Eucalyptol Inhibits Amyloid–Induced Barrier Dysfunction in Glucose

antioxidants

Article Eucalyptol Inhibits Amyloid-β-Induced Barrier Dysfunction in Glucose-Exposed Retinal Pigment Epithelial Cells and Diabetic Eyes

Dong Yeon Kim †, Min-Kyung Kang †, Eun-Jung Lee, Yun-Ho Kim, Hyeongjoo Oh, Soo-Il Kim, Su Yeon Oh, Woojin Na and Young-Hee Kang * Department of Food Science and Nutrition and The Korean Institute of Nutrition, Hallym University, Chuncheon 24252, Korea; [email protected] (D.Y.K.); [email protected] (M.-K.K.); [email protected] (E.-J.L.); [email protected] (Y.-H.K.); [email protected] (H.O.); [email protected] (S.-I.K.); [email protected] (S.Y.O.); [email protected] (W.N.) * Correspondence: [email protected]; Tel.: +82-33-248-2132; Fax: +82-33-254-1475 Dong Yeon Kim and Min-Kyung Kang contributed equally to this study. † Received: 9 August 2020; Accepted: 29 September 2020; Published: 15 October 2020

Abstract: Hyperglycemia elicits tight junction disruption and blood-retinal barrier breakdown, resulting in diabetes-associated vison loss. Eucalyptol is a natural compound found in eucalyptus oil with diverse bioactivities. This study evaluated that eucalyptol ameliorated tight junctions and retinal barrier function in glucose/amyloid-β (Aβ)-exposed human retinal pigment epithelial (RPE) cells and in db/db mouse eyes. RPE cells were cultured in media containing 33 mM glucose or 5 µMAβ for 4 days in the presence of 1–20 µM eucalyptol. The in vivo animal study employed db/db mice orally administrated with 10 mg/kg eucalyptol. Nontoxic eucalyptol inhibited the Aβ induction in glucose-loaded RPE cells and diabetic mouse eyes. Eucalyptol reversed the induction of tight junction-associated proteins of ZO-1, occludin-1 and matrix metalloproteinases in glucose- or Aβ-exposed RPE cells and in diabetic eyes, accompanying inhibition of RPE detachment from Bruch’s membrane. Adding eucalyptol to glucose- or Aβ-loaded RPE cells, and diabetic mouse eyes reciprocally reversed induction/activation of apoptosis-related bcl-2, bax, cytochrome C/Apaf-1 and caspases. Eucalyptol attenuated the generation of reactive oxygen species and the induction of receptor for advanced glycation end products in Aβ-exposed RPE cells and diabetic eyes. Eucalyptol may ameliorate RPE barrier dysfunction in diabetic eyes through counteracting Aβ-mediated oxidative stress-induced RPE cell apoptosis.

Keywords: amyloid-β; apoptosis; blood-retinal barrier; db/db mice; eucalyptol; glucose; retinal pigment epithelium; tight junction

1. Introduction Retinitis pigmentosa (RP) is an outer retinal degenerative disorder in which rod photoreceptors and retinal pigment epithelial (RPE) cells are damaged, leading to peripheral and night-time vision loss [1,2]. Diabetic retinopathy (DR) is characterized by abnormal permeability of the blood-retinal barrier, pericyte loss, basement thickening and retinal neuronal abnormalities [3,4]. Ultimately, the disruption of blood-retinal barrier and leakage of the blood contents accompany severe vision loss in both RP and DR [5–7]. The RPE, a monolayer of end part of retina, locates between the neural retina and the choroid, involving the outer blood-retinal barrier, and supports the structural and functional integrity of the retina [8,9]. The RPE barrier takes in adherent junctions, tight junctions and transporters, [6,8]. Tight junctions are intricate complexes of both transmembrane and cytoplasmic proteins of zonula occludens-1 (ZO-1) and occludin-1 [10,11]. These junction proteins form a selective

Antioxidants 2020, 9, 1000; doi:10.3390/antiox9101000 www.mdpi.com/journal/antioxidants Antioxidants 2020, 9, 1000 2 of 19 permeable barrier between adjacent RPE cells and anchor the actin cytoskeleton components, which is essential for the maintenance of visual function of eyes [3,4,12]. Under hyperglycemic conditions, the collapse of the blood-retinal barrier arises owing to disruption of tight junctions, resulting in diabetic macular edema, a major cause of vision loss [7,8,13]. Macular edema can contribute to visual loss in the RP [14]. On the other hand, hyperglycemia-associated inflammatory pathways lead to the impairment of blood-retinal barrier [15,16]. However, the involved mechanisms are mostly unknown. Alzheimer’s disease and RP are neurodegenerative diseases stemming from the same basic cell death mechanisms [17]. Recent evidence suggests that hyperglycemia is an increased risk for developing dementia, especially Alzheimer’s disease pathologically characterized by amyloid-β (Aβ) plaque deposits and neurofibrillary tangle in the brain [18,19]. However, the cellular and molecular mechanisms by which glucose toxicity can promote pathology of Alzheimer’s disease remains unknown. It has been shown that diabetes mellitus causes an increase in Aβ peptide levels [20,21]. A potential relationship between Aβ-derived neurotoxins and retinal degeneration has been implicated in diabetes as well as aging and Alzheimer’s disease [21]. Aβ is found within RPE cells and in extracellular drusen deposits present between the RPE and Bruch’s membrane [22,23]. The accumulation of Aβ peptide in retina is responsible for the development of retinal cell apoptosis [24]. In addition, the Aβ treatment leads to pathophysiological alternations of barrier damage, apoptotic cell death and Aβ production-triggered events in an Aβ-induced retinal degeneration in mice [25]. The NLRP3 inflammasome activation is induced by Aβ via reactive oxygen species (ROS)-dependent oxidative stress, which may be responsible for RPE dysfunction in age-related macular degeneration [26]. However, the involvement of Aβ in the development of DR remains vague. While the unfavorable effects of Aβ on retinas have been broadly evaluated, Aβ-induced RPE dysfunction and barrier impairment need to be fully investigated. Defining the molecular mechanisms and signals manipulating the blood-retinal barrier and accumulation of Aβ following RPE injury will contribute to the development of new therapeutic strategies preventing and treating eye diseases. Eucalyptol (1,8-cineole) is a natural organic compound chiefly found in eucalyptus oil, rosemary, tea tree, bay leaves and other aromatic foliage [27]. Eucalyptol exhibits multiple pharmacological effects including anti-microbial, antioxidant and anti-inflammatory activities [28,29]. Our previous studies show that multifunctional eucalyptol exerts the renoprotection through inhibiting diabetes-associated disruption of podocyte slit junctions and disjunction of renal tubular epithelial cells [30,31]. However, it is elusive whether eucalyptol ameliorates blood-retinal barrier breakdown through inhibiting accumulation and its malicious effects of Aβ in DR. The present study investigated that eucalyptol improved disruption of blood-retinal barrier through maintaining robust cell-cell tight junctions in high glucose/Aβ-exposed RPE cells and in db/db mouse eyes. In diabetic eyes was examined the mechanism(s) germane to RPE dysfunction, and the contribution of Aβ-RAGE system and oxidative stress to the blood-retina impairment.

