3′-Sialyllactose Protects SW1353 Chondrocytic Cells From Interleukin-1β-Induced Oxidative Stress and Inflammation

Osteoarthritis (OA) is a major degenerative joint disease. Oxidative stress and inflammation play key roles in the pathogenesis of OA. 3′-Sialyllactose (3′-SL) is derived from human milk and is known to regulate a variety of biological functions related to immune homeostasis. This study aimed to investigate the therapeutic mechanisms of 3′-SL in interleukin-1β (IL-1β)-treated SW1353 chondrocytic cells. 3′-SL potently suppressed IL-1β-induced oxidative stress by increasing the levels of enzymatic antioxidants. 3′-SL significantly reversed the IL-1β mediated expression levels of reactive oxygen species in IL-1β-stimulated chondrocytic cells. In addition, 3′-SL could reverse the increased levels of inflammatory markers such as nitrite, prostaglandin E2, inducible nitric oxide synthase, cyclooxygenase-2, IL-1β, and IL-6 in IL-1β-stimulated chondrocytic cells. Moreover, 3′-SL significantly inhibited the apoptotic process, as indicated by the downregulation of the pro-apoptotic protein Bax, upregulation of the anti-apoptotic protein Bcl-2 expression, and significant reduction in the number of TUNEL-positive cells in the IL-1β-treated chondrocytic cells. Furthermore, 3′-SL reversed cartilage destruction by decreasing the release of matrix metalloproteinases (MMP), such as MMP1, MMP3, and MMP13. In contrast, 3′-SL significantly increased the expression levels of matrix synthesis proteins, such as collagen II and aggrecan, in IL-1β-treated chondrocytic cells. 3′-SL dramatically suppressed the activation of mitogen-activated protein kinases (MAPK) and phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT)/nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathways, which are related to the pathogenesis of OA. Taken together, our data suggest that 3′-SL alleviates IL-1β-induced OA pathogenesis via inhibition of activated MAPK and PI3K/AKT/NF-κB signaling cascades with the downregulation of oxidative stress and inflammation. Therefore, 3′-SL has the potential to be used as a natural compound for OA therapy owing to its ability to activate the antioxidant defense system and suppress inflammatory responses.


INTRODUCTION
Osteoarthritis (OA) is a complex progressive degenerative joint disorder that accompanies cartilage degradation and physical disability (Sanchez et al., 2005;Sanchez et al., 2008;Jeon et al., 2017). The development and progression of OA are related to oxidative stress-induced cartilage damage and an imbalance between catabolic and anabolic factors in joints (Loeser, 2009;Appleton, 2018). Although the occurrence and development of OA have been studied extensively, there is currently no efficient therapy to prevent OA progression.
The accumulation of reactive oxygen species (ROS) causes an increase in oxidative stress, and these reactive products are detoxified by the anti-oxidative defensive system (Betteridge, 2000;Jones, 2008). ROS are free radicals that are mainly generated by mitochondria, in the form of nonmitochondrial membrane-bound nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and xanthine oxidase (XO) (Turrens, 2003). Several studies have indicated that elevated oxidative stress and excessive generation of ROS are observed in OA patients (Altindag et al., 2007;Erturk et al., 2012;Altay et al., 2015).
Recent studies revealed that excessive generation of ROS occurs as OA develops, leading to increased inflammation (Bolduc et al., 2019;Xie et al., 2019;Ansari et al., 2020). Interleukin-1β (IL-1β) and IL-6 are highly upregulated in OA joints and play important roles in the pathogenesis of OA by modulating oxidative stress, apoptosis, cartilage extracellular matrix degradation extracellular matrix (ECM) synthesis, and intracellular signaling pathways in OA (Kapoor et al., 2011;Lepetsos and Papavassiliou, 2016;Collins et al., 2018). In particular, mitogen-activated protein kinases (MAPK) and phosphoinositol 3 kinase (PI3K)/protein kinase B (AKT)/nuclear factor-κ light chain enhancer of activated B cells (NF-κB) pathways play vital roles in OA pathogenesis (Ahmed et al., 2005;Lu et al., 2018;Huang et al., 2019). Thus, oxidative stress and inflammation are closely related, and their regulation should be considered as therapeutic strategies of OA.
Human SW1353 chondrocytic cells and human chondrocytes have similar phenotypes. Previous studies have shown that IL-1β can mimic the pathological microenvironment of OA chondrocytes (Jia et al., 2013;Bao et al., 2016). Herein, we investigated the antioxidant and anti-inflammatory activities and mechanisms of action of 3′-SL on IL-1β-treated human SW1353 chondrocytic cells and explored the mechanisms underlying potential therapeutic effects in OA. (JNK), JNK, p-PI3K, PI3K, p-AKT, AKT, p-inhibitor of nuclear factor kappa B (p-IκBα), IκBα, p-P65, P65, glyceraldehyde-3phosphate dehydrogenase (GAPDH), and horseradish peroxidase-conjugated secondary antibodies were obtained from Santa Cruz Biotechnology (Dallas, TX, United States). p-PI3K antibody and enhanced chemiluminescence detection system were purchased from Cell Signaling Technologies (Beverly, MA, United States) and Amersham Pharmacia Biotech (Little Chalfont, United Kingdom), respectively.

