**Preface to "Oxidative Stress Modulators and Functional Foods"**

Many years of research have elucidated the role of natural antioxidants and dietary supplements as functional foods with the potential to prevent oxidative stress due to the suppressing of reactive oxygen species (ROS) and reactive nitrogen species (RNS). Recent studies have also revealed that cell signalling pathways, such as Nrf2-ARE, signalling with antioxidant protein (heme oxygenase-1, HO-1) expression and coexisting anti-apoptotic cell signaling, play significant roles to avoid cell damage by the excessive production of ROS, RNS, or electrophiles. Therefore, natural products extending beyond the traditional antioxidant role are gaining a great deal of attention in functional foods, which can protect against various diseases related to oxidative stress. This book of "Oxidative Stress Modulators and Functional Foods" consists of 16 articles including 1 review article related to the antioxidant role of natural products and their ability to modulate oxidative stress and/or reverse disease both in vitro and in animal models. Additionally, the molecular mechanisms of these actions and the modulation of the signalling pathways in the redox system by natural products are included.

I would like to thank all the authors for their valuable contributions and all the reviewers for their availability to review the papers involving their useful suggestions to elevate scientific quality. Appreciate also to the journal's publishing team for their help in disseminating the call for papers and in every step of the publishing process.

> **Junsei Taira** *Editor*

### *Editorial* **Oxidative Stress Modulators and Functional Foods**

**Junsei Taira**

Department of Bioresources Technology, Okinawa College, National Institute of Technology, 905 Henoko, Okinawa, Nago 905-2192, Japan; taira@okinawa-ct.ac.jp

Many years of research have seen the investigation of natural antioxidants and dietary supplements as functional foods with the potential to prevent oxidative stress due to the scavenging of reactive oxygen species (ROS) and reactive nitrogen species (RNS). Recent studies have also revealed that cell signalling pathways, such as Nrf2-ARE, signalling with antioxidant protein (heme oxygenase-1, HO-1) expression, play significant roles in the cell's survival response to avoid cell damage by the excessive production of ROS, RNS, or electrophiles. Therefore, natural products extending beyond the traditional antioxidant role are gaining a great deal of attention in functional foods, which can protect against various diseases related to oxidative stress. This Special Issue consists of 15 articles related to the antioxidant role of natural products, but also their ability to modulate oxidative stress and/or reverse disease both in vitro and in animal models. Additionally, the molecular mechanisms of these actions and the modulation of the signalling pathways in the redox system by natural products are included.

Folic acid (FA) is known as a dietary supplement that can prevent neural tube defects (NTDs), involving the failure of neural tube (NT) closure in the developing embryo, especially spina bifida and anencephaly in the periconceptional period. Previous study indicated that moderate levels of nitric oxide (NO) and nitric oxide synthase (NOS) play a critical role in normal embryonic development. NO inhibits methionine synthase (MS), involving the interference transfer of the methyl group from the methyl donor, 5-methyltetrahydrofolate through the FA metabolite system (Folate pathway), to homocysteine during methionine production. Taira et al. [1] elucidated that FA can directly scavenge NO, suggesting that NO modulation, due to FA, may contribute to alleviation from failure in neural tube formation, causing the high level of NO production.

Understanding the structure–activity relationships of antioxidants and their mechanisms of action is important for designing more potent antioxidants for potential use as therapeutic agents as well as preservatives. The kinetic studies of antioxidative action of ellagic acid (EA) under physiological conditions elucidated that the hydroxyl radical (•OH) with EA conforms to hydrogen atom transfer and radical adduct formation mechanisms, whereas the sequential proton loss electron transfer mechanism is responsible for the scavenging of the CCl3OO• radical, generating in the organism during the metabolism of CCl4. In addition, compared to trolox, EA was found to be more reactive toward •OH, but less reactive toward CCl3OO• in which their calculated rate constants are in very good agreement with the corresponding experimental values [2]. From another viewpoint of antioxidant mechanisms, the computational study for antioxidant mechanisms was carried out using total enthalpy values on the electronic effects of ortho-substituents in dendritic tri-phenolic antioxidants, comprising a common phenol moiety and two other phenol units with electron-donating or electron-withdrawing substituents. As the preferred antioxidant mechanism, in sequential proton loss electron transfer (SPLET) it was found that electron-donating groups, such as the OCH3 group, are useful for designing potent dendritic antioxidants, while the nitro and halogens do not add value to the radical scavenging antioxidant activity [3]. Furthermore, to predict the antioxidant potentiality in vivo, Boix et al. [4] proposed that the zebrafish model assay has the capability to predict in vivo protective activity or to determine their underlying mechanisms of action. This

**Citation:** Taira, J. Oxidative Stress Modulators and Functional Foods. *Antioxidants* **2021**, *10*, 191. https:// doi.org/10.3390/antiox10020191

Received: 26 January 2021 Accepted: 27 January 2021 Published: 29 January 2021


**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the author. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

article showed a useful experimental system to evaluate the in vivo protective effects of different antioxidant compounds, based on the zebrafish embryo test under oxidative stress conditions using tert-butyl hydroperoxide, tetrachlorohydroquinone and lipopolysaccharide chemicals. This system was also applied to the study of the effects of well-known antioxidants, such as vitamin E, quercetin, and lipoic acid, and confirmed the zebrafish model as a useful in vivo tool to test the protective effects of antioxidant compounds.

