{"id":4903,"date":"2024-01-15T06:33:58","date_gmt":"2024-01-15T06:33:58","guid":{"rendered":"https:\/\/corp.farlong.com\/protective-effect-of-tisochrysis-lutea-on-dry-eye-syndrome-via-nf-%ce%bab-inhibition\/"},"modified":"2024-01-27T10:30:56","modified_gmt":"2024-01-27T10:30:56","slug":"protective-effect-of-tisochrysis-lutea-on-dry-eye-syndrome-via-nf-%ce%bab-inhibition","status":"publish","type":"post","link":"https:\/\/directcm.com\/fr\/protective-effect-of-tisochrysis-lutea-on-dry-eye-syndrome-via-nf-%ce%bab-inhibition\/","title":{"rendered":"Effet protecteur de Tisochrysis lutea sur le syndrome de l'\u0153il sec via l'inhibition de NF-\u03baB"},"content":{"rendered":"
\n

Materials<\/h3>\n

In vivo\/in vitro materials<\/h4>\n

T. lutea<\/i> 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\u2019s Modified Eagle\u2019s Medium (DMEM):F12, Dulbecco\u2019s phosphate-buffered saline (DPBS), fetal bovine serum (FBS), and 0.25% trypsin\u2013EDTA solution were obtained from Life Technologies (Carlsbad, CA, USA). Antibiotic solution, comprising 10,000\u00a0U\/mL penicillin and 10,000\u00a0\u03bcg\/mL streptomycin, was purchased from Hyclone Laboratories Inc. (South Logan, UT, USA). Recombinant human TNF-\u03b1 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-\u03b1-tubulin, and anti-\u03b2-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\u20134,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA).<\/p>\n

Chemical analysis materials<\/h3>\n

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<\/i>-hexane, Na2<\/sub>SO4<\/sub>, CH3<\/sub>Cl, MeOH, and NaCl, used to extract PUFAs from T. lutea<\/i> were purchased from Sigma-Aldrich (St. Louis, MO, USA).<\/p>\n

Ethical statement<\/h3>\n

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.<\/p>\n

Animal experiments and induction of DES model<\/h3>\n

Mice were housed in an air-conditioned animal room and maintained at 22\u00a0\u00b0C\u2009\u00b1\u20092\u00a0\u00b0C, 50%\u2009\u00b1\u200910% humidity, and a 12\u00a0h light\/12\u00a0h 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 \u03bcL (2.5\u00a0mg\/mL) of scopolamine diluted in phosphate-buffered saline (PBS; Sigma-Aldrich, St. Louis, MO, USA). T. lutea<\/i> was administered orally to groups of mice daily at concentrations of 0 (vehicle control), 100, 150, or 300\u00a0mg\/kg in 200\u00a0\u03bcL of distilled water. The control group received PBS without scopolamine. When T. lutea<\/i> was administered orally, 200 \u03bcL distilled water was used in the control and DES group. Tear production was quantified after two weeks using a standard Schirmer\u2019s test strip placed in the lower one-third of the temporal eyelid before closing the eye for 1\u00a0min. The strip was then removed and the length of the wet point in millimeters was measured to determine Schirmer\u2019s 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\u2009\u2212\u200980\u00a0\u00b0C. The lacrimal gland was removed from the mouse\u2019s 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\u2009\u2212\u200980\u00a0\u00b0C protected from light.<\/p>\n

Histology<\/h3>\n

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\u2009\u00d7\u2009magnification using a microscope (TE-2000U, Nikon, Tokyo, Japan). the central corneal epithelial thickness was determined by dividing each cornea into five sections.<\/p>\n

DAB staining for immunohistochemical (IHC) analysis<\/h3>\n

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\u00a0\u03bcm), and the paraffin was removed three times for 3\u00a0min using xylene (Junsei, Tokyo, Japan). The sections were hydrated for 1\u00a0min in 100, 95, 70, and 50% ethanol. Lacrimal gland antigen was extracted by microwave with TRIS\u2013EDTA buffer (pH 9.0). After applying 3% hydrogen peroxide to tissue slides for 15\u00a0min, the sections were blocked with 2% BSA for 1\u00a0h at room temperature. The sections were treated overnight at 4\u00a0\u00b0C with CD45 (Bio-RAD, MCA 1258GT, 1:100 dilution). After washing with PBS, the cells were treated for 2\u00a0h at room temperature with anti-rabbit IgG-HRP (Santa Cruz Biotechnology, Dallas, TX, USA, SC-2357, 1:100 dilution) and stained for 1\u00a0min with 3,3\u2032-diaminobenzidine tetrahydrochloride (Vector Laboratories, Burlingame, CA, USA). The slides were stained for 30\u00a0s with hematoxylin and rinsed with tap water. The slices were then cover-slipped and mounted with Permount\u2122 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<\/a><\/sup> was used to perform quantitative data analysis.<\/p>\n

