Synthesis of the Opioid Agonist
Biphalin was synthesized by Adriano Mollica at his laboratory in Università degli Studi G. d’Annunzio Chieti e Pescara, Department of Pharmacy, Chieti, Italy. The peptide’s chemical properties were in full agreement with those already reported in the literature (Fig. 1). [11]
Corneal Epithelial Cell Culture
Human immortalized corneal epithelial cells (HCECs) were a generous gift from Dr. James Jester (Irvine, CA, USA). The HCECs were cultured in a keratinocyte serum-free medium (KSFM; Gibco, NY, USA) supplemented with bovine pituitary extract (BPE; 25 μg/mL), epidermal growth factor (EGF; 50 ng/mL), penicillin (100 IU/mL), and streptomycin (100 μg/mL). The cells were maintained in 75 cm2 flasks until experimentation. The HCECs did not differentiate in the keratinocyte serum-free medium. To differentiate HCECs, we followed the protocol described by Sahin et al. [12] We changed the medium with DMEM with 10% FBS. In this way, the HCECs were differentiated and stratified.
Cytotoxicity Assay
The HCECs were treated with different concentrations of biphalin (from 1 pM to 100 µM) in 96-well culture dishes (Corning, NY, USA) for 24 hours. The cytotoxicity of exposure was measured with the MTT (3-(4,5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) assay (Thermo Fisher Scientific, MA, USA). The color of the MTT tetrazole salt was measured with a spectrophotometer at a wavelength of 570 nm.
In Vitro Scratch Assay
The HCECs were grown to confluence on 12-well culture dishes (Corning, NY, USA). On reaching confluence, the cells were rinsed with a phosphate-buffered saline solution (PBS) and exposed to a differentiation medium consisting of Dulbecco’s Modified Eagle Medium (DMEM; Gibco, NY, USA) with 10% fetal bovine serum (FBS), penicillin (100 IU/mL), and streptomycin (100 μg/mL) for one day. Two perpendicular linear scratches were made using a sterile 200 µL pipette tip, and the wells were washed with PBS. Immediately after the scratch, all groups were incubated in KSFM. Biphalin (in two different concentrations; 1 µM [10-6 M] and 10 µM [10-5 M]) or biphalin plus naloxone solution (in two different concentrations; 1 µM and 10 µM) or their vehicle (PBS) was added to the cultures. Naloxone, which is a selective opioid receptor antagonist, was added to inhibit the opioid receptor-related effects of biphalin. The scratch area was captured hourly for 24 hours using live-cell microscopy (DMi 8; Leica, Wetzlar, Germany). The relative wound area (RWA) was measured using ImageJ software (National Institutes of Health [NIH], MD, USA) (Fig. 2).
Transwell Migration Assay
After wounding, the cells were used to do a Transwell Migration Assay. This is because during confluency, gene expressions of cells are different from the wound-healing situation. By creating a wound, we simulated the wound process in epithelial cells. Immediately after the wounding process was complete, as described above, the HCECs were trypsinized, washed, and plated (2.5×105 cells per insert) in 8.0 µm pore size Transwell inserts (Corning, NY, USA) in KSFM.
The lower compartment was filled with DMEM with 10% FBS or DMEM with 10% FBS plus either biphalin (in two different concentrations; 1 µM and 10 µM) or biphalin plus naloxone solution (in two different concentrations; 1 µM and 10 µM) or their vehicle (PBS). After 24 hours, the cells on the upper side of the insert were removed by scraping, and the cells that had migrated through were fixed on the lower side of the membrane with 4% paraformaldehyde, then stained with hematoxylin-eosin and quantified by counting the number of cells in 10 separate fields. The data were expressed as the number of migrated cells per micrograph field for each sample well.