Recent studies have revealed that cell signalling pathways, such as Nrf2-ARE signalling with HO-1 expression play significant roles to avoid cell damage causing oxidativerelated various diseases. Taira et al. [5] focused on exploring the Nrf2 active compound and over five hundred various edible medicinal herbs were evaluated by a reporter assay, and the highest Nrf2 activity was found in the ethanol extract of *Peucedanum japonicum* leaves. The active compound in the extract was identical to pteryxin based on 1H, 13C-NMR spectra and liquid chromatography/time-of-fright/mass spectrometry (LC/TOF/MS). Pteryxin accumulated the transcription factor Nrf2 in the nucleus and resulted in the expression of the HO-1. This study also suggested that the electrophilicity, due to α,β-carbonyl and/or substituted acyl groups in the molecule, modulates the cysteine residue in Keap1 via the Michel reaction, at which point the Nrf2 is dissociated from the Keap1. Furthermore, the Nrf2 activator in bioresource reported the function in relation to disease. Osteoarthritis (OA) is a common joint degenerative disease induced by oxidative stress in chondrocytes. Kim et al. [6] demonstrated the inhibitory effects of cudratricusxanthone O (CTO), isolated from the *Maclura tricuspidata* Bureau (Moraceae), on the H2O2-induced damage of SW1353 chondrocytes. CTO induced HO-1 expression, involving the translocation of Nrf2 into the nucleus. Pretreatment with CTO in H2O2-treated cells regulated ROS production by inducing, expression of antioxidant enzymes (SOD, catalase, glutathione peroxidase (GSH-Px), glutathione reductase, and HO-1, and also prevented H2O2-induced apoptosis by regulating the expression of anti-apoptotic proteins, such as Bcl-2 and Bax. Environmental stress, involving oxidative stress, due to Ultraviolet (UV) and air pollutants contributing fine dust (FD) containing hazardous chemicals, induces triggering allergic reactions and inflammation of the skin, which lead to thickening of the epidermis, discoloration, skin wrinkling, loss of elasticity and skin-cell growth retardation [7,8]. Fernando et al. [7] reported that a low molecular weight fucoidan fraction (SHC4, 60 kDa, with 37.43% fucose and 28.01% sulfate), isolated from *Sargassum horneri*, reduced intracellular ROS levels and increased the cell viability on UVB (280–320 nm) exposed HaCaT keratinocytes and inhibited UVB-induced apoptotic body formation, sub-G1 accumulation of cells through the mitochondria-mediated pathway. The UVB protective effect of SHC4 was facilitated by enhancing intracellular antioxidant defence via Nrf2-HO-1 signalling. The FD in air pollutants also produced the ROS in human HaCaT keratinocytes. (–)-loliolide (HTT), isolated from *Sargassum horneri*, has the potential to increase cell viability by reducing the ROS production in FD-stimulated keratinocytes, involving the mitochondria-mediated apoptosis pathway. HTT suppressed FD-stimulated DNA damage and the formation of apoptotic bodies, and it reduced the population of cells in the sub-G1 apoptosis phase. The cytoprotective effects of the HTT against FD-stimulated oxidative damage is mediated through squaring the Nrf2-HO-1 pathway involved in increasing HO-1 and NAD(P)H dehydrogenase (quinone) 1 in the cytosol [8].

In this Special Issue, the approach to find new biological activities of antioxidants in relation to various diseases and the different bioactivities, due to habitat-derived conditions, were reported by the following: (1) Type 2-diabetes mellitus (T2-DM) is caused by hyperglycaemic abnormalities in controlling blood glucose and insulin resistance. This article showed the mechanism of ameliorative effects due to quamoclit angulata (QA) on diabetes. QA supplementation (5 or 10 mg/kg/day) for 12 weeks reduced homeostasis model assessment insulin resistance, kidney malfunction, and glomerular hypertrophy in T2-DM. Moreover, the QA treatment significantly attenuated renal NLRP3 inflammasome-dependent hyper-inflammation and the consequential renal damage caused by oxidative stress, apoptosis, and fibrosis in T2-DM [9]. (2) In a similar model animal experiment, the high sugar-fat

(HSF) diet induced obesity, insulin resistance, cardiac dysfunction, and oxidative damage. When tomato-oleoresin supplementation (containing 10 mg lycopene/kg body weight (BW) per day) was given orally every morning for a 10-week period, the insulin resistance, cardiac remodelling, and dysfunction were improved by regulating the β-adrenergic response. [10]. (3) The antioxidant properties of epigallocatechin-gallate (EGCG), a green tea compound, have been already studied in various diseases. To improve the bioavailability of EGCG, this article demonstrated the result of the comparative effect of liposomal EGCG (L-EGCG) with EGCG solution in experimental DM induced by streptozotocin in rats. L-EGCG indicated a better efficiency regarding the improvement of oxidative stress parameters for malondialdehyde (MDA), NO, and total oxidative status; antioxidant status for total antioxidant capacity of plasma, thiols, and catalase and matrix-metalloproteinase-2 were also significantly reduced in the L-EGCG-treated group, compared with the EGCG group [11]. (4) Myricetin is present in many natural foods with various biological activities, such as anti-oxidative and anti-cancer activities. Benzo[a]pyrene (B[a]P), a group 1 carcinogen, induces mutagenic DNA adducts. B[a]P is metabolized by phase I enzymes, cytochrome P450 (CYP), and CYP1A1 also produces the metabolites conjugated with the DNA-BPDE (B[a]P-7,8-dihydrodiol-9,10-epoxide) adduct and 8-hydroxy-2'-deoxyguanosine formation. This article showed that myricetin reduces B[a]P-induced toxicity by inhibiting those metabolites by the reduction of the B[a]P metabolism via reduced CYP1A1 expression, and the elimination of B[a]P metabolites via enhanced GST expression [12]. (5) The variations in the phenolic profile for 21 compounds and bioactivities for antioxidant and antimutagenic activities between bilberry and lingonberry leaves different from three locations due to different altitude, solar exposure and temperature range were investigated. As a result, flavonols, hydroxycinnamic acids, and anthocyanins, due to habitat-derived conditions, could be clearly distinguished in these species [13].

