Zinc Protects Articular Chondrocytes through Changes in Nrf2-Mediated Antioxidants, Cytokines and Matrix Metalloproteinases

Osteoarthritis (OA) is an age-related degenerative joint disease characterized by high oxidative stress, chondrocyte death and cartilage damage. Zinc has been implicated in the antioxidant capacity of the cell, and its deficiency might inhibit chondrocyte proliferation. The present study examined the potential of zinc as a preventive supplement against OA using the in vitro chondrosarcoma cell line SW1353 and an in vivo Wistar rat model to mimic OA progress induced by monosodium iodoacetate (MIA). The results demonstrated that, in SW1353 cells, 5 μM MIA exposure increased oxidative stress and decreased the expression of GPx1 and Mn-SOD but still increased GSH levels and HO-1 expression and enhanced the expression of interleukin (IL)-10, IL-1β, and matrix metalloproteinase (MMP)-13. Zinc addition could block these changes. Besides, the expression of Nrf2 and phosphorylated (p)-Akt was dramatically increased, implicating the p-Akt/Nrf2 pathway in the effects of zinc on MIA-treated cells. A rat model achieved similar results as those of cell culture, and 1.6 mg/kg/day of zinc supplementation is sufficient to prevent OA progress, while 8.0 mg/kg/day of zinc supplementation does not have a better effect. These findings indicate that zinc supplementation exerts a preventive effect with respect to MIA-induced OA progress.


Introduction
Osteoarthritis (OA) is a common degenerative joint disease in the knees, hips, spine, and fingers that is characterized by the progressive abrasion of articular cartilage and remodeling of the underlying bone in the synovial joints, potentially resulting in disability in the aging population [1]. The cartilage of NY, USA) and 10% fetal bovine serum (FBS) (HyClone, Auckland, NZ, USA) in a 5% CO 2 incubator. The cells were seeded at 4 × 10 5 onto dishes in DMEM for 16 h to enable attachment and subsequently treated with 25 µM zinc in the presence or absence of 5 µM MIA for a further indicated period, followed by analysis of the influences.

Cell Viability Assay
SW1353 cells (8 × 10 4 /well) were seeded in 24-well plates and incubated with 0-6 µM MIA or 0-100 µM zinc for 24 h or 48 h. The cells were harvested, and the viable cells were counted using a dye exclusion technique with 0.4% trypan blue (GibcoBRL, Grand Island, NY, USA) in a hemocytometer. All counts were performed in triplicate.

Measurement of ROS
SW1353 cells (4 × 10 5 ) were seeded in a 6-cm dish and treated with MIA and/or zinc for 24 h, and intracellular ROS were detected by using 10 µM 2 ,7 -dichlorofluorescein diacetate (DCFH-DA) (Molecular Probes, Eugene, OR, USA) for 30 min at 37 • C as described in our previous study [18]. Each experiment was carried out in triplicate.

Measurement of GSH
SW1353 cells (4 × 10 5 ) were seeded in a 6-cm dish and treated with MIA and/or zinc for 48 h, and GSH levels were measured using a GSH assay kit (Cayman Chemical, Ann Arbor, MI, USA) according to the manufacturer's instructions. Each experiment was carried out in triplicate.

Western Blot Analysis
SW1353 cells (4 × 10 5 ) were seeded in a 6-cm dish and treated with MIA and/or zinc for 48 h, and then the cell extracts were prepared for Western blot analyses were as previously described [18]. Each experiment was carried out in triplicate. Herein, the proteins were visualized using chemiluminescence detection (PerkinElmer Life Sciences, Inc., Boston, WA, USA). Actin was used as the internal control, each targeted band was calibrated by respective actin. Afterwards, the data of study group were quantitatively analyzed relative to the control group.

