Sargassum serratifolium Extract Attenuates Interleukin-1β-Induced Oxidative Stress and Inflammatory Response in Chondrocytes by Suppressing the Activation of NF-κB, p38 MAPK, and PI3K/Akt

Abstract

Osteoarthritis (OA) is a degenerative joint disease that is characterized by irreversible articular cartilage destruction by inflammatory reaction. Among inflammatory stimuli, interleukin-1β (IL-1β) is known to play a crucial role in OA pathogenesis by stimulating several mediators that contribute to cartilage degradation. Recently, the marine brown alga Sargassum serratifolium has been reported to exhibit antioxidant and anti-inflammatory effects in microglial and human umbilical vein endothelial cell models using lipopolysaccharide and tumor necrosis factor-α, but its beneficial effects on OA have not been investigated. This study aimed to evaluate the anti-osteoarthritic effects of ethanol extract of S. serratifolium (EESS) in SW1353 human chondrocytes and, in parallel, primary rat articular chondrocytes. Our results showed that EESS effectively blocked the generation of reactive oxygen species in IL-1β-treated SW1353 and rat primary chondrocytes, indicating that EESS has a potent antioxidant activity. EESS also attenuated IL-1β-induced production of nitric oxide (NO) and prostaglandin E₂, major inflammatory mediators in these cells, which was associated with the inhibition of inducible NO synthase and cyclooxygenase-2 expression. Moreover, EESS downregulated the level of gene expression of matrix metalloproteinase (MMP)-1, -3 and -13 in SW1353 chondrocytes treated with IL-1β, resulting in their extracellular secretion reduction. In addition, the IL-1β-induced activation of nuclear factor-kappa B (NF-κB) was restored by EESS. Furthermore, EESS reduced the activation of p38 mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-kinase (PI3K)/Akt signaling pathways upon IL-1β stimulation. These results indicate that EESS has the potential to exhibit antioxidant and anti-inflammatory effects through inactivation of the NF-κB, p38 MAPK, and PI3K/Akt signaling pathways. Collectively, these findings demonstrate that EESS may have the potential for chondroprotection, and extracts of S. serratifolium could potentially be used in the prevention and treatment of OA.

1. Introduction

Osteoarthritis (OA), characterized by progressive articular cartilage destruction, is the most common joint disease to be associated with joint trauma and aging [1,2]. Among the various causes of OA, inflammation is the most important contributor of imbalance of the cartilage metabolic pathway, which promotes the reduction of homeostasis and the degradation of extracellular matrix (ECM). In addition, the pathogenesis of OA involves oxidative stress due to the overproduction of reactive oxygen species (ROS), and changes in redox signaling pathways [3,4,5].

Inflammatory cytokines such as interleukin-1 beta (IL-1β) and tumor necrosis factor-α play a pivotal role in OA pathogenesis as potent inducers of many catabolic factors [6,7,8]. Among these cytokines, the levels of IL-1β have been reported to be significantly higher in synovial fluid, as well as serum, than in normal individuals [9,10]. When chondrocytes are exposed to IL-1β, the production of ROS and the release of inflammatory mediators, such as nitric oxide (NO) and prostaglandin E₂ (PGE₂), are increased. IL-1β also induces cartilage degradation by promoting the expression of major proteases, such as matrix metalloproteinases (MMPs), in chondrocytes [11,12,13]. Currently, non-steroidal anti-inflammatory drugs are commonly used for the symptomatic relief of OA. However, some side effects from long-term use have been reported, and reliable and efficient drugs still remain to be studied [14,15].

