Arctigenin Prevents Retinal Edema in a Murine Retinal Vein Occlusion Model

Abstract

Macular edema causes vision loss in patients with retinal vein occlusion (RVO) and diabetic macular edema (DME). The intravitreal injection of anti-vascular endothelial growth factor (VEGF) agents is used for treatment; however, this therapy is invasive, and recurrence occurs in some cases. The establishment of a non-invasive treatment would help to solve these problems. Here, we focused on arctigenin, a lignan polyphenol found in burdock sprout, and has effects on inflammatory and microcirculatory when taken orally. We hypothesized that oral intake of arctigenin could be effective against retinal edema in RVO and DME. In this study, the degree of retinal edema by measuring the total retinal thickness using optical coherence tomography (OCT) and the thickness of the inner nuclear layer (INL) by hematoxylin–eosin (H&E) staining were investigated. Oral administration of arctigenin ameliorated retinal edema in an RVO murine model by inhibiting the decrease in occludin and vascular endothelial (VE)-cadherin. Moreover, in retinas with edema, arctigenin suppressed the induction of VEGF, tumor necrosis factor α (TNFα), and matrix metallopeptidase 9 (MMP9). Next, the effects of arctigenin on barrier function were assessed in human retinal microvascular endothelial cells (HRMECs) by measuring the trans-endothelial electrical resistance (TEER) and conducting fluorescein isothiocyanate (FITC)-dextran permeability assays. Arctigenin showed a protective effect against VEGF-induced barrier dysfunction. In addition, arctigenin inhibited the TNFα-mediated activation of the nuclear factor-kappaB (NF-κB)/p38 mitogen-activated protein kinase (MAPK) pathway. These results suggested that oral administration of arctigenin has beneficial effects on retinal edema by inhibiting vascular hyperpermeability in endothelial cells.

INTRODUCTION

Macular edema is the common cause of vision loss in patients with retinal vein occlusion (RVO)¹⁾ and diabetic macular edema (DME).²⁾ An accumulation of fluid from hyperpermeable blood vessels induces retinal edema. The fluid primarily accumulates in the inner nuclear layer (INL) and outer plexiform layer (OPL) in retinas with RVO resulting of visual dysfunction.³⁾ Vascular endothelial growth factor (VEGF) induces vascular permeability and anti-VEGF therapies are available to treat macular edema; however, intravitreal administration of anti-VEGF drugs is highly invasive.⁴⁾ Moreover, in some cases, anti-VEGF therapy needs to be administered continually due to the recurrence of macular edema.⁵⁾ Therefore, it was considered that the supplement which can be taken orally was useful as complementary therapy and preventive treatment of the anti-VEGF therapy.

Arctigenin (Fig. 1A) and its glycoside (arctiin) are bioactive lignans derived from Arctium lappa L. and Forsythia suspensa (Thunb.) Vahl. Arctigenin and arctiin have many therapeutic properties including anti-tumor and anti-inflammatory activities.^6,7) Since arctiin is metabolized into arctigenin by intestinal bacteria,^8,9) the physiological effects of arctiin in vivo may be attributed to the physiological activity of arctigenin. Arctigenin may affect the vascular endothelial cells because it has reported to suppresses tumor growth via inducing changes of vascular structure.¹⁰⁾ Arctigenin also enhances intestinal epithelial barrier function by upregulating the expression of tight junction proteins.¹¹⁾ These reports demonstrated the potential effects of arctigenin on inflammation and permeability in retinal microvascular endothelial cells. Interestingly, it has been reported that arctigenin acts in the brain after oral administration.¹²⁾ Therefore, oral administration of arctigenin may exhibit ocular effects.

