Protective Effects of Fermented Paprika (Capsicum Annuum L.) on Sodium Iodate-Induced Retinal Damage

nutrients

Article Protective Effects of Fermented Paprika (Capsicum annuum L.) on Sodium Iodate-Induced Retinal Damage

Ha-Rim Kim 1, Sol Kim 1, Sang-Wang Lee 2, Hong-Sig Sin 2 and Seon-Young Kim 1,*

1 Jeonju AgroBio-Materials Institute, Wonjangdong-gil 111-27, Deokjin-gu, Jeonju-si, Jeollabuk-do 54810, Korea; [email protected] (H.-R.K.); [email protected] (S.K.) 2 Chebigen Co., Ltd., Jeonju 54853, Korea; [email protected] (S.-W.L.); [email protected] (H.-S.S.) * Correspondence: [email protected]; Tel.: +82-63-711-1053

Abstract: Diseases of the outer retina, including age-related macular degeneration (AMD), are major cause of permanent visual damage. The pathogenesis of AMD involves oxidative stress and damage of the retinal pigment epithelium. Capsicum annuum L. (paprika) fruits have been known as a source of vitamins, carotenoids, phenolic compounds, and metabolites with a well-known antioxidant activity, which have positive effects on human health and protection against AMD and cataracts. In this study, we investigated whether paprika (fermented (FP), yellow, and orange colored) fermented with Lactobacillus (L.) plantarum could increase the protective effect of retinal degeneration using in vitro and in vivo models. FP signiﬁcantly increased cell survival and reduced levels of lactate dehydrogenase as well as intracellular reactive oxygen species (ROS) increase in SI (sodium iodate,

NaIO3)-treated human retinal pigment epithelial (ARPE-19) cells. We developed a model of retinal damage in C57BL/6 mice using SI (30 mg/kg) via intraperitoneal injection. Seven days after SI administration, deformation and a decrease in thickness were observed in the outer nuclear layer, but improved by FP treatment. FP administration protected the SI-mediated reduction of superoxide dismutase and glutathione levels in the serum and ocular tissues of mice. The overproduction

of cleaved poly(ADP-Ribose) Polymerase (PARP)1, caspase-3 and -8 proteins were signiﬁcantly protected by FP in SI-treated cells and ocular tissues. In addition, we evaluated the potentiating effects

Citation: Kim, H.; Kim, S.; Lee, S.; of FP on antioxidants and their underlying mechanisms in RAW 264.7 cells. Lipopolysaccharide (LPS)- Sin, H.; Kim, S. Protective Effects of induced nitrite increase was markedly blocked by FP treatment in RAW 264.7 cells. Furthermore, FP Fermented Paprika (Capsicum annuum reduced LPS-induced inducible nitric oxide synthase and cyclooxygenase-2 activation. The FP also L.) on Sodium Iodate-Induced Reti- enhanced the inhibitory effects on mitogen activated kinase signaling protein activation in ARPE-19 nal Damage. Nutrients 2021, 13, 25. and RAW 264.7 cells and ocular tissues. There was no significant difference in total phenol and https://dx.doi.org/10.3390/nu1301 flavonoid content in paprika by fermentation, but the vitamin C content was increased in orange 0025 colored paprika, and protective effect against oxidative stress-mediated retinal damage was enhanced after fermentation. These results suggest that FP may be a potential candidate to protect against Received: 31 October 2020 retinal degenerative diseases through the regulation of oxidative stress. Accepted: 21 December 2020 Published: 23 December 2020 Keywords: age-related macular degeneration; oxidative stress; Capsicum annuum L. (paprika); fermentation; antiiflammation Publisher’s Note: MDPI stays neu- tral with regard to jurisdictional claims in published maps and institutional affiliations. 1. Introduction Age-related macular degeneration (AMD) is a progressive eye disease caused by the degeneration of photoreceptor and retinal pigment epithelial (RPE) cells adjacent in the Copyright: © 2020 by the authors. Li- central part of the macula [1]. censee MDPI, Basel, Switzerland. This Oxidative stress is one of the most important causes of aging process. Studies indicate article is an open access article distributed that oxidative stress contributes to the pathophysiology of AMD. The retina is the most under the terms and conditions of the oxygen-consuming tissue, indicating that RPE cells are more susceptible to oxidative stress, Creative Commons Attribution (CC BY) especially damage caused by reactive oxygen species (ROS) [2–4]. ROS are generated license (https://creativecommons.org/ from aerobic metabolism under pathophysiologic conditions that can induce oxidative licenses/by/4.0/).

Nutrients 2021, 13, 25. https://dx.doi.org/10.3390/nu13010025 https://www.mdpi.com/journal/nutrients Nutrients 2021, 13, 25 2 of 15

damage. Imbalanced intracellular ROS can alter essential physiological signaling, such as regulatory feedback related to metabolism, hypoxia, and inflammation, that can impair RPE cell function [5,6]. Cells exposed to stress initially induce activation of compensatory pathways that restore homeostasis; however, chronic or intense stress activates death pathways such as cell death and necrosis [7]. Caspase has an important role in regulating cell apoptosis, and induction of the transformed apoptotic signal cascade and programed cell death. It has been reported that caspase-dependent apoptosis of RPE can be induced by oxidative stress, and inhibition of oxidative stress can effectively protect RPE’s apoptosis. The SI (sodium iodate, NaIO3)-induced AMD animal model is widely used to study the molecular mechanism of oxidative stress associated cell death in AMD and induces consistent and selective damage to the RPE. PI3K/AKT has been implicated in a various cellular signaling pathway, including cell survival, differentiation, and death. It has been reported that PI3K/AKT can be involved in SI-induced AMD pathophysiology [8–10]. The mitogen- activated protein kinases (MAPKs) are also known to regulate survival and apoptosis [11]. Interestingly, studies indicated that oxidative stress induces RPE cell death and ocular damage via AKT and MAPKs protein kinase cascade signaling [9,10]. ROS induces an inflammatory response that is the expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) [11]. Paprika (Capsicum annuum L.) is considered a healthy vegetable that contains phy- tochemicals including carotenoids, ascorbic acid, tocopherols, flavonoids and phenolic compounds, which are important antioxidant components that may reduce the risk of diseases [12–14]. Studies have revealed that paprika displays pharmacological effects such as antioxidant, antimicrobial, anti-inflammatory, anticancer, and antiviral effects [14]. Fermentation is a technology that improves the activity of raw materials through bio- chemical changes. This bioprocessing is increasingly used in the pharmaceutical, chemical, and food industries as these altered properties appear to produce beneficial effects such as increased antioxidant activity [15,16]. It has been reported that during fermentation, biological activity is enhanced due to the release of flavonoids and phenols, respectively, increased by microbial enzymes in plant chemicals [16]. Despite many reports on useful pharmacological properties of paprika, no studies have been considered using fermented paprika (FP) to improve ocular degeneration and inflammation caused by oxidative stress. In this study, we investigated whether fermented paprika have protective effects on oxidative stress and retinal degeneration. We also tested the expression of proteins involved in apoptosis and MAPK signaling in cells and ocular tissues.

2. Materials and Methods 2.1. Chemicals and Antibodies Chemicals and cell culture materials were obtained from the following sources: LPS (lipopolysaccharide), SI (sodium iodate), MTT [3-(4,5)-dimethylthiazolyl-2-yl-2,5- diphenyl tetrazolium bromide], BSA (bovine serum albumin), Folin Ciocalteu reagent, quercetin, gallic acid, and vitamin C (L-ascorbic acid) were purchased from Sigma-Aldrich (St Louis, MO, USA); PBS (phosphate-buffered saline), DMEM (Dulbecco’s modified Ea- gle’s medium), DME/F12 medium, FBS (fetal bovine serum), penicillin–streptomycin solution, DCF-DA (20,70-dichlorofluoresin diacetate) were from Invitrogen (Carlsbad, CA, USA); antibodies against COX-2 and β-actin were from Santa Cruz Biotechnology, Inc.; the protein assay kit was from Thermo Scientific (Waltham, MA, USA); primary antibodies [anti-phospho-ERKl/2 (Thr202/Tyr204), anti-phospho-p38 (Thr180/Tyr182), and anti- phospho-SAPK/JNK (Thr183/Tyr185), anti-AKT/-phospho-AKT (Ser473), anti- (cleaved- )caspase3/8, anti-(cleaved-)PARP1, and secondary antibody (HRP-linked anti-rabbit and anti-mouse IgG) were from Cell Signaling Technology Inc. (Beverly, MA, USA); ECL chemiluminescence system and PVDF (polyvinylidene difluoride) membrane were from Nutrients 2021, 13, 25 3 of 15

Amersham Pharmacia Biotech (Piscataway, NJ, USA). All other chemicals were of analytical grade or complied with the standards required for cell culture experiments.

