Human retinal microvascular endothelial cells (HRMECs; Shanghai Zhong Qiao Xin Zhou Biotechnology Co., Ltd., China) were cultured in M199 medium (Procell, Wuhan, China) containing 10% fetal bovine serum (FBS; Excell Bio, Shanghai, China) at 37 °C in humidified 95% room air with 5% CO2. To detect the expression of GHSR-1a in cells, HRMECs were randomly divided into a control group (C group; cultured in M199 medium with 5.5 mM glucose) and a high-glucose group (HG group; cultured in M199 medium with 30 mM glucose); the cells in both groups were treated for 48 h.

Then, the cells were treated with high glucose and different concentrations of ghrelin (0 nM, 2.5 nM, 5 nM, 10 nM, 20 nM, and 40 nM) (Glpbio, Montclair, CA, USA) for 12 h, 24 h, 48 h and 72 h. The optimal concentration and action time of ghrelin based on the results of the cell counting kit-8 (CCK-8) assay (10 nM, 48 h) was used in the subsequent experiments. To evaluate cell viability, migration and tube formation, HRMECs were randomly divided into five groups and incubated for 48 h: the C group, HG group, ghrelin group (cultured in M199 medium with 30 mM glucose and 10 nM ghrelin), ghrelin + siGHSR-1a group (transfected with GHSR-1a-specific siRNA for 24 h and then cultured in M199 medium with 30 mM glucose and 10 nM ghrelin) and ghrelin + NC-siGHSR-1a group (transfected with a nonspecific siRNA sequence for 24 h and then cultured in M199 medium with 30 mM glucose and 10 nM ghrelin).

Finally, tunicamycin (MedChemExpress, Newark, NJ, USA), an inducer of ER stress, was utilized in the ghrelin + tunicamycin group (cultured in M199 medium with 30 mM glucose, 10 nM ghrelin and 10 μM tunicamycin). Cell viability, migration, and tube formation of HRMECs in the C group, HG group, ghrelin group and ghrelin + tunicamycin group were compared.

HRMECs on a climbing slide were rinsed with PBS and fixed with 4% paraformaldehyde for 15 min. The slide was then treated with 0.5% Triton X-100 at room temperature for 20 min after rinsing with PBS. After washing and drying, goat serum was dripped onto the slide (Boster, Wuhan, China) at room temperature for 30 min. Then, a primary antibody against GHSR-1a (1:100, Affinity, OH, USA) was dripped onto each slide and incubated at 4 °C overnight prior to incubation with Cy3-labelled goat anti-rabbit IgG (1:100, Boster, Wuhan, China) as a secondary antibody at 37 °C for 1 h. The slide was then incubated with DAPI (Beyotime, Shanghai, China) in the dark for 5 min and sealed with an anti-fluorescence quenching sealing solution. Images were acquired under a fluorescence microscope (Olympus BX53, Japan), and three images acquired under high-power (400 ×) magnification were selected from each group and used for analysis of the mean optical density with the IPP6.0 software (Media Cybernetics, Inc., USA).

The viability of HRMECs was determined by a CCK-8 assay (Elabscience, Wuhan, China) according to manufacturer’s instructions. In brief, cells were seeded in each well of a 96-well plate at a density of 5 × 103 cells/mL. Then, after different treatments for 48 h, 10 μL of CCK-8 reagent was added to each well. After incubation at 37 °C for 4 h, the absorbance value (A) of each well at 450 nm was estimated in a microplate reader. Cell viability (%) was calculated as (Aexperiment group − Ablank group)/(Acontrol group − Ablank group) × 100, where Aexperiment group is the absorbance value of treated cells, Ablank group is the absorbance value of cell-free medium, and Acontrol group is the absorbance value of untreated cells.