Fig. 1. Oral Administration of Arctigenin (30, 100 mg/kg) Ameliorated Retinal Edema in a Murine RVO Model

(A) The chemical structure of arctigenin. (B) The protocol for investigating the effects of arctigenin on the intraretinal edema in RVO model mice. (C) The OCT images of the retinal layer. The graph shows the thickness of the whole retinal layer. The data represent the mean ± S.E.M. (n = 5–7). ^##p < 0.01 versus Normal group and, * p < 0.05 versus Vehicle group (Student’s t-test). (D) Typical images of retinas stained with hematoxylin and eosin. Scale bar = 5 µm. (E) Quantitation of the INL thickness in the retinas from normal, vehicle, and orally administered arctigenin (30, 100 mg/kg) treatment groups. The data represent the mean ± S.E.M. (n = 5–10). ^##p < 0.01 versus Normal group (Student’s t-test), * p < 0.05, and ** p < 0.01 versus Vehicle group (Dunnett’s test). Scale bar = 50 µm. INL, inner nuclear layer. ONL, outer nuclear layer. (F) Quantitation of the cell counts in the INL from the retinas from normal, vehicle, and orally administered arctigenin (30, 100 mg/kg) treatment groups. The data represent the mean ± S.E.M. (n = 5–10). ^##p < 0.01 versus Normal group (Student’s t-test), ** p < 0.01 versus Vehicle group (Dunnett’s test).

The blood–retinal barrier (BRB) is essential for maintaining the permeability of retinas and its disruption induced retinal edema.¹³⁾ The BRB is comprised of tight junctions and adherens junctions between retinal capillary endothelial cells.¹⁴⁾ Vascular endothelial (VE)-cadherin is an endothelial-specific adhesion molecule localized to the junctions between endothelial cells, and occludin is a tight junction component of endothelial and epithelial cells.^15,16) A decrease in VE-cadherin and occludin is involved in hyperpermeability and retinal edema.^17,18) Thus, the protection of these junction proteins may suppress retinal edema.

Inflammation is also involved in the formation of retinal edema.^19,20) Inflammatory cytokines such as tumor necrosis factor α (TNFα) and interleukin-6 (IL-6) contribute to an increase in vascular permeability.²¹⁾ The expression of inflammatory cytokines is increased in the eyes of RVO patients.²²⁾ In endothelial cells, TNFα induces an increase in vascular permeability though a reduction in VE-cadherin.²³⁾ TNFα induces the expression of matrix metalloproteinase 9 (MMP9), which disrupts the tight junction in endothelial cells such as occludin.^24,25) However, TNFα does not seem to be directly involved in retinal hyperpermeability induced by VEGF.^26,27) This suggests that the inhibition of both VEGF and inflammatory cytokine have synergistic effects on retinal edema. Therefore, the development of new treatment focusing on not only VEGF but inflammation cytokines are beneficially for treating macular edema.

Since arctigenin potentially affects inflammation and permeability in retinal microvascular endothelial cells, we hypothesized that arctigenin may be an effective non-invasive oral supplement for treating retinal edema. In order to address this hypothesis, we utilized an in vivo RVO mouse model and an in vitro vascular permeability model using human retinal microvascular endothelial cells (HRMECs). These studies revealed that arctigenin has protective effects against retinal vascular hyperpermeability. Thus, an arctigenin-based supplement or drug may be developed for the treatment of various vascular disorders, including RVO and DME.

MATERIALS AND METHODS

Animals

Male albino ddY mice (8 weeks old; 35–45 g body weight) were obtained from Japan SLC (Hamamatsu, Japan). The mice were housed under a 12 h light–dark cycle at 24 ± 2 °C and 55 ± 15% humidity and fed with free access to food (CLEA rodent diet CE-2; CLEA Japan, Inc., Tokyo, Japan) and water. Experimental procedures were consistent with the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research. All experiments were approved and monitored by the Institutional Animal Care and Use Committee of Gifu Pharmaceutical University (Approval No.2020-043).

Mouse Model of RVO

The mouse model of RVO was established as previously described.²⁸⁾ Briefly, we used a mixture of ketamine (120 mg/kg; Daiichi-Sankyo, Tokyo, Japan) and xylazine (6 mg/kg; Bayer, Health Care, Osaka, Japan) to anesthetize mice. After the injection of rose bengal (8 mg/mL; FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) into a tail vein, three retinal veins were photocoagulated by laser light. (Condition of laser irradiation: 50 mW, 5 s, and 50 µm, Meridian AG, Bierigustrasse, Switzerland).

Drug Administration

Arctigenin (30, 100 mg/kg) was orally administrated at 1 h before, 1, 6, and 12 h after laser irradiation (Fig. 1B). Arctigenin was suspended in 0.5% carboxymethylcellulose sodium aqueous solution (CMC) (FUJIFILM Wako Pure Chemical Corporation). The vehicle-treatment group was administrated CMC.