2.2. Fermentation and Preparation of Paprika Samples L. plantarum JBMI F5 (KACC91638P) was prepared following a previously described method [6]. The raw materials thus prepared were mixed using a crusher. The crushed yellow or orange colored paprika (P-Y or P-O) was ﬁltered using a mesh. The prepared paprika solution was adjusted to pH 7.0 after acidiﬁcation (pH 4.5) for fermentation, steril- ization and then inoculated with 5% L. plantarum and cultured overnight. The fermentation process was carried out for 18 h while stirring at 150 rpm, 37 ◦C. After the fermentation, the fermentation broth was recovered and added with dextrin, followed by lyophilization. Raw paprika (-Y or -O), which was inoculated with L. plantarum but not fermented, was compared with fermented paprika. The raw or fermented paprika powder (1 mg) was treated with 70% ethanol (10 mL) and sonicated several times at room temperature for 2 h to produce extract. The solvent was evaporated under N2 gas atmosphere and freeze-dried. The lyophilized powder and extract was stored at −80 ◦C for in vivo and in vitro studies, respectively.

2.3. Analysis Physicochemical and Antioxidant Effect Total polyphenol and ﬂavonoids and vitamin C content was analyzed using a previously described method [6,17]. DPPH (2,2-diphenyl-1-picrylhydrazyl) and ABTS (2,20- azino-bis-3-ethylbezthiazoline-6-sulphonic acid) radical scavenging activity was determined as previously described [6,17].

2.4. Cell Culture Mouse macrophages (RAW 264.7), and human RPE cells (ARPE-19) were obtained from ATCC (American Type Culture Collection). Cells were cultured in DMEM or DMEM/ F12 medium (Invitrogen): supplemented with 10% FBS, 100 U/mL penicillin, and 100 µg/mL ◦ streptomycin at 37 C with 5% CO2 in a humidiﬁed atmosphere.

2.5. Cell Viability Assay The ARPE-19 cells were divided into six groups: Normal (N), SI (sodium iodate), P-Y, FP-Y, P-O, and FP-O pretreated group. ARPE-19 cells were plated at a density of 1 × 104 cells/well in 96-well plates. After cell attachment, the cells were pre-treated with P-Y, FP-Y, P-O, and FP-O for 1 h before SI (1200 µg/mL) treating. After another 24 h, 10 µL of MTT (5 mg/mL) reagent was added to each well, and incubated for 3 h at 37 ◦C. Then the cell culture medium was removed, and 100 µL of DMSO (dimethyl sulfoxide) was added to each well. The lactate dehydrogenase (LDH) leakage into the culture medium as determined using a commercially available kit from Sigma-Aldrich (St Louis, MO, USA). The absorbance was measured on a microplate reader (Multiskan GO, Thermo Scientiﬁc, Waltham, MA, USA) at 570 nm for MTT and 340 nm for LDH assay.

2.6. Measurement of Intracellular Reactive Oxygen Species (ROS) Increase ARPE-19 cells were pretreated with SI for 24 h, followed with or without 24 h exposure to P-Y, FP-Y, P-O, and FP-O extract (500 µg/mL). The intracellular ROS levels in ARPE-19 cells were detected using DCF-DA (Invitrogen. Brieﬂy, cells were incubated with 50 µM DCFH-DA reagent from the kit for 30 min in the dark at 37 ◦C, then cells were washed twice with PBS. The intracellular ROS was analyzed using a ﬂuorescence microscope (BX53, Olympus, Tokyo, Japan).

2.7. Measurement of NO Production in RAW 264.7 Cells Nitrite, which accumulated in culture supernatants, was measured using a method based on the Griess reaction as an indicator of NO production [18]. Cells (5 × 105 cells/well) were incubated in 6-well plates, and treated with P-Y, FP-Y, P-O, and FP-O ex- Nutrients 2021, 13, 25 4 of 15

tract (500 µg/mL), and then stimulated with LPS (1 µg/mL) for 18 h. The culture medium was mixed with Griess reagent (equal volumes of 1% sulfanilamide in 5% phosphoric acid and 0.1% N-(1-naphtyl) ethylenediamine dihydrochloride), and further incubated for 20 min at room temperature (RT). The concentration of nitrite was determined using the sodium nitrite (NaNO2) standard curve at 540 nm with a microplate reader.

2.8. Animals and Treatment C57BL/6J male mice weighing 20–25 g (Damul Science, Deajeon, Korea) were housed in cages with free access to food and water and maintained in temperature and light controlled rooms (23 ± 2 ◦C, 55 ± 10%, 12/12 h light/dark cycle with lights on at 8:00) at least one week before the experiments. The in vivo experiment was performed in accordance with the Jeonju AgroBio-Materials Institute (JAMI) guidelines under a protocol approved by the Institutional Animal Care and Use Committee (IACUC) of institute and all experiments strictly followed committee guidelines (JAMI IACUC 2019001). Mice were randomly divided into the following six groups (n = 6 per group), which were N (normal control), C (control), P-Y (195 mg/kg), FP-Y (1 × 108 CFU for L. plantarum and 195 mg/kg for P-Y), P-O (195 mg/kg), or FP-O (1 × 108 CFU for L. plantarum and 195 mg/kg for P-O) treatment group. The raw or fermented paprika powder was suspended in saline. Each of the samples were administered orally once a day for 1 week following SI (sodium iodate, Sigma-Aldrich) treatment. SI was dissolved in sterile normal saline and treated for 5 days by intraperitoneal injection. C group were administered with the normal saline instead of samples. At the end of the study, blood and tissues were harvested from the sacriﬁced mice after being anesthetized with tribromoethanol (Avertin, Sigma-Aldrich).

2.9. Determination of Antioxidant Activity Superoxide dismutase (SOD) activities and glutathione (GSH) levels were measured analyzed using a SOD (Sigma-Aldrich, St Louis, MO, USA) and GSH (Cayman, Ann Arbor, MI, USA) assay kit, according to the manufacturer’s instructions.

2.10. Histology of Ocular Tissues Ocular tissues were removed and immediately placed in 10% formalin solution, embedded in parafﬁn and cut into 5-µm sections. Specimens were stained with hematoxylin and eosin (H and E) to identify morphological changes. Histological images were acquired by light microscopy (Nikon, Tokyo, Japan).

2.11. Immunoblotting RAW 264.7 and ARPE-19 cells and ocular tissues were lysed in ice-cold lysis buffer [10 mM Tris-HCl (pH 7.4), 0.1 M ethylenediaminetetra acetic acid (EDTA), 10 mM NaCl, and 0.5% Triton X-100] supplemented with a protease and a phosphatase inhibitor cocktail (Sigma-Aldrich). Protein concentrations were determined by protein assay (ThermoFisher Scientiﬁc) using BSA (Sigma-Aldrich) as a standard. Lysates (20 µg) were resolved on a 10–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and then transferred to PVDF membranes (BioRad, Hercules, CA, USA). After blocking with Tris-buffered saline (TBS) containing 5% nonfat dry milk and 0.1% (w/v) Tween 20, membranes were probed with primary antibody. The following speciﬁc antibodies were used to characterize protein expression: iNOS, COX-2, (p-)AKT, (p-)ERK1/2, (p-)JNK, (p-)p38, (cleaved-)Caspase 3/8, (cleaved-)PARP1, and β-actin. After overnight incuba- tion at 4 ◦C, then incubated with secondary antibody (anti-rabbit IgG-HRP or anti-mouse IgG-HRP). Immunoblots were visualized using chemiluminescent (ECL) Western blotting detection reagents (GE Healthcare, Buckinghamshire, UK) and immunoreactive signals were analyzed by densitometry scanning (Amersham imager 600, GE Healthcare, Bucking- hamshire, UK). Nutrients 2021, 13, 25 5 of 15

2.12. Statistical Analysis Data represent means ± standard deviation (SD) of at least three separate experiments. Data were analyzed using Sigmaplot v14.0 software (Systat Software Inc., San Jose, CA, USA). Student’s t-test was performed to compare the parameters between two groups, while the analysis of variance (ANOVA) test followed by Duncan’s multiple range test. Data with p < 0.05 were considered statistically signiﬁcant.