As a large molecule antioxidant, a novel pectic polysaccharide, SAZMP4 (M.W, 28.94 kDa), mainly containing 1,4-linked galacturonic acid (GalA, 93.48%), with side chains of various neutral sugars, such as rhamnose, arabinose was isolated from Jujube pomace and the structure was determined by GC, FI-IR, GC-MS, NMR for molecule analysis, and SEM (scanning electron microscope) and AFM (atomic force microscope) for molecular morphological analysis. In addition, the antioxidant activity of SAZMP4 against H2O2-induced oxidative stress in Caco-2 cells demonstrated SOD activity and GSH-Px, MDA. Additionally, a better water retention capacity and the thermal stability of SAZMP4 indicated a potential application in the food industry as an additive [14].

The review article in this Special Issue discussed cellular and genetic factors that increase oxidative stress in Parkinson's disease (PD). PD is a neurodegenerative disorder caused by the depletion of dopaminergic neurons in the basal ganglia, the movement centre of the brain. The accumulation of oxidative stress-induced neuronal damage, due to the increased production of ROS or impaired intracellular antioxidant defences, invariably occurs at the cellular levels. The dopaminergic prodrugs and agonists can alleviate some of the symptoms of PD, but they could not be completely prohibited by the progression of PD pathology. The progress of PD takes a long time for the neurodegenerative process; therefore, the authors proposed that strategies to prevent or delay PD pathology may be well suited to lifestyle changes, such as dietary modification with antioxidant-rich foods, including vitamin C, vitamin E, carotenoids, selenium, and polyphenols [15].

**Funding:** This research received no external funding.

**Conflicts of Interest:** The author declares no conflict of interest.

#### **References**


### *Article* **Cudratricusxanthone O Inhibits H2O2-Induced Cell Damage by Activating Nrf2**/**HO-1 Pathway in Human Chondrocytes**

#### **Eun-Nam Kim** †**, Hyun-Su Lee** † **and Gil-Saeng Jeong \***

College of Pharmacy, Keimyung University, 1095 Dalgubeol-daero, Daegu 42601, Korea;

enkimpharm@gmail.com (E.-N.K.); hyunsu.lee@kmu.ac.kr (H.-S.L.)

**\*** Correspondence: gsjeong@kmu.ac.kr; Tel.: +82-53-580-6649

† These two authors contributed equally to this work.

Received: 24 July 2020; Accepted: 23 August 2020; Published: 25 August 2020

**Abstract:** Osteoarthritis (OA) is a common joint degenerative disease induced by oxidative stress in chondrocytes. Although induced-heme oxygenase-1 (HO-1) has been found to protect cells against oxygen radical damage, little information is available regarding the use of bioactive compounds from natural sources for regulating the HO-1 pathway to treat OA. In this study, we explored the inhibitory effects of cudratricusxanthone O (CTO) isolated from the *Maclura tricuspidata* Bureau (*Moraceae*) on H2O2-induced damage of SW1353 chondrocytes via regulation of the HO-1 pathway. CTO promoted HO-1 expression by enhancing the translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) into the nucleus without inducing toxicity. Pretreatment with CTO-regulated reactive oxygen species (ROS) production by inducing expression of antioxidant enzymes in H2O2-treated cells and maintained the functions of H2O2-damaged chondrocytes. Furthermore, CTO prevented H2O2-induced apoptosis by regulating the expression of anti-apoptotic proteins. Treatment with the HO-1 inhibitor tin-protoporphyrin IX revealed that these protective effects were exerted due to an increase in HO-1 expression induced by CTO. In conclusion, CTO protects chondrocytes from H2O2-induced damages—including ROS accumulation, dysfunction, and apoptosis through activation of the Nrf2/HO-1 signaling pathway in chondrocytes and, therefore, is a potential therapeutic agent for OA treatment.

**Keywords:** cudratrixanthone O; reactive oxygen species; nuclear transcription factor erythroid-2-like factor 2; hemeoxygenase-1; apoptosis

#### **1. Introduction**

Osteoarthritis (OA) is a chronic joint degenerative disease that affects normal movements due to loss of articular cartilage, particularly in older adults [1,2]. OA is accompanied by decomposition and destruction of the mesochondrium and cartilage, as well as synovial inflammation, but the main pathological reasons include oxidative stress, aging, and expression of inflammation-related genes [3]. A previous study showed that the degradation of the extracellular matrix (ECM) by the inflammatory response is essential for OA progression, and the damaged joint tissues produce severe cytokines and ECM degradation by-products [4]. Chondrocytes play an important role in maintaining the function of the joints and can generate tissue ECM—including collagen II and proteoglycan—to maintain tissue homeostasis and joint movement [5]. Reactive oxygen species (ROS)—such as hydrogen peroxide (H2O2)—are crucial modulators of the redox-sensitive cell signaling pathway, and involved in biological processes—such as host defense, oxygen sensing, proliferation, and apoptosis. However, from a pathological point of view, the overproduction of ROS is associated with inflammation, atherosclerosis, diabetes, high blood pressure, tumor formation, and OA [6]. Therefore, suppressing cell damage to chondrocytes by regulating ROS production in osteoarthritis is an important treatment strategy.