Optical Coherence Tomography Imaging

OCT images were taken one day after laser irradiation using a Micron IV fundus camera and an OCT Scan Head equipped with a mouse objective lens (Phoenix Research Labs, Pleasanton, CA, U.S.A.). Right eyes had previously been dilated with 0.5% tropicamide and phenylephrine hydrochloride (Santen Pharmaceutical Co., Ltd., Osaka, Japan). Images were captured from 20 positions for each eye using StreamPix 6 and Micron OCT commercial software (Phoenix Research Labs). Captured images were quantitatively analysed using “In Sight” software, which can automatically detect and measure each retinal layer.

Histological Analysis

Mice were scarified by cervical dislocation after producing RVO model mice. The eyes were enucleated and fixed in 4% paraformaldehyde (PFA; FUJIFILM Wako Pure Chemical Corporation) in 0.1 M phosphate buffer (PB; pH 7.4) for 48 h at 4 °C. Paraffin-embedded tissues were cut (5 µm) using microtome and stained with hematoxylin and eosin. The stained sections were imaged with the All-in-One BZ-X710 fluorescent microscope (Keyence, Osaka, Japan). The inner nuclear layer (INL) thickness was measured every 240 µm from the optic nerve head using the Image-J software (National Institutes of Health, Bethesda, MD, U.S.A.). Retinal edema has been reported to form in the INL in RVO patients and in our RVO mouse model.^3,28) Therefore, the evaluation index for retinal edema was the INL thickness. Also, the cells in the INL were counted up to 500 μm from the optic disc.

Immunoblotting Analysis

Isolated retinas were frozen at −80 °C. Whole retina was homogenized in cell-lysis buffer using a Physcotron homogenizer (Microtec Co., Ltd., Chiba, Japan) and centrifuged at 12000 × g for 20 min at 4 °C. Cells were lysed in RIPA buffer (Sigma-Andrich, St. Louis, MO, U.S.A.) containing protein inhibitor cocktail (Sigma-Andrich), phosphatase inhibitor cocktails 2 and 3 (Sigma-Andrich). For in vitro study, HRMECs were seeded at a density of 10000 cells/mL in 24-well plates and incubated overnight. Arctigenin was added an hour before addition of recombinant human TNFα (50 ng/mL; R&D System, Minneapolis, MN, U.S.A.) and incubated for 24 h. Cells were collected using cell lysis buffer. Proteins were separated by 5–20% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gradient gel (FUJIFILM Wako Pure Chemical Corporation), and transferred to polyvinylidene difluoride membranes (Immobilon-P; Millipore Co., Billerica, MA, U.S.A.). After blocking with Blocking One-P (Nacalai Tesque, Inc., Kyoto, Japan) at room temperature for 30 min, the transferred membranes were incubated with antibody against the specific target overnight at 4 °C. The following primary antibodies were used for immunoblotting; β-actin (mouse monoclonal antibody 1 : 2000; Sigma-Aldrich), Occludin (rabbit polyclonal antibody; 1 : 200; Santa Cruz biotechnology, CA, U.S.A.), VE-cadherin (rabbit polyclonal antibody; 1 : 1000; Abcam, Cambridge, MA, U.S.A.), MMP9 (rabbit polyclonal antibody; 1 : 1000; Abcam), VEGF (mouse monoclonal antibody; 1 : 200; Santa Cruz), TNFα (mouse monoclonal antibody; 1 : 1000; Santa Cruz), Phospho-nuclear factor-kappaB (NF-κB) p65 (Ser536) (rabbit monoclonal antibody; 1 : 1000; Cell Signaling, Danvers, MA, U.S.A.), total NF-κB p65 (rabbit monoclonal antibody; 1 : 1000; Cell Signaling), Phospho-p38 mitogen-activated protein (MAP) Kinase (rabbit polyclonal antibody; 1 : 1000; Cell Signaling), total p38 MAP Kinase Antibody (rabbit polyclonal antibody; 1 : 1000; Cell Signaling). After the membranes were washed with Tris-buffered saline containing 0.05% Tween 20, they were incubated with secondary antibody. The secondary antibodies were goat anti-rabbit horseradish peroxidase (HRP)-conjugated immunoglobulin G (IgG) or goat anti-mouse HRP-conjugated IgG (1 : 1000, Pierce Biotechnology, Inc., Waltham, MA, U.S.A.).