3. Results 3.1. Physicochemical Content and Radical Sacavenging Ability In this study, we tested using yellow and orange paprika (P-Y and P-O) on eye damage protection. The raw and fermented paprika was extracted using 70% ethanol for in vitro study. After fermentation, the physicochemical contents and antioxidant effects was compared with raw paprika. As shown in Table1, the total phenolic and flavonoid levels and their antioxidant activity in paprika was increased by fermentation. The phenolic compounds usually have glycosidic bonds with various glycosides, the bonds of which were hydrolyzed by microorganisms for the release of the total phenolic and flavonoid [19] content. The phenolic compounds are known to have many potential benefits for human health, especially as strong antioxidants [20,21]. The total phenol content of fermented yellow or orange paprika (FP-Y or FP-O) was measured at the level of 84.17 or 81.53 mg GAE (gallic acid equivalent) g−1 and the flavonoid content was 9.59 or 8.69 mg QE (quercetin equivalent) g−1. Each level did not increase significantly compared to raw paprika. On the other hand, vitamin C content was increased in FP-O compared to P-O. The antioxidant activity was significantly increased by fermentation compared to raw paprika.

Table 1. Physicochemical content and radical scavenging ability.

DPPH Radical ABTS Radical TPC TFC VC Scavenging Scavenging (GAE, mg/g) (QE, mg/g) (mg/100 g) Activity (%) Activity (%) P-Y 81.59 ± 0.01 8.78 ± 0.04 1.98 ± 0.02 24.29 ± 0.56 48.25 ± 1.21 P-O 70.52 ± 0.11 8.00 ± 0.09 1.88 ± 0.02 25.09 ± 0.86 50.70 ± 0.39 FP-Y 84.17 ± 0.54 9.59 ± 0.85 2.05 ± 0.07 34.51 ** ± 0.54 55.14 ** ± 1.28 FP-O 81.53 ± 0.40 8.69 ± 0.29 2.03 # ± 0.03 28.26 ## ± 1.22 53.45 # ± 0.24 AA - - 95.52 ± 0.12 - Trolox - - - 94.66 ± 0.06 Values are means ± SD of three independent experiments. ** p < 0.01 vs. P-Y; # p < 0.05, ## p < 0.01 vs. P-O. TPC, total polyphenol content; GAE, gallic acid equivalent; TFC, total ﬂavonoid content; QE, quercetin equivalent; VC, vitamin C content; DPPH; 2,2-diphenyl-1-pycrylhydrazyl; ABTS, 2,20- azino-bis (3-ethylbenzothiazoline-6-sulphonic acid); P-Y, Yellow Paprika; P-O, Orange Paprika; FP-Y, Fermented P-Y; FP-O, Fermented P-O; AA, Ascorbic acid.

3.2. Effects of Fermented Paprika (FP) on Sodium Iodate (SI)-Induced Damage in Human Retinal Pigment Epithelial Cell line (ARPE-19) The effect of FP on SI-mediated cytotoxicity was determined using dimethylthiazolyl- 2-yl)2,5-diphenyl tetrazolium bromide (MTT) and lactate dehydrogenase (LDH) leakage assay. ARPE-19 cells were exposed to SI (1200 µg/mL) for 24 h in presence or absence of various concentrations ranging from 1 to 1000 µg/mL of P (-Y or -O) and FP (-Y or -O) extracts (Figure1A,B). As shown in Figure1A,B, SI (1200 µg/mL) significantly decreased the viability of ARPE-19 cells compared with the normal (N) group. Otherwise, SI-induced cell damage was protected by P (-Y or -O) and FP (-Y or -O) extracts in a dose dependent manner. Cell viability was increased significantly at doses of 500 µg/mL FP (-Y or -O) compared to the raw extract. The effects of P and FP extract on SI-induced cell damage was further confirmed using LDH leakage into the cell culture medium. As shown in Figure1C, P (-Y or -O) and FP (-Y or -O) significantly suppressed SI-induced LDH leakage at a dose of 500 µg/mL. In addition, FP-O showed the most potent effect compared to other groups. The cytotoxicity was not observed in all samples at a dose from 1 to 1000 µg/mL. As shown Nutrients 2021, 13, 25 6 of 15 Nutrients 2021, 13, x FOR PEER REVIEW 6 of 15

compared to other groups. The cytotoxicity was not observed in all samples at a dose from 1 into Figure1000 μg/mL1D, no. signiﬁcantAs shown differencein Figure was1D, no observed significant among difference various was groups. observed Therefore, among the variousmost effective groups. andTherefore, non-cytotoxic the most concentration effective and ofnon P(-Y-cytotoxic or -O) concentration and FP (-Y or -O)of P( extracts,-Y or - O)500 andµg/mL, FP (-Y wasor -O) used extracts, in subsequent 500 μg/mL, studies. was used in subsequent studies.

FigureFigure 1. 1. SodiumSodium iodate iodate ( (SI)-inducedSI)-induced damage damage of human RPERPE cellscells(ARPE-19) (ARPE-19) was was decreased decreased by by fermented fermented yellow yellow or or orange or- angepaprika paprika (P-Y (P or-Y -O) or extracts.-O) extracts. Cells Cells were were exposed exposed to SI to (1200 SI (1200µg/mL) μg/mL) in presence in presence or absence or absence of various of various doses doses of P (-Yof P or (- -O)Y orand -O) FP and (-Y FP or ( -O)-Y or extracts -O) extracts for 24 h.for Protective 24 h. Protective effects ofeffects (A) P-Y, of ( FP-Y,A) P-Y, (B )FP P-O,-Y, and(B) P FP-O-O, and extracts FP-O on extracts SI-induced on SI damage-induced in damageARPE-19 in cells.ARPE (C-19) Cell cells. viability (C) Cell was viability determined was determined using LDH leakageusing LDH assay. leakage The SI-induced assay. The cell SI- damageinduced wascell reduceddamage atwas 500 reduced at 500 μg/mL of P (-Y or -O) and FP (-Y or -O) extracts. (D) Cell cytotoxicity was analyzed using the MTT assay. µg/mL of P (-Y or -O) and FP (-Y or -O) extracts. (D) Cell cytotoxicity was analyzed using the MTT assay. *** p < 0.001 vs. *** p < 0.001 vs. N, # p < 0.05, ## p < 0.01 vs. C) † p < 0.05 vs. P-O. Values are means ± SEM. Significant differences between N, # p < 0.05, ## p < 0.01 vs. C) † p < 0.05 vs. P-O. Values are means ± SEM. Signiﬁcant differences between the groups were the groups were determined by analysis of variance (ANOVA) followed by Duncan’s multiple range test. Conc., Concen- tration;determined N, Normal; by analysis C, Control; of variance P-Y, Yellow (ANOVA) Paprika; followed P-O, by Orange Duncan’s Paprika; multiple FP-Y, range Fermented test. Conc., P-Y; Concentration; FP-O, Fermented N, Normal;P-O. C, Control; P-Y, Yellow Paprika; P-O, Orange Paprika; FP-Y, Fermented P-Y; FP-O, Fermented P-O. 3.3. Effects of FP on SI-Induced Intracellular ROS Generation in ARPE-19 Cells 3.3.The Effects mechanism of FP on SI-Induced of SI-induced Intracellular retinal damage ROS Generation is associated in ARPE-19 with oxidative Cells stress, but the protectiveThe mechanism effect of FP of SI-inducedon the SI-induced retinal damageROS generation is associated in RPE with cells oxidative was not revealed. stress, but Inthe order protective to investigate effect of the FP effect on the of SI-induced FP on oxidative ROS generation stress in ARPE in RPE-19 cellscells, was the notSI-induced revealed. intracellularIn order to investigateROS generation the effect were of determined FP on oxidative using stress DCF- inDA. ARPE-19 As shown cells, in the Figure SI-induced 2, in- tracellularintracellular ROS ROS was generationmarkedly increased were determined in APPE-19 using cells DCF-DA. by SI treatment, As shown while in the Figure P (-Y2, orintracellular -O) and FP ROS(-Y or was -O) markedly extract reduced increased the in intracellular APPE-19 cells ROS by increase. SI treatment, Moreover, while the FP P(- (-YY oror -O) -O) extract and FP caused (-Y or a -O) significant extract reduced (p < 0.05) the decrease intracellular in the ROS ROS increase. levels, compared Moreover, to FP raw (-Y materialsor -O) extract (Figure caused 2B). These a signiﬁcant results (suggestedp < 0.05) decrease that the inenhanced the ROS protective levels, compared effect against to raw cellmaterials damage (Figure by SI of2B). P ( These-Y or - resultsO) is due suggested to increased that antioxidant the enhanced capacity protective by fermentation. effect against cell damage by SI of P (-Y or -O) is due to increased antioxidant capacity by fermentation.