ARPE-19 cell culture<\/h3>\n

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\u2009\u00d7\u2009105<\/sup> per well in 6-well plates and cultured to induce an inflammatory and endoplasmic response.<\/p>\n

Cell viability<\/h3>\n

The effect of T. lutea<\/i> on cell viability was evaluated by MTT assay as described previously study49<\/a><\/sup> but with slight modifications. Cells were plated in 96-well culture plates at a density of 1.5\u2009\u00d7\u2009104<\/sup> cells\/well and incubated for 24\u00a0h. Cells were then treated with various concentrations of T. lutea<\/i> and incubated for a further 24\u00a0h. The supernatant of each well was replaced with a fresh growth medium containing 0.5\u00a0mg\/mL of MTT and incubated for 1\u00a0h. The supernatant was removed and generated formazan deposits were dissolved in 200\u00a0\u03bcL of dimethyl sulfoxide. Absorbance was measured at 570\u00a0nm. Cell viability was relatively calculated as a percentage compared to negative control groups.<\/p>\n

Western blot analysis<\/h3>\n

Western blotting was conducted to determine cellular signaling pathways underlying the anti-inflammatory effect of T. lutea<\/i> as described previously study49<\/a><\/sup>, but with minor modifications. Confluent ARPE-19 cells were starved in DMEM:F12 containing 1% FBS and 1% antibiotics for 24\u00a0h. Cells were then treated with T. lutea<\/i> (50 and 100\u00a0\u03bcg\/mL) for 2\u00a0h and then stimulated with 20\u00a0ng\/mL of TNF-\u03b1 for 15\u00a0min. 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\u00d7g<\/i>, 30\u00a0min, 4\u00a0\u00b0C) and their protein concentration was estimated using a DCTM protein assay kit (Bio-Rad, Hercules, CA, USA). Equal amounts (15\u201325\u00a0\u03bcg) 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-I\u03baB-\u03b1, anti-I\u03baB-\u03b1, anti-phospho-p65, anti-p65, anti-\u03b1-tubulin and anti-\u03b2-action) at 4\u00a0\u00b0C and 12\u00a0h. The membranes were then rinsed with TBS-T and treated 1\u00a0h 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<\/a><\/sup>.<\/p>\n

Total PUFA extraction from T. lutea<\/i><\/h3>\n

Total PUFAs were extracted from T. lutea<\/i> using the Folch technique50<\/a><\/sup>. Briefly, 100\u00a0mg of the sample was placed into a vial containing 5\u00a0mL of CH3<\/sub>Cl:MeOH (2:1, v\/v) solution and ultrasonicated for 15\u00a0min. The suspension was clarified by centrifugation at 10,000\u00a0rpm for 5\u00a0min. Na2<\/sub>SO4<\/sub> 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.<\/p>\n

Fatty acid methylation<\/h3>\n

Fatty acid methylation was carried out to prepare lipid samples for gas chromatography (GC) analysis. Extracted material was added to a 10\u00a0mL vial followed by 1\u00a0mL of 0.5\u00a0N NaOH:MeOH mixture. This solution contains 400\u00a0\u03bcg\/mL of internal strandard (valeric acid). The mixture was shaken for 30\u00a0s, and then incubated for 20\u00a0min at 80\u00a0\u00b0C. The mixture was then cooled at room temperature for 5\u00a0min, and then 2\u00a0mL of boron trifluoride:MeOH was added, and then agitated for 30\u00a0s. The mixture was incubated for 20\u00a0min at 80\u00a0\u00b0C followed by cooling for 10\u00a0min. A volume of 2\u00a0mL of supersaturated NaCl was added to induce phase separation and fatty acids collection. Next, 1.5\u00a0mL of n<\/i>-hexane was added and the mixture was vortexed for 30\u00a0s before cooling at room temperature for 20\u00a0min. The layers were separated and the liquid supernatant was removed from the mixture and the water was completely removed using a saturated Na2<\/sub>SO4<\/sub> filter.<\/p>\n