Quantitative Real-Time PCR Analysis (qPCR)
SW1353 cells (4 × 10 5 ) were seeded in a 6-cm dish and treated with MIA and/or zinc for 24 h, and then the total RNA was extracted by using REzol reagent (Protech, Taipei, Taiwan) according to the manufacturer's instructions, as previously described [19]. Each experiment was carried out in triplicate. The complementary DNA (cDNA) was synthesized from random primed reverse transcription from 2 µg of total RNA using M-MLV reverse transcriptase (Promega Corporation, Madison, WI, USA) according to the manufacturer's instructions. Real-time PCR was performed on a MiniOpticon TM Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA) using iQ TM SYBR ® Green Supermix (Bio-Rad Laboratories, Hercules, CA, USA) as previously described [20], was used to confirm the results of real-time PCR. The mRNAs encoding GCLC, GCLM, IL-10, and IL-1β were measured using real-time PCR, with RPS18 mRNA as the housekeeping gene. The primers and amplified products of each gene used in the present study are shown in Table 1. The cycle threshold (C t ) value of the target gene was normalized to RPS18. The data were calculated and expressed as 2 −∆∆Ct [21] using MJ Opticon Monitor Analysis software version 3.1 (Bio-Rad Laboratories, Hercules, CA, USA).

Animals and Treatments
Male Wistar rats at 4 weeks of age were purchased from BioLASCO Taiwan Co., Ltd. (Charles River Technology, Taipei, Taiwan). Wistar rats at 5 weeks of age (150-170 g) were used. The present study was performed in accordance with the Guide for the Care and Use of Laboratory Animals of the United States National Institutes of Health. The protocol for animal use was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Kaohsiung Medical University (Approval No. 104059; Approval date: 13 August 2015). Sixty male Wistar rats were randomly assigned to six groups containing 10 rats each. The rats were anesthetized using a Tiletamine and Zolazepam mixture (Zoletil 50) (Virbac, Carros, France) and a single intra-articular injection of either sterile saline or MIA (Sigma-Aldrich, St. Louis, MO, USA) at a dose of 3 mg MIA in 20 µL of 0.9% sterile saline was administered through the infrapatellar ligament of the left knee using a 31-gauge needle. Control animals were administered a single intra-articular injection of 20 µL 0.9% sterile saline into the left knee. After the single dose of MIA injection to mimic the OA progress, rats were randomly assigned to three of the six treatment groups, which were one untreated or two zinc supplemented groups. Zinc supplemented groups received water-dissolved zinc of the recommendations of daily dose of 1.6 mg zinc/kg/day [22] or a high dose of 8.0 mg zinc/kg/day by gavage for 2 weeks. All rats were fed standard rodent chow with zinc 70 ppm (Altromin, Lage, Germany). At the end of the experiment period, the rats were sacrificed using CO 2 , and the left leg was removed for histomorphometric analyses. All samples were stored at −80 • C until further analysis.

Histopathology of Joint Tissues: Safranin O and Fast Green Staining
The histopathology of the rat left joints, stained with safranin O and fast green (Sigma-Aldrich, St. Louis, MO, USA), was analyzed as previously described [23]. The histological score of knee joints was assessed by the OARSI cartilage degeneration score [24].

Serum Biomarkers Measurements
The rats (n = 10 per group) were sacrificed, and the serum was obtained by centrifuging the blood samples at 3000× g for 15 min. The resulting serum samples were divided into aliquots and frozen at −80 • C until further use. There was no repeated freezing and thawing of specimens prior to obtaining measurements. The inflammatory marker IL-1β and anti-inflammatory maker IL-10 were assayed using rat IL-1β and IL-10 ELISA kits (Elisa kit, Antibody-Sunlong Biotech Co., Ltd., Hangzhou, China), respectively. The ECM degrading enzymes MMP-1 and MMP-13 were assayed using rat MMP-1 and MMP-13 ELISA kits, respectively (Elisa kit, Antibody-Sunlong Biotech Co., Ltd., Hangzhou, China). The antioxidant and GSH activities were assayed using an antioxidant assay kit and a GSH assay kit (Cayman Chemical, Ann Arbor, MI, USA), respectively. All samples were examined in triplicate within each assay.