2. Materials and Methods

2.1. Materials Dulbecco’s modiﬁed eagle medium (DMEM, low glucose), mannitol, D-glucose and eucalyptol were provided by Sigma-Aldrich Chemical (St Louis, MO, USA). Fetal bovine serum (FBS), trypsin-ethylenediaminetetraacetic acid and penicillin-streptomycin were obtained from Lonza (Walkersvillle, MD, USA). Aβ protein was obtained from Calbiochem (San Diego, CA, USA). Mouse monoclonal antibodies of matrix metalloproteinase (MMP)-2, MMP-9, receptor for advanced glycation end products (RAGE), Aβ and cytochrome C, and rabbit polyclonal antibody of occludin-1 were supplied by Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit polyclonal antibody of ZO-1 was obtained from Thermo Fisher Scientiﬁc (Waltham, MA, USA). Rabbit monoclonal Apaf-1 antibody was supplied by Abcam Biochemicals (Cambridge, UK). Antibodies of mouse polyclonal cleaved caspase-9 and rabbit polyclonal cleaved caspase-3 were purchased from Cell Signaling Technology AntioxidantsAntioxidants2020 2020, 9, 9 1000, x FOR PEER REVIEW 3 of 18 19

mouse polyclonal cleaved caspase-9 and rabbit polyclonal cleaved caspase-3 were purchased from (Denver,Cell Signaling MA, USA). Technology Mouse bax (Denver, antibody MA, was USA). obtained Mouse from bax BD antibody Transduction was Laboratoriesobtained from (West BD Grove,Transduction PA, USA). Laboratories Rabbit amyloid (West precursor Grove, protein PA, USA). (APP) antibodyRabbit amyloid was purchased precursor from protein MyBioSource (APP) (Sanantibody Diego, was CA, purchased USA). Mouse from monoclonal MyBioSourceβ-actin (San and Diego, rabbit CA, polyclonal USA). Mouse bcl-2 antibodymonoclonal were β-actin provided and fromrabbit Sigma-Aldrich polyclonal Chemical.bcl-2 antibody Horseradish were provi peroxidaseded from (HRP)-conjugated Sigma-Aldrich goat Chemical. anti-rabbit Horseradish IgG, goat anti-mouseperoxidase and (HRP)-conjugated donkey anti-goat goat IgG anti-rabbit were received IgG, from goat Jackson anti-mouse ImmunoResearch and donkey anti-goat Laboratories IgG (Westwere Grove,received PA, from USA). Jackson ImmunoResearch Laboratories (West Grove, PA, USA). EucalyptolEucalyptol (Figure (Figure1 A)1A) was was dissolved dissolved inin dimethyl sulfoxide sulfoxide (DMSO, (DMSO, <0.5%<0.5% concentration) concentration) for forexperiments. experiments.

FigureFigure 1. 1.Chemical Chemical structure structure of of eucalyptol eucalyptol ( A(A),), toxicity toxicity of of eucalyptol-treated eucalyptol-treated humanhuman retinalretinal pigmentpigment epithelialepithelial (RPE) (RPE) cells cells (B ),(B inhibitory), inhibitory eff ectseffects ofeucalyptol of eucalyptol on proliferationon proliferation of glucose-exposed of glucose-exposed RPE cellsRPE (Ccells), temporal (C), temporal responses responses of epithelial of epithelial induction induction of ZO-1 and of MMP-2ZO-1 and (D) andMMP-2 effects (D) of and eucalyptol effects onof tighteucalyptol junction on proteins tight junction of ZO-1, proteins occludin-1, of ZO-1, MMP-2 occludin-1, and MMP-9 MMP-2 injured and byMMP- glucose9 injured (E and byF ).glucose An MTT (E assayand wasF). An conducted MTT assay for measurementwas conducted of for cell measurement viability (B and of Ccell, 100% viability viability (B and with C 5.5, 100% mM viability glucose control).with 5.5 Bar mM graphs glucose for viabilitycontrol). (meanBar graphsSEM, forn viability= 5) was (mean expressed ± SEM, as the n percentage= 5) was expressed of cell survival, as the ± comparedpercentage to theof cell glucose survival, control. compared Cell lysates to the wereglucose subject control. to western Cell lysates blot analysiswere subject with to a primarywestern antibodyblot analysis against with ZO-1, a primary occludin-1, antibody MMP-2, against MMP-9 ZO-1, and occludin-1,β-actin for MMP-2, a cellular MMP-9 internal and control β-actin ( Dfor–F a). Barcellular graphs internal (mean controlSEM, (nD=–F3)). inBar the graphs panels (mean represent ± SEM, densitometric n = 3) in the results panels of represent blot bands. densitometric * Values in ± respectiveresults of bar blot graphs bands. indicate * Values a in significant respective di barfference graphs at indicatep < 0.05. a significant difference at p < 0.05.

2.2.2.2. RPE RPE Cell Cell Culture Culture PrimaryPrimary human human RPE RPE cells cells (Lonza) (Lonza) were were cultured cultured in DMEMin DMEM containing containing 100 U100/mL U/mL penicillin, penicillin, and 100 µg/mL streptomycin at 37 C humidiﬁed atmosphere of 5% CO in air. To mimic a hyperglycemic and 100 μg/mL streptomycin◦ at 37 °C humidified atmosphere2 of 5% CO2 in air. To mimic a microenvironment,hyperglycemic microenvironment, RPE cells were incubated RPE cells inwere media incubated containing in media 33 mM containing D-glucose 33 mM in the D-glucose absence andin presencethe absence of 1–20and µpresenceM eucalyptol of 1–20 for μ upM eucalyptol to 5 days. Additionally,for up to 5 days. RPE Additionally, cells were cultured RPE cells in media were containingcultured in 5.5 media mM D-glucosecontaining as5.5 a mM glucose D-glucose control, as or a glucose 27.5 mM control, mannitol or 27.5 (+5.5 mM mM mannitol D-glucose) (+5.5 as mM an osmoticD-glucose) control. as an In osmotic another setcontrol. of experiments, In another RPEset of cells experiments, were incubated RPE cells for 4were days incubated in media containingfor 4 days 5 µinMA mediaβ in containing the presence 5 μ ofM 1–20Aβ inµ theMeucalyptol. presence of 1–20 μM eucalyptol. TheThe RPERPE cellcell viabilityviability waswas determineddetermined withwith MTTMTT (3-(4,5-dimethylthiazol-2-yl)-2,5-(3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltertrazoliumdiphenyltertrazolium bromide) bromide) [30 ,[30,31]31]. Eucalyptol. Eucalyptol at the at doses the ofdoses 1–20 ofµM 1–20 did notμM show did cytotoxicitynot show Antioxidants 2020, 9, 1000 4 of 19

(Figure1B). The RPE cell growth was observed in 33 mM glucose-containing media for 4 days (Figure1C). However, eucalyptol at 1–20 µM tended to lessen hyperglycemic proliferation.

2.3. In Vivo Animal Experiments All the experiments were approved by the Committee on Animal Experimentation of Hallym University and conducted in compliance with the University’s Guidelines for the Care and Use of Laboratory Animals (hallymR1 2016-10). Adult male db/db mice (C57BLKS/+Leprdb Iar; Jackson Laboratory, Sacramento, CA, USA) and their age-matched non-diabetic db/m littermates (C57BLKS/J; Jackson Laboratory, Sacramento, CA, USA) were introduced in the present study [30,31]. Mice were raised at 23 1 C with 50 10% relative ± ◦ ± humidity (12 h light/12 h dark cycle) under speciﬁc pathogen-free conditions, and supplied with water ad libitum at the animal facility of Hallym University. Animals were divided into three subgroups (n = 9–10 for each subgroup), as described in previous studies [30,31]. The ﬁrst group of mice was non-diabetic db/m control mice, and db/db mice were divided into two groups. One group of db/db mice was daily supplied 10 mg/kg BW eucalyptol via gavage for 8 weeks. The fasting blood glucose levels and glycated hemoglobin were measured every other week from mouse tail veins during the 8 week-supplementation of eucalyptol [30]. The plasma insulin level was reduced in eucalyptol-challenged db/db mice [30].

2.4. Western Blot Analysis Western blot analysis was carried out using whole cell lysates prepared in RPE cells (3.5 105 × cells/culture dish) and eye tissue extracts. Whole cell lysates and eye tissue extracts were prepared in a lysis buffer [30,31]. Cell lysates and tissue extracts containing equal amounts of proteins were electrophoresed on 8–15% SDS-PAGE and transferred onto a nitrocellulose membrane. Blocking nonspecific binding was achieved by using either 3% fatty acid-free bovine serum albumin (BSA) or 5% nonfat dry skim milk for 3 h. The membrane was incubated overnight at 4 ◦C with each primary antibody of the target proteins (dilution in 5% BSA, 1:1000) and washed in a Tris-buffered saline-Tween 20 for 10 min. The membrane was then incubated for 1 h with a secondary antibody conjugated to HRP (dilution in 3% BSA, 1:5000). Target protein levels were determined with immobilon Western chemiluminescent HRP substrate (Millipore, Merck KGaA, Darmstadt, Germany). Incubation with mouse monoclonal β-actin antibody was also conducted for comparison.