Recently, great attention has been focused on marine resources for the prevention and treatment of various diseases. Among them, seaweeds are rich in active substances having various pharmacological actions, which may offer great potential for the treatment of OA [16]. Sargassum is a genus of marine brown algae (Phaeophyceae) that is found in most seas around the world, and many coastal people, especially in South Korea, Japan, and China, use it as food and a medicine source [17]. Recently, extracts or isolated compounds of Sargassum serratifolium belonging to Sargassum spp. have been reported to have a variety of potent antioxidant, anti-inflammatory, and anti-cancer effects [18,19,20,21]. However, the effects of extracts and constituents from S. serratifolium on OA have not previously been investigated. Therefore, as part of a study to identify potent anti-OA agents from seaweeds, this study was designed to examine the protective effects of ethanol extract of S. serratifolium (EESS) and the underlying mechanisms on oxidative and inflammatory responses to IL-1β-stimulated chondrocytes.

2. Results

2.1. Effect of EESS on SW1353 and Rat Articular Chondrocyte Cytotoxicity

To determine the treatment concentration of EESS in SW1353, a well-established chondrocyte model, and primary cultures of rat articular chondrocytes isolated from the knee joints of 6-week-old Sprague-Dawley male rats, we first examined the cytotoxicity of EESS. According to the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay results in Figure 1A,B, EESS at the concentration range of 100–400 μg/mL did not show cytotoxic effect on these cells, but significant cytotoxicity was observed at concentrations above 600 μg/mL. In addition, when EESS was administered to SW1353 and rat articular chondrocytes treated with 40 ng/mL IL-1β, at a concentration of 300 μg/mL or less, no adverse effect on cell viability was shown (Figure 1B,D). Therefore, the maximum treatment concentration of EESS was set at 300 μg/mL in all of the following experiments.

2.2. EESS Suppresses the IL-1β-Mediated Generation of ROS

Because oxidative stress plays an important role in the degradation of ECM, we investigated whether EESS could inhibit ROS production by IL-1β using the 5,6-carboxy-2′,7′-dichlorofluorescin diacetate (DCF-DA) probe. Our flow cytometry results showed that the level of ROS gradually increased with the treatment of IL-1β, and decreased after reaching the peak at 1 h (Figure 2A). However, the increase in ROS content in IL-1β-stimulated SW1353 cells was dramatically reduced by the addition of EESS (Figure 2B). Also, as expected, pretreatment of N-acetyl-l-cysteine (NAC), an ROS scavenger, completely blocked ROS production by IL-1β. The effect of inhibiting ROS formation was confirmed by fluorescence microscopy, and the increase in DCF-DA fluorescence intensity observed in IL-1β-treated SW1353 cells was weakened by EESS pretreatment consistent with the results of flow cytometry (Figure 2C). In addition, IL-1β could significantly induce ROS production in rat articular chondrocytes, while EESS blocked IL-1β-induced ROS accumulation (Figure 3A,B).

2.3. EESS Reduces IL-1β-Induced NO and PGE₂ Production

The effects of EESS on the production of inflammatory mediators, such as NO and PGE₂, were detected to evaluate the anti-inflammatory effects of EESS. For this study, SW1353 and rat articular chondrocytes were pretreated with various concentrations of EESS for 1 h, before stimulation with 40 ng/mL IL-1β for 24 h, and then the NO concentration in the cell suspension was assayed by Griess reaction, and the level of PGE₂ was measured using an enzyme-linked immunosorbent assay (ELISA) kit, respectively. Figure 4A,B shows that IL-1β stimulation significantly increased the production of NO and PGE₂ in SW1353 cells compared to the unstimulated control, but their amounts were significantly decreased in EESS-pretreated cells in a concentration-dependent manner. Similar results were obtained in experiments using rat articular chondrocytes (Figure 4C,D).

2.4. EESS Inhibits IL-1β-Induced iNOS and COX-2 Expression

We next examined whether EESS could inhibit the expression of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2), which are key to the production of NO and PGE₂. According to our immunoblotting results, the enhanced expression of iNOS and COX-2 protein by IL-1β was concentration dependently reduced in both cell lines cultured under EESS pretreatment conditions (Figure 5A,C). Similarly, reverse transcriptase-polymerase chain reaction (RT-PCR) results showed that the expression of both genes at the transcription level was also markedly inhibited by pretreatment with EESS in SW1353 and rat articular chondrocytes (Figure 5B,D) indicating that EESS inhibits NO and PGE₂ production via downregulation of iNOS as well as COX-2 mRNA and protein levels.