Statistical Analysis
All data are presented as the means ± standard deviation (S.D.). The differences between control and treated groups were analyzed using ANOVA, followed by Fisher's Exact Test. All statistical analyses were performed using SAS version 6.011 software (SAS Institute Inc., Cary, NC, USA). A p value < 0.05 was considered statistically significant.

Cell Viability
To obtain an initial insight into the responses of SW1353 cells to MIA treatment, the cells were incubated for 24 h alone or in the presence of 0-6 µM MIA. Cell viability was not affected by 2 or 3 µM MIA but showed dose-dependent cell death at higher MIA concentrations ( Figure 1A). Cell viability was approximately 50% after treatment with 5 µM MIA; thus, 5 µM MIA was selected to treat cells, mimicking OA conditions. However, to determine whether zinc treatment affected cell viability, the cells were incubated for 24 h alone or in the presence of 0-100 µM zinc. As shown in Figure 1B, cell viability was not altered by zinc, even at a concentration of 100 µM. Moreover, the addition of 25 µM of zinc to 5 µM MIA-treated cells showed an increase in cell viability ( Figure 1C), but higher concentrations were not more effective (data not shown). Therefore, we selected 5 µM MIA and 25 µM zinc for the following cell culture experiments.

Oxidative Stress and Antioxidants
The results of a previous study indicated that MIA toxicity in chondrocytes reflects MIA-induced oxidative stress [23]. Herein, we examined oxidative stress by evaluating ROS production in SW1353 cells treated with 5 µM MIA in the presence or absence of 25 µM zinc for 24 h. As shown in Figure 2, MIA induced ROS production, while zinc addition would decrease ROS production. Furthermore, we examined whether the levels of antioxidants were changed. The GSH levels were not changed by zinc, but were increased by MIA. Interestingly, the addition of zinc augments the increase of GSH by MIA ( Figure 3A). Analysis of the mRNA expression of GCLC and GCLM, key enzymes of GSH synthesis, using real-time PCR showed that the expression of both GCLC and GCLM was increased after MIA treatment, and the addition of zinc augmented this MIA-increased expression ( Figure 3B), suggesting that the increase in GCLC and GCLM expression contributes to increased GSH levels. The expression of the antioxidative enzymes GPx1, Zn/Cu-SOD, Mn-SOD, and HO-1 was analyzed using Western blotting, and Figure 3C shows that zinc alone significantly decreased Mn-SOD expression and increased HO-1 expression; MIA decreased GPx1 and Mn-SOD expression but increased HO-1 expression; and the addition of zinc and MIA could massively increase the expression of GPx1, Zn/Cu-SOD, Mn-SOD, and HO-1 as compared with MIA-treated group. These results demonstrate that zinc reduces MIA-induced oxidative stress, reflecting an enhancement of antioxidative enzyme expression. To examine this theory, an inhibitor of GSH or SOD was added to zinc and MIA-treated cells, and, subsequently, cell viability was analyzed. As shown in Figure 3D, the addition of either BSO, a GSH inhibitor, or DETC, a SOD inhibitor, would abate the effects of zinc, indicating that antioxidants are important for the effects of zinc on MIA-treated cells.

Expression of Cytokines and MMPs
Fernández et al. [25] reported that the anti-inflammatory cytokine, IL-10, could enhance HO-1 protein expression in human osteoarthritic chondrocytes. In addition, IL-10 exhibits protective effects in the course of OA [26]. We further examined whether the increased HO-1 expression was associated with IL-10 mRNA expression. As shown in Figure 4A, MIA increased IL-10 mRNA expression, and the addition of zinc to MIA-treated cells could augment this increase, similar to the trend of HO-1 changes. Studies also reported that IL-10 blocked pro-inflammatory cytokine secretion and inhibited MMPs production [27,28]. Therefore, we examined the mRNA expression of IL-1β, a pro-inflammatory cytokine, using real-time PCR and characterized active MMP-13 protein expression using Western blotting. Figure 4B shows that IL-1β mRNA expression was increased by MIA, while zinc decreased MIA-induced IL-1β mRNA expression. Figure 4C shows a similar trend of active MMP-13 expression.
These results indicate that IL-10 may play an important role in the changes of HO-1, IL-1β, and MMP-13 by MIA in the absence or presence of zinc treatment.