2.5. Hemotoxylin and Eosin (H&E) Staining For the histological observation, eye tissue specimens were fixed in 4% buffered-formaldehyde. The paraffin-embedded eye tissue sections were deparaffinized and stained with H&E for 3 min and dehydrated in 95% alcohol. The H&E-stained tissue sections were observed using an optical microscope Axio Iimager system equipped with fluorescence illumination (Zeiss, Gottingen, Germany). Five images were captured for each tissue section.

2.6. RT-PCR Analysis Total RNA was prepared from RPE cells using a Trizol reagent kit (Molecular Research Center, Cincinnati, OH, USA). The RNA (5 µg) was reversibly transcribed with 200 units of reverse transcriptase (Promega Co., Madison, WI, USA) and 0.5 mg/mL oligo-(dT)15 primer (Bioneer, Daejeon, Korea). The RT-PCR analysis was carried out for semi-quantifying the mRNA transcript levels of ZO-1 and MMP-2. The PCR condition for ZO-1 [50-AGCCTGCAAAGCCAGCTCA-30 (forward), 50-AGTGGCCTGGATGGGTTCATAG-30 (reverse, 104 bp)] and the condition for MMP-2 [50-TGGCAAGTACGGCTTCTGTC-30 (forward), 50-TTCTTGTCGCGGTCGTAGTC-30 (reverse, 180 bp)] were 94 ◦C (5 min), with 30 cycles at 94 ◦C (30 s), 55 ◦C (30 s) and 72 ◦C (30 s). The housekeeping gene of β-actin [50-GACTACCTCATGAAGATC-30 (forward), 50-GATCCACATCTGCTGGAA-30 (reverse, 500 bp)] was used for the co-ampliﬁcation with respective gene. Antioxidants 2020, 9, 1000 5 of 19

2.7. ROS Generation Dihydroethidium (DHE, Invitrogen, Carlsbad, CA, USA) staining was conducted for ROS production in RPE cells. RPE cells (0.5 104 cells/slide) were fixed with 4% formaldehyde for 10 min × and permeated by 0.1% Triton-X100 for 10 min on ice. RPE cells were stained by incubating with 20 µM DHE for 1 h. For the identification of nuclei, 40,6-diamidino2-phenylindole (DAPI) was treated for 10 min. Stained cells on slides were mounted in a mounting solution. In addition, eye tissue specimens were fixed. The eye tissue sections (10 µm thickness) were stained with DHE. Slide images and stained tissue sections were photographed using an optical microscope system. Oxidant generation in RPE cells was also assessed by staining of cell permeant reagent 2’,7’-dichlorofluorescin diacetate (DCF-DA). After challenge with 33 mM glucose, cells were loaded for 30 min with 10 µM DCF-DA in pre-warmed DMEM. After dye loading at 37 ◦C, the cells were washed thoroughly in phosphate-buffered saline, and the nuclear DAPI staining was performed. The culture images were photographed with an optical microscope system.

2.8. Immunocytochemical Staining RPE cells (0.5 104 cells/slide) were ﬁxed with 4% formaldehyde for 10 min, permeated in 0.1% × Triton-x 100 for 10 min on ice and blocked with a 4% FBS for 1 h. Immunoﬂuorescent cytochemical staining of Aβ in RPE cells was performed using its primary antibody and FITC-conjugated IgG. DAPI was used for the nuclear staining. Images on slides were taken using an optical microscope system.

2.9. Hoechst 33258 Staining After the ﬁxation of RPE cells for 15 min on a glass-covered 24 well plates, 10 µg/mL Hoechst 33258 (Promega Co., Madison, WI, USA) was treated for the nuclear staining. Cells with fragmented or condensed nuclei were considered to be apoptotic. Each slide image was taken for detecting nuclear morphology with an optical microscope system.

2.10. Data Analysis The data are presented as mean SEM for each treatment group in all the experiments. Statistical ± analyses were carried out using Statistical Analysis Systems (SAS Institute, Cary, NC, USA). Significance was evaluated by one-way analysis of variance and the following Duncan range test for multiple comparisons. Differences were regarded to be significant at p < 0.05.

3. Results

3.1. Inhibition of Glucose-Induced Loss of Epithelial Junction Proteins by Eucalyptol Tight junctions enable the RPE monolayer to form the outer blood-retinal barrier that regulates solute transports between the fenestrated choroid capillaries and the photoreceptor layer of the retina [10,11]. The current study investigated that eucalyptol inhibited loss of RPE tight junction proteins under diabetic condition. The induction of the tight junction marker of ZO-1 was attenuated in RPE cells exposed to 33 mM glucose for 5 days in a temporal manner (Figure1D). When 1–20 µM eucalyptol was added to glucose-loaded RPE cells for 4 days, the epithelial induction of ZO-1 and occludin-1 was highly reversed (Figure1E). The MMP proteins cause barrier disruption via aberrant proteolysis of epithelial tight junction proteins, leading to visual dysfunctions [13,32]. The induction of the MMP-2 was temporally enhanced in glucose-loaded RPE cells (Figure1D). In contrast, the glucose-induced expression of epithelial MMP-2 and MMP-9 was dose-dependently attenuated by treating eucalyptol to RPE cells (Figure1F). Accordingly, eucalyptol may block barrier disruption in diabetic retina through dampening the MMP induction. Antioxidants 2020, 9, x FOR PEER REVIEW 6 of 18

(Figure 1F). Accordingly, eucalyptol may block barrier disruption in diabetic retina through dampening the MMP induction.

3.2. Blockade of Diabetic Disruption of Blood-Retinal Barrier by Eucalyptol Aberrant alterations of the blood-retinal barrier are involved in the development of macular edema [4,6]. Consistent with the RPE cell culture results (Figure 1E,F), the in vivo animal data Antioxidantssupported2020 ,that9, 1000 eucalyptol inhibited diabetic disruption of cell-to-cell tight junction.6 ofThe 19 FITC-immunohistochemical data revealed that the ZO-1 induction (yellow arrows) declined in diabetic retinal tissues of mice (Figure 2A). Oral administration of 10 mg/kg eucalyptol restored the 3.2.ZO-1 Blockade induction of Diabetic reduced Disruption in diabetic of Blood-Retinal retinal tissues Barrier (Figure by Eucalyptol 2A). Consistently, the western blot data showedAberrant that alterationseucalyptol induced of the blood-retinal the retinal expressi barrieron are of involved ZO-1 and in theoccludin-1 development in diabetic of macular animals edema(Figure [ 42B).,6]. When Consistent 10 mg/kg with eucalyptol the RPE was cell ora culturelly supplemented results (Figure to 1db/dbE,F), mice, the inthe vivo inductionanimal of dataMMP-2 supported and MMP-9 that eucalyptolwas diminished inhibited (Figure diabetic 2C). disruptionThus, eucalyptol of cell-to-cell may block tight the junction. breakdown The of FITC-immunohistochemicalblood-retinal barrier of RPE. data revealed that the ZO-1 induction (yellow arrows) declined in diabeticHistological retinal tissues alterations of mice of (Figure RPE 2areA). major Oral administrationfactors causing of disrupti 10 mg /onkg of eucalyptol ocular malfunction restored the of ZO-1visual induction cycle in reducedDR [33]. inThe diabetic detachment retinal of tissues Bruch’ (Figures membrane2A). Consistently, from RPE cells the was western observed blot datain eye showedtissues thatof db/db eucalyptol mice,induced evidenced the by retinal the expressionhistological of H&E ZO-1 staining and occludin-1 (Figure in2D). diabetic However, animals oral (Figureadministration2B). When of 10 10 mg mg/kg/kg eucalyptol eucalyptol was apparently orally supplemented inhibited tosuch db/ dbpathological mice, the inductiondetachment. of MMP-2Accordingly, and MMP-9 eucalyptol was may diminished ameliorate (Figure diabetic2C). degeneration Thus, eucalyptol of RPE may and block Bruch’s the membrane breakdown for of the blood-retinalrobust blood-retinal barrier of barrier. RPE.