The same quantitative method was used as previously described.²⁸⁾ The immunoreactive bands were imaged by Immuno Star^® LD (FUJIFILM Wako Pure Chemical Corporation), and the band intensity was measured by Amersham Imager 680 (GE healthcare, Chicago, IL, U.S.A.).

Cell Culture

HRMECs were purchased from Cell Systems (Kirkland, WA, U.S.A.) and cultured in complete medium (Cell Systems) including with Culture Boost-R (Cell Systems), 100 U/mL penicillin (Meiji Seika Pharma Co., Ltd., Tokyo, Japan) and 100 µg/mL streptomycin (Meiji Seika Pharma). The culture dishes were coated with attachment factor (Cell Systems) before HRMECs are seeded. Cells used in all experiments were 5–10 passages and were incubated under a humidified atmosphere of 5% CO₂ at 37 °C.

Evaluation of Permeability

Endothelial permeability was evaluated by trans-endothelial electrical resistance (TEER) and fluorescein isothiocyanate (FITC)-dextran permeability rate. HRMECs were seeded in Transwell^® inserts (Hanging Cell Culture Inserts; pore size 0.4 µm, membrane area 0.33 cm²; Corning, Inc., NY, U.S.A.) at a density of 1.0 × 10⁵ cells/well. Transwell^® inserts were put in 24-well plate.

To measure TEER, Epithelial Volt-Ohm Meter (Millicell ERS-2, Millipore, Merck Millipore Co., Darmstart, Germany) and cup-shaped electrode (Endohm-6, World Precision Instruments, Inc., Sarasota, FL, U.S.A.) were used. After pre-TEER value was measured, cells were treated with arctigenin (Kracie Holdings, Ltd., Tokyo, Japan) at 0.1, 1, 10 µM for 1 h and then treated with recombinant VEGF (10 ng/mL; R&D Systems) for 24 h. TEER value measurements were performed at 24 h after VEGF treatment.

After TEER measurement, FITC-dextran (average MW = 2000000; 1 mg/mL; Sigma-Aldrich) was added to the upper insert and incubated for 6 h. After that, the medium from a lower chamber was placed in 96-well plate. The florescence intensity was measured by using Varioskan Flash (Thermo Fisher Scientific, Waltham, MA, U.S.A.).

Immunostaining

HRMECs were seeded at a density of 3.0 × 10⁴ cells/well and incubated for 3 d. Arctigenin (10 µM) was treated an hour before treatment of TNFα (50 ng/mL). Twenty-four hours after, cells were fixed with 4% PFA for 10 min at room temperature. Cells were incubated with 0.3% triton X-100 (Bio-Rad, Hercules, CA, U.S.A.) for 10 min, and blocked with 3% goat serum (Vector Laboratories, Burlingame, CA, U.S.A.). Then, cells were incubated overnight at 4 °C with anti–VE-cadherin antibody (rabbit polyclonal antibody; 1 : 250; Abcam). The cells were covered with a secondary Alexa Fluor 488 goat anti-rabbit IgG antibody (1 : 1000; Thermo Fisher Scientific) for 1 h. The nuclei were stained with Hoechst 33342 (1 : 1000, Thermo Fisher Scientific) for 10 min. Cells were enclosed using Fluoromount (Diagnostic BioSystems) and imaged by a confocal microscope (FLUOVIEW FV3000; Olympus, Tokyo, Japan).

Statistical Analyses

Statistical analyses were performed using the 16 Statistical Package for the Social Sciences 15.0 J for Windows software (SPSS Japan Inc, Tokyo, Japan). Statistical comparisons were made using Student’s t-test or ANOVA with Dunnett’s test and Tukey’s test. All results were presented as mean ± standard error of the means (S.E.Ms.), and p < 0.05 were considered significant.