Nutrients 2021, 13, 25 7 of 15

Nutrients 2021, 13, x FOR PEER REVIEW 7 of 15

Figure 2. EffectsFigure of 2. fermentedEffects of fermented paprika (FP) paprika on SI-induced (FP) on SI- ROSinduced generation ROS generation in ARPE-19 in ARPE cells.-19 Cells cells. were Cells exposed to SI after incubatedwere with exposed 500 µ tog/mL SI after of Pincubated (-Y or -O) with and 500 FP (-Yμg/mL or -O) of extractP (-Y or for -O) 24 and h. IntracellularFP (-Y or -O) ROS extract were for measured 24 using DCFH-DAh. (2 0Intracellular,70-dichlorofluoresin ROS were diacetate) measured assay. using The DCFH magnification-DA (2′,7′-dichlorofluoresin was ×200. Scale diacetate) bar = 20 µ assay.m. (A) ( RepresentativeA) fluorescenceRepresentative micrographs depictingfluorescence ROS micrographs generation indepicting SI-exposed ROS ARPE-19 generation cells in forSI-exposed 24 h after ARPE pretreatment-19 cells with P (-Y or -O) and FPfor (-Y 24 or h -O) after extract pretreatment at 500 µg/mL with P (upper (-Y or panel).-O) and Total FP (- (DAPIY or -O) stained) extract numberat 500 μg/mL of cells (upper were panel). presented in lower Total (DAPI stained) number of cells were presented in lower panel. (B) DCF-DA intensity was panel. (B) DCF-DA intensity was quantified using Image J. *** p < 0.001 vs. N, ### p < 0.01 vs. C, † p < 0.05 vs. P-Y, ‡ p < 0.01 quantified using Image J. The magnification was × 200. *** p < 0.001 vs. N, ### p < 0.01 vs. C, † p < vs. P-O. Values are means ± SEM. Significant differences between the groups were determines by ANOVA followed by 0.05 vs. P-Y, ǂ p < 0.01 vs. P-O. Values are means ± SEM. Significant differences between the groups Duncan’s multiplewere determines range test. by N, ANOVA Normal; followed C, Control; by P-Y, Duncan’s Yellow multiple Paprika; range P-O, Orange test. N, Paprika; Normal; FP-Y, C, Control; Fermented P- P-Y; FP-O, FermentedY, P-O. Yellow Paprika; P-O, Orange Paprika; FP-Y, Fermented P-Y; FP-O, Fermented P-O.

3.4. Effect of FP on Lipopolysaccharide (LPS)-Induced Nitric Oxide (NO) Production in RAW 3.4. Effect of FP on Lipopolysaccharide (LPS)-Induced Nitric Oxide (NO) Production in RAW 264.7 Macrophages264.7 Macrophages Reactive nitrogenReactive species nitrogen (RNS) such species as nitric (RNS) oxide such (NO) as nitric also contribute oxide (NO) to alsothe oxi- contribute to the dative stress. NOoxidative is produced stress. by NOthree is diffe producedrent isoforms by three ofdifferent nitric oxide isoforms synthases of nitric (NOs), oxide synthases which catalyze(NOs), L-arginine which to L catalyze-citrulline L-arginine by releasing to L-citrulline the NO. Cellular by releasing NO cause the NO.s the Cellularpro- NO causes duction of severalthe productionRNS that are of implicated several RNS in thatoxidative are implicated damage [22] in oxidative. In this regard, damage RAW [22]. In this regard, 264.7 macrophagesRAW were 264.7 included macrophages to further were confirm included the to furthereffect of confirm fermented the effectpaprika offermented ex- paprika tract (FPE) on oxidativeextract (FPE) stress. on LPS oxidative-induced stress. NO production LPS-induced was NO suppressed production by was P (- suppressedY or - by P (-Y O) and FP (-Y oror - -O)O) extracts and FP pretreatment (-Y or -O) extracts at 500 pretreatment μg/mL (Figure at 3A). 500 µTheg/mL inhibitory (Figure effect3A). The inhibitory of LPS-inducedeffect NO increase of LPS-induced was more NO effective increase in cells was pretreated more effective with inFPcells (-Y or pretreated -O) ex- with FP (-Y tracts than theiror raw -O) counterparts extracts than (Figure their raw 3A). counterparts The expression (Figure levels3A). of NO The produc expressiontion- levels of NO related proteins,production-related including iNOS and proteins, COX- including2, were significantly iNOS and COX-2, up-regulated were significantly in the LPS- up-regulated in treated group (C,the control) LPS-treated by 2.65 group- and (C, 3.46 control)-fold by(p < 2.65- 0.001), and compared 3.46-fold (withp < 0.001), the N comparedgroup with the N (Figure 3B). Rawgroup or fermented (Figure3B). paprika Raw or extract fermented significantly paprika reduced extract significantly LPS-stimulated reduced iNOS LPS-stimulated and COX-2 proteiniNOS expression and COX-2 levels protein in expressionRAW 264.7 levels cells (Figure in RAW 3B). 264.7 Consistent cells (Figure with3B). the Consistent with results in Figure 2, fermented paprika extracts exhibited a more potent inhibitory effect on LPS-mediated NO production and signaling protein expression in RAW264.7 cells.

Nutrients 2021, 13, 25 8 of 15

Nutrients 2021, 13, x FOR PEER REVIEW 8 of 15 the results in Figure2, fermented paprika extracts exhibited a more potent inhibitory effect on LPS-mediated NO production and signaling protein expression in RAW264.7 cells.

Figure 3. Effects of FP on LPS-induced NO production and iNOS, and COX-2 protein expression in RAW 264.7 cells. Figure 3. Effects of FP on LPS-induced NO production and iNOS, and COX-2 protein expression in RAW 264.7 cells. RAW264.7 cells were pretreated with P (-Y or -O) and FP (-Y or -O) extracts (500 µg/mL) for 1 h, and then incubated with RAW264.7 cells were pretreated with P (-Y or -O) and FP (-Y or -O) extracts (500 μg/mL) for 1 h, and then incubated with LPSLPS (1 (1μg/mL)µg/mL) for for 18 18 h. h.(A ()A The) The nitrite nitrite levels levels in in culture culture media media were were measured measured using using Griess Griess reagent. (B) TheThe proteinprotein levelslevels of ofiNOS iNOS and and COX-2 COX-2 were were analyzed analyzed by by immunoblotting immunoblotting in in cell cell lysates. lysates. The The band band density density was was normalized normalized to β to-Actin β-Actin followed fol- lowedby statistical by statistical analysis. analysis. *** p ***< 0.001p < 0.001 vs. vs. N; #N;p #< p 0.05, < 0.05, ## ##p < p 0.01,< 0.01, ### ###p p< < 0.001 0.001 vs. vs. C.C.† † pp << 0.05 vs. P P ( (-Y-Y or or - -O),O), ǂ ‡p p< <0.01 0.01 vs.vs. P- P-Y.Y. Values Values are are means means ± ±SEM.SEM. Significant Signiﬁcant differences differences between between the the groups groups were were determines determines by by ANOVA ANOVA followed followed by by Duncan’sDuncan’s multiple multiple range range test test.. LPS, LPS, Lipopolysaccharides; Lipopolysaccharides; iNOS, iNOS, inducible inducible nitric nitric oxide oxide synthase; synthase; COX COX,, cyclooxygenase; cyclooxygenase; N, N, Normal;Normal; C, C, Control; Control; P- P-Y,Y, Yellow Yellow Paprika; Paprika; P P-O,-O, Orange Orange Paprika; Paprika; FP FP-Y,-Y, Fermented Fermented P P-Y;-Y; FP FP-O,-O, Fermented Fermented P P-O.-O.