FigureFigure 2. 2. Immunohistochemical data data (A (A) )and and western western blot blot data data (B (andB and C) showingC) showing eye tissue eye tissue levels levelsof ZO-1, of ZO-1, occludin-1, occludin-1, MMP-2 MMP-2 and and MMP-9 MMP-9 in in eu eucalyptol-supplementedcalyptol-supplemented dbdb/db/db mice.mice. ForFor thethe immunohistochemicalimmunohistochemical analysis analysis of of ZO-1, ZO-1, green green FITC-conjugated FITC-conjugated secondary secondary antibody antibody was was used used for for visualizingvisualizing ZO-1 ZO-1 induction induction (A). ( YellowA). Yellow arrows arrows indicate indicate retinal retinal pigment pigment epithelial epithelial ZO-1. Tissue ZO-1. extracts Tissue wereextracts subject were to western subject blotto western analysis withblot aanalysis primary with antibody a primary against antibody ZO-1, occludin-1, against ZO-1, MMP-2, occludin-1, MMP-9 andMMP-2,β-actin MMP-9 for an internaland β-actin control for ( Banand internalC). The control bar graphs (B and (mean C). TheSEM, barn graphs= 3) represent (mean ± quantitative SEM, n = 3) ± results of blots in the panels. * Values in respective bar graphs indicate a signiﬁcant diﬀerence at p < 0.05. Inhibition of retinal pigment epithelial detachment from Bruch’s membrane by eucalyptol in db/db mice (D, black arrow). Histological sections of mouse retina were H&E-stained. Each microphotograph is representative of four mice (A and D). Scale bar: 200 µm.

Histological alterations of RPE are major factors causing disruption of ocular malfunction of visual cycle in DR [33]. The detachment of Bruch’s membrane from RPE cells was observed in eye tissues of db/db mice, evidenced by the histological H&E staining (Figure2D). However, oral administration of Antioxidants 2020, 9, x FOR PEER REVIEW 7 of 18

represent quantitative results of blots in the panels. * Values in respective bar graphs indicate a Antioxidants 2020, 9, 1000 7 of 19 significant difference at p < 0.05. Inhibition of retinal pigment epithelial detachment from Bruch’s membrane by eucalyptol in db/db mice (D, black arrow). Histological sections of mouse retina were 10 mgH&E-stained./kg eucalyptol Each apparently microphotograph inhibited is suchrepresentative pathological of four detachment. mice (A and Accordingly, D). Scale bar: eucalyptol200 μm. may ameliorate diabetic degeneration of RPE and Bruch’s membrane for the robust blood-retinal barrier. 3.3. Anti-Apoptotic Role of Eucalyptol in Diabetic Rpe Cells and Retina 3.3. Anti-Apoptotic Role of Eucalyptol in Diabetic Rpe Cells and Retina This study examined whether apoptosis of glucose-loaded RPE cells was responsible for loss of epithelialThis study tight examined junction whetherproteins. apoptosis In RPE ofcell glucose-loadeds exposed to RPE33 mM cells wasglucose, responsible the induction for loss ofof epithelialpro-apoptotic tight junctionbax and proteins.anti-apoptotic In RPE bcl-2 cells was exposed temporally to 33 mM reciprocal glucose, the(Figure induction 3A). The of pro-apoptotic induction of baxbax andand anti-apoptoticbim by glucose bcl-2 was wasreduced temporally by treating reciprocal 1–20 μM (Figure eucalyptol3A). The to RPE induction cells for of 4 bax days and (Figure bim by3B). glucose On the was contrary, reduced the by treatingreduced 1–20 bcl-2µ Minduct eucalyptolion was to restored RPE cells in for eucalyptol-added 4 days (Figure3B). RPE On thecells contrary,exposed theto glucose reduced (Figure bcl-2 induction 3B). In addition, was restored the ac intivation eucalyptol-added of caspase-3 RPEand cellscaspase-9 exposed was to enhanced glucose (Figurein diabetic3B). InRPE addition, cells, which the activation was highly of caspase-3 attenuated and in caspase-9 20 μM eucalyptol-treated was enhanced in diabetic RPE cells RPE (Figure cells, which3C). was highly attenuated in 20 µM eucalyptol-treated RPE cells (Figure3C).

FigureFigure 3. 3.Time Time course course responsesresponses of of induction induction of of bax bax andand bcl-2bcl-2 ((AA),), and and reciprocal reciprocal e effectsffects of of eucalyptol eucalyptol onon induction induction ofof bax, bax, bim, bim, bcl-2, bcl-2, cleaved cleaved caspase-3 caspase-3 andand cleavedcleaved caspase-9caspase-9 (B(Band andC C).). RPE RPE cell cell lysates lysates werewere subject subject to westernto western blot blot analysis analysis with awith primary a pr antibodyimary antibody against bax,against bcl-2, ba bim,x, bcl-2, cleaved bim, caspase-3, cleaved cleaved caspase-9 and β-actin as an internal control. The bar graphs (mean SEM, n = 3) represent caspase-3, cleaved caspase-9 and β-actin as an internal control. The bar graphs± (mean ± SEM, n = 3) quantitative results of blots in the panels. * Values in respective bar graphs indicate a significant represent quantitative results of blots in the panels. * Values in respective bar graphs indicate a difference at p < 0.05. significant difference at p < 0.05. This study further confirmed that retinal epithelial apoptosis led to diabetes-associated injury of epithelial tight junction and blood-retinal barrier. As expected, the tissue bax level was elevated Antioxidants 2020, 9, x FOR PEER REVIEW 8 of 18

Antioxidants 2020, 9, 1000 8 of 19 This study further confirmed that retinal epithelial apoptosis led to diabetes-associated injury of epithelial tight junction and blood-retinal barrier. As expected, the tissue bax level was elevated in indiabetic diabetic eyes, eyes, while while the the bcl-2 bcl-2 level level was was abolishe abolishedd (Figure (Figure 4A).4A). However, thethe inductioninduction ofof thesethese apoptosis-relatedapoptosis-related proteinsproteins was was reciprocally reciprocally restored restored by supplementing by supplementing 10 mg /10kg eucalyptolmg/kg eucalyptol to diabetic to mice.diabetic Consistently,eucalyptol mice. Consistently, eucalyptol reduced the reduced expression the ofexpression mitochondrial of mitochondrial apoptotic proteins apoptotic of cytochrome proteins Cof andcytochrome Apaf-1 elevated C and inApaf-1 diabetic elevated eyes (Figure in diabetic4B). Furthermore, eyes (Figure the4B). activation Furthermore, of both the caspase-3 activation and of caspase-9both caspase-3 in eye and tissues caspase-9 of db /dbin ey micee tissues were of diminished db/db mice by were providing diminished 10 mg by/kg providing eucalyptol 10 to mg/kg these animalseucalyptol (Figure to these4C). animals (Figure 4C).