To eliminate cell damage caused by excessive ROS production, most cells, including chondrocytes, have endogenous defense strategies that protect cells from oxidative stress through the nuclear factor erythroid-2-related factor 2 (Nrf2) pathway [7,8]. The defense system activated by Nrf2 leads to induction of superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione (GSH), and heme oxygenase-1 (HO-1) [9]. Heme oxygenases (HOs) are a group of enzymes that catalyze heme breakdown, and four metabolites have been identified: iron, carbon monoxide (CO), and biliverdin. Three types of HO have been discovered, including HO-1, HO-2, and HO-3. HO-1 plays a key role in the defense mechanism against oxidative damage [10–12]. Although activation of Nrf2 and HO-1 as endogenous defense mechanisms in chondrocytes is important, little is known about whether bioactive small molecules isolated from natural products promote the Nrf2/HO-1 pathway to defend cells from oxidative damage.

*Maclura tricuspidata* Bureau (*Moraceae*) is a deciduous broad-leaved tree that is common in China, Korea, and Japan. It has been used in Korean traditional medicine to treat inflammation, gastritis, cancer, and liver damage [13–15]. The major components of *M. tricuspidata* are xanthones, flavonoids, isoflavonoids, and benzylated flavonoids. Among them, prenylated xanthones exhibit antioxidative, anti-inflammatory, antiatherosclerotic, and neuroprotective activities [16–18]. Moreover, prenylated xanthones have shown anti-inflammatory effects in RAW264.7 cells stimulated with lipopolysaccharides (LPS) by inhibiting the expression of pro-inflammatory mediators through HO-1 expression [19]. However, despite these various biological activities, prenylated xanthones have not been studied for OA. Previous studies have shown that hypoxia or inflammatory induction of SW1353 chondrocytes stimulated by IL-1β, monosodium iodoacetate (MIA), and H2O2 is known as a representative in vitro model for OA studies [20–23]. In this study, we investigated the role of prenylated xanthones, cudratricusxanthone O (CTO), isolated from *M. tricuspidata*, on the suppression of H2O2-induced cell damage by promoting the Nrf2/HO-1 pathway in SW1353 cells.

#### **2. Materials and Methods**

#### *2.1. Chemicals and Reagents*

Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum (FBS), penicillin, and streptomycin were purchased from Welgene Inc. (Korea). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) was purchased from Amresco Inc. (Solon, OH, USA). The primary antibodies of Nrf2, HO-1, β-actin, Bcl-2, superoxide dismutase (SOD) and catalase (CAT) were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA), Bax and Caspase-3 were purchased from Cell Signaling Technology (Danvers, MA, USA). Hydrogen peroxide solution (H2O2), Protoporphyrin IX (SnPP), Cobalt protoporphyrin (CoPP) and DCF-DA (2- , 7- -dichloroflourescin diacetate) were bought from Sigma Aldrich (St. Louis, MO, USA). RIPA buffer and ECL Western blotting detection reagents were purchased from Fisher Scientific Inc. (Waltham, MA, USA).

#### *2.2. Plant Materials and Isolation of Compounds*

A voucher specimen (accession number KMU 002019-0116) was deposited at the College of Pharmacy, Keimyung University, Daegu, Korea. Cudratrixanthone O (CTO) was isolated from the bark extract of *M. tricuspidata*, and the structure of CTO was identified using nuclear magnetic resonance (NMR) and electrospray ionization mass spectrometry (ESIMS) compared with previously reported literature [24].

#### *2.3. Cell Culture*

The human chondrosarcoma cell line SW1353 was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in Dulbecco's modified Eagle medium (DMEM) (Welgene, Gyeongsangbuk-do, Korea) containing 10% (v/v) fetal bovine serum, 10 μg/mL streptomycin, and 100 U/mL penicillin (Gibco BRL, Grand Island, NY, USA) in incubated on at 37 ◦C in 5% CO2.

#### *2.4. Cell Viability and Coe*ffi*cient Assays*

SW1353 cells (5 <sup>×</sup> 103 cells/well) were seeded in 96-well plates for 24 h, and cultured with or without CTO (1, 2, 5 μM) for 24 h. Then, 50 μL of MTT (5 mg/mL in PBS, Sigma-Aldrich) was treated to each well for 4 h. Four hours later, supernatant was aspirated, and 150 μL of DMSO was added to each well. The absorbance values were measured at 540 nm on a microplate reader (TECAN, Austria), and for confluency assays cells were seeded in 24-well plates for 24 h, after then, with CTO (1, 2, 5 μM), and the cells counted with Incucyte® Live-Cell analysis systems (Göttingen, Germany).

#### *2.5. Western Blot Analysis*

Western blot analysis was performed to examine the expression levels of indicated proteins in SW1353 chondrocytes. Cells were lysed in RIPA buffer containing protease inhibitors and centrifuged at 14,000 rpm for 30 min and quantitate by Bradford assay using a Bio-Rad Bradford assay reagent (Hercules, CA, USA). Then, proteins were separated using 8–12% SDS/polyacrylamide gel electrophoresis and transferred on to PVDF membranes, After blocking with TBS-T buffer containing skim milk (5%), The membranes were incubated with the primary antibodies overnight at 4 ◦C, after with a secondary antibody. PVDF membrane was detected with Healthcare Life Science ECL-plus (Tokyo, Japan), the images were taken by ImageQuant LAS 4000 (GE Healthcare Life Science, Tokyo, Japan). The expressional value of cytosolic proteins was normalized to the intensity level of β-actin and proteins compared with the untreated cells (control) using image J software.