RESULTS

The Effects of Arctigenin on Retinal Edema in an RVO Mouse Model

In order to evaluate the inhibitory effects of arctigenin on retinal edema, we administrated arctigenin orally to RVO model mice. In the murine RVO model, edema forms in the INL. According to our previous study, the INL thickness as well as the thickness of all retinal layers can be used for the evaluation of edema formation in the murine RVO model. The thickness of all the retinal layers was investigated by OCT. Our analysis revealed that thickness of all retinal layer was increased in the RVO model and oral administration of arctigenin at 100 mg/kg for four intervals significantly reduced the retinal thickness (Fig. 1C). Next, we measured the thickness and the cell number of the INL using retinal tissue sections. We found that oral administration of arctigenin at 100 mg/kg suppressed the increase of the INL thickness and the reduction in the cell number in the RVO model (Figs. 1D–F).

Four Oral Treatments of Arctigenin Ameliorated the Decrease in the Expression of Junction Proteins in the Retinas from RVO Model Mice

Next, we investigated the effects of arctigenin on the expression of junction proteins in the retinas from RVO model mice. The expression of occludin and VE-cadherin was reduced in the retinas from RVO model mice at 1 d after laser irradiation (Figs. 2A–C). The oral administration of arctigenin (100 mg/kg) at four intervals significantly suppressed the decrease in expression of both occludin and VE-cadherin (Figs. 2A–C).

Fig. 2. The Expression of Cell–Cell Junctions and Related Factors in the Retina

The (A) representative blots and (B) quantitation of occludin, (C) VE-cadherin, (D) MMP9, (E) VEGF, and (F) TNFα expression in the retinas of RVO model mice administrated arctigenin (100 mg/kg). The data were normalized to β-actin. The data represent the mean ± S.E.M. (n = 8). ^##p < 0.01 versus Normal group, * p < 0.05, and ** p < 0.01 versus Vehicle group (Student’s t-test).

The expression of the integrity modulator tight junction proteins, MMP9 (Fig. 2D) and VEGF (Fig. 2E), as well as an inflammatory factor, TNFα (Fig. 2F) were all increased in the retinas from RVO model mice at 24 h after laser irradiation. The oral administration of arctigenin (100 mg/kg) significantly reduced the induction of these factors (Figs. 2D–F).

Arctigenin Suppressed the Hyperpermeability of HRMECs Induced by VEGF

In order to determine the effects of arctigenin on the permeability of HRMECs, we first assessed the barrier function of these endothelial cells by determining the TEER values and FITC-dextran permeability rates. VEGF significantly reduced the TEER value and treatment with arctigenin at 1 and 10 µM significantly suppressed this VEGF-induced decrease in the TEER value (Fig. 3A). In addition, VEGF increased the FITC-dextran permeability rate, and arctigenin treatments of 0.1–10 µM significantly inhibited this increase induced by VEGF (Fig. 3B). There is no significant difference between control group and arctigenin at 10 µM single treated group (Figs. 3A, B).

Fig. 3. Arctigenin Suppressed the Increase in Permeability Induced by VEGF

(A) The TEER values from HRMECs at 24 h after arctigenin (0.1, 1, 10 µM) and/or VEGF (10 ng/mL) treatment. The data represent the mean ± S.E.M. (n = 4). ^##p < 0.01 versus Control group (Student’s t-test), and ** p < 0.01 versus VEGF treatment group (Dunnett’s test). (B) FITC-dextran permeability assay in HRMECs at 24 h after arctigenin (0.1, 1, 10 µM) and/or VEGF (10 ng/mL) treatment. The data represent the mean ± S.E.M. (n = 4). ^##p < 0.01 versus Control group (Student’s t-test), and ** p < 0.01 versus VEGF treatment group (Dunnett’s test).

Arctigenin Suppressed the Breakdown of VE-Cadherin via Inhibition of the TNFα-Induced Activation of the NF-κB/p38 MAPK Pathway

To explore the effects of arctigenin on the barrier dysfunction induced by TNFα in HRMECs, we examined the expression of VE-cadherin (one of the endothelial specific adhesion molecules) by immunostaining. TNFα induced the breakdown of VE-cadherin; however, treatment of arctigenin at 10 µM prevented the decrease in VE-cadherin (Figs. 4A, B). To clarify the mechanism underlying the protective effects of arctigenin against VE-cadherin breakdown, the effects of arctigenin on the NF-κB/p38 MAPK pathway were investigated by Western blotting. TNFα induced the phosphorylation of NF-κB and p38, which activates the NF-κB/p38 MAPK pathway. In contrast, treatment with arctigenin (10 µM) significantly inhibited these phosphorylation events and thus, the NF-κB/p38 MAPK pathway was inactive (Figs. 4C–E).