FigureFigure 4.4. InhibitoryInhibitory eeffectsffects ofof eucalyptoleucalyptol onon retinalretinal apoptosisapoptosis inin diabeticdiabetic eyes.eyes. RetinalRetinal corticalcortical tissuetissue extractsextracts werewere subjectsubject to to western western blot blot analysis analysis with with a a primary primary antibody antibody against against bax, bax, bcl-2, bcl-2, cytochrome cytochrome C, Apaf-1,C, Apaf-1, cleaved cleaved caspase-3, caspase-3, cleaved cleaved caspase-9 caspase and-9 βand-actin β-actin for an for internal an internal control control (A–C). The(A–C bar). The graphs bar (mean SEM, n = 3) represent the quantitative results of blots in the panels. * Values in respective bar graphs± (mean ± SEM, n = 3) represent the quantitative results of blots in the panels. * Values in graphsrespective indicate bar graphs a significant indicate di ffaerence significant at p

FigureFigure 5.5. InhibitionInhibition ofof ROSROS productionproduction byby eucalyptoleucalyptol inin RPERPE cellscells ((AA andand BB)) andand inin retinaretina ((CC).). ForFor thethe measurementmeasurement ofof ROSROS production,production, thethe DHEDHE stainingstaining was conducted in RPE cells (A) and in retina ( C). INL,INL, innerinner nuclearnuclear layer;layer; ONL,ONL, outerouter nuclearnuclear layer;layer; PRL,PRL, photophoto receptorreceptor layer;layer; RPE,RPE, retinalretinal pigmentpigment epitheliumepithelium monolayer.monolayer. TheThe yellowyellow arrowsarrows indicateindicate RPERPE layer.layer. Furthermore,Furthermore, thethe DCF-DADCF-DA stainingstaining forfor ROSROS productionproduction ((BB)) werewere carriedcarried outout inin eucalyptol-treatedeucalyptol-treated RPERPE cells,cells, andand thethe DCFDCF stainingstaining intensityintensity (mean(mean ± SEM, nn == 3) was measured. Nuclear staining was donedone withwith 44′,6-diamidino-2-phenylindole.,6-diamidino-2-phenylindole. ± 0 DHE-DHE- oror DCF-stainedDCF-stained RPERPE cellscells oror retinaretina werewere visualizedvisualized underunder fluorescentfluorescent microscopy. Scale bars: 50–20050–200 µμm.m. ** BarBar graphgraph valuesvalues indicateindicate aa significantsignificant didifferencefference atatp p< < 0.05.0.05.

3.5.3.5. Inhibitory EEffectsﬀects ofof EucalyptolEucalyptol onon Glucose-InducedGlucose-Induced aaββ FormationFormation BasedBased onon direct direct evidence evidence that th diabetesat diabetes mellitus mellitus causes caus anes increase an increase in the in Aβ thepeptide Aβ peptide levels [ 20levels,21], this[20,21], study this examined study examined whether whether glucose perglucose se induced per se Ainducedβ formation Aβ formation in RPE cells, in RPE which cells, was which deterred was bydeterred treating by eucalyptol. treating eucalyptol. When RPE When cells were RPE exposedcells were to 33exposed mM glucose to 33 formM 5 days,glucose the for cellular 5 days, levels the ofcellular APP andlevels Aβ ofwere APPtemporally and Aβ were prompted, temporally with prompted, 50–60% increasewith 50–60% of these increase proteins of these by glucose proteins for by 4 daysglucose (Figure for 64A). days However, (Figure such 6A). an However, increase wassuch reversed an increase by treating was reversed 1–20 µM by eucalyptol, treating 1–20 in which μM theeucalyptol, cellular levelsin which of APP the and cellular Aβ were levels signiﬁcantly of APP reduced and inAβ a dose-dependentwere significantly manner reduced (Figure in6B). a Indose-dependent addition, western manner blot data(Figure revealed 6B). In that addition, eye tissue west levelsern blot of APPdata andrevealed Aβ were that elevated eye tissue in levels diabetic of miceAPP and (Figure Aβ 6wereC). When elevated 10 in mg diabetic/kg eucalyptol mice (Figure was 6C). orally When given 10 tomg/kg these eucalyptol animals forwas 8 orally weeks, given the inductionto these animals of these for proteins 8 weeks, was the diminished induction inof dbthese/db proteins mouse eyes. was Furthermore,diminished in thedb/db serum mouse Aβ leveleyes. enhancedFurthermore, in diabetic the serum mice A wasβ level reduced enhanced by supplying in diabetic 10 mgmice/kg was eucalyptol reduced to by the supplying animals (Figure 10 mg/kg6C). eucalyptol to the animals (Figure 6C). The FITC-green staining data of Aβ confirmed that eucalyptol reduced the epithelial induction of Aβ enhanced by glucose (Figure 6B). There was weak cytoplasmic green-staining in RPE cells under glucose control condition, while the 4 day-glucose stimulation caused a strong FITC staining of Aβ (Figure 6D). Thus, eucalyptol may inhibit diabetes-associated RPE amyloidosis, ultimately leading to disruption of the blood-retinal barrier. Antioxidants 2020, 9, 1000 10 of 19 Antioxidants 2020, 9, x FOR PEER REVIEW 10 of 18

Figure 6. Temporal induction of APP and Aβ (A) and the inhibitory effects of eucalyptol on their Figure 6. Temporal induction of APP and Aβ (A) and the inhibitory effects of eucalyptol on their induction (B–D). RPE cell lysates, eye tissue extracts and sera were subject to western blot analysis induction (B–D). RPE cell lysates, eye tissue extracts and sera were subject to western blot analysis with a primary antibody against APP, Aβ, and β-actin for an internal control. The bar graphs (mean ± with a primary antibody against APP, Aβ, and β-actin for an internal control. The bar graphs (mean SEM, n = 3) represent quantitative results of blot bands (A–C). * Values in respective bar graphs SEM, n = 3) represent quantitative results of blot bands (A–C). * Values in respective bar graphs indicate± a significant difference at p < 0.05. For the immunocytochemical analysis of Aβ, green indicate a significant difference at p < 0.05. For the immunocytochemical analysis of Aβ, green FITC-conjugated secondary antibody was used for visualizing Aβ induction, being counterstained FITC-conjugated secondary antibody was used for visualizing Aβ induction, being counterstained with 4′,6-diamidino-2-phenylindole for the nuclear staining (D). Each microphotograph (mean ± with 40,6-diamidino-2-phenylindole for the nuclear staining (D). Each microphotograph (mean SEM, SEM, n = 3) was obtained by using a microscope system. Scale bar: 50 μm. ± n = 3) was obtained by using a microscope system. Scale bar: 50 µm.

3.6. SuppressiveThe FITC-green Effects stainingof Eucalyptol data ofon Aaββ-Inducedconﬁrmed Disruption that eucalyptol of Tight reducedJunctions the epithelial induction of Aβ enhancedWhen 0.25–5 by glucoseμM Aβ (Figure was loaded6B). There to RPE was cells, weak no cytoplasmic noticeable green-staining RPE cytotoxicity in RPE was cells observed under (Figureglucose 7A). control The condition, induction whileof ZO-1 the and 4 day-glucose occludin-1 stimulationwas gradually caused diminished a strong in FITC 5 μ stainingM Aβ-loaded of Aβ RPE(Figure cells6D). in Thus,a temporal eucalyptol manner may inhibit(Figure diabetes-associated7B). In contrast, 1–20 RPE μ amyloidosis,M eucalyptol ultimately dose-dependently leading to prompteddisruption the of theexpression blood-retinal of these barrier. proteins dampened by 5 μM Aβ (Figure 7C). On the other hand, this study found that the secretion of MMP-2 and MMP-9 was upregulated in Aβ-exposed RPE cells, 3.6. Suppressive Eﬀects of Eucalyptol on aβ-Induced Disruption of Tight Junctions which was reduced by adding 1–20 μM eucalyptol to these cells (Figure 7D). Furthermore, nontoxic eucalyptolWhen enhanced 0.25–5 µMA the ZO-1β was transcription loaded to RPE reduced cells, by no A noticeableβ, indicating RPE that cytotoxicity the ZO-1 wasinduction observed by eucalyptol(Figure7A). was The regulated induction ofat ZO-1its transcriptional and occludin-1 levels was gradually(Figure 7E). diminished In contrast, in 5 µtheMA transcriptionalβ-loaded RPE levelcells inof a temporalMMP-2 elevated manner (Figureby Aβ7B). was In contrast,downregulated 1–20 µM by eucalyptol the presence dose-dependently of eucalyptol prompted in a dose-dependentthe expression ofmanner. these proteins dampened by 5 µMAβ (Figure7C). On the other hand, this study found that the secretion of MMP-2 and MMP-9 was upregulated in Aβ-exposed RPE cells, which was reduced by adding 1–20 µM eucalyptol to these cells (Figure7D). Furthermore, nontoxic eucalyptol enhanced the ZO-1 transcription reduced by Aβ, indicating that the ZO-1 induction by eucalyptol was regulated at its transcriptional levels (Figure7E). In contrast, the transcriptional level of MMP-2 elevated by Aβ was downregulated by the presence of eucalyptol in a dose-dependent manner. Antioxidants 2020, 9, 1000 11 of 19 Antioxidants 2020, 9, x FOR PEER REVIEW 11 of 18