2.5. EESS Decreases IL-1β-Mediated Increase of MMP-1, -3, and -13 Production

Since the catabolic factors play an important role in OA pathogenesis, we investigated whether EESS can avoid the generation of MMPs that play an important role in cartilage degradation. The results showed that increased release of MMPs (MMP-1, -3, and -13) in SW1353 chondrocytes was detected in the culture supernatants after stimulation with IL-1β; however the enhanced production of these MMPs by IL-1β was suppressed by EESS pretreatment, which was dependent on the EESS concentration (Figure 6).

2.6. EESS Attenuates IL-1β-Induced Expression of MMP-1, -3, and -13

Next, we investigated whether inhibition of MMPs production by EESS in IL-1β-treated SW1353 chondrocytes is associated with decreased expression of these genes. Figure 7A shows that the expression of the MMP-1, -3 and -13 protein in SW1353 chondrocytes was dramatically increased by IL-1β treatment alone, but their expression was greatly reduced in cells cultured under EESS pretreatment conditions. In addition, pretreatment with EESS resulted in a concentration-dependent inhibition of IL-1β-induced mRNA expression of the MMPs (Figure 7B). These results suggest that EESS also modulates MMPs expression at the transcriptional level.

2.7. EESS Alleviates IL-1β-Induced Nuclear Accumulation of NF-κB p65

We further investigated whether EESS inhibits IL-1β-induced NF-κB activation, because it is an important mediator of IL-1β signaling in chondrocytes, and the key factor controlling the transcription of inflammatory mediators and MMPs. Figure 8A shows that when SW1353 cells were stimulated with IL-1β, NF-κB p65 expression in the nucleus was increased, compared to the control. At the same time, the amount of IκB-α total protein expression decreased with increasing phosphorylation of IκB-α in the cytoplasm (Figure 8B). However, EESS markedly blocked IL-1β-induced expression of NF-κB p65 in the nucleus, and suppressed the phosphorylation and decreased the expression of IκB in the cytoplasm. We have also demonstrated that pyrrolidine dithiocarbamate (PDTC), a specific inhibitor of NF-κB, prevented IL-1β-induced iNOS and MMP-1 expression (Figure 8C), indicating that EESS enhances inflammatory mediators and MMPs expression by inducing NF-κB activation.

2.8. Effects of EESS on IL-1β-Induced Activation of MAPKs and PI3K/Akt Signaling Pathways

Mitogen-activated protein kinases (MAPKs) and the phosphatidylinositol-3-kinase (PI3K)/Akt signaling pathways, along with the NF-κB, also play critical roles in IL-1β-stimulated OA chondrocytes. Figure 9 shows that phosphorylation of all three kinases belonging to MAPKs was increased by IL-1β in SW1353 chondrocytes, indicating that MAPKs were activated by IL-1β. We therefore investigated the effect of EESS on the activation of MAPKs by IL-1β, and found that phosphorylation of p38 MAPK was highly inhibited, although phosphorylation of extracellular signal-regulated kinase (ERK) and c-jun N-terminal kinase (JNK) was not blocked by pretreatment of EESS. In addition, phosphorylation of PI3K and Akt, a downstream target of PI3K, was increased in SW1353 chondrocytes treated with IL-1β, but phosphorylation of these proteins was almost reduced to the control level by pretreatment of EESS (Figure 9).

