2.1 Chemicals
Fetal bovine serum (FBS), trypsin-EDTA, and penicillin–streptomycin was provided from Lonza (Walkersville, MD, USA). M199 media, mannitol, and D-glucose and Eucalyptol), and all other regents not mentioned elsewhere were obtained from Sigma-Aldrich Chemical (St. Louis, MO, USA). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin (Ig)G, goat anti-mouse and donkey anti-goat IgG were obtained from Jackson Immumno Reserch Laboratories (West Grove, PA, USA). Essentially fatty acid free bovine serum albumin (BSA) and skim milk were supplied by Becton Dickinson Company (Sparks, MD, USA).
Eucalyptol was dissolved in dimethyl sulfoxide (DMSO) for living culture with cells.
2.2 Cell Culture
Human primary retinal microvascular endothelial cells (HRMVECs) from Cell System Corporation (Kirkland, WA, USA) were cultured in M199 media containing 10% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM glutamine, 0.75 µg/ml human epidermal growth factor, and 75 µg/ml hydrocortisone at 37⁰C in a humidified incubator containing 5% CO2. The cells were maintained in 5.5 mM normal glucose or 33 mM glucose for hyperglycemia condition. In addition, HRMVEC were incubated for 3 days in media containing 5 µM amyloid-β in the absence and presence of 1–20 µM eucalyptol. Cell viability or cytotoxicity was measured by assaying with MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltertrazolium bromide). Cells were incubated with 1 mg/ml MTT solution at 37⁰C for 3 h, forming insoluble purple formazan product that was dissolved in isopropanol prior to reading the absorbance in a microplate reader at 570 nm.
2.3 In vivo Animal Experiments
Male, 7-week-old db/db mice C57BLKS/+Leprdb Iar were employed and randomly divided into two groups after 1 week of acclimation to the environment. Non-diabetic control db/m mice were also obtained from Jackson Laboratory (Sacramento, CA, USA). Mice were kept on a 12 h light/12 h dark cycle at 23 ± 1°C with 50 ± 10% relative humidity under specific pathogen-free conditions, fed a standard laboratory chow diet (CJ Feed, Seoul, Korea), and were supplied with water ad libitum at the animal facility of Hallym University. All experimental protocol was approved by the Institutional Animal Care and Use Committee of Hallym University (Approval number: HallymR1 2016-10). Mice were divided into three groups (n = 9–10 for each group). Control group was non-diabetic db/m mice, and db/db mice were divided into two groups. One group of db/db mice was daily supplemented 10 mg/kg per day eucalyptol via gavage for 8 weeks.
The measurement of fasting blood levels of glucose and glycated hemoglobin HbA1C, a biomarker of development of diabetic complication, was conducted every other week from mouse tail veins during the 8 week-supplementation of eucalyptol [17].
2.4 Western Blot Analysis
Protein was extracted for Western blot analysis from retinal endothelial cells and eye tissues after it was homogenized in a lysis buffer containing 1 M β-glycerophosphate, 1% β-mercaptoethanol, 0.5 M NaF, 0.1 M Na3VO4 and protease inhibitor cocktails.
Lysed protein extract was electrophoresed on 8–15% SDS-PAGE and transferred onto a nitrocellulose membrane and followed by immunoblotting analysis. [18] The following antibodies were used: anti-rabbit vascular endothelial cadherin (Abcam Biochemicals, Cambridge, UK), anti-goat angiopoietin (Ang)-1 and Ang-2, anti-rabbit occludin-1 and phospho-PERK, and anti-mouse amyloid-β antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-goat vascular endothelial growth factor (VEGF) (R&D systems, Minneapolis, MN, USA), anti-rabbit phospho-Tie-2 (Millipore Corporation, Temecula, CA, USA), anti-mouse β-actin and anti-rabbit bcl-2 antibody (Sigma-Aldrich Chemical), anti-mouse bax (BD Transduction Laboratories, West Grove, PA, USA), anti-rabbit caspase-12 and phospho-eIF2α, anti-mouse CHOP and anti-rabbit ATF4 (Cell Signaling Technology, Danvers, MA, USA).
2.5 Detection of Cellular DNA Fragmentation
For measuring HRMVEC apoptosis, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was verified. HRMVEC were seeded on a chamber slide and treated with 20 µM eucalyptol in amyloid-β-containing media. The DNA fragmentation was detected by the using a commercial DeadEnd™ Florometric TUNEL kit (Promega Corporation, Madison, WI, USA). The cells were fixed with 4% formaldehyde for 30 min and permeabilized with 0.2% Triton X-100 and fluorescein isothiocyanate (FITC)-12-dUTP was added. For the nuclear counterstaining, cells were treated with 4 mg/ ml 4’,6-diamidino-2-phenylindole (DAPI, Santa Cruz Biotechnology). DNA fragmentation was analyzed using an Axiomager microscope system (Carl Zeiss, Oberkochen, Germany).
2.6 In vitro Permeability Assay
The permeability of albumin was measured by using transwell inserts with 8 µM pore size filters (Costar, Corning, NY, USA), in accordance with the manufacturer’s instructions. HRMVEC were plated on the transwell inserts at (7 x 104 cells/well) in media containing 5.5 mM glucose, 33 mM glucose or 5 µM amyloid-β in absence and presence of eucalyptol for 3 days. Subsequently, 50 µg/ml FITC-labeled BSA was added onto the upper inserts with serum-free media. After 2 h, in the lower compartments of the media were collected, and the fluorescent intensity was measured by using Fluoroskan reader (Thermo Fisher Scientific, Waltham, MA, USA) with 495 nm excitation and 520 nm emission filters.
2.7 Immunostaining
After dissection, part of the eye tissue was fixed in 4% buffered formalin, eye tissues were paraffin-embedded, cut into 5 µm thickness, deparaffinized, and dehydrated with xylene and graded ethanol solutions. Tissue sections of eye were incubated with a primary antibody of amyloid-β overnight and FITC-conjugated anti-mouse IgG. In addition, immunofluorescent histochemical staining for Bcl-2 was done with FITC-conjugated anti-rabbit IgG and Bax was done with CY3-conjugated anti-mouse IgG. For nuclear staining, 4’,6-diamidino-2-phenylindole (DAPI) was used to labeling nuclei. Each slide was taken using an optical Axiomager microscope system (Zeiss, Oberkochen, Germany). The protein levels of amyloid-β, Bcl-2 and Bax were quantified with an image analysis program from the microscope system.
2.8 Data Analysis
All experiments were repeated at least three times. Each analysis, results from triplicate samples were presented as as mean ± standard deviation (SD). Statistical analyses were performed using Statistical Analysis Systems statistical software package (SAS Institute, Cary, NC, USA). Significance between the groups was determined with by one-way analysis of variance, followed by Dunca n range test for multiple comparisons. Differences were considered significant at P < 0.05.