FigureFigure 7.7. Dose-responsesDose-responses ofof AAββ toto humanhuman retinalretinal pigmentpigment epithelialepithelial (RPE)(RPE) cellcell toxicitytoxicity ((AA),), temporaltemporal inductioninduction ofof tighttight junctionjunction markersmarkers ofof ZO-1ZO-1 andand occludin-1occludin-1 byby AAββ ((BB)) andand eeffectsffects ofof eucalyptoleucalyptol onon cellularcellular inductioninduction of of ZO-1 ZO-1 and and occludin-1 occludin-1 and and secretion secretion of MMP-2 of MMP-2 and MMP-9 and MMP-9 (C and D(C). and Cell viabilityD). Cell wasviability measured was measured by using by an using MTT an assay MTT (A, assay 100% (A, viability100% viability with untreatedwith untreated control). control). Bar graphsBar graphs for viability (mean SEM, n = 5) was expressed as percent cell survival. RPE cell lysates were subject for viability (mean± ± SEM, n = 5) was expressed as percent cell survival. RPE cell lysates were subject β toto westernwestern blotblot analysisanalysis usingusing aa primaryprimary antibodyantibody ofof ZO-1,ZO-1, occludin-1,occludin-1, MMP-2,MMP-2, MMP-9MMP-9 andand β-actin-actin for an internal control (B–D). The bar graphs (mean SEM, n = 3) represent quantitative results of for an internal control (B–D). The bar graphs (mean± ± SEM, n = 3) represent quantitative results of blotblot bands.bands. ** ValuesValues inin respectiverespective barbar graphsgraphs indicateindicate aa significantsignificant didifferencefference at atp p< <0.05. 0.05. TheThe mRNAmRNA β transcriptionaltranscriptional levels levels of of ZO-1 ZO-1 and and MMP-2 MMP-2 were were measured measured using usin RT-PCRg RT-PCR analysis. analysis.-Actin β-Actin was was used used as aas housekeeping a housekeeping gene gene for for the the co-amplification co-amplification with with ZO-1 ZO-1 and and MMP-2 MMP-2 (E). (E). 3.7. Blockade of aβ-Induced Apoptosis by Eucalyptol 3.7. Blockade of aβ-Induced Apoptosis by Eucalyptol This study examined whether Aβ stimulated apoptotic death of RPE cells, which was inhibited by This study examined whether Aβ stimulated apoptotic death of RPE cells, which was inhibited supplementing eucalyptol. Consistent with 33 mM glucose-loading data (Figure3A), A β influenced by supplementing eucalyptol. Consistent with 33 mM glucose-loading data (Figure 3A), Aβ the expression of bax and bcl-2 proteins in RPE cells (Figure8A). However, 1–20 µM eucalyptol influenced the expression of bax and bcl-2 proteins in RPE cells (Figure 8A). However, 1–20 μM dose-dependently restored the induction of anti-apoptotic bcl-2 dampened by amyloid-β, while the eucalyptol dose-dependently restored the induction of anti-apoptotic bcl-2 dampened by amyloid-β, cellular level of pro-apoptotic bax was reduced in eucalyptol-supplied RPE cells. In addition, treating while the cellular level of pro-apoptotic bax was reduced in eucalyptol-supplied RPE cells. In RPE cells with eucalyptol reduced the induction of cytochrome C and Apaf-1 by Aβ (Figure8B). addition, treating RPE cells with eucalyptol reduced the induction of cytochrome C and Apaf-1 by Furthermore, Aβ highly activated caspase-3 and caspase-9 in RPE cells, which was significantly Aβ (Figure 8B). Furthermore, Aβ highly activated caspase-3 and caspase-9 in RPE cells, which was diminished in 1 µM eucalyptol-treated RPE cells (Figure8C). significantly diminished≥ in ≥1 μM eucalyptol-treated RPE cells (Figure 8C). Antioxidants 2020, 9, 1000 12 of 19 Antioxidants 2020, 9, x FOR PEER REVIEW 12 of 18

FigureFigure 8. 8.Blockade Blockade of of A Aββ-induced-induced apoptosis apoptosis byby eucalyptoleucalyptol inin humanhuman retinalretinal pigment pigment epithelial epithelial (RPE) (RPE) cells.cells. RPE RPE cell cell lysates lysates were were subject subject to westernto western blot bl analysisot analysis with with a primary a primary antibody antibody against against bax, bcl-2, bax, cytochromebcl-2, cytochrome C, Apaf-1, C, Apaf-1, cleaved cleave caspase-3,d caspase-3, cleaved cleaved caspase-9 caspase-9 and β-actin and forβ-actin an internal for an internal control (controlA–C). Bar(A–C graphs). Bar (mean graphs (meanSEM, n ±= SEM,3) in then = panels3) in the represent panels represent densitometric densitometric results of blotresults bands. of blot * Values bands. in * ± respectiveValues in barrespective graphs bar indicate graphs a signiﬁcant indicate a disignificantﬀerence at differencep < 0.05. at p < 0.05.

HoechstHoechst 3325833258 stainingstaining revealedrevealed thatthat 55 µμMAM Aββ highlyhighly elicitedelicited nuclearnuclear condensationcondensation andand fragmentationfragmentation ofof RPERPE cells,cells, proving that A Aββ promotedpromoted RPE RPE cell cell apoptosis apoptosis (Figure (Figure 9A).9A). In In contrast, contrast, 1– 1–2020 μµMM eucalyptol eucalyptol blocked blocked A Aββ-induced-induced nuclear nuclear condensation condensation of RPERPE cells.cells. ToTo determinedetermine whetherwhether AAββ-induced-induced retinal retinal epithelial epithelial apoptosis apoptosis entailed entailed ROS generation,ROS generation, superoxide superoxide production production was detected was asdetected DHE ﬂuorescence as DHE fluorescence in RPE cells in exposed RPE cells to A βexposedfor 4 days. to A Thereβ for was 4 days. strong There DHE was ﬂuorescence strong DHE in 5 µMAβ-loaded RPE cells (Figure9B). However, the supply of 1 µM eucalyptol to RPE cells attenuated fluorescence in 5 μM Aβ-loaded RPE cells (Figure 9B). However,≥ the supply of ≥1 μM eucalyptol to cellularRPE cells ROS attenuated generation. cellular ROS generation. Antioxidants 2020, 9, 1000 13 of 19 Antioxidants 2020, 9, x FOR PEER REVIEW 13 of 18