#### *2.6. Cytosolic and Nuclear Protein Extraction*

SW1353 cells were seeded at 5 <sup>×</sup> 105 cells/mL in a 6-well plate. The harvested cells were then lysed on ice for 20 min with radioimmunoprecipitation assay (RIPA) buffer (Thermo Fisher Scientific, Waltham, MA, USA) and the isolated cytoplasm and nuclei were removed using the NE-PER nuclear and cytoplasmic extraction reagent kit (Pierce Biotechnology, Rockford, IL, USA) according to the manufacturer's instructions.

#### *2.7. Measurement of ROS Generation*

The production of intracellular ROS was assessed using a cell-permeable fluorogenic probe, 2- , 7- -dichlorodihydrofluorescein diacetate (DCF-DA). SW-1353 cells were seeded in 6-well plate at <sup>1</sup> <sup>×</sup> 105 cells/well for 24 h, and after pretreated with different concentrations of CTO for 6 h and then cultured for 2 h in the presence or absence of 0.5 mM H2O2. Then, the cells were washed twice with PBS to and DCF-DA was incubated in a dark place at 37 ◦C for 20 min. After 20 min, cells were washed with PBS and fixed with 4% paraformaldehyde (pH 7.4) for 20 min. After observation, the ROS was detected by a fluorescence Olympus IX microscope 71-F3 2PH (Tokyo, Japan).

#### *2.8. RT-qPCR Analysis*

After treatment, total RNA was extracted from the SW-1353 cells using TRIzol/chloroform reagent (Bioneer, Korea) according to the manufacturer's instructions. Total RNA was transcribed into cDNA by PrimeScript-RT reagent kit, then the cDNA was amplificated by the SYBR Premix Ex Taq (Sangon). The cycling conditions were 40 cycles at 50 ◦C for 2 min, 95 ◦C initial denaturation for 10 min, 95 ◦C denaturation for 15 s, and 60 ◦C annealing for 30 s. The mRNA encoding each target was measured using real-time PCR and GAPDH was used as the housekeeping gene. The cycle threshold (Ct) value of the target gene was normalized to GAPDH. The primers and amplification products of each gene used in this study are shown in Table 1.


**Table 1.** Primer sequences.

#### *2.9. Annexin V-FITC*/*PI Apoptosis Assay*

SW-1353 cells were seeded in six-well plates for 24 h, then pretreated with CTO (0, 1, 2, 5 μM) for 6 h. The positive control group and the CTO-treated groups were then exposed to H2O2 to a final concentration of 0.5 mM for 2 h. Cells were collected by centrifugation and washed twice with PBS, and apoptotic incidence was analyzed by the Annexin V-FITC/PropidiumIodide (PI) detection kit (BD Biosciences, San Diego, CA, USA) according to the manufacturer's instructions. The rate of apoptosis was analyzed using a Incucyte® Live-Cell analysis systems.

#### *2.10. Statistical Analysis*

Each experiment was performed in triplicate and expressed as mean value and standard deviation. Statistical analysis was conducted using SPSS Statistics 19.0 software. Differences among groups were analyzed by one-way analysis of variance (ANOVA) followed by Tukey's test or Student's *t*-test. *p* < 0.05 were considered to indicate statistical significance.

#### **3. Results**

#### *3.1. CTO Is Not Cytotoxic to SW1353 Chondrocytes*

To investigate whether CTO (Figure 1A) showed cytotoxicity in chondrocytes, SW1353 chondrocytes were treated with 0, 1, 2, and 5 μM of CTO for 24 h, and cell viability was examined by MTT analysis. No cytotoxicity was observed in the CTO-treated cells, and no morphological change in CTO-treated SW1353 cells (Figure 1B). Moreover, treatment with CTO (0–5 μM) did not affect cellular confluency in SW1353 cells (Figure 1C). CTO showed no statistically significant changes in cytotoxicity or cellular confluency.

**Figure 1.** Cudratricusxanthone O (CTO) is not cytotoxic to SW1353 chondrocytes. (**A**) The chemical structure of CTO. (**B**) SW1353 chondrocytes were seeded at a density of 1 <sup>×</sup> 104 cells/well and treated with CTO at the indicated concentrations (0–5 μM) for 24 h, then after CTO cytotoxicity was evaluated by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay. (**C**) The confluency of cells was determined using IncuCyte imaging system. The Student's *t*-test was used for statistical analysis.

#### *3.2. CTO Induces HO-1 Expression by Promotion of Nrf2 Translocation*

Nrf2 and HO-1, the major downstream antioxidant signaling pathway, play a major role in regulating oxidative stress. Therefore, the protein expression of HO-1 was measured by Western blot to confirm that CTO activates the Nrf2/HO-1 signaling pathway. After treatment with 5 μM of CTO in SW1353 cells, the expression of HO-1 was induced in a time- and dose-dependent manner. HO-1 was expressed in cells treated with CTO for 6 h and showed the highest expression at 18 h (Figure 2A, top). Concentration-dependent experiments showed that treatment with CTO promoted HO-1 expression in a dose-dependent manner (Figure 2A, bottom). Western blot was used to verify whether Nrf2 translocation into the nucleus was involved in HO-1 expression by CTO. Dose-dependent treatment with 5 μM CTO reduced Nrf2 in the cytosol but enhanced its expression in the nucleus (Figure 2B). HO-1 expression is induced by the activation of MAPK, of which ERK1/2, JNK, and p38 act as upstream regulators of the Nrf2 cascade. We investigated whether CTO promotes Nrf2 translocation and HO-1 induction through activation of the MAPK pathway. Western blotting was performed on SW1353 cells treated with CTO 5 μM in a time-dependent manner for 15, 30, and 60 min. Gradually enhanced phosphorylation of MAPKs was observed after CTO treatment (Figure 2C). These results suggested that CTO was involved in Nrf2 translocation and HO-1 expression by activation of MAPKs in SW1353 cells.