Expression of Phosphorylated-Akt and Nrf2 Expression
Nrf2 is a sensitive mild oxidative stress sensor that regulates antioxidant enzyme expression [29,30]. As shown above, oxidative stress and antioxidative enzyme expression in SW1353 cells were changed by MIA and/or zinc treatment; thus, we determined whether these changes were associated with the expression of Nrf2 or its upstream regulator Akt. Figure 5A shows that zinc alone increased the expression of Nrf2 and active/phosphorylated Akt, while MIA increased Nrf2 expression but did not affect active/phosphorylated Akt expression. The addition of zinc to MIA-treated cells intensively increased both Nrf2 and active/phosphorylated Akt expression, suggesting that Akt/Nrf2 pathways are involved in the effects of zinc and/or MIA on SW1353 cells. To further investigate the role of Akt/Nrf2, LY294002, an inhibitor of the upstream Akt regulator, phosphoinositide 3-kinase (PI3K), was added to zinc and/or MIA-treated cells, and, subsequently, cell viability was assayed. Figure 5B shows that the effect of zinc on the viability of MIA-treated cells disappeared after LY294002 addition. This effect further suggests that PI3K/Akt/Nrf2 pathways participate in the zinc effect on MIA-treated cells.

MIA-Induced OA Progression in Rats, with/without Zinc Supplementation
We further examined the effects of zinc on MIA-induced OA in rats to confirm whether the results obtained in cell culture were the same in vivo. After two weeks of experimental treatment, the morphology of the articular cartilage in the zinc supplemented group was same as that of the control group, whereas the MIA-treated group showed severe erosion, synovial hypertrophy and cartilage defect indicating marked arthritic progression ( Figure 6A). In addition, zinc supplementation could reduce the arthritic progression in the MIA-treated group. Next, we further examined the histological results of cartilage extracellular matrix proteoglycan ( Figure 6B,C). The MIA-treated group had higher Osteoarthritis Research Society International (OARSI) scores and was negative for safranin O staining (red color) and positive for fast green staining indicating no acidic proteoglycan cartilage. The MIA-treated group with zinc supplementation showed smooth joint surfaces, lower OARSI scores, and positive safranin O staining, indicating that proteoglycan was not lost. Notably, the effects of 1.6 mg/kg/day zinc supplementation were not apparently different from those of 8.0 mg/kg/day zinc supplementation. The antioxidative capacity of the serum as well as the serum levels of GSH, IL-10, MMP-1, MMP-13, and IL-1β in treated rats were evaluated, and the results are shown in Figure 7, demonstrating that the zinc supplemented group had higher GSH levels and antioxidative capacity, increased IL-10 and MMP-13 levels, and decreased IL-1β levels compared with the control group. There was no significant difference between 1.6 and 8.0 mg/kg/day of zinc supplementation. The MIA-treated group had massively decreased GSH and antioxidative capacity and lower IL-10 levels but higher MMP-1, MMP-13, and IL-1β levels compared with the control group. Zinc supplementation of MIA-treated rats could inhibit the changes induced by MIA. Notably, IL-10 levels were dramatically increased with zinc supplementation compared to the MIA-treated group, suggesting that IL-10 may play a significant role in the effects of zinc on the MIA-induced OA progress of rats. The in vitro data showed zinc supplementation increased the mRNA expression of GCLC, GCLM, and pro-inflammatory cytokines as well as the protein expressions of antioxidative enzymes, while in vivo rat model zinc supplementation increased serum antioxidative capacity, GSH levels, and IL-10 levels. Taken together, the results of the present study demonstrated that zinc supplementation could prevent MIA-induced OA progress, particularly through the enhancement of antioxidative capacity.