FigureFigure 9. 9.BlockadeBlockade of ofAβ A-inducedβ-induced nuclear nuclear condensation condensation (A (),A ),cellular cellular ROS ROS production production (B ()B and) and cellular cellular RAGERAGE induction induction (C (C–E–)E by) by eucalyptol eucalyptol in in human human retinal retinal pigm pigmentent epithelial epithelial (RPE) (RPE) cells. cells. Hoechst Hoechst 33258 33258 stainingstaining was was done done for for the the measurement measurement of nuclearnuclear condensationcondensation ofof RPE RPE cells cells exposed exposed to to 5 µ5MA μMβ A(βA ). (AFor). For the the measurement measurement of ROSof ROS production production (B), (B DHE), DHE staining staining was was conducted conducted in eucalyptol-treatedin eucalyptol-treated RPE RPEcells. cells. Nuclear Nuclear staining staining was done was with done 40 ,6-diamidino-2-phenylindole.with 4′,6-diamidino-2-phenylindole. Hoechst 33258-Hoechst and 33258- DHE-stained and RPE cells were visualized under fluorescent microscopy. Each microphotograph (mean SEM, n = 3) DHE-stained RPE cells were visualized under fluorescent microscopy. Each microphotograph± (mean ± wasSEM, obtained n = 3) was byusing obtained a microscope by using system.a microscope Scale bars: system. 50 µ Scalem. RPE bars: cell 50 lysates μm. RPE were cell subject lysates to western were subjectblot analysis to western with blot a primary analysis antibody with a againstprimary RAGE, antibody and against with β -actinRAGE, as and an internal with β-actin control as (C an–E ). Bar graphs (mean SEM, n = 3) in the panel represent densitometric results of blot bands. * Values in internal control (C–E±). Bar graphs (mean ± SEM, n = 3) in the panel represent densitometric results of blotrespective bands. * bar Values graphs in respective indicate a significantbar graphs diindicatefference a atsignificantp < 0.05. difference at p < 0.05. 3.8. Blockade of Aβ-RAGE Interaction by Eucalyptol 3.8. Blockade of Aβ-RAGE Interaction by Eucalyptol There is much evidence that the AGE-RAGE-oxidative stress axis place a role in diabetic vascular There is much evidence that the AGE-RAGE-oxidative stress axis place a role in diabetic complications [34]. We found that glucose strongly enhanced AGE formation in RPE cells (unpublished vascular complications [34]. We found that glucose strongly enhanced AGE formation in RPE cells data). RAGE is a multi-ligand receptor capable of binding diverse molecules including AGE and (unpublished data). RAGE is a multi-ligand receptor capable of binding diverse molecules including Aβ [35,36]. High glucose elevated the RAGE induction in RPE cells (Figure9C). In contrast, 1–20 µM AGE and Aβ [35,36]. High glucose elevated the RAGE induction in RPE cells (Figure 9C). In contrast, eucalyptol attenuated the retinal epithelial induction of RAGE enhanced by glucose. In addition, 1–20 μM eucalyptol attenuated the retinal epithelial induction of RAGE enhanced by glucose. In the eye tissue level of RAGE was elevated in db/db mice (Figure9D). When diabetic animals were addition, the eye tissue level of RAGE was elevated in db/db mice (Figure 9D). When diabetic administered with 10 mg/kg eucalyptol, the tissue level of RAGE was reduced. As expected, the RAGE animals were administered with 10 mg/kg eucalyptol, the tissue level of RAGE was reduced. As induction was promoted in Aβ-loaded RPE cells. When RPE cells were treated with 1 µM eucalyptol, expected, the RAGE induction was promoted in Aβ-loaded RPE cells. When RPE cells≥ were treated the epithelial induction of RAGE by Aβ was highly abrogated (Figure9E). with ≥1 μM eucalyptol, the epithelial induction of RAGE by Aβ was highly abrogated (Figure 9E).

4. Discussion Nine major findings were obtained from this study. (1) Nontoxic eucalyptol lessened the induction of APP and Aβ elevated in 33 mM glucose-loaded RPE cells and in db/db mouse eyes. (2) Antioxidants 2020, 9, 1000 14 of 19

4. Discussion Nine major ﬁndings were obtained from this study. (1) Nontoxic eucalyptol lessened the induction Antioxidants 2020, 9, x FOR PEER REVIEW 14 of 18 of APP and Aβ elevated in 33 mM glucose-loaded RPE cells and in db/db mouse eyes. (2) Eucalyptol restoredEucalyptol the restored induction the of induction the tight junctionof the tight proteins junction of ZO-1proteins and of occludin-1 ZO-1 and in occludin-1 RPE cells in diminished RPE cells µ β bydiminished glucose andby glucose 5 MA and. (3)5 μM Eucalyptol Aβ. (3) Eucalyptol attenuated attenuated the induction the induction of MMP-2 of andMMP-2 MMP-9 and MMP-9 in RPE µ β cellsin RPE by cells glucose by glucose and 5 andMA 5 .μ (4)M A Oralβ. (4) administration Oral administration of 10 mg of/ 10kg mg/kg eucalyptol eucalyptol restored restored retinal retinal tissue levelstissue oflevels ZO-1, of ZO-1, occludin-1, occludin-1, MMP-2 MMP-2 and MMP-9and MMP-9 in diabetic in diabetic mice, mice, accompanying accompanying the the inhibition inhibition of µ detachmentof detachment of of RPE RPE from from Bruch’s Bruch’s membrane. membrane. (5) (5) While While treating treating 1–20 1–20 μMM eucalyptoleucalyptol toto RPERPE cellscells β reducedreduced glucose-glucose- andand AAβ-induced-induced pro-apoptoticpro-apoptotic proteins ofof bax,bax, bimbim andand cleavedcleaved caspase-3caspase-3 andand cleavedcleaved caspase-9,caspase-9, the the anti-apoptotic anti-apoptotic bcl-2 bcl-2 was upregulatedwas upregulated in eucalyptol-treated in eucalyptol-treated RPE cells stimulatedRPE cells β bystimulated glucose orby A glucose. (6) Supplementing or Aβ. (6) Supplementing eucalyptol to eucalyptol diabetic mice to di reciprocallyabetic mice reversed reciprocally retinal reversed tissue levelsretinal oftissue apoptosis-related levels of apoptosis-related proteins of bcl-2, proteins bax, of cytochrome bcl-2, bax, cytochrome C, Apaf-1 caspase-3 C, Apaf-1 and caspase-3 caspase-9. and β (7)caspase-9. Eucalyptol (7) attenuatedEucalyptol theattenuated ROS generation the ROS in generation glucose- or in A glucose--exposed or RPEAβ-exposed cells and RPE in diabetic cells and RPE. in β (8)diabetic Submicromolar RPE. (8) eucalyptolSubmicromolar inhibited eucalyptol A -elicited inhibited nuclear condensationAβ-elicited nuclear and fragmentation condensation of RPEand β cells.fragmentation (9) Eucalyptol of RPE attenuated cells. (9) the Eucalyptol RPE induction attenuated of RAGE the enhanced RPE induction by glucose of RAGE and A enhancedand also by in dbglucose/db mouse and A eyes.β and All also together, in db/db these mouse results eyes. indicated All together that, eucalyptolthese results may indica ameliorateted that RPEeucalyptol injury β andmay barrier ameliorate integrity RPE ininjury diabetic and retinabarrier through integrity combating in diabetic A retina-triggered through ROS combating generation Aβ and-triggered RAGE inductionROS generation (Figure and 10). RAGE induction (Figure 10).

FigureFigure 10.10.Schematic Schematic ﬁgure figure outlining outlining the inhibitionthe inhibition of epithelial of epithelial tight junction tight disruptionjunction disruption by eucalyptol by followingeucalyptol exposure following of exposure glucose and of glucose Aβ to human and Aβ retinal to human pigment retinal epithelial pigment (RPE) epithelial cells. (RPE) cells.

TheThe RPERPE isis situatedsituated betweenbetween neuro-retinaneuro-retina andand choroidchoroid capillaries,capillaries, where it formsforms thethe outerouter blood-retinalblood-retinal barrier.barrier. Dynamic barrier functions of RPE are crucialcrucial forfor maintainingmaintaining optimaloptimal retinalretinal healthhealth [[1,3,4].1,3,4]. The tight junctions involving ZO-1 andand occludin-1occludin-1 proteinsproteins betweenbetween neighboringneighboring RPERPE cellscells areare anan integralintegral structuralstructural componentcomponent ofof thethe blood-retinalblood-retinal barrierbarrier [[4,11].4,11]. Loss of RPE cellcell toto cellcell junctionjunction andand impairmentimpairment ofof blood-retinalblood-retinal barrier are typical events in retinal degenerativedegenerative diseases,diseases, resultingresulting inin vascularvascular leakageleakage andand retinalretinal edemaedema in DR and RPRP [[2,7,8].2,7,8]. Furthermore, the RPERPE glucoseglucose transporttransport toto photoreceptors photoreceptors is is diminished diminished in RP,in RP and, and metabolic metabolic deregulation deregulation of the of blood-outer the blood-outer retinal barrierretinal occursbarrier [5occurs]. Accordingly, [5]. Accordingly, the maintenance the maintenance of the RPE of integritythe RPE isintegrity vital in is the vital visual in the functions. visual Thisfunctions. study foundThis study that thefound levels that of the ZO-1 levels and of occludin-1 ZO-1 and were occludin-1 greatly reducedwere greatly in diabetic reduced RPE in cells diabetic and inRPE db /cellsdb mouse and in eyes. db/db In addition,mouse eyes. the MMPIn addition, proteins the of RPEMMP were proteins upregulated of RPE in were glucose-loaded upregulated RPE in cellsglucose-loaded and in diabetic RPE mouse cells and eyes. in Elevateddiabetic expressionmouse eyes. of MMPElevated proteins expression in the retinaof MMP increases proteins vascular in the permeabilityretina increases by avascular mechanism permeability related to by proteolytic a mechanism decomposition related to ofproteolytic the tight junctiondecomposition proteins of [ 13the]. tight junction proteins [13]. A study reported that MMP-2-mediated degradation of occludin contributes to blood-brain barrier damage in early ischemic stroke [37]. In this study, the H&E staining showed that hyperglycemia resulted in an increase in separation between the RPE and the Bruch’s membrane in retina. On the other hand, several retinal cells experienced cell death in a diabetic environment [38]. This study showed that glucose instigated oxidative stress-mediated apoptotic signaling in RPE cells. In addition, hyperglycemia stimulated apoptotic bax signaling, Antioxidants 2020, 9, 1000 15 of 19