Ki67 Proliferation Assay
The effect of biphalin on in vitro proliferation was assessed by immunofluorescence staining for Ki67. The Ki67 protein is present during the G1, S, G2, and M phases of the cell cycle and is strictly associated with cell proliferation. 2.5 x 105 HCECs were plated in equal numbers in 24-well culture dishes (Corning, NY, USA). After reaching confluence, cells were rinsed twice with PBS and exposed to a stratification medium consisting of DMEM with 10% FBS. Two perpendicular linear scratches were made using a sterile 200 µL pipette tip, and the wells were washed three times with PBS and incubated with KSFM without EGF and BPE. Immediately after the scratch, biphalin (in two different concentrations; 1 µM and 10 µM) or biphalin plus naloxone solution (in two different concentrations; 1 µM and 10 µM) or their vehicle (PBS) were added to the cell culture medium. The cells were incubated for 6 hours at 37°C. After the treatment, the cells grown on 24-well culture dishes were fixed in 4% paraformaldehyde for 20 min. After three washes with PBS, the cells were incubated with 0.1% TritonX-100 in PBS for 8 min. The cells were incubated with Superblock (Thermo Fisher Scientific, MA, USA) for 10 min at room temperature and then overnight at 4°C with the rabbit anti-Ki67 primary antibody (Abcam, MA, USA) at optimal dilutions in a blocking solution. After three washes with PBS, the cells were incubated with the FITC-conjugated secondary antibody (Abcam, MA, USA) for 90 min at 37°C, then washed, counterstained with 406-diamidino-2-phenylindole (DAPI), and mounted. Negative controls were stained in a similar fashion (DMi 8; Leica, Wetzlar, Germany). The Ki67 proliferation index is the proportion of Ki67 stained cell nuclei to DAPI stained cell nuclei from 20 different micrograph areas. The Ki67 proliferation index is calculated by dividing the number of Ki67 stained cell nuclei by the number of DAPI stained cell nuclei using ImageJ software (National Institutes of Health [NIH], MD, USA)
Gene Expression Analysis of Opioid Receptors with Quantitative Reverse Transcription PCR
The presence and expression levels of MOR, DOR, and KOR in the HCECs were measured quantitatively using a real-time polymerase chain reaction (qRT-PCR). The presence of OPRM1, OPRD1, and OPRK1 mRNAs, which are related with mu, delta, and kappa opioid receptor proteins, respectively, was studied in differentiated and undifferentiated HCECs and in SH-SY5Y cell lines. SH-SY5Y cell lines are human neuronal cancer cell lines that have demonstrated expressions of these three opioid receptors in previous literature.[13] To show the effect of the serum on HCECs, HCECs cultured in KSFM and in DMEM+10% FBS were used in qRT-PCR analysis. The qRT-PCR primers for the genes that are responsible for the opioid receptor expressions selected is shown in Table 1. RNA isolation from cells was performed via a Quick-RNA MicroPrep Kit (Zymo Research, Irvine, CA, USA). Extracted RNAs were quantified with Nanodrop 2000 spectrophotometry (Thermo Fisher Scientific, Waltham, MA, USA), and 1000 ng cDNA was prepared using M-MLV Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA). Quantitative real-time expressions of mRNAs were detected and compared with a Light Cycler 480 SYBR Green I Master (Roche, Basel, Switzerland).
Statistical Analysis
Each experiment was performed at least two times. For blind analysis, collection of images was made by E.Y., and each image was assigned a number. Then images were analyzed anonymously by K.K. Values were displayed as a mean ± standard deviation. For the in vitro scratch assay, we measured time-dependent change in the wound area. For other assays, we collected data only at one time point. Since two-way ANOVA is used to examine the interaction between two independent variables (treatment and time), and one-way ANOVA tests the effect of one independent variable (treatment), statistical analysis was performed using two-way ANOVA for in vitro scratch assay results, and one-way ANOVA with a Tukey’s Honest Significant Difference test was used for other results to determine the degree of significance (R; R-Project, Vienna, Austria).
Results were considered statistically significant when the p-value was less than 0.05.