Fig. 4. Arctigenin Suppressed the Increase in Permeability Induced by TNFα

(A) VE-cadherin immunostaining and (B) graphical representation of the VE-cadherin area. The data represent the mean ± S.E.M. (n = 3–4). ^##p < 0.01 versus Control group, and * p < 0.05 versus TNFα treatment group (Student's t-test). Scale bar = 50 µm. (C) The representative blots and (D) quantitation of phosphorylated (p)-NF-κB and NF-κB as well as (E) p-p38 and p38 from protein samples derived from HRMECs treated with arctigenin (10 mM) and TNFα (50 ng/mL). The data represent the mean ± S.E.M. (n = 6). ^##p < 0.01 versus Control group, * p < 0.05, and ** p < 0.01 versus vehicle-treated group (Tukey’s test).

DISCUSSION

This study suggested that oral administration of arctigenin has protective effects on retinal vascular hyperpermeability. Thus, our hypothesis that arctigenin may be an effective ocular supplement for treating retinal edema is fully supported by our data. This data includes the following observations: (1) orally administered arctigenin ameliorated the decrease in the expression of retinal endothelial tight junction proteins, which resulted in the suppression of retinal edema in an RVO murine model; (2) arctigenin suppressed the hyperpermeability induced by VEGF in HRMECs; and (3) arctigenin suppressed the breakdown of VE-cadherin via inhibition of the TNFα-induced activation of the NF-κB/p38 MAPK pathway. Arctigenin is bioactive lignans derived from Arctium lappa L. and Forsythia suspensa (Thunb.) Vahl. Burdock sprout and the leaves of Forsythia spp. are readily available and suitable for the extraction and isolation of arctigenin. Therefore, arctigenin has potential utility as an ocular supplement for prophylactic and/or complementary treatment for patients at risk for retinal vascular hyperpermeability.

Oral administration of arctigenin has anti-inflammatory effects in many tissues such as brain, kidney, and skin.^12,29,30) Orally administered arctigenin is mainly absorbed in the small intestine. It is converted into glucuronate conjugate by UDP-glucuronosyltransferase in the liver and intestine followed by its excretion.³¹⁾ Although the glucuronide conjugate of arctigenin is inactive, it is reactivated by cleavage, which is mediated by the inflammatory cell-derived β-glucuronidase at the inflamed site.³²⁾ Therefore, arctigenin is expected to have an inflammatory site-specific activity with few systemic effects. Macular edema triggers the recruitment of macrophage cells, that induce local inflammation within the eye, which is associated with the pathogenesis of edema.³³⁾ For this reason, arctigenin is considered safe because it has the potential for acting specifically on retinal edema that is often accompanied by inflammation. In previous reports, oral administration of arctigenin (25–80 mg/kg) was reported to exhibit neuroprotective effects in the brain.^12,34) From these reports, when arctigenin is administrated orally at a dose of 100 mg/kg, enough arctigenin is present in the blood in order to act peripherally. Even though we did not determine whether the administered arctigenin reached the retinas we did show that oral administration of arctigenin (100 mg/kg) suppressed retinal edema. Considering the site-specific effects of arctigenin on inflammation, and a previous report which demonstrated that the oral administration of arctigenin at a dose of 25–80 mg/kg exhibited protective effects in the brain, orally administrated arctigenin likely suppresses retinal edema by reaching the retina via the bloodstream.

The expression of VEGF and inflammatory cytokines are involved in the formation of edema in patients with RVO and DME.^19,20,26) It has been reported that these factors are produced by microglial cells, macrophage cells, and vascular endothelial cells in hypoxic tissue and their expression leads to hyperpermeability.³⁵⁾ Our results showed that oral administration of arctigenin decreased the expression of VEGF, TNFα, and MMP9 in retinas from RVO model mice. Previous reports have suggested that arctigenin is involved in the downregulation of VEGF expression in cancer-bearing mice.³⁶⁾ It has been reported that arctigenin suppressed the activation of microglia cells and decreased the expression of TNFα in a middle cerebral artery occluded rat model.³⁴⁾ In these reports, it was suggested that arctigenin potentially regulates cytokine expression. Moreover, we indicated that arctigenin inhibits NF-κB/p38 MAPK pathway in HRMECs. NF-κB regulates the expression of genes involved in inflammation.³⁷⁾ P38 MAPK is also associated with inflammation through modulation of NF-κB transcriptional activity.³⁸⁾ Arctigenin has been reported to suppress NF-κB translocation and DNA binding activity.³⁹⁾ Moreover, it has been reported that a powerful binding capacity between ATG and phosphatidylinositol 3-kinase (PI3K) which one of the upstream factors of NF-κB and p38 MAPK.⁴⁰⁾ These observations implied that arctigenin may suppress the expression of cytokines via inhibition of the NF-κB/p38 MAPK pathway. These data indicated that arctigenin not only inhibits the hyperpermeability induced by VEGF and TNFα, but also reduces the expression of these factors.