3. Discussion

It is well known that oxidative stress plays an important role in OA development. As reported in many clinical studies, ROS levels in OA cartilage are significantly increased compared to normal cartilage [22,23]. High levels of ROS in chondrocytes cause ECM degradation, and cell dysfunction and death. In addition, ROS stimulates the production of inflammatory cytokines and alters the function of various signaling pathways [24,25], suggesting that oxidative stress acts as a risk factor for the pathogenesis of OA. To investigate the effect of EESS on IL-1β-induced oxidative stress in SW1353 and rat articular chondrocytes, we examined whether EESS inhibits IL-1β-induced ROS production, and found that EESS effectively repressed the production of ROS by IL-1β. Along with oxidative stress, inflammation is a major cause of OA development. In particular, the increase of inflammatory mediators such as NO and PGE₂ by IL-1β may accelerate the development of OA [1,8], and inhibiting the production of these inflammatory mediators may inhibit OA development. Therefore, we examined the effect of EESS on the secretion of NO and PGE₂ to investigate the inhibitory effect of EESS on IL-1β-mediated inflammatory response. Our results, similar to a previous study in the microglial cell model [19], showed that EESS significantly reduced IL-1β-induced production of NO and PGE₂ in SW1353 and rat articular chondrocytes, which was associated with decreased expression of iNOS and COX-2. These results suggest that the anti-inflammatory effect of EESS is due to the decreased expression of enzymes involved in the production of NO and PGE₂.

A number of previous studies have shown that some types of MMPs among proteases produced by chondrocytes in response to IL-1β are important proteolytic factors in degradation of ECM [5,6]. In addition, the expression of these enzymes is increased by oxidative and inflammatory stresses [3,4]. Therefore, candidate substances that inhibit the expression of MMPs can be applied as new therapeutic strategies for OA. In the present study, MMP-1, -3, and -13, which are critical enzymes for cartilage degradation, were significantly increased in IL-1β-exposed SW1353 chondrocytes; however, the elevated levels of the MMPs by IL-1β were obviously suppressed by EESS treatment, and their mRNA and protein expression were also potently inhibited. These results indicate that EESS can protect the ECM degradation, by inhibiting the expression of MMPs.

As noted in many studies, various intracellular signaling systems are also involved in the development and progression of OA. Among them, NF-κB plays a most important role in the regulation of inducible inflammatory mediators, cytokines, and MMPs expression in OA pathogenesis [13,26]. Normally, NF-κB is retained in the cytoplasm in an inactive form associated with IκB-α, an inhibitory subunit. When IκB-α is degraded through phosphorylation by inflammatory stimuli, NF-κB translocates from the cytosol into the nucleus, and triggers the transcriptional activation of inflammatory mediators and cytokines, and catabolic enzymes [27,28]. Therefore, the efficacy of EESS on the activation of NF-κB induced by IL-1β was evaluated, because blocking the activity of NF-κB may be effective in treating OA. Our results confirmed that nuclear accumulation of NF-κB and degradation of IκB-α were promoted in IL-1β-treated chondrocytes, and this phenomenon is inhibited by EESS pretreatment, indicating that the nuclear translocation of NF-κB, which is an essential step of NF-κB activation, was effectively blocked. Therefore, it can be inferred that inhibition of IL-1β-induced expression of iNOS, COX-2, and MMPs by EESS is due to the blocking of nuclear translocation of NF-κB. Furthermore, to investigate whether EESS can regulate upstream NF-κB signaling pathways, we examined the effects of EESS on MAPKs and PI3K/Akt pathways. Consistent with the results of previous studies [29,30,31], it was found that ERK, JNK, and p38 MAPK were activated in IL-1β treated chondrocytes, as evidenced by their phosphorylation. However, by pretreatment of EESS, only the activation of p38 MAPK was attenuated, and EESS did not affect the activation of ERK and JNK. In addition, phosphorylation of PI3K and Akt was increased by treatment with IL-1β, but their activation was significantly inhibited by EESS. These results suggest that p38 MAPK among MAPKs, and PI3K/Akt are involved at least in the inhibition of IL-1β-induced NF-κB activation by EESS.

