Protective effect of Tisochrysis lutea on dry eye syndrome via NF-κB inhibition

Materials

In vivo/in vitro materials

T. lutea was obtained from Microalgae ask us Corp. (Gangneung, Korea). Scopolamine, fluorescein, and hematoxylin and eosin (H&E) were purchased from Sigma-Aldrich (St. Louis, MO, USA) for animal experiments. Dulbecco’s Modified Eagle’s Medium (DMEM):F12, Dulbecco’s phosphate-buffered saline (DPBS), fetal bovine serum (FBS), and 0.25% trypsin–EDTA solution were obtained from Life Technologies (Carlsbad, CA, USA). Antibiotic solution, comprising 10,000 U/mL penicillin and 10,000 μg/mL streptomycin, was purchased from Hyclone Laboratories Inc. (South Logan, UT, USA). Recombinant human TNF-α was obtained from R&D systems (Minneapolis, MN, USA). Primary antibodies including anti-extracellular signal-regulated kinase (ERK), anti-c-jun N-terminal kinase (JNK), anti-p38, anti-α-tubulin, and anti-β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Other primary antibodies and horseradish peroxidase (HRP)-conjugated secondary antibodies were obtained from Cell Signaling Technology, Inc (Beverly, MA, USA). Chemicals including 3–4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Chemical analysis materials

PUFAs were identified using a solution containing 37 PUFA standard substances, which was purchased from Sigma-Aldrich (St. Louis, MO, USA). Valeric acid was obtained from Sigma-Aldrich (St. Louis, MO, USA) for use as an internal standard. Solvents, including boron trifluoride methanol solution, n-hexane, Na₂SO₄, CH₃Cl, MeOH, and NaCl, used to extract PUFAs from T. lutea were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Ethical statement

This research was approved by the Institutional Animal Care and Use Committee (IACUC) of the Korea Institute of Science and Technology (KIST): KIST No. 2020-002, Gangneung Institute, and followed the Association for Research in Vision and Ophthalmology’s statement on the Use of Animals in Ophthalmic and Vision Research. In addition, we follow the Animal Research: Reporting of In Vivo Experiments (ARRIVE) recommendations. All procedures were conducted in compliance with applicable regulations and guidelines.

Animal experiments and induction of DES model

Mice were housed in an air-conditioned animal room and maintained at 22 °C ± 2 °C, 50% ± 10% humidity, and a 12 h light/12 h dark circadian cycle. Food and water were provided ad libitum. Mice were acclimated for one week before being randomly divided into five groups of seven male 6-week-old BALB/c mice (Orientbio, Gyunggi-do, Korea). DES was induced experimentally in mice twice daily via intraperitoneal injection of 200 μL (2.5 mg/mL) of scopolamine diluted in phosphate-buffered saline (PBS; Sigma-Aldrich, St. Louis, MO, USA). T. lutea was administered orally to groups of mice daily at concentrations of 0 (vehicle control), 100, 150, or 300 mg/kg in 200 μL of distilled water. The control group received PBS without scopolamine. When T. lutea was administered orally, 200 μL distilled water was used in the control and DES group. Tear production was quantified after two weeks using a standard Schirmer’s test strip placed in the lower one-third of the temporal eyelid before closing the eye for 1 min. The strip was then removed and the length of the wet point in millimeters was measured to determine Schirmer’s test value. Staining of the corneal surface was used to determine the extent of corneal surface changes. The corneal surface was observed and scored after injecting one drop of 3% fluorescein into the inferior lateral conjunctival. Corneal staining was evaluated blindly. Mice were euthanized by cervical dislocation. The corneas were removed by flicking the eyelid of the mouse and using forceps to cut out the optic nerve from the eyeball and remove the cornea. The crystalline lens was removed from within the cornea and stored at − 80 °C. The lacrimal gland was removed from the mouse’s lower jaw using forceps, and the epidermis of the pulled area was cut away to reveal the lower half of the face. The lacrimal gland near the lower jaw was secured and removed then stored at − 80 °C protected from light.

Histology

Corneal epidermal and lacrimal gland tissues were collected and fixed in 10% formaldehyde, followed by processing for paraffin embedding and sectioning. Sections were stained with H&E and examined at 40 × magnification using a microscope (TE-2000U, Nikon, Tokyo, Japan). the central corneal epithelial thickness was determined by dividing each cornea into five sections.

DAB staining for immunohistochemical (IHC) analysis

Tissues from the lacrimal glands of BALB/c mice were taken and fixed with 10% formaldehyde. Fixed lacrimal gland tissues were paraffin-embedded, sectioned (5 μm), and the paraffin was removed three times for 3 min using xylene (Junsei, Tokyo, Japan). The sections were hydrated for 1 min in 100, 95, 70, and 50% ethanol. Lacrimal gland antigen was extracted by microwave with TRIS–EDTA buffer (pH 9.0). After applying 3% hydrogen peroxide to tissue slides for 15 min, the sections were blocked with 2% BSA for 1 h at room temperature. The sections were treated overnight at 4 °C with CD45 (Bio-RAD, MCA 1258GT, 1:100 dilution). After washing with PBS, the cells were treated for 2 h at room temperature with anti-rabbit IgG-HRP (Santa Cruz Biotechnology, Dallas, TX, USA, SC-2357, 1:100 dilution) and stained for 1 min with 3,3′-diaminobenzidine tetrahydrochloride (Vector Laboratories, Burlingame, CA, USA). The slides were stained for 30 s with hematoxylin and rinsed with tap water. The slices were then cover-slipped and mounted with Permount™ Mounting Medium (Thermo Fisher Scientific, Waltham, MA, USA). All slides were examined and photographed using a light microscope (Olympus, CX43, Olympus Optical Co., Tokyo, Japan). ImageJ software (version 1.53, National Institutes of Health, Bethesda, MD, USA)⁴⁸ was used to perform quantitative data analysis.