3.5. Preservation Efect of Fermented Paprika Extract (FPE) on Retinal Structure and Oxidative Stress3.5. Preservationin SI-Induced Efect Retinal of Fermented Degeneration Paprika Mose Extract Model(FPE) on Retinal Structure and Oxidative Stress in SI-Induced Retinal Degeneration Mose Model Oxidative stress could be involved in the photo-aging process as a primary factor. The aboveOxidative in vitro stress results could suggest be involved that infermented the photo-aging paprika process extract as may a primary be an factor.effective The above in vitro results suggest that fermented paprika extract may be an effective means means of protecting retinal damage from oxidant insult and consequent damage. To vali- of protecting retinal damage from oxidant insult and consequent damage. To validate date whether the FP provides protection against SI-induced retinal degeneration, we es- whether the FP provides protection against SI-induced retinal degeneration, we established tablished a mouse model of SI-induced retinal degeneration. SI is an oxidative toxic agent a mouse model of SI-induced retinal degeneration. SI is an oxidative toxic agent and and widely used because of its reproducibility and controllable retinal damage degree widely used because of its reproducibility and controllable retinal damage degree [23,24]. [23,24]. For in vivo studies, lyophilized P (-Y or -O) and FP (-Y or -O) powders were sus- For in vivo studies, lyophilized P (-Y or -O) and FP (-Y or -O) powders were suspended pended in sterile saline and administered orally once a day for 1 week. The dosage for in sterile saline and administered orally once a day for 1 week. The dosage for each each sample was 1 × 108 CFU for L. Plantarum, 159 mg/kg for paprika, and 500 mg/kg for sample was 1 × 108 CFU for L. Plantarum, 159 mg/kg for paprika, and 500 mg/kg for total total sample amount. sample amount. Representative images from histologic sections stained with hematoxylin and eosin Representative images from histologic sections stained with hematoxylin and eosin are presented in Figure 4A. As expected, the outer nuclear layer (ONL) cells of SI-induced are presented in Figure4A. As expected, the outer nuclear layer (ONL) cells of SI-induced retinalretinal degeneration degeneration control control mice mice (C) (C) were were disordered disordered and and th thinner,inner, and and the the structure structure of of thethe RPE RPE layer layer was was not not clear clear compared compared to to normal normal mice mice (N). (N). In In contrast, contrast, administration ofof P P (-Y(-Y or - -O)O) and FP FP ( (-Y-Y or or - -O)O) rescued rescued RPE RPE degeneration degeneration and and retinal retinal structure structure (Figure (Figure 4A).4A). ComparedCompared to tothe the control control (C, (C, SI SI-treatment-treatment group), group), the the FP FP (-Y (-Y or or -O) -O) group group was was effective effective in in preventingpreventing ONL ONL thickness thickness reduction reduction and and improving improving shape shape change change (Figure (Figure 4A).4A). Because Because oxidativeoxidative stress stress has has been been identified identified as as a major a major inducer inducer of of RPE RPE injury injury in ina SI a SI-induced-induced retinal retinal degenerationdegeneration model model [25] [25, ],we we further further assessed assessed the the antioxidant antioxidant capacity capacity of of FP FP in in the the serum serum andand tissue tissue on on the the basis basis of of the the superoxide superoxide dismutase (SOD)(SOD) andand glutathioneglutathione (GSH) (GSH) assay. assay. As Asshown shown in in Figure Figure4B,C, 4B SOD,C, SOD and GSHand GSH content content significantly significantly decreased decreased in serum in and serum tissues and of tissuesthe mice of the with mice SI injection with SI (injectionp < 0.001), (p whereas < 0.001), FP whereas (-Y or -O) FP treatment (-Y or -O) significantly treatment increasessignifi- cantlythe levels increases of SOD the andlevels GSH of SOD (p < and 0.001) GSH (Figure (p < 40.001)B,C). (Figure These data 4B,C). suggest These thatdata FP suggest shows thatincreased FP shows anti-oxidative increased anti effects-oxidative against effects SI-induced against oxidative SI-induced stress oxidative in mice stress ocular in tissue. mice ocular tissue.

Nutrients 2021, 13, 25 9 of 15

Nutrients 2021, 13, x FOR PEER REVIEW 9 of 15

FigureFigure 4. 4. FPFP protect protect SI SI-induced-induced retinal retinal morphological morphological impairment impairment in in mice. mice. (A (A) )The The enucleated enucleated eyes eyes were were stained stained with with hematoxylinhematoxylin and and eosin eosin (H (H and and E). E). Histological Histological images images showing showing the the alterations alterations in in retinal retinal mor morphology.phology. The The magnification magnification waswas ×× 200.200. (B Scale) Superoxide bar = 20 µdismum. (B)tase Superoxide (SOD) and dismutase (C) glutathione (SOD) and (GSH) (C) levels glutathione were measured (GSH) levels in serum were measured and tissue in lysates serum fromand tissueall groups lysates using from an allenzyme groups-linked using immunosorbent an enzyme-linked assay immunosorbent (ELISA) kit. * p assay < 0.05, (ELISA) *** p < 0.001 kit. * pvs.< N; 0.05, # p *** < 0.05,p < 0.001 ### p vs.< 0.001N; # pvs.< C. 0.05, † p ###< 0.05p < vs. 0.001 P (-Yvs. or C. -O).† p Values< 0.05 are vs. means P (-Y or ± -O).SEM. Values Significant are means differences± SEM. between Significant the groups differences were betweendetermines the bygroups ANOVA were followed determines by byDuncan’s ANOVA multiple followed range by Duncan’s test. GCL, multiple Ganglion range cell test. layer; GCL, INL, Ganglion Inner cellnuclear layer; layer; INL, InnerONL, nuclearOuter nuclearlayer; ONL, layer; Outer RPE, nuclearRetinal layer;pigment RPE, epithelium; Retinal pigment N, Normal; epithelium; C, Control; N, Normal; P-Y, Yellow C, Control; Paprika; P-Y, P Yellow-O, Orange Paprika; Paprika; P-O, Orange FP-Y, Fermented P-Y; FP-O, Fermented P-O. Paprika; FP-Y, Fermented P-Y; FP-O, Fermented P-O. 3.6. FP Inhibits SI-Induced Apoptosis in ARPE-19 Cells and Mouse Retina 3.6.SI FP treatment Inhibits SI-Induced reduces vision Apoptosis and in follows ARPE-19 RPE Cells damage and Mouse with Retinaphotoreceptor degeneration [25,26]SI treatment. We also reduces observed vision similar and results follows in RPE which damage SI treatment with photoreceptor induces cells degenera-death, as measuredtion [25,26 by]. WeLDH also release observed in ARPE similar-19 resultscells (Figure in which 1D). SI The treatment cell death induces process cells of death,apopto- as sismeasured is responsible by LDH for release the cell in loss ARPE-19 seen in cells several (Figure disorders1D). The of cell retina, death glaucoma, process of and apoptosis macu- laris responsibledegeneration for [27,28] the cell. Evidence loss seen indicate in severals that disorders caspases of play retina, a key glaucoma, role in both and the macular initi- ationdegeneration and execution [27,28 ].pathways Evidence of indicates apoptosis that [29,30] caspases. Similarly, play a key therole cleavage in both of the PARP initiation-1 by caspasesand execution is also pathways considered of to apoptosis be a hallmark [29,30]. of Similarly, apoptosis the [31] cleavage. of PARP-1 by caspases is alsoTo consideredidentify whether to be athe hallmark FP could of affect apoptosis SI-induced [31]. apoptosis in ARPE-19 cells and in mouseTo retina, identify we whether analyzed the cell FP death could-related affect SI-induced signaling mechanisms.apoptosis in ARPE-19 Indeed, our cells data and showin mouse that retina,SI induces we analyzedproteolytic cell cleavage death-related and activation signaling of mechanisms. PARP-1 and Indeed, both caspas our datae-8 andshow -3 thatin the SI ARPE induces-19 proteolytic cells and mouse cleavage retina and (Figure activation 5). However, of PARP-1 no and significant both caspase-8 decrease and was-3 in observed the ARPE-19 in the cells expression and mouse of cleaved retina- (FigurePARP-51). and However, caspase no-8 and significant -3 by treatment decrease with was Pobserved (-Y or -O) in the and expression FP-Y extract of cleaved-PARP-1 whereas PF-O and extract caspase-8 significantly and -3 by inhibited treatment SI with-induced P (-Y cleavageor -O) and of FP-YPARP extract-1 and whereascaspase-8 PF-O and extract-3 in ARPE significantly-19 cells (Figure inhibited 5A; SI-induced p < 0.001). cleavageSimilar toof the PARP-1 results and in cells, caspase-8 FP-O and administration -3 in ARPE-19 showed cells (Figurethe most5A; significantp < 0.001). effect Similar on the to SI the- inducedresultsin apoptosis cells, FP-O in mouse administration retina. Additionally, showed the mostFP-Y significantshowed an effect effect on on the the SI-induced cleavage ofapoptosis caspase in3 (Figure mouse retina.5B). These Additionally, results suggest FP-Y showed that FP, an especially effect on theFP- cleavageO, could ofinhibit caspase SI- 3 induced(Figure5 retinalB). These apoptosis results suggestvia the PARP that FP,-1 especiallyand caspases FP-O, pathways. could inhibit SI-induced retinal apoptosis via the PARP-1 and caspases pathways.

Nutrients 2021, 13, 25 10 of 15 Nutrients 2021, 13, x FOR PEER REVIEW 10 of 15

FigureFigure 5. 5. FPFP inhibits inhibits SI SI-induced-induced apoptosisapoptosis inin APRE-19APRE-19 cells cells and and mouse mouse retina. retina. Representative Representative blot blot and and quantitative quantitative analysis analysisof PARP-1, of PARP caspase-1, caspase 3, and 3, and caspase caspase 8 protein 8 protein expression expression levels levels in (A in) ARPE-19(A) ARPE cells-19 cells and and (B) mouse(B) mouse retina. retina. Each Each band band was wasdensitometrically densitometrically quantiﬁed quantified by imageby image analysis. analysis. The The band band density density was was normalized normalized using using each each total total protein.protein. **** p < 0.01, 0.01, ****** p < 0.001 0.001 vs.vs. N; N; # # pp << 0.05, 0.05, ## ## pp << 0.01, 0.01, and and ### ### pp << 0.001 0.001 vs. vs. C. C. Values Values are are means means ±± SEM.SEM. Significant Signiﬁcant differences differences between between thethe groups groups were were determines by ANOVA followedfollowed by by Duncan’s Duncan’s multiple multiple range range test. test. PARP, PARP, poly poly (ADP-ribose) (ADP-ribose) polymerase; polymerase; N, N,Normal; Normal; C, C, Control; Control; P-Y, P- YellowY, Yellow Paprika; Paprika; P-O, P- OrangeO, Orange Paprika; Paprika; FP-Y, FP Fermented-Y, Fermented P-Y; P FP-O,-Y; FP Fermented-O, Fermented P-O. P-O.