Discussion
GSH is the first line defense against oxidative damage and can destroy ROS and other free radicals via enzymatic and non-enzymatic mechanisms [31]. The SOD catalyzes oxygen anion to hydrogen peroxide, which is subsequently detoxified by GPx or catalase to water. HO-1 catalyzes the conversion of heme into biliverdin, carbon monoxide and free iron. All these are known as the intracellular defense system against oxidative stress [32,33]. Kloubert and Rink [34] reported that zinc plays an important role in the activation of these defense enzymes. In addition, the production of various pro-inflammatory cytokines, such as IL-1β and IL-6 by macrophages requires zinc signals.
Reports further indicated that the enhancing or inhibiting pro-inflammatory cytokines release by zinc depends on zinc dosage used, cell types and experimental condition [35]. Moreover, zinc is recognized as a regulator in signal pathways in immunity and redox metabolism, and is called as zinc signals [36,37].
The results of the present study showed that exposure of 5 µM MIA to SW1353 cells can increase oxidative stress, leading to cell cytotoxicity as previously described [23]. MIA exposure also intensively decreases GPx1 and Mn-SOD expression but increased GSH levels and HO-1 expression. This finding indicates that some antioxidative capacities are induced in defense against MIA-induced oxidative stress. In addition, the expression of IL-10, IL-1β, and MMP-13 is increased. Furthermore, these MIA-induced changes are blocked by supplementation with zinc. Interestingly, the results ( Figure 5A) showed that MIA or zinc can stimulate Nrf2 expression, while only zinc stimulates the upstream active regulator, p-Akt. Co-addition of MIA and zinc dramatically increases Nrf2 and p-Akt expression, consistent with the responses of antioxidants, indicating that the p-Akt/Nrf2 pathway is involved in the effects of zinc and/or MIA on SW1353 cells.
The MIA-induced OA rat model generated results similar to those obtained in cell culture, and 1.6 mg/kg/day zinc supplementation was sufficient to prevent OA progress, while 8.0 mg/kg/day zinc supplementation does not show a better effect. Notably, zinc supplementation to rats without MIA treatment decreased the serum IL-1β levels indicating immunosuppression was induced by the supplemented dosage of either 1.6 mg/kg/day or 8.0 mg/kg/day. In contrast, MIA treatment dramatically increased serum IL-1β levels indicating the immunity and inflammation would be activated. Therefore, we proposed that the zinc supplementation to the MIA-treated rats could lessen inflammation due to its immunosuppression effect. These findings indicate that zinc supplementation inhibits MIA-induced OA progress in vitro and in vivo through changes in antioxidative capacity, pro-inflammatory cytokines and MMPs (decrease protein level of MMP-13 in vitro, and MMP-1 and MMP-13 in vivo). In addition, according to in vitro study, the protective effects exert through the p-Akt/Nrf2 signaling pathway.
The increase in oxidative stress of MIA-treated chondrocytes observed in the present study is consistent with the results of a previous study [23] and other reports [38]. GSH is an important antioxidant that can destroy ROS and other free radicals through enzymatic and non-enzymatic mechanisms [31]. Studies have reported that patients with osteoarthritis have lower GSH levels in erythrocytes [39] and joint fluids [40]. In addition, the levels of GPx, Mn-SOD, and GCLC and GCLM, rate-limiting enzymes for GSH synthesis, were decreased in OA cells [41][42][43][44], in contrast to the results obtained in cell culture showing increased GSH levels after MIA treatment. However, in vivo experiments showed a decrease of GSH levels in the serum of rats in MIA-induced OA progress. We propose that the increase in GSH levels of cells is a necessary response to MIA-induced oxidative stress, since GSH is the first-line defense and may gradually be decreased as the other lines of defense do not work efficiently or decay. Consistently, GPx1 and Mn-SOD expression was decreased in SW1353 cells after MIA treatment. Importantly, this study showed that zinc supplementation to MIA-treated cells can enormously increase the protein expression of GPx1, Zn/Cu-SOD, Mn-SOD, and HO-1, and the mRNA expression of GCLC and GCLM, rate-limiting enzymes for GSH synthesis. Among these factors, Zn/Cu-SOD, GCLC, GCLM, and GPx participate in the antioxidant activity in the cytosol, and Mn-SOD and GPx participate in mitochondrial antioxidant activity [45].