Endothelial cells are connected via both adherens and tight junctions which are essential for the regulation of permeability.^13,14) Endothelial dysfunction results from degradation of junction proteins induced by VEGF and/or inflammatory cytokines.^21,41) Using an inflammatory bowel disease mice model, it has been previously reported that arctigenin maintains the function of the intestinal epithelial barrier by promoting the expression of tight junction proteins.¹¹⁾ In this study, we demonstrated that arctigenin suppressed hyperpermeability after treatment of HRMECs with VEGF. We also showed that the expression of VE-cadherin and occludin were increased in the retinas of RVO model mice. Thus, we hypothesized that arctigenin has the potential to suppress hyperpermeability through upregulation of endothelial junction proteins. In addition, we found that in HRMECs, arctigenin protects against VE-cadherin degradation induced by TNFα. TNFα activates NF-κB/p38 MAPK pathway, which are play roles for the regulation of permeability in endothelial cells. Several reports have shown that in human endothelial cells, the activation of NF-κB is involved in VE-cadherin degradation induced by inflammatory cytokines.^42,43) The p38 MAPK pathway, activated by TNFα is also involved in the endocytosis of VE-cadherin and plays a role in the transcriptional activity of NF-κB.^38,44) It has been reported that the inhibition of the NF-κB/p38 MAPK pathway maintains BRB function in a mouse diabetes model.⁴⁵⁾ Thus, inhibition of the NF-κB/p38 MAPK pathway can be targeted for the suppression of retinal edema. Arctigenin was previously reported to suppress the inflammation by inhibiting the transfer of NF-κB to the nucleus and the activation of the NF-κB/p38 MAPK pathway in microglia cells and macrophage cells.^46,47) Our results showed that in HRMECs, arctigenin inhibited the TNFα-induced activation of the NF-κB/p38 MAPK pathway. Taken together, our investigation suggested that arctigenin inhibited the breakdown of endothelial junctions via its anti-inflammatory activity mediated by the inhibition of the NF-κB/p38 MAPK pathway.

Macular edema is caused by RVO and diabetic retinopathy (DR).²⁶⁾ Orally administered medicine which is simpler and less invasive than existing anti-VEGF therapy, is required because the number of these patients is rapidly increaseing because of our aging society. The prevention of retinal edema is also important for reducing the number of patients requiring treatment. Our investigation demonstrated that oral administration of arctigenin suppressed retinal edema in RVO model mice. Arctigenin likely acts on both VEGF and inflammatory cytokines, which is useful for the treatment of macular edema. Administration of arctigenin has the potential to ameliorate edema in DR patients. It has been reported that oral administration of arctigenin decreased blood glucose in a mouse model of diabetes.⁴⁸⁾ For this report, arctigenin is particularly expected to have protective effects on retinal edema of DR patients. The number of diabetic patients is increasing, and DR may also increase in the future, which underscores the need for DME preventions. Oral administration of arctigenin shows promise as a novel preventative agent or treatment for retinal edema. Importantly, arctigenin has the distinct advantage of being far less invasive than traditional therapies for retinal edema. Further studies are needed to determine whether or not arctigenin has a therapeutic effect after the onset of RVO.

In conclusion, these findings suggested that oral administration of arctigenin has protective effects towards retinal vascular hyperpermeability. Thus, we anticipate that arctigenin will be developed as a drug or supplement for the prevention and/or treatment of retinal vascular disorders such as RVO and DME.

Conflict of Interest

We received financial support from Kracie Holdings, Ltd., as collaborative research.

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