3.7. Effect of FP on SI-Mediated AKT and Mitogen-Activated Protein Kinases (MAPK) Signaling3.7. Effect In of Vitro FP on and SI-Mediated In Vivo AKT and Mitogen-Activated Protein Kinases (MAPK) Signaling In Vitro and In Vivo To further confirm the capacity of FP, the effects on SI-induced oxidative stress-related Tosignaling further pathways confirm the were capacity investigated of FP, the in effects cellular on and SI-induced retina tissue. oxidative Oxidative stress-related stress issignaling known to pathways be a potent were activator investigated of PI3K/AKT in cellular and and MAPKs retina [9,32] tissue.. The Oxidative phosphorylation stress is known to be a potent activator of PI3K/AKT and MAPKs [9,32]. The phosphorylation of the of the signaling molecules, which are AKT, ERK1/2, JNK, and p38, were significantly in- signaling molecules, which are AKT, ERK1/2, JNK, and p38, were significantly increased creased by SI administration in ARPE-19 cells (Figure 6A) and mouse retina (Figure 6B). by SI administration in ARPE-19 cells (Figure6A) and mouse retina (Figure6B). There There is no significant difference between C and P (-Y or -O) and FP-Y extract treatment is no significant difference between C and P (-Y or -O) and FP-Y extract treatment group group in ARPE-19 cells. Interestingly, FP-O extract showed an inhibitory effect on SI-inin ARPE-19 cells. Interestingly, FP-O extract showed an inhibitory effect on SI-induced duced p38 phosphorylation in ARPE-19 cells (Figure 6A). Furthermore, FP-O extract p38 phosphorylation in ARPE-19 cells (Figure6A). Furthermore, FP-O extract markedly markedly decreased the phosphorylation of AKT, JNK, and p38 compared to the C group decreased the phosphorylation of AKT, JNK, and p38 compared to the C group (Figure6A). (Figure 6A). On the other hand, SI-induced ERK1/2 phosphorylation was not affected by On the other hand, SI-induced ERK1/2 phosphorylation was not affected by FP (-Y or -O) as FP (-Y or -O) as well as its raw materials extract treatment in ARPE-19 cell. Further anal- well as its raw materials extract treatment in ARPE-19 cell. Further analysis in mouse retina ysis in mouse retina showed that the FP (-Y or -O) treatment significantly downregulated showed that the FP (-Y or -O) treatment significantly downregulated the phosphorylated the phosphorylated AKT, ERK1/2, JNK, and p38 compared to SI treatment group (Figure AKT, ERK1/2, JNK, and p38 compared to SI treatment group (Figure6B) , indicating that 6B),fermentation indicating ofthat paprika, fermentation may be of helpfulpaprika, for may regulation be helpful of for SI-induced regulation PI3K/AKT of SI-induced and PI3K/AKTMAPK activation. and MAPK activation.

Nutrients 2021, 13, 25 11 of 15

Nutrients 2021, 13, x FOR PEER REVIEW 11 of 15

FigureFigure 6. 6.EffectEffect of FP of FPon ultraviolet on ultraviolet (UVB (UVB)-induced)-induced AKT AKTand MAPKs and MAPKs phosphorylation phosphorylation in APRE in-19 APRE-19 cells and cells mouse and retina. mouse Representativeretina. Representative blot and blotquantitative and quantitative analysis analysisof AKT, ofp-AKT, AKT, ERK, p-AKT, p-ERK1/2, ERK, p-ERK1/2, JNK, p-JNK, JNK, p38, p-JNK, and p38,p-p38 and in p-p38(A) ARPE in (A- ) 19ARPE-19 cells and cells (B) andmouse (B) retina. mouse β retina.-Actinβ was-Actin used was as useda loading as a loading control. control. The band The density band density was normalized was normalized using each using total each protein. Each band was densitometrically quantified by image analysis. ** p < 0.01, *** p < 0.001 vs. N; # p < 0.05, ## p < 0.01, total protein. Each band was densitometrically quantified by image analysis. ** p < 0.01, *** p < 0.001 vs. N; # p < 0.05, and ### p < 0.001 vs. C. Values are means ± SEM. Significant differences between the groups were determined by ANOVA ## p < 0.01, and ### p < 0.001 vs. C. Values are means ± SEM. Significant differences between the groups were determined followed by Duncan’s multiple range test. N, Normal; C, Control; P-Y, Yellow Paprika; P-O, Orange Paprika; FP-Y, Fer- mentedby ANOVA P-Y; FP followed-O, Fermented by Duncan’s P-O. multiple range test. N, Normal; C, Control; P-Y, Yellow Paprika; P-O, Orange Paprika; FP-Y, Fermented P-Y; FP-O, Fermented P-O. We further examined whether FP affects LPS-induced MAPK phosphorylation in We further examined whether FP affects LPS-induced MAPK phosphorylation in RAW 264.7 macrophages. RAW 264.7 cells were pretreated with P (-Y or -O) and FP-Y RAW 264.7 macrophages. RAW 264.7 cells were pretreated with P (-Y or -O) and FP-Y extract (500 μg/mL) and then treated with LPS (1 μg/mL) for 18 h. As shown in Figure 7, extract (500 µg/mL) and then treated with LPS (1 µg/mL) for 18 h. As shown in Figure7, treatment with LPS caused a significant increase in phospho-ERK1/2, JNK, and p-38 treatment with LPS caused a significant increase in phospho-ERK1/2, JNK, and p-38 MAPK. MAPK. FP extract significantly decreased the LPS-stimulated phosphorylation of JNK and FP extract significantly decreased the LPS-stimulated phosphorylation of JNK and p-38, p-38, whereas it had no effect on the expression level of p-ERK1/2. In both cellular levels, whereas it had no effect on the expression level of p-ERK1/2. In both cellular levels, we we obtained similar results that ERK1/2 phosphorylation was not affected by FP treat- obtained similar results that ERK1/2 phosphorylation was not affected by FP treatment. ment. These results suggest that the ability of fermented paprika to inhibit phosphorylation of JNKThese and results p38 MAPK suggest mediated that the ability by LPS of in fermented the RAW 264.7paprika macrophage. to inhibit phosphorylation of JNK and p38 MAPK mediated by LPS in the RAW 264.7 macrophage.

Nutrients 2021, 13, 25 12 of 15

Nutrients 2021, 13, x FOR PEER REVIEW 12 of 15

Figure 7. EffectFigure of FP on 7. EffectLPS-induced of FP on MAPKs LPS-induced activation MAPKs in RAW activation 264.7 macrophages. in RAW 264.7 Levels macrophages. of Levels of phosphorylatedphosphorylated ERK1/2, JNK, and ERK1/2, p38 were JNK, assessed and p38 by were immunoblot assessed byanalysis. immunoblot β-Actin analysis. was usedβ-Actin was used as a loading control.as a loading Each band control. was Eachdensitometrically band was densitometrically quantified by image quantiﬁed analysis. by ** image p < 0.01, analysis. *** ** p < 0.01, p < 0.001 vs. N; ## p < 0.01, and ### p < 0.001 vs. C; ǂ p < 0.01 vs. P-Y or P-O. Values are means ± *** p < 0.001 vs. N; ## p < 0.01, and ### p < 0.001 vs. C; ‡ p < 0.01 vs. P-Y or P-O. Values are means ± SEM. Significant differences between the groups were determines by ANOVA followed by Dun- SEM. Signiﬁcant differences between the groups were determines by ANOVA followed by Duncan’s can’s multiple range test. LPS, Lipopolysaccharides; N, Normal; C, Control; P-Y, Yellow Paprika; P-O, Orange Paprika;multiple FP range-Y, Fermented test. LPS, P Lipopolysaccharides;-Y; FP-O, Fermented P N,-O. Normal; C, Control; P-Y, Yellow Paprika; P-O, Orange Paprika; FP-Y, Fermented P-Y; FP-O, Fermented P-O.