In conclusion, the current study clearly indicated that EESS has the potential to inhibit IL-1β-induced oxidative stress and inflammatory responses in chondrocytes, and its effect is associated with the suppression of NF-κB, p38 MAPK, and PI3K/Akt signaling pathways. Therefore, we suggest that EESS or its components may have cartilage protective properties, and may be used as therapeutic agents for OA therapy.

4. Materials and Methods

4.1. Reagents and Antibodies

Dulbecco’s modified Eagle’s medium (DMEM), DMEM/Ham’s Nutrient Mixture F-12 (DMEM/F-12) medium and other reagents for cell culture were obtained from WelGENE Inc. (Daegu, Republic of Korea). Recombinant human IL-1β and ELISA kits were purchased from R&D Systems (St. Paul, MN, USA). MTT, NAC and DCF-DA were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). TRIzol reagent and nuclear and cytoplasmic extraction reagents were obtained from Invitrogen Life Technologies (Carlsbad, CA, USA) and Pierce (Rockford, IL, USA), respectively. Primary antibodies were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA) and Cell Signaling Technology Inc. (Beverly, MA, USA). Appropriate horseradish-peroxidase (HRP)-linked secondary antibodies were purchased from Santa Cruz Biotechnology Inc. An ECL solution was purchased from Amersham Corp. (Arlington Heights, IL, USA), and reagents not specifically mentioned were purchased from Sigma-Aldrich Chemical Co.

4.2. Preparation of EESS

The EESS used in this study was provided by the National Marine Biodiversity Institute of Korea (Seocheon, Korea). For the preparation of EESS, S. serratifolium was collected from offshore Jeju island, Republic of Korea in March 2016. Collected S. serratifolium was washed with tap water to remove slats, epiphytes, and sand attached to the surface of the samples, and then lyophilized. The dried sample of S. serratifolium (170 g) was pulverized, and extracted with 70% EtOH (1:10 w/v) for 1 h (five times) by sonication. The S. serratifolium extract (EESS) was obtained by evaporation under vacuum. The extract was dissolved in dimethylsulfoxide (DMSO), before use in the experiment.

4.3. Cell Culture and Viability Assay

SW1353 cells were obtained from the American Type Culture Collection (Manassas, VA, USA), and were cultured in DMEM containing 10% fetal bovine serum (FBS), and 100 U/mL penicillin and streptomycin at 37 °C in humidified air with 5% CO₂. For the isolation of rat articular chondrocytes from the knee joints of 6-week-old Sprague-Dawley male rats (Samtako Bio Korea, Osan, Korea), articular cartilage was isolated from rat femoral grooves and femoral condyles, and then aseptically dissected according to Rao and colleagues [32]. After digestion in 0.25% trypsin at 37 °C for 40 min, two times, the tissue was digested with 0.3% collagenase II for 3 h at 37 °C. The chondrocytes were filtered through a mesh screen to prepare a single-cell suspension, and then centrifuged at 1500× g for 10 min, transferred into a culture flask. Rat articular chondrocytes were cultured at 37 °C in a 5% CO₂-humidified incubator in DMEM/F12 medium containing 10% FBS, and 100 U/mL penicillin and streptomycin. The procedures used in the present study complied with the guidelines of the Institutional Animal Care and Use Committee at Dongeui University, Busan, Republic of Korea. The cytotoxicity of EESS to SW1353 and rat articular chondrocytes was determined by MTT assay. In brief, cells were treated with EESS or IL-1β (40 ng/mL) alone, or pretreated with different concentrations of EESS for 1 h before IL-1β treatment. Afterward, the medium was removed, and 0.5 mg/mL of MTT was added to each well, and incubated at 37 °C for 3 h. The supernatant was then replaced with an equal volume of DMSO, to dissolve blue formazan crystals for 10 min. The optical density was measured at a wavelength of 540 nm by microplate reader (Dynatech Laboratories, Chantilly VA, USA). All experiments were performed in triplicate.