ARPE-19 cell culture

Human retinal epithelial ARPE-19 cells (American Type Culture Collection, ATCC, Manassas, VA, USA) were cultured in DMEM/F-12 media (Gibco, Carlsbad, CA, USA) supplemented with 10% FBS (HyClone Laboratories, Logan, UT, USA) and 1% penicillin/streptomycin (HyClone Laboratories). Cells were seeded at a density of 2 × 10⁵ per well in 6-well plates and cultured to induce an inflammatory and endoplasmic response.

Cell viability

The effect of T. lutea on cell viability was evaluated by MTT assay as described previously study⁴⁹ but with slight modifications. Cells were plated in 96-well culture plates at a density of 1.5 × 10⁴ cells/well and incubated for 24 h. Cells were then treated with various concentrations of T. lutea and incubated for a further 24 h. The supernatant of each well was replaced with a fresh growth medium containing 0.5 mg/mL of MTT and incubated for 1 h. The supernatant was removed and generated formazan deposits were dissolved in 200 μL of dimethyl sulfoxide. Absorbance was measured at 570 nm. Cell viability was relatively calculated as a percentage compared to negative control groups.

Western blot analysis

Western blotting was conducted to determine cellular signaling pathways underlying the anti-inflammatory effect of T. lutea as described previously study⁴⁹, but with minor modifications. Confluent ARPE-19 cells were starved in DMEM:F12 containing 1% FBS and 1% antibiotics for 24 h. Cells were then treated with T. lutea (50 and 100 μg/mL) for 2 h and then stimulated with 20 ng/mL of TNF-α for 15 min. The cells were rinsed three times with ice-cold DPBS and then lysed in protein extraction buffer (iNtRON Biotechnology, Gyeonggi-do, Korea) supplemented with a cocktail of protease and phosphatase inhibitors. Supernatants of lysates were obtained by centrifugation (14,000×g, 30 min, 4 °C) and their protein concentration was estimated using a DCTM protein assay kit (Bio-Rad, Hercules, CA, USA). Equal amounts (15–25 μg) of cellular proteins were separated by SDS-PAGE and transferred onto PVDF membranes. Membranes were incubated with 5% bovine serum albumin or skim milk, and then incubated with a 1:2000 dilution of primary antibodies (anti-phopho-ERK, anti-ERK, anti-phospho-JNK, anti-JNK, anti-phospho-p38, anti-p38, anti-phospho-AKT, anti-AKT, anti-phospho-IκB-α, anti-IκB-α, anti-phospho-p65, anti-p65, anti-α-tubulin and anti-β-action) at 4 °C and 12 h. The membranes were then rinsed with TBS-T and treated 1 h at room temperature with anti-rabbit HRP-conjugated secondary antibodies at a dilution of 1:10,000. Protein bands were detected by using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific) and visualized in an iBright CL1000 gel documentation system (Thermo Fisher Scientific). The density of each protein blots was determined by using Image J software (version 1.53, NIH)⁴⁸.

Total PUFA extraction from T. lutea

Total PUFAs were extracted from T. lutea using the Folch technique⁵⁰. Briefly, 100 mg of the sample was placed into a vial containing 5 mL of CH₃Cl:MeOH (2:1, v/v) solution and ultrasonicated for 15 min. The suspension was clarified by centrifugation at 10,000 rpm for 5 min. Na₂SO₄ was added to the supernatant which was then filtered to eliminate any remaining water. This step was repeated two more times. The solution was then concentrated under nitrogen gas to obtain pure algal lipids.

Fatty acid methylation

Fatty acid methylation was carried out to prepare lipid samples for gas chromatography (GC) analysis. Extracted material was added to a 10 mL vial followed by 1 mL of 0.5 N NaOH:MeOH mixture. This solution contains 400 μg/mL of internal strandard (valeric acid). The mixture was shaken for 30 s, and then incubated for 20 min at 80 °C. The mixture was then cooled at room temperature for 5 min, and then 2 mL of boron trifluoride:MeOH was added, and then agitated for 30 s. The mixture was incubated for 20 min at 80 °C followed by cooling for 10 min. A volume of 2 mL of supersaturated NaCl was added to induce phase separation and fatty acids collection. Next, 1.5 mL of n-hexane was added and the mixture was vortexed for 30 s before cooling at room temperature for 20 min. The layers were separated and the liquid supernatant was removed from the mixture and the water was completely removed using a saturated Na₂SO₄ filter.

Identification of fatty acids

Fatty acid methyl esters (FAMEs) were examined using a GC equipped with a flame ionization detector (FID; Agilent 7890A, Agilent Technologies, Wilmington, DE, USA). Samples (1 µL) were injected into the HP-88 column of the GC (J&W 112-88A7, Agilent Technologies, Wilmington, DE, USA) (100 m × 250 μm I.D., 0.2 μm film). GC analysis was performed as follows: samples were initially held at 140 °C for 5 min, and then the temperature was ramped up at 4 °C/min to 240 °C for 15 min. The flow rate of the column was fixed at 1 mL/min. Nitrogen gas was employed as the carrier gas for the analysis, the FID was set at 280 °C, and the split ratio was 30:1. FAMEs were identified by comparing them to known standards. Each sample was measured by GC analysis in triplicate.

Statistical analysis

All data were analyzed statistically using one-way ANOVA with Tukey’s post hoc analysis by SPSS version 18.0 (IBM Co., Armonk, NY, USA). At least three independent replicates were performed for each experiment.