4. Discussion4. Discussion In an attemptIn to an understand attempt to the understand protective the role protective of fermented role of paprika fermented in oxidative paprika in oxidative stress-inducedstress-induced RPE degeneration, RPE degeneration, we have investigated we have investigatedthe effect of suppression the effect of suppressionof FP on of FP on apoptosis andapoptosis have studied and havethe signaling studied themechanisms signaling mechanismsassociated with associated this effect. with For this this effect. For this purpose, we used a murine model of NaIO3-induced retinal degeneration in vivo and in purpose, we used a murine model of NaIO3-induced retinal degeneration in vivo and in human RPE human and LPS RPE-induced and LPS-induced oxidative oxidativestress in stressRAW264.7 in RAW264.7 macrophages. macrophages. Oxidative Oxidative stress stress has longhas been long beenrecognized recognized as a contribas a contributinguting factor factor to the to the pathogenesis pathogenesis of AMD of AMD [2,4,5]. A [2,4,5]. A commonlycommonly used used exp experimentalerimental model model to study to study the the link link between between oxidative oxidative stress stress and AMD and AMD involvesinvolves the the use use of cultured of cultured human human RPE RPE(ARPE19) (ARPE19) cells [4,8,11] cells [4,.8 Thus,,11]. Thus,it would it would be of be of great significancegreat significance to explore to explorethe oxidative the oxidative damage damageof RPE cells of RPE in order cells into orderexamine to examine the the pathogenesispathogenesis and treatment and treatment of AMD [5,24,33] of AMD. [5,24,33]. Studies have Studiesreported have that Capsicum reported annuum that Capsicum L. exhibit annuum a rangeL. of exhibit bioactivities a range includ- of bioactivities in- ing antioxidant,cluding antimicrobial, antioxidant, antiviral, antimicrobial, anti-inflammatory, antiviral, anti-inflammatory, and anticancer andproperties anticancer proper- [12–14]. Healthties-linked[12–14 functionality]. Health-linked improves functionality by fermentation improves of by nutrients, fermentation which of is nutrients, use- which is ful technologyuseful for enhancing technology antioxidative for enhancing and antioxidative biological activity and biological [15,20]. activityL. plantarum [15,20 is]. L. plantarum a lactic acid speciesis a lactic that acid improves species thatthe content improves of thephenolic content compounds of phenolic and compounds subsequently and subsequently enhanced antioxidantenhanced capacity antioxidant during capacity fermentation during fermentation[34]. Recently, [34 fermentation]. Recently, fermentation has been has been reported as onereported of the as effective one of the means effective to meansimprove to improvethe antioxidant the antioxidant properties properties of plants of plants [6,34]. [6,34]. We alsoWe reported also reported that antioxidant that antioxidant effects effectsof blackberry of blackberry were increased were increased by fermen- by fermentation tation using L.using plantarumL. plantarum JBMI F5JBMI [6]. However, F5 [6]. However, studies studieson the beneficial on the beneficial effects of effects fermen- of fermentation tation to improve biological efficacy have been reported using various materials, but there is no research on fermented paprika.

Nutrients 2021, 13, 25 13 of 15

to improve biological efficacy have been reported using various materials, but there is no research on fermented paprika. Based on this background, we tested whether paprika fermented with L. plantarum could increase its antioxidant capacity and the ability to regulate retinal degeneration which caused by oxidative stress. Total antioxidant capacity after fermentation was compared to non-fermented paprika. The radical scavenging effect of the fermented paprika was increased by fermentation (Table1) . Intracellular oxidative damage and consequent dysfunction in various pathologi- cal conditions is caused by increased cellular production of ROS and RNS [22]. ARPE-19 cells and RAW 264.7 cells were used to determine the potential of FP for intracellular oxidative stress. FP significantly improved the effect of reducing intracellular ROS production in ARPE-19 cells and NO production in RAW 264.7 cells (Figures2 and3). SI as a retinotoxin that is directly toxic to RPE by oxidative stress and consequently induces retinal degeneration [2,4,5]. To confirm that paprika fermentation is more useful for treating oxidative stress-induced ocular degeneration, we used a SI-induced retinal degeneration model in ARPE-19 cells and mice. Figure4A shows that SI treatment induced extensive disruption in the ONL and RPE. In contrast, pretreatment with FP ameliorated SI-induced retinal degeneration by counteracting oxidative stress through decreasing the SOD and GSH levels in serum and tissues (Figure4B,C). These in vivo experimental results suggested that, FP may protect the retinal oxidative stress through those functions mentioned in this study. SI has been reported to induce RPE cell death, which is associated with PARP and caspase-dependent cell death [24,26,31]. In order to evaluate if FP has the anti-apoptotic effects on SI-induced apoptosis, we analyzed apoptosis-related signaling proteins using Western blot analysis in APRE-19 cells and mouse retina tissues. The results showed that FPE had an anti-apoptotic effect on reducing the levels of cleaved PARP-1 and caspase-8 and -3 (Figure5). Next, we further examined the FP effect on ROS signaling pathway in vitro and in vivo. We found that FP administration inhibits AKT, JNK, and p38 phosphorylation, which are induced by SI treatment in ARPE-19 cells and retinal tissues (Figure6). ROS modulated various signaling pathways involved in inflammation, proliferation, differentiation, and apoptosis. Apoptosis is regulated by several signaling mechanisms, including the AKT and mitogen activated kinases in mammalian cells. Studies have reported that ROS can activate PI3K/AKT as well as MAPK subfamilies including ERK1/2, JNK, and P38 [9,11]. Hao et al. have reported that piceatannol protects H2O2-induced ARPE-19 cells’ apoptosis through inhibiting AKT phosphorylation [10]. Accumulating evidence reported that MAPKs are implicated in the pathogenesis of many disease [11,32,35]. In RPE cells, inhibition of MAPK pathway was able to prevent oxidative stress-induced RPE apoptosis [11]. Three conventional MAPKs subfamilies, ERKs, JNK, and p38-MAPK, play an important role in modulating the apoptotic pathway. The MAPKs pathway also plays important role in the transcriptional regulation of the LPS-induced expression of iNOS and COX-2 in macrophages [35]. Studies have shown that phenolic compounds increase during fermentation. However, in our results, TPC and TFC did not change significantly by fermentation (Table1). These results suggest that fermentation by L. plantarum contributed to glycoside cleavage and then depolymerization of additional polymer compounds [34]. However, our hypothesis will have to be proved through further research. In summary, our in vitro and in vivo results have shown that fermented paprika can increase the ability to protect macrophages and retinal damage caused by oxidative stress. The protective effect of FP on oxidative stress-mediated damage is the result of its anti-apoptotic effect and regulation of PI3K/AKT and MAPK signaling. In conclu- sion, FP can be expected as a potent therapeutic candidate for the treatment of retinal degenerative diseases. Nutrients 2021, 13, 25 14 of 15

Author Contributions: H.-R.K. and S.K. performed the experiments and analyzed the data. S.-W.L. and H.-S.S. discussed the result. S.-Y.K. designed the project and wrote the manuscript. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by the Technology development Program (S2653090) funded by the Ministry of SMEs and Startups (MSS, Korea). Institutional Review Board Statement: The in vivo experiment was performed in accordance with the Jeonju AgroBio-Materials Institute (JAMI) guidelines under a protocol approved by the Institu- tional Animal Care and Use Committee (IACUC) of JAMI (JAMI IACUC 2019001). Informed Consent Statement: Not applicable. Data Availability Statement: Data available on request due to ethical restrictions. Conﬂicts of Interest: The authors declare no potential conﬂict of interests.