4.4. Detection of the Intracellular ROS Levels

Production of intracellular ROS was monitored using DCF-DA, a cell-permeable fluorogenic probe. Briefly, SW1353 and rat articular chondrocytes were treated with IL-1β (40 ng/mL) for the indicated times, or pretreated with 300 μg/mL EESS or NAC (10 mM) for 1 h, and then cultured for 1 h in the presence or absence of IL-1β. Cells were harvested, and stained with 10 μM DCF-DA in the dark at 37 °C for 15 min. The cells were then rinsed twice with phosphate buffered saline (PBS), and a total of 10,000 events were immediately analyzed by flow cytometer (BD Biosciences, San Jose, CA, USA) with an excitation wavelength of 480 nm and an emission wavelength of 525 nm [33]. To observe the degree of ROS production by fluorescence microscopic observation, cells attached to glass coverslips were stimulated with or without IL-1β after EESS treatment. The cells were stained with 10 μM DCF-DA at 37 °C for 15 min, washed twice with PBS, and then fixed with 4% paraformaldehyde (pH 7.4) for 20 min. The fixed cells were washed twice with PBS, and analyzed by fluorescence microscopy (Carl Zeiss, Oberkochen, Germany), as previously described [34].

4.5. NO Measurement and ELISA Assay

SW1353 and rat articular chondrocytes were pretreated with different concentrations of EESS for 1 h, followed by treatment with IL-1β (40 ng/mL) for 24 h, and the levels of NO in the supernatant of the medium were assessed by Griess reaction, as described previously [35]. Protein levels of MMP-1, MMP-3, and MMP-13 released from cultured chondrocytes under the same conditions were quantified by commercially available ELISA kits, according to the manufacturer’s instructions.

4.6. RNA Isolation and RT-PCR

Cells were pretreated with various concentrations of EESS for 1 h, followed by treatment with IL-1β (40 ng/mL) for 24 h. Total RNA from isolated chondrocytes was prepared using TRIzol reagent, following the manufacturer’s instructions, and quantified. For mRNA expression analysis, cDNA was synthesized from 1 μg of total RNA using AccuPower^® RT PreMix (Bioneer, Daejeon, Korea) containing moloney murine leukemia virus reverse transcriptase. The RT-generated cDNA was amplified using selected primers (Bioneer), under the following conditions: 10 min 95 °C, followed by 40 cycles of 15 s 95 °C and 1 min 60 °C. GAPDH was used as an internal control. Bands were quantified using ImageJ (Ver. 1.46; NIH, Bethesda, MD, USA) and normalized to GAPDH and the ratio was determined.

4.7. Western Blot Analysis

To extract the total cellular proteins, the cells were collected, washed twice with ice-cold PBS, and then lysed using the cell lysis buffer (25 mM Tris-Cl (pH 7.5), 250 mM NaCl, 5 mM ethylenediaminetetraacetic acid, 1% Nonidet-P40, 1 mM phenylmethylsulfonyl fluoride, and 5 mM dithiothreitol) for 1 h, before cell debris was removed by centrifugation. The nuclear and cytoplasmic proteins were isolated using the extraction kit, as recommended by the manufacturer. Protein concentration was measured using a Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA), and the same amounts of protein were resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to polyvinylidene difluoride membranes (Schleicher and Schuell, Keene, NH, USA). After blocking with 5% non-fat dry milk for 1 h at room temperature, the cells were subsequently incubated with the primary antibodies overnight with gentle agitation at 4 °C. After washing three times with Tris-buffered saline containing 0.1% Tween-20 for 5 min, the membranes were incubated with respective HRP-linked secondary antibodies for 2 h at room temperature. The membranes were visualized by an ECL solution, and exposed to X-ray films. The images of Western blotting were also analyzed with Image J program.

4.8. Statistical Analysis

All data are expressed as means ± SD. Statistically significant values were analyzed using one-way ANOVA with Dunnett’s test. Statistical analysis was performed using the Statistical Package for the Social Sciences (SPSS) statistical analysis software (Version 19.0, IBM, New York, NY, USA). A p-value below 0.05 was considered significant.