A study reported that MMP-2-mediated degradation of occludin contributes to blood-brain barrier damage in early ischemic stroke [37]. In this study, the H&E staining showed that hyperglycemia resulted in an increase in separation between the RPE and the Bruch’s membrane in retina. On the other hand, several retinal cells experienced cell death in a diabetic environment [38]. This study showed that glucose instigated oxidative stress-mediated apoptotic signaling in RPE cells. In addition, hyperglycemia stimulated apoptotic bax signaling, mitochondrial pathways of cytochrome C/Apaf-1 apoptosomes and proteolytic caspase activation in mouse eyes. Similarly, diabetic insult of high glucose promotes mitochondrial pathways of cell apoptosis in RPE by regulating ROS-mediated inhibition of mitophagy [39]. A recent report highlights the consequence of autophagy and oxidative stress as two related mechanisms involved in RP [40]. Identifying mechanisms of cell death will lead to a more targeted approach in the development of new therapies to treat DR and RP. Several retina pathologies, such as DR and secondary cone photoreceptor death in RP, may entail mitochondrial dysfunction [40]. Several studies have shown that naturally-occurring compounds inhibit oxidative stress-induced RPE cell damage through enhancing antioxidant activity and anti-apoptotic function [41,42]. For both RP and DR, botanical compounds and plant extracts may have significant effects on treatment and prevention [43]. Efficient treatment of RP can be antioxidant agents and autophagy modulators, which curtails the excessive ROS levels and regulates autophagy [40]. Diphlorethohydroxycarmalol and kaemperol protect RPE cells from oxidative stress-induced DNA damage and apoptosis [41,42]. Curcumin inhibits N-methyl-N-nitrosourea-induced photoreceptor cell apoptosis by attenuating DNA oxidative stress, indicating that curcumin may inhibit the onset and progression of human RP [44]. This study revealed that eucalyptol suppressed ROS generation and modulated apoptosis-related protein induction in RPE cells and eye tissues in parallel with cytochrome C/Apaf-1 apoptosome formation and proteolytic caspase activation under the glucotoxic condition. Accordingly, eucalyptol blocked hyperglycemia-triggered oxidative stress-mediated RPE apoptosis through disturbing the bax-caspase signaling pathway. Furthermore, eucalyptol ameliorated the glucose-damaged barrier function of RPE by restoration of loss of the tight junction-associated proteins of ZO-1 and occludin-1 and by inhibition of RPE detachment from Bruch’s membrane. Thus, it can be assumed that oxidative stress may be one of the pathways implicated in RPE barrier dysfunction and retinal degeneration promoted by glucose. However, whether the primary target of oxidative stress by hyperglycemia in DR is RPE cells and/or photoreceptors remains unclear. Retinal Müller cells are shown to undergo inflammation-driven cell death in the diabetic retina [38]. Considering that DR and RP are chronic inflammatory diseases [43], retinal inflammation-induced cell death might play an important role in the RPE barrier dysfunction. The RPE dysfunction in age-related macular degeneration entails the NLRP3 inflammasome activation induced by Aβ-responsive oxidative stress [26]. Unfortunately, this study did not examine whether glucose-induced retinal inflammation was responsible for RPE barrier dysfunction. It is shown that eucalyptol exerts anti-inflammatory activity [27]. A rich literature describes how hyperglycemia affects RPE-specific barrier functions [6,8]. Recent work identifies possible mechanisms for new targets and therapeutic strategies to reverse retinal dysfunction and blood-derived content leakage in DR and RP [5,45]. However, the underlying mechanisms still remain to be elucidated. Accumulating epidemiologic evidence suggests that people with diabetes mellitus are at increased risk of Alzheimer’s disease [46,47]. Neurodegenerative diseases such as Alzheimer’s disease and RP induce spontaneous cell death via same basic cell death mechanisms [17]. Several studies have reported a potential mechanistic linking between diabetes mellitus and increase in Aβ peptide levels [20,21]. A proteomic study reveals that amyloid processing pathways are enhanced in diabetic retinas without glial activation [48]. Thus, it was necessary to elucidate the relationship between hyperglycemia and Aβ accumulation in diabetic retina. This study found that APP and Aβ were induced in diabetic RPE cells and diabetic eyes, possibly contributing to retinal cell apoptosis and loss of tight junction proteins. Eucalyptol is reported to reduce Aβ-induced inflammation in differentiated PC12 cells and to act as an anti-inflammatory agent in neurodegenerative diseases including Alzheimer’s disease [49]. The treatment of Aβ-induced Antioxidants 2020, 9, 1000 16 of 19 impairment of RPE barrier with eucalyptol led to a new target and therapeutic strategy during glucotoxic conditions. One investigation shows that hyperglycemia inhibits APP degradation and enhances Aβ production via a reduction of APP turnover rate [50]. In fact, eucalyptol suppressed the generation of APP and Aβ and the RAGE induction in glucose-loaded RPE cells, and reduced the Aβ level locally in eyes and systemically in blood. Accordingly, it can be speculated that eucalyptol may increase Aβ degradation and elimination. In summary, this study attempted to determine the capability of eucalyptol in counteracting Aβ-mediated malfunction of RPE tight junction and barrier in glucose-loaded RPE and diabetic mouse eyes. Oral supplementation of eucalyptol inhibited the proteolytic MMP induction in diabetic eyes, thus maintaining a strong RPE adhesion to Bruch’s membrane. Nontoxic eucalyptol deterred glucose-mediated Aβ-induced loss of cell to cell junction proteins of RPE in parallel with inhibition of RAGE induction. Furthermore, eucalyptol inhibited the generation of APP and Aβ in diabetic retina, consequently leading to oxidative stress and caspase-dependent RPE apoptosis. Therefore, eucalyptol may be a potent retinoprotective agent ameliorating diabetes-associated blood-retinal barrier disruption via inhibition of Aβ-mediated oxidative stress-dependent apoptotic signaling pathway. The amyloid plaques in brains of patients with Alzheimer’s disease resemble the amyloid deposits in pancreatic islet of diabetic patients with decreased β-cell mass [51]. Elevated insulin level in diabetes promotes Aβ accumulation by competing with Aβ for insulin-decomposing enzyme [51,52]. Although further studies are needed, compromised glucose metabolism and insulin signaling in diabetic eyes may be attributed to Aβ accumulation and RPE barrier dysfunction.

Author Contributions: D.Y.K., M.-K.K. and Y.-H.K. designed the research; D.Y.K., M.-K.K., Y.-H.K. (Yun-Ho Kim), E.-J.L., H.O., S.-I.K., S.Y.O. and W.N. conducted the research; D.Y.K. and M.-K.K. analyzed the data; D.Y.K. and Y.-H.K. (Young-Hee Kang) wrote the paper. Y.-H.K. (Young-Hee Kang) had primary responsibility for final content. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by National Research Foundation of Korea (NRF) grants funded by the Korean government (2019R1A2C1003218 and 2017R1A6A3A04011473). Conflicts of Interest: The authors declare that they have no conflict of interest.

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