References 1. van Lookeren Campagne, M.; LeCouter, J.; Yaspan, B.L.; Ye, W. Mechanisms of age-related macular degeneration and therapeutic opportunities. J. Pathol. 2014, 232, 151–164. [CrossRef] 2. Anderson, R.E.; Rapp, L.M.; Wiegand, R.D. Lipid peroxidation and retinal degeneration. Curr. Eye Res. 1984, 3, 223–227. [CrossRef] 3. Pujol-Lereis, L.M.; Schäfer, N.; Kuhn, L.B.; Rohrer, B.; Pauly, D. Interrelation between oxidative stress and complement activation in models of age-related macular degeneration. Adv. Exp. Med. Biol. 2016, 854, 87–93. [CrossRef] 4. Beatty, S.; Koh, H.; Phil, M.; Henson, D.; Boulton, M. The role of oxidative stress in the pathogenesis of age-related macular degeneration. Surv. Ophthalmol. 2000, 45, 115–134. [CrossRef] 5. Jabbehdari, S.; Handa, J.T. Oxidative stress as a therapeutic target for the prevention and treatment of early age-related macular degeneration. Surv. Ophthalmol. 2020, in press. [CrossRef] 6. Kim, H.-R.; Jeong, D.-H.; Kim, S.; Lee, S.-W.; Sin, H.-S.; Yu, K.-Y.; Jeong, S.-I.; Kim, S.-Y. Fermentation of blackberry with L. plantarum JBMI F5 enhance the protection effect on UVB-mediated photoaging in human foreskin fibroblast and hairless mice through regulation of MAPK/NF-κB signaling. Nutrients 2019, 11, 2429. [CrossRef][PubMed] 7. Chinskey, N.D.; Besirli, C.G.; Zacks, D.N. Retinal cell death and current strategies in retinal neuroprotection. Curr. Opin. Ophthalmol. 2014, 25, 228–233. [CrossRef][PubMed] 8. Ao, J.; Wood, J.P.; Chidlow, G.; Gillies, M.C.; Casson, R.J. Retinal pigment epithelium in the pathogenesis of age-related macular degeneration and photobiomodulation as a potential therapy? Clin. Exp. Ophthalmol. 2018, 46, 670–686. [CrossRef][PubMed] 9. Faghiri, Z.; Bazan, N.G. PI3K/Akt and mTOR/p70S6K pathways mediate neuroprotectin D1-induced retinal pigment epithelial cell survival during oxidative stress-induced apoptosis. Exp. Eye Res. 2010, 90, 718–725. [CrossRef][PubMed] 10. Hao, Y.; Liu, J.; Wang, Z.; Yu, L.L.; Wang, J. Piceatannol protects human retinal pigment epithelial cells against hydrogen peroxide induced oxidative stress and apoptosis through modulating PI3K/Akt signaling pathway. Nutrients 2019, 11, 1515. [CrossRef] 11. Kyosseva, S.V. Targeting MAPK signaling in age-related macular degeneration. Ophthalmol. Eye Dis. 2016, 8, 23–30. [CrossRef] [PubMed] 12. Silva, L.R.; Azevedo, J.; Pereira, M.J.; Valentão, P.; Andrade, P.B. Chemical assessment and antioxidant capacity of pepper (Capsicum annuum L.) seeds. Food Chem. Toxicol. 2013, 53, 240–248. [CrossRef][PubMed] 13. Sun, T.; Xu, Z.; Wu, C.T.; Janes, M.; Prinyawiwatkul, W.; No, H.K. Antioxidant activities of different colored sweet bell peppers (Capsicum annuum L.). J. Food. Sci. 2007, 72, S98–S102. [CrossRef][PubMed] 14. Khan, F.A.; Mahmood, T.; Ali, M.; Saeed, A.; Maalik, A. Pharmacological importance of an ethnobotanical plant: Capsicum annuum L. Nat. Prod. Res. 2014, 28, 1267–1274. [CrossRef] 15. Hur, S.J.; Lee, S.Y.; Kim, Y.-C.; Choi, I.; Kim, G.-B. Effect of fermentation on the antioxidant activity in plant-based foods. Food Chem. 2014, 160, 346–356. [CrossRef] 16. Torino, M.I.; Limón, R.I.; Martínez-Villaluenga, C.; Mäkinen, S.; Pihlanto, A.; Vidal-Valverde, C.; Frias, J. Antioxidant and antihypertensive properties of liquid and solid state fermented lentils. Food Chem. 2013, 136, 1030–1037. [CrossRef] 17. Floegel, A.; Kim, D.-O.; Chung, S.-J.; Chun, O.K. Comparison of ABTS/DPPH assays for the detection of antioxidant capacity in foods. FASEB J. 2010, 24, 535–539. [CrossRef] 18. Eigler, A.; Moeller, J.; Endres, S. Exogenous and endogenous nitric oxide attenuates tumor necrosis factor synthesis in the murine macrophage cell line RAW 264.7. J. Immunol. 1995, 154, 4048–4054. 19. Wei, X.; Luo, M.; Xu, L.; Zhang, Y.; Lin, X.; Kong, P.; Liu, H. Production of fibrinolytic enzyme from Bacillus amyloliquefaciens by fermentation of chickpeas, with the evaluation of the anticoagulant and antioxidant properties of chickpeas. J. Agric. Food Chem. 2011, 59, 3957–3963. [CrossRef] 20. Yan, Y.; Zhang, F.; Chai, Z.; Liu, M.; Battino, M.; Meng, X. Mixed fermentation of blueberry pomace with L. rhamnosus GG and L. plantarum-1: Enhance the active ingredient, antioxidant activity and health-promoting benefits. Food Chem. Toxicol. 2019, 131, 110541. [CrossRef] Nutrients 2021, 13, 25 15 of 15

21. Stanisavljević,N.; Samardžić,J.; Janković,T.; Šavikin, K.; Mojsin, M.; Topalović,V.; Stevanović,M. Antioxidant and antiprolifera- tive activity of chokeberry juice phenolics during in vitro simulated digestion in the presence of food matrix. Food Chem. 2015, 175, 516–522. [CrossRef][PubMed] 22. He, L.; He, T.; Farrar, S.; Ji, L.; Liu, T.; Ma, X. Antioxidants maintain cellular redox homeostasis by elimination of reactive oxygen species. Cell. Physiol. Biochem. 2017, 44, 532–553. [CrossRef][PubMed] 23. Enzmann, V.; Row, B.W.; Yamauchi, Y.; Kheirandish, L.; Gozal, D.; Kaplan, H.J.; McCall, M.A. Behavioral and anatomical abnormalities in a sodium iodate-induced model of retinal pigment epithelium degeneration. Exp. Eye Res. 2006, 82, 441–448. [CrossRef][PubMed] 24. Du, W.; An, Y.; He, X.; Zhang, D.; He, W. Protection of Kaempferol on oxidative stress-induced retinal pigment epithelial cell damage. Oxidative Med. Cell. Longev. 2018, 2018.[CrossRef] 25. Wang, J.; Iacovelli, J.; Spencer, C.; Saint-Geniez, M. Direct effect of sodium iodate on neurosensory retina. Invest. Ophthalmol. Vis. Sci. 2014, 55, 1941–1953. [CrossRef] 26. Balmer, J.; Zulliger, R.; Roberti, S.; Enzmann, V. Retinal cell death caused by sodium iodate involves multiple caspase-dependent and caspase-independent cell-death pathways. Int. J. Mol. Sci. 2015, 16, 15086–15103. [CrossRef] 27. Liu, C.; Li, Y.; Peng, M.; Laties, A.M.; Wen, R. Activation of caspase-3 in the retina of transgenic rats with the rhodopsin mutation s334ter during photoreceptor degeneration. J. Neurosci. 1999, 19, 4778–4785. [CrossRef] 28. Sanvicens, N.; Gómez-Vicente, V.; Masip, I.; Messeguer, A.; Cotter, T.G. Oxidative stress-induced apoptosis in retinal photoreceptor cells is mediated by calpains and caspases and blocked by the oxygen radical scavenger CR-6. J. Biol. Chem. 2004, 279, 39268–39278. [CrossRef] 29. Gastman, B.R.; Johnson, D.E.; Whiteside, T.L.; Rabinowich, H. Tumor-induced apoptosis of T lymphocytes: Elucidation of intracellular apoptotic events. Blood 2000, 95, 2015–2023. [CrossRef] 30. Yang, M.; So, K.F.; Lam, W.C.; Lo, A.C.Y. Novel programmed cell death as therapeutic targets in age-related macular degeneration? Int. J. Mol. Sci. 2020, 21, 7279. [CrossRef] 31. Singh, P.K.; Kumar, A. Mitochondria mediates caspase-dependent and independent retinal cell death in Staphylococcus aureus endophthalmitis. Cell Death Discov. 2016, 2, 16034. [CrossRef] 32. Wang, Z.; Bartosh, T.; Ding, M.; Roque, R. MAPKs Modulate RPE response to oxidative stress. J. Med. Bioeng. 2014, 3, 67–73. 33. Yang, Y.; Qin, Y.J.; Yip, Y.W.Y.; Chan, K.P.; Chu, K.O.; Chu, W.K.; Ng, T.K.; Pang, C.P.; Chan, S.O. Green tea catechins are potent anti-oxidants that ameliorate sodium iodate-induced retinal degeneration in rats. Sci. Rep. 2016, 6, 29546. [CrossRef][PubMed] 34. Li, Z.; Teng, J.; Lyu, Y.; Hu, X.; Zhao, Y.; Wang, M. Enhanced antioxidant activity for apple juice fermented with Lactobacillus plantarum ATCC14917. Molecules 2018, 24, 51. [CrossRef][PubMed] 35. Chen, C.-C.; Lin, M.-W.; Liang, C.-J.; Wang, S.-H. The anti-inflammatory effects and mechanisms of Eupafolin in Lipopolysaccharide- induced inflammatory responses in RAW264.7 macrophages. PLoS ONE 2016, 11, e0158662. [CrossRef] [PubMed]