#### *3.3. CTO Suppresses ROS Production by Inducing SOD and CAT in SW1353 Cells*

It has been reported that treatment with H2O2 of chondrocytes produces ROS, which leads to cell apoptosis. To evaluate the effect of CTO on the production of ROS from cells treated with H2O2 in chondrocytes, the production of ROS and the expression of antioxidant enzymes such as SOD and CAT were evaluated in H2O2-stimulated SW1353 cells. In DCF-DA fluorescence staining, H2O2-stimulated cells showed an increase in ROS, but pretreatment with dose-dependent CTO reduced ROS production (Figure 3A). Expression levels of antioxidant proteins SOD and CAT were inhibited by H2O2 treatment but significantly protected by CTO treatment (Figure 3B). Furthermore, the effect of CTO on mRNA levels of SOD and CAT was evaluated by real-time PCR. We confirmed that expression levels of both antioxidant protein and mRNA were protected (Figure 3C). These results suggest that CTO regulates ROS production by preventing H2O2-induced apoptosis and retaining the expression of antioxidant proteins in chondrocytes.

**Figure 2.** CTO induces heme oxygenase-1 (HO-1) expression by the promotion of Nrf2 translocation. (**A**) The cells (5 <sup>×</sup> <sup>10</sup><sup>5</sup> cells/mL) were treated with 5 <sup>μ</sup>M for the indicated time (0, 6, 12, 18, 24 h) or with the indicated concentrations of CTO (0.5, 1, 2, and 5 μM) and cobalt protoporphyrin (CoPP) (20 μM) for 18 h. The induced HO-1 expression was detected by Western blot analysis. (**B**) The translocation of Nrf2 was analyzed by Western blot analysis from cells treated with 5 μM of CTO for the indicated time. (**C**) The cells were treated with 5 μM CTO for the indicated times (0–60 min) and phosphorylation of ERK1/2, JNK and p38 were determined by Western blot analysis. \* *p* < 0.05 was considered significant differences between groups are indicated.

#### *3.4. CTO Up-Regulates Chondrocytes-Specific Genes but Inhibits the Expression of MMPs in H2O2 Treated SW1353 Cells*

Accumulating evidence suggests that H2O2-induced ROS production degrades the function of chondrocytes. Cartilage-specific genes such as *col2a1* and *aggrecan* play an essential role in the creation and maintenance of cartilage tissue, and proteolytic enzymes such as MMPs induce cartilage tissue loss. Therefore, to explore whether CTO pretreatment prevents functional loss of chondrocytes by H2O2, the mRNA levels of essential genes for cartilage tissue formation (*col2a1, aggrecan*), *timps, mmps*, and *adamts* were measured using real-time PCR in H2O2-treated SW1353 chondrocytes. We found that the mRNA levels of *col2a1, aggrecan*, *timp1*, and *timp3* were suppressed by H2O2, but protected in a concentration-dependent manner by CTO (Figure 4A). Moreover, we confirmed that the mRNA levels of *mmps* and *adamts* (a group of secreted proteinases and proteolytic enzymes), which suppressed the functions of chondrocytes, were increased by H2O2 treatment but significantly decreased by CTO treatment (Figure 4B). These results suggest that CTO protects the function of chondrocytes by upregulating essential genes for cartilage tissue formation as well as downregulating *mmps* and *adamts* upon H2O2 treatment.

**Figure 3.** CTO suppresses ROS production by inducing superoxide dismutase (SOD) and catalase (CAT) in SW1353 cells. (**A**) The cells were pre-treated with the indicated concentrations of CTO for 6 h, and then stimulated with or without 0.5 mM H2O2 for 2 h. The cells were incubated at 37 ◦C in the dark for 20 min with culture medium containing 2 μM 2- , 7- -dichloroflourescin diacetate (DCF-DA) to monitor reactive oxygen species (ROS) production. The degree of ROS production was measured by fluorescence microscope. (**B**) The expression of SOD and CAT proteins were measured by Western blot analysis from cells pre-treated with CTO and stimulated by 0.5 mM H2O2. (**C**) The mRNA level of these genes was measured by real-time PCR. \* *p* < 0.05 was considered significant compared to only H2O2 treat group.

#### *3.5. CTO Inhibits Apoptotic Pathway Induced by Treatment with H2O2 in SW1353 Cells*

To evaluate whether pretreatment with CTO of chondrocytes prevents H2O2-induced apoptosis, we measured the confluency of H2O2-stimulated cells. Cellular confluency revealed that pretreatment

with CTO protected cells from the cytotoxicity of H2O2 in chondrocytes in a concentration-dependent manner (Figure 5A). To confirm whether pretreatment with CTO prevents cells to undergo apoptosis, Annexin V and caspase 3/7 from SW1353 cells pretreated with CTO and stimulated with H2O2 were detected by IncuCyte imaging system. In the CTO-pretreated cells, the previously increased intensity of Annexin V by H2O2 was then significantly reduced, and the previously decreased intensity of caspase 3/7 by H2O2 was then enhanced. (Figure 5B). To examine whether CTO pretreatment affected the mRNA levels of anti-apoptotic genes such as *bcl2* and *caspases3* or pro-apoptotic genes including *bax* in H2O2-treated conditions, we performed real-time PCR for SW1353 cells incubated with the indicated condition. The expression of *bcl2* and *caspases3* was reduced by H2O2 treatment, but retained by CTO pretreatment. On the other hand, the enhanced expression of the pro-apoptotic gene *bax* by H2O2 treatment decreased in a concentration-dependent manner by CTO pretreatment (Figure 5C). Protein levels of these genes were confirmed through Western blotting (Figure 5D). These results suggest that CTO pretreatment effectively prevents chondrocytes from H2O2-induced apoptosis by regulating the expression of apoptosis-related genes.