Nrf2, a transcription factor, is a master regulator of antioxidant defense genes which regulate the redox imbalance through the upregulation of antioxidant response element (ARE)-responsive antioxidant enzymes [46]. Reports have shown that Nrf2-knockout mice had more severe cartilage damage in MIA-induced OA models [9], and Nrf2 activation could decrease MIA-induced oxidative stress in chondrocytes [47]. Studies have also reported that the activation of the PI3K-Akt signaling pathway promotes matrix synthesis [48] and enhances the survival of chondrocytes [49]. These results are consistent with reports that the activation of the p-Akt/Nrf2 pathway can reduce damage to chondrocytes through the regulation of downstream effectors, such as antioxidant enzymes. Bach1 −/− mice with deficiencies in the transcriptional repressor of HO-1 showed severe OA-like changes, and these changes could be reduced through the upregulation of HO-1 expression [50]. In addition, HO-1 expression has been associated with the expression of MMPs and pro-inflammatory cytokines in human articular chondrocytes [51]. IL-10 stimulates HO-1 expression in human osteoarthritic chondrocytes [25]. In addition, IL-10 blocks pro-inflammatory cytokine secretion and inhibits MMPs production in chondrocytes [25,26]. The results of the present study provide consistent evidence that zinc supplementation to MIA-treated cells massively increases IL-10 mRNA expression and HO-1 protein expression and decreased IL-1β mRNA expression and MMP-13 protein expression. Moreover, in the MIA-induced OA rat model, zinc supplementation also dramatically increases serum IL-10 levels and decreases MMP and IL-1β levels. These results suggest that IL-10 participates in the effect of zinc on OA progress.
In summary, as shown in Figure 8, the present study demonstrates that in SW1353 cells, MIA exposure induces oxidative stress, decreases the expression of the antioxidative enzymes GPx1 and Mn-SOD, and increases the levels of pro-inflammatory cytokines IL-1β and MMP-13. The addition of zinc to MIA-treated cells activates the PI3K/Akt/Nrf2 pathway to increase antioxidative activity in defense against oxidative stress and decrease IL-1β and MMP-13 levels. Notably, the results observed in vitro were similar to those observed in vivo. These results indicate that zinc can prevent against MIA-induced OA progress. The results of the present study demonstrates that zinc can protect against MIA-increased oxidative stress, pro-inflammatory cytokines (IL-1β), and MMPs through the activation of the Akt/Nrf2 pathway, which upregulates the gene expression of antioxidants, such as Cu/Zn-SOD, Mn-SOD, GPx1, GSH, GCLC and GCLM, and HO-1, leading to the increased antioxidative capacity in defense against MIA-induced oxidative stress. In addition, IL-10 expression is relatively slightly increased by MIA and massively increased after zinc addition, leading to decreased IL-1β and MMPs expression and increased HO-1 expression. Red↑: enhanced by MIA; Red↓: decreased by MIA; Red=: did not change by MIA; Green↑: enhanced by zinc; Green↓: decreased by zinc.

Conclusions
In conclusion, the results of the present study demonstrate, both in vitro and in vivo, zinc can prevent against MIA-induced changes in cartilage degradation similar to human OA. It suggests that zinc has the potential to be a preventive supplement for OA in humans.
Acknowledgments: This work was partially supported by a grant from the Ministry of Science and Technology, Taiwan (104-2320-B-037-024-MY3).
Author Contributions: All authors made a significant contribution to the study and are in agreement with the content of the manuscript. Tzu-Ching Huang, Jeng-Hsien Yen, and Kee-Lung Chang conceived and designed the study. Tzu-Ching Huang, Wen-Tsan Chang, Yu-Chen Hu, Bau-Shan Hsieh, Hsiao-Ling Cheng, and Pu-Rong Chiu performed the experiments. Tzu-Ching Huang and Yu-Chen Hu analyzed the data. Tzu-Ching Huang, Pu-Rong Chiu, and Kee-Lung Chang interpreted the data and drafted the manuscript.

Conflicts of Interest:
The authors have declared no conflict of interest.