Identification of fatty acids<\/h3>\n

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\u00a0\u00b5L) were injected into the HP-88 column of the GC (J&W 112-88A7, Agilent Technologies, Wilmington, DE, USA) (100\u00a0m\u2009\u00d7\u2009250\u00a0\u03bcm I.D., 0.2\u00a0\u03bcm film). GC analysis was performed as follows: samples were initially held at 140\u00a0\u00b0C for 5\u00a0min, and then the temperature was ramped up at 4\u00a0\u00b0C\/min to 240\u00a0\u00b0C for 15\u00a0min. The flow rate of the column was fixed at 1\u00a0mL\/min. Nitrogen gas was employed as the carrier gas for the analysis, the FID was set at 280\u00a0\u00b0C, 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.<\/p>\n

Statistical analysis<\/h3>\n

All data were analyzed statistically using one-way ANOVA with Tukey\u2019s post hoc analysis by SPSS version 18.0 (IBM Co., Armonk, NY, USA). At least three independent replicates were performed for each experiment.<\/p>\n<\/div>\n

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The prevalence of dry eye syndrome (DES) has increased worldwide, affecting around 50% of all adults1. DES is a multifactorial ocular surface disease characterized by tear film instability and hyperosmolarity, ocular surface inflammation and damage, neurosensory abnormalities<\/p>","protected":false},"author":1,"featured_media":5363,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"site-sidebar-layout":"default","site-content-layout":"","ast-site-content-layout":"default","site-content-style":"default","site-sidebar-style":"default","ast-global-header-display":"","ast-banner-title-visibility":"","ast-main-header-display":"","ast-hfb-above-header-display":"","ast-hfb-below-header-display":"","ast-hfb-mobile-header-display":"","site-post-title":"","ast-breadcrumbs-content":"","ast-featured-img":"","footer-sml-layout":"","theme-transparent-header-meta":"default","adv-header-id-meta":"","stick-header-meta":"","header-above-stick-meta":"","header-main-stick-meta":"","header-below-stick-meta":"","astra-migrate-meta-layouts":"set","ast-page-background-enabled":"default","ast-page-background-meta":{"desktop":{"background-color":"var(--ast-global-color-4)","background-image":"","background-repeat":"repeat","background-position":"center center","background-size":"auto","background-attachment":"scroll","background-type":"","background-media":"","overlay-type":"","overlay-color":"","overlay-gradient":""},"tablet":{"background-color":"","background-image":"","background-repeat":"repeat","background-position":"center center","background-size":"auto","background-attachment":"scroll","background-type":"","background-media":"","overlay-type":"","overlay-color":"","overlay-gradient":""},"mobile":{"background-color":"","background-image":"","background-repeat":"repeat","background-position":"center center","background-size":"auto","background-attachment":"scroll","background-type":"","background-media":"","overlay-type":"","overlay-color":"","overlay-gradient":""}},"ast-content-background-meta":{"desktop":{"background-color":"var(--ast-global-color-5)","background-image":"","background-repeat":"repeat","background-position":"center center","background-size":"auto","background-attachment":"scroll","background-type":"","background-media":"","overlay-type":"","overlay-color":"","overlay-gradient":""},"tablet":{"background-color":"var(--ast-global-color-5)","background-image":"","background-repeat":"repeat","background-position":"center center","background-size":"auto","background-attachment":"scroll","background-type":"","background-media":"","overlay-type":"","overlay-color":"","overlay-gradient":""},"mobile":{"background-color":"var(--ast-global-color-5)","background-image":"","background-repeat":"repeat","background-position":"center center","background-size":"auto","background-attachment":"scroll","background-type":"","background-media":"","overlay-type":"","overlay-color":"","overlay-gradient":""}},"footnotes":""},"categories":[9],"tags":[],"class_list":["post-4903","post","type-post","status-publish","format-standard","has-post-thumbnail","hentry","category-supplements"],"_links":{"self":[{"href":"https:\/\/directcm.com\/fr\/wp-json\/wp\/v2\/posts\/4903","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/directcm.com\/fr\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/directcm.com\/fr\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/directcm.com\/fr\/wp-json\/wp\/v2\/users\/1"}],"replies":[{"embeddable":true,"href":"https:\/\/directcm.com\/fr\/wp-json\/wp\/v2\/comments?post=4903"}],"version-history":[{"count":0,"href":"https:\/\/directcm.com\/fr\/wp-json\/wp\/v2\/posts\/4903\/revisions"}],"wp:featuredmedia":[{"embeddable":true,"href":"https:\/\/directcm.com\/fr\/wp-json\/wp\/v2\/media\/5363"}],"wp:attachment":[{"href":"https:\/\/directcm.com\/fr\/wp-json\/wp\/v2\/media?parent=4903"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/directcm.com\/fr\/wp-json\/wp\/v2\/categories?post=4903"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/directcm.com\/fr\/wp-json\/wp\/v2\/tags?post=4903"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}