Cells in the logarithmic growth phase were seeded in 6-well plates and placed in an incubator at 37 °C for 24 h. At approximately 50% confluence, HRMECs were transfected with a GHSR-1a-specific siRNA (20 μM; Tsingke Biotechnology Co., Ltd., Beijing, China) using Lipofectamine™ 2000 Reagent (Invitrogen, Carlsbad, CA, USA) in accordance with the manufacturer’s instructions. Cells transfected with the GHSR-1a-specific siRNA and cells transfected with the nonspecific siRNA sequence (negative control) were set as the GHSR-1a-siRNA group and NC-siRNA group, respectively. Untransfected cells were used as the control group. The expression of GHSR-1a was examined 24 h after transfection by using reverse transcription polymerase chain reaction (RT-PCR).

To determine the mRNA level of GHSR-1a, total RNA was extracted from HRMECs using an RNA extraction kit (Thermo Fisher Scientific, USA) according to the manufacturer’s protocol. To synthesize cDNA, a total of 2 μg of mRNA was subjected to RT-PCR using HiScript® II Q RT SuperMix (Vazyme, Nanjing, China). Quantitative real-time PCR was performed using SYBR Green Master Mix (Vazyme, Nanjing, China) in a PCR system (QuantStudio 6.0, ABI, USA). The primers (Tsingke Biotechnology Co., Ltd., Beijing, China) used for target mRNA detection were as follows: GHSR (forward 5′-CTACAGTCTCATCGGCAGGA-3′, reverse 5′-GAGAGAAGGGAGAAGGCACA-3′); GAPDH (forward 5′-TCAAGAAGGTGGTGAAGCAGG-3′, reverse 5′-TCAAAGGTGGAGGAGTGGGT-3′). The results are presented as values normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression using the 2−ΔΔCt method.

Cell migration was analyzed using Transwell chambers (Corning, USA). HRMECs from each group were digested with 0.25% trypsin, and a single-cell suspension was prepared with serum-free M199 medium at a density of 3 × 105 cells/mL. Two hundred microlitres of the cell suspension was added to the upper compartment of the Transwell chamber, and M199 medium containing 10% FBS was added to the lower compartment. After 24 h of incubation, the Transwell chamber was removed. The cells were carefully cleaned with PBS, fixed with 70% iced ethanol solution for 1 h and stained with 0.5% crystal violet for 20 min. Nonadherent cells were removed by wiping with a swab, and the chamber was observed under a microscope. Five high magnification fields were randomly selected for counting the cells that crossed the membrane.

Each well of 24-well plates was coated with 200 μL of Matrigel (Corning, USA) and placed at 37 °C for 60 min. Treated cells were digested with 0.25% trypsin, and single-cell suspensions were prepared with serum-free M199 medium. After counting, the cells were seeded evenly into a 24-well plate precoated with Matrigel at 1 × 105 cells/well and cultured at 37 °C for 12 h. Images were acquired (IX51 Olympus, Japan), and the tube-like structures were analyzed using ImageJ (NIH, USA) at 100 × magnification.

Total protein was extracted from cells with RIPA lysis buffer (Beyotime, China) after incubation for 48 h. After quantification of the protein concentration with a BCA protein assay kit (Beyotime, China), proteins in the samples (40 μg) were separated by SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes. Then, the membranes were blocked with 5% fat-free milk and incubated with diluted primary rabbit polyclonal antibodies, including anti-GHSR-1a (1:1000, Affinity, USA), anti-PERK (1:1000, Affinity, USA), anti-p-PERK (1:2000, Affinity, USA), anti-AFT4 (1:1000, Affinity, USA), anti-CHOP (1:1000, Affinity, USA), and anti-GAPDH (1:1000, Hangzhou Xianzhi Biology Co., Ltd., China) antibodies, overnight at 4 °C. Then, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:10,000, Boster, China) as the secondary antibody for 2 h at room temperature. The bands were visualized by ECL and quantified using Bandscan software 5.0 (Glyko Inc., CA, USA).

Quantitative data are presented as the mean ± standard deviation (SD) of three independent experiments. SPSS 19.0 (IBM, USA) was used for conducting a pairwise t test or one-way analysis of variance (ANOVA) followed by a LSD post hoc test. Differences between or among the groups were statistically significant when P < 0.05.