**Figure 5.** CTO inhibits the apoptotic pathway induced by treatment with H2O2 in SW1353 cells. (**A**, **B**) The cells were pre-treated with or without the indicated concentration of CTO (0, 1, 2, and 5 μM) for 6 h and then incubated with 0.5 mM H2O2 for 2 h. The confluency of cells was measured by IncuCyte imaging system (**A**), and apoptotic cells were evaluated by staining with Annexin V and caspase-3/7 (**B**). (**C**, **D**) The mRNA levels of *bcl-2*, *bax* and *caspase3* were measured by real-time PCR (**C**) and protein expressions were confirmed by Western blot analysis (**D**). The results were normalized to *gapdh* (**C**) or β–actin (**D**) expression. \* *p* < 0.05 was considered significant compared to only H2O2 treat group.

#### *3.6. Upregulated HO-1 by Pre-Treatment with CTO Protects SW1353 Cells from ROS Production Induced by H2O2*

In Section 3.2, we showed that CTO effectively induces HO-1 expression by promoting Nrf2 translocation in SW1353 cells. We next investigated the effect of CTO on ROS production and antioxidant enzymes induced by H2O2 treatment in suppressing the expression of HO-1 by treatment with tin protoporphyrin IX (SnPP), an inhibitor of heme oxygenase enzyme. CTO treatment suppressed ROS generated by H2O2 stimulation in a concentration-dependent manner; however, the inhibitory effect of CTO on ROS production was reversed in the presence of SnPP (Figure 6A). Moreover, the recovery of SOD and CAT expression by pretreatment with CTO in H2O2-treated cells was inhibited in the SnPP-treated group (Figure 6B) and mRNA (Figure 6C). These results suggested that HO-1 induced by CTO pretreatment in H2O2-stimulated SW1353 cells is involved in the regulation of ROS production and recovery of antioxidant enzymes.

**Figure 6.** Upregulated HO-1 by pre-treatment with CTO protects SW1353 cells from ROS production induced by H2O2. (**A**) SW1353 cells were seeded at a density of 5 <sup>×</sup> 10<sup>4</sup> cell/well and pre-treated with or without 20 μM Protoporphyrin IX (SnPP) for 1 h. Then cells were treated with the indicated concentrations of CTO (0, 1, 2, and 5 μM) for 6h, and incubated with 0.5 mM of H2O2 for 2h. The cells were incubated with culture medium containing 20 μM DCF-DA to monitor ROS production at 37 ◦C in the dark for 20 min. The images were obtained by fluorescent microscope system. (**B**, **C**) Protein expressions and mRNA levels of *sod* and *cat* were detected by Western blot analysis (**B**) and real-time PCR (C) from cells cultured with the indicated conditions. The results were normalized with β–actin (**B**) or *gapdh* (**C**) expression. \* *p* < 0.05 vs. only H2O2 treat group; # *p* < 0.05 vs. only CTO treated group.

#### *3.7. Induced HO-1 by CTO Regulates Chondrocytes-Specific Genes and Expression of mmps in H2O2 Treated SW1353 Cell*

We evaluated the effects of CTO on the specific genes and its function regulation of chondrocytes. Therefore, we additionally evaluated whether HO-1 expression by pretreatment with CTO protects chondrocyte-specific genes and functions in SW1353 cells in H2O2-treated conditions. CTO dose-dependently recovered the mRNA levels of *col2a1, aggrecan, timp1* and *timp3*; however, SnPP-treated cells did not show recovery of *col2a1, aggrecan, timp1* and *timp3* in H2O2-treated conditions (Figure 7A). We subsequently evaluated the effect of HO-1 expression by CTO treatment in H2O2-stimulated SW1353 cells on the mRNA levels of *mmps* and *adamts*, components of the extracellular matrix. We confirmed that the enhanced mRNA levels of *mmp3*, *mmp13*, and *adamts* by H2O2 were significantly blocked by CTO treatment, but recovered by treatment with SnPP (Figure 7B). These results suggest that the CTO-induced HO-1 protects SW1353 cells against dysfunction caused by H2O2 treatment, such as restoring cartilage-specific core proteins and inhibiting proteolytic enzymes.

#### *3.8. Enhanced HO-1 by CTO Shows Protective E*ff*ect from Apoptosis Induced by H2O2 in SW1353 Cells*

In Section 3.7, we confirmed that CTO protected SW1353 cells H2O2-induced apoptosis. To investigate whether induced HO-1 expression by CTO pretreatment plays a protective role in apoptosis induced by H2O2 treatment, the expression of annexin V and caspase 3/7 were assessed in cells treated with SnPP. Suppression of Annexin V by CTO pretreatment was significantly increased by SnPP treatment. The level of caspase-3/7 upregulated by CTO treatment was re-inhibited by SnPP

treatment (Figure 8). These results suggest that the induction of HO-1 by CTO pretreatment prevents H2O2-induced apoptosis in SW1353 cells.

