The stained slices were analyzed under a Zeiss LSM 880 microscope (Carl Zeiss Meditec, Jena, Germany).

On the seventh postoperative day, corneas from the normal, control, and resveratrol groups were excised and cut into pieces. An RNeasy Fibrous Tissue Mini Kit (Qiagen, Duesseldorf, Germany) was used to extract RNA from the corneas. An RNA Quick Purification Kit (ESscience, Shanghai, China) was used to extract RNA from macrophages. The RNA was then reverse-translated into cDNA using HiScript II QRT SuperMix (Vazyme, Nanjing, Jiangsu, China). A LightCycler 480 SYBR Green I Master (Roche, Basel, Switzerland) was used to perform a real-time quantitative polymerase chain reaction (qRT-PCR). The primers are shown in Tables 2, 3.

On the seventh postoperative day, whole corneas from the control and resveratrol groups were excised and fixed with 4% PFA for 1 h. The corneas were then digested by protease K and permeated with methanol. Subsequently, they were blocked at 4°C overnight with 10% Bovine Serum Albumin (BSA; Epizyme, Shanghai, China) blocking solution containing 0.5% Triton X-100. The corneas were then dyed with rabbit anti-rat lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1; 1:200, 11-036; AngioBio, San Diego, CA, United States) at 4°C overnight. Finally, they were stained with Goat anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody (Alexa Fluor Plus 594, 1:200, Invitrogen, Carlsbad, CA, United States) at room temperature for 2 h.

On the 10th postoperative day, the right cervical lymph nodes on the same side as the surgical eyes of the rats in the normal, control, and resveratrol groups were removed and ground. The obtained cells were resuspended using stain buffer and surface-stained with CD103 (Integrin alpha E) Monoclonal Antibody (OX62), PE, eBioscience (12-1030-82, 0.25 μg per test; Thermo Fisher Scientific, Waltham, MA, United States) for 20 min. Analyses were then performed using a BD LSRFortessa flow cytometer (BD Biosciences, San Jose, CA, United States).

Total RNA was extracted from corneas in the control and resveratrol groups using the method described above. A NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, United States) was used to evaluate the purity and quantification of the extracted RNA. The libraries were sequenced on a NovaSeq 6000 platform (Illumina, San Diego, CA, United States). DESeq25 (Siemens, Berlin, Germany) was used to analyze differential expression. Q-values of <0.05 were considered to indicate significant differences in differentially expressed genes (DEGs). R (v 3.2.0) was used to perform the gene ontology (GO) 6 and Kyoto encyclopedia of genes and genomes (KEGG) 7 pathways of DEGs to identify significant enrichment terms and draw a histogram based on the hypergeometric distribution. All RNA sequencing and analyses were performed by Ouyi Biomedical Technology Co., Ltd. (Shanghai, China).

On the seventh postoperative day, proteins were isolated from corneas in the control and resveratrol groups. After measuring protein concentrations using the bicinchoninic acid assay (BCA) method, the samples were prepared and denatured at 95°C for 5 min. Following the protocol described in the manual for the 12–230 kDa Wes Separation Module (SM-W004; ProteinSimple, Bio-Techne, San Jose, CA, United States), an automated capillary electrophoresis system was used to separate and detect the proteins. Antibodies targeting the proteins GAPDH, PI3K, Akt and Phospho-Akt (p-Akt) were used [GAPDH (1:50, 5174S; Cell Signaling Technology, Danvers, MA, United States), PI3K (1:50, 4249S; Cell Signaling Technology, Danvers, MA, United States), AKT (1:600, 4691S; Cell Signaling Technology, Danvers, MA, United States), and p-Akt (1:10, 4060S; Cell Signaling Technology, Danvers, MA, United States)]. Horseradish peroxidase (HRP)-conjugated secondary anti-rabbit antibody was used to detect signals. Compass software (ProteinSimple) was used for the quantitative analysis.

The cell supernatants of the five groups of cells were extracted separately. Cytokines secreted by macrophages were measured using enzyme-linked immunosorbent assays (ELISA; TNF-α ELISA Kit BMS2034, IL-6 ELISA Kit BMS213-2, MCP-1 ELISA Kit BMS281INST, and VEGF-C ELISA Kit BMS297-2; Thermo Fisher Scientific, Waltham, MA, United States).

The data were analyzed using GraphPad Prism 7 software. Kaplan–Meier survival curves were drawn to analyze corneal graft survival. Differences between the two groups were assessed using an independent-samples t-test. p-values of <0.05 were considered statistically significant.

Graft survival time curves were plotted based on three scores, including corneal graft clarity, edema, and neovascularization (Figure 1). At the end of the 30 days observation period, all corneal grafts in the autograft group were still alive. In the control group, all grafts were rejected between days 8 and 12. The median survival time (MST) was 9 days. In the resveratrol group, six grafts were rejected between days 10 and 24. The MST was 17 days. The other four grafts in this group survived until the end of the observation period (Figure 1A). During the observation period, the average corneal graft clarity, edema, and corneal neovascularization scores in the resveratrol group were significantly lower than those in the control group (Figures 1B–E).

Most corneal grafts in the control group showed inflammatory responses soon after surgery, which gradually worsened. In this group, neovascularization reached the periphery of almost all grafts around 7 days after surgery. In contrast, in the resveratrol group, neovascularization grew to the edge of most grafts on the 14th postoperative day. In this group, the grafts remained relatively transparent, with no significant edema and significantly milder rejection reactions (Figure 1F). On the 14th day after surgery, the corneas were used for histological analysis. The H&E staining results showed that inflammatory cells were significantly infiltrated in the corneas of rats with CGR. However, in the allografts of the resveratrol group, inflammatory cell infiltration was significantly decreased (Figure 1G).