**Figure 7.** Induced HO-1 by CTO regulates chondrocytes-specific genes and expression of MMPs in H2O2 treated SW1353 cells. (**A**, **B**) SW1353 cells were pre-treated with or without 20 μM SnPP for 1h. Then cells were treated with the indicated concentration of CTO (2 or 5 μM) for 6 h, and incubated with 0.5 mM H2O2 for 2 h. The mRNA levels of cartilage-specific core genes and *timps* (**A**) and *mmps* genes (**B**) were determined by real-time PCR analysis. The results were normalized to *gapdh* expression. \* *p* < 0.05 vs. only H2O2 treat group; # *p* < 0.05 vs. only CTO treated group.

**Figure 8.** Enhanced HO-1 by CTO shows a protective effect from apoptosis induced by H2O2 in SW1353 cells. (**A**) SW1353 cells were pre-treated with or without 20 μM SnPP for 1h. Then cells were treated with the indicated concentration of CTO (2 or 5 μM) for 6 h, and incubated with 0.5 mM H2O2 for 2 h. Apoptotic cells were evaluated by staining with caspase 3/7. Reagents using IncuCyte imaging system. \* *p* < 0.05 vs. only H2O2 treat group; # *p* < 0.05 vs. only CTO treated group.

#### **4. Discussion**

Promoting the expression of antioxidant enzymes in articular chondrocytes and inhibiting oxidative stress has been considered as a potential therapeutic approach in OA [25,26]. Hydrogen peroxide (H2O2) is a general type of ROS, and enzymes such as SOD, CAT, GPX, glutathione reductase (GR), and HO-1 protect the oxides caused by oxidative stress [27]. Nrf2 is a nuclear transcription factor that promotes the expression of antioxidant-related enzymes, such as HO-1, through binding to antioxidant response elements (AREs) and shows a protective role against cellular damages [28]. The MAPK pathway is involved in the translocation of Nrf2 to regulate oxidative stress [29]. Western blot analysis showed that CTO increased HO-1 expression depending on the concentration and time of translocation of Nrf2 to the nucleus. Moreover, CTO enhanced the phosphorylation of MAPKs in a time-dependent manner. These results suggest that CTO defends the cells against oxidative damage by up-regulating HO-1 expression via the MAPK pathway.

As one of several antioxidant mechanisms to prevent ROS-induced damage, SOD and CAT play vital roles in reducing oxidative stress by removing H2O2 [30]. In these mechanisms, CTO down-regulated the level of ROS stimulated by H2O2, and Western blot analysis showed the recovery of expression levels of antioxidant enzymes previously inhibited by H2O2. Reduced expression of Col2A1 and Aggrecan in chondrocytes is an important feature of cartilage degeneration; MMP-3 and MMP-13 are responsible for the degradation of the extracellular matrix that damages cartilage structures and properties, and TIMPs inhibit activities of these MMPs [31,32]. Inhibition of MMPs is recognized as a major treatment strategy to block joint cartilage loss in OA, and H2O2 promotes the expression and secretion of enzymes such as MMP-3, MMP-13, and ADAMTS [33]. CTO effectively inhibited the MMP-3, MMP-13, and ADAMTS enzymes secreted from H2O2 stimulated chondrocytes, and recovered Col2A1, Aggrecan, and TIMP mRNA, which play an essential role in cartilage degeneration caused by H2O2. From the previous results, CTO is an HO-1 inducer. Therefore, to investigate the protective effect of CTO-induced HO-1 expression on the H2O2-stimulated chondrocytes, we reversed the protective effect of chondrocytes by treatment with SnPP, an HO-1 inhibitor.

H2O2 causes apoptosis by increasing the production of ROS and destroying the mitochondrial membrane potential, which activates Bax of apoptosis and inactivates antiapoptosis Bcl-2 [34,35]. It also sequentially activates stimulators of apoptosis, such as caspase-3 and 7 [36]. In this study, CTO recovered the mRNA and protein expression levels of the anti-apoptotic protein BCL-2 in H2O-treated conditions and further inhibited those of apoptotic stimulants, such as BAX, induced by H2O2. The protein and mRNA levels of caspase-3 were also reversed. Previous studies showed that hypoxia leads to the formation of ROS, which induces oxidative stress and partially activates the transcription factor Nrf2, resulting in HO-1 expression and hypoxia-inducible factors through the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway [37]. In this study, H2O2-stimulated SW1353 chondrocytes were treated with SnPP, an HO-1 inhibitor, to suppress HO-1 expression induced by CTO. Therefore, the ROS inhibitory and anti-apoptotic effects of CTO as shown above, and the expression of antioxidant enzymes, were reversed. These results suggest that the protective effect on chondrocytes appears through HO-1 expression by the activity of Nrf2 by CTO rather than by Nrf2 activity by H2O2. These results suggest that, through activation of Nrf2 and HO-1, CTO could suppress ROS production and apoptosis as well as regulate antioxidant enzyme expression stimulated by H2O2 in SW1353 cells.

#### **5. Conclusions**

In this study, we investigated the effects of CTO on apoptotic, antioxidant enzymes and cartilage-specific proteins caused by ROS, a major cause of osteoarthritis in H2O2 stimulated SW1353 cells. We found that CTO effectively regulated ROS generation by H2O2 and apoptosis of SW1353 through Nrf2/HO-1 activity, and recovered lost antioxidant enzymes. This study revealed a new pharmacological effect of prenylated xanthones CTO, isolated from *M. tricuspidata*, and suggests its potential as a novel natural treatment for osteoarthritis.

**Author Contributions:** E.-N.K. and H.-S.L. performed the experiments and wrote the manuscript, performed the statistical analysis. G.-S.J. participated in study design and coordination as well as drafting the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2016R1A6A1A03011325).

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

#### *Article*