Corneal thickness after corneal transplantation is related to transplant rejection. The average CCT in the control group was 0.345 ± 0.015 mm, whereas the average CCT in the resveratrol group was 0.194 ± 0.012 mm. (Normal rat CCT is 0.158 ± 0.004 mm.) The corneal grafts in the resveratrol group were significantly thinner than those in the control group (p < 0.01) (Figures 1H,I).

To investigate the recruitment and polarization of macrophages in corneas, macrophages were labeled with CD11b, and M1 macrophages were labeled with iNOS on the seventh postoperative day (Figure 2A). No macrophage infiltration was observed in normal corneal tissue. In the control group, the average number of CD11b⁺-labeled macrophages was 30 cells per field, and the proportion of CD11b⁺ iNOS⁺-labeled M1 macrophages was approximately 76.67% (Figure 2B). In the resveratrol group, the average number of CD11b⁺-labeled macrophages was 17 cells per field, and the proportion of CD11b⁺ iNOS⁺-labeled M1 macrophages was approximately 58.82% (Figure 2C). Resveratrol treatment resulted in significantly lower of CD11b⁺-labeled macrophage recruitment (p < 0.01) and a significantly lower polarization ratio of CD11b⁺ iNOS⁺-labeled M1 macrophages (p < 0.01) than saline treatment (Figures 2B,C). M1 macrophages secreted multiple types of pro-inflammatory cytokines, including TNF-α, iNOS, IL-1β, monocyte chemoattractant protein-1 (MCP-1), IL-6, and VEGF-C. The mRNA expressions of TNF-α, iNOS, IL-1β, MCP-1, IL-6, and VEGF-C were significantly lower in the resveratrol group than in the control group (Figures 2D–I).

Previous PCR-based studies have shown that resveratrol can reduce inflammatory cytokines related to the formation of corneal lymphatic vessels, including TNF-α, IL-1β, and VEGF-C. Seven days after surgery, the rats’ corneas were removed, and LYVE-1 was used as a lymphatic-specific marker for whole-mount corneal immunofluorescence to clarify the influence of resveratrol on the formation of new lymphatic vessels after corneal transplantation. In the control group, LYVE-1-labeled lymphatic vessels grew considerably from the corneal limbus to the grafts. In contrast, lymphatic vessels grew only slightly in the resveratrol group (Figure 3A).

After corneal transplant rejection, DCs, a main type of APCs, can be transported to the local lymph nodes on the same side through lymphatic vessels (32). The results reported above confirmed that resveratrol reduced the generation of corneal lymphatic vessels. The changes in APCs in cervical lymph nodes were further investigated using flow cytometry. DCs from the ipsilateral lymph nodes from the different treatment groups were labeled with OX62 (Figure 3B). The results showed a significant increase in the proportion of DCs in the cervical lymph nodes after corneal transplantation. However, on the 10th day after surgery, the resveratrol group showed significantly fewer DCs than the control group (Figure 3C).

Total RNA was extracted from three corneas from the control group and three corneas from the resveratrol group. RNA transcriptome sequencing was performed, and the data were analyzed. A total of 234 DEGs, including 21 upregulated and 213 downregulated genes, were detected (Figure 4A). A differential gene expression heat map was created to visualize the grouping and clustering of DEGs (Figure 4B). The GO analysis showed that among the DEGs in the resveratrol group, immune response (biological process), extracellular matrix (cellular component), and MHC class II protein complex connection (molecular function) exhibited the greatest enrichment (Figure 4C). These results provided references for further research on related signaling pathways, and DEGs were analyzed for this purpose. The results showed that the downregulated genes in the resveratrol group regulated a total of 201 signaling pathways, of which 50 showed significant differences. Notably, in the KEGG analysis, one of the most important pathways of downregulated DEG enrichment in the resveratrol group was allograft rejection, suggesting that resveratrol may act against CGR (Figure 4D). Moreover, antigen processing and presentation of inflammatory-related diseases, Th1 and Th2 cell differentiation, Th17 cell differentiation, PI3K-Akt signaling pathway, and other related pathways were significantly enriched in the downregulation pathway of the resveratrol group. These results suggest that resveratrol may interfere with CGR through multiple pathways, indicating possible directions for follow-up research.

Based on the RNA sequencing results reported above, the PI3K/Akt pathway was selected for protein-level pathway validation. The expressions of the PI3K and Akt proteins in the corneas of the rats in the control and resveratrol groups were analyzed using Simple Western. The protein table indicated that the expression levels of PI3K, Akt and p-Akt were significantly lower in the resveratrol group than in the control group (Figure 4E). These results suggest that resveratrol can effectively regulate the PI3K/Akt signaling pathway after corneal transplantation in rats, which may be related to its action against CGR.

THP-1 was used to validate the effect of resveratrol on macrophages in vitro. After being treated with PMA and LPS, THP-1 cells can be considered M1 macrophages, which express various pro-inflammatory cytokines. The qRT-PCR results showed that resveratrol at concentrations of 10, 25, and 50 μM reduced the production of pro-inflammatory cytokines, such as TNF-α, IL-6, IL-1β, MCP-1, and VEGF-C, by M1 macrophages (Figures 5A–E). The subsequent ELISA results were consistent with the qRT-PCR results (Figures 5F–I). Therefore, it can be concluded that resveratrol can inhibit the polarization of human M1 macrophages in vitro.