Cell Culture
Human chondrocytic cells were maintained in DMEM-HG with 10% FBS and 1% penicillin-streptomycin at 37°C. When approximately 80% confluency was achieved, the cells were washed with PBS and harvested with 0.05% trypsin-EDTA. Next, the cells were washed, centrifuged (1,000 g, 5 min, 25°C), resuspended, and finally seeded in new plates. The medium was replaced every 2-3 days. The cells were treated with IL-1β (10 ng/ml) under hypoxic conditions for 24 h, as previously described (Baek et al., 2018a;Baek et al., 2018b) to mimic the pathological microenvironment of OA chondrocytes. Then, 50 or 100 μM of 3′-SL was added and incubation continued for 24 h. In the NF-κB inhibitor experiment, the cells were pretreated with Bay 11-7082 (10 µM), which was dissolved in dimethyl sulfoxide, before the cells were exposed to IL-1β with or without 3′-SL. The culture supernatants were harvested, centrifuged (2,000 g, 5 min, 25°C), and stored at −70°C until further analysis. The cell pellets were washed with ice-cold PBS, centrifuged (2,000 g, 5 min, 4°C), and stored at −70°C until further analysis.

Enzyme-Linked Immunosorbent Assay
Cell culture supernatants were collected, centrifuged (12,000 ×g, 5 min, 4°C), and stored at −70°C until use. The production of ROS, activities of TAC, SOD, and CAT, and the levels of IL-1β, IL-6, nitrite, and PGE2 in cell culture supernatants were measured according to the manufacturer's instructions.

Terminal dUTP Nick End-Labeling Assay
For analysis of apoptosis, SW1353 human chondrocytic cells were seeded in gelatin-coated slides in 6-well cell plates. IL-1β and 3′-SL treatments were performed as described in Cell Culture. The DeadEnd ™ Fluorometric TUNEL System was conducted according to the manufacturer's protocol. Samples were mounted on glass slides with fluorescent mounting medium with DAPI for imaging, using the Zeiss Axio Imager M2 (Carl Zeiss, Gottingen, Germany) fluorescence microscope. The number of positively stained cells over the total number of cells per specimen field was measured, and the percentage of positive cells was calculated. Four individual specimens per group were analyzed.

Quantitative Real-Time Reverse Transcription Polymerase Chain Reaction
Total RNA extraction from the cell pellets was carried out using the TRIzol reagent, according to the manufacturer's protocol. Total RNA was used in cDNA synthesis with ReverTra Ace ® qPCR RT Master Mix with gDNA Remover. The mRNA expression of genes such as iNOS, COX-2, MMP1, MMP3, MMP13, collagen II, aggrecan, and GAPDH was profiled with qPCRBIO SyGreen Mix Hi-ROX in a StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA, United States). Data analysis was performed using the 2 -ΔΔCT method. Primers used for qRT-PCR are described in Table 1.

Statistical Analysis
All data are expressed as mean ± standard error of the mean from at least three independent experiments. Statistical analyses were conducted using the Statistical Package for Social Sciences (SPSS) version 25.0 (SPSS, Inc, Chicago, IL, United States). To confirm statistically significant results, one-way analysis of variance, followed by a post hoc Bonferroni comparison, was conducted. Statistical significance was set at p < 0.05.

39-Sialyllactose Suppressed Interleukin-1β-Induced Oxidative Stress in the Chondrocytic Cells
To determine whether 3′-SL has antioxidant effects, generation of ROS in the chondrocytic cells was induced by IL-1β. Treatment with Frontiers in Pharmacology | www.frontiersin.org April 2021 | Volume 12 | Article 609817 IL-1β elevated ROS levels in the chondrocytic cells. This elevation was significantly attenuated by treatment with 3′-SL in a dosedependent manner ( Figure 1A, **p < 0.01, and ***p < 0.001). Next, total antioxidant capacity and antioxidant enzyme activities were examined. 3′-SL potently suppressed IL-1β-induced oxidative stress, as revealed by the significant increase in total antioxidant capacity and levels of antioxidant enzymes, such as SOD and catalase, in a dose-dependent manner (Figures 1B-D, *p < 0.05, **p < 0.01, and ***p < 0.001). Similarly, thioredoxin, which was decreased by IL-1β, was significantly increased after 3′-SL treatment of the chondrocytic cells (Figures 1C,D, *p < 0.05, **p < 0.01, and ***p < 0.001). Taken together, these findings suggest that 3′-SL could suppress IL-1βinduced oxidative stress in chondrocytic cells via the reduction of ROS and the upregulation of antioxidant enzyme activities.

39-Sialyllactose Suppressed Interleukin-1β-Induced Cartilage Matrix Degradation in the Chondrocytic Cells
To assess whether 3′-SL can prevent the production of MMPs, which are catabolic factors in OA pathogenesis, the expression of MMPs was examined in IL-1β-treated chondrocytic cells. 3′-SL significantly suppressed IL-1β-induced expression of MMP1, MMP3, and MMP13 ( Figure 4, *p < 0.05, **p < 0.01, and ***p < 0.001). Collagen II and aggrecan are the two major components of the matrix, and they are considered as ECM synthesis genes. To explore whether 3′-SL mitigates IL-1βinduced ECM degradation in the cell model of OA, the expression of ECM synthesis genes was examined. As shown in Figure 4, IL-1β decreased the expression levels of collagen II and aggrecan, while 3′-SL treatment significantly suppressed IL-1βinduced cartilage degradation (*p < 0.05, **p < 0.01, and ***p < 0.001). Taken together, these results indicate that 3′-SL could inhibit cartilage matrix degradation by alleviating ECM degradation in IL-1β-induced chondrocytic cells.

DISCUSSION
In the present study, we evaluated the underlying mechanisms of the therapeutic effects of 3′-SL using IL-1β-treated chondrocytic cells. Our data revealed that 3′-SL efficiently protects the cells from oxidative stress, inflammation, apoptosis, and cartilage matrix degradation by suppressing the activated MAPK and PI3K/AKT/NF-κB signaling pathways (Figure 7). These results provide novel insights into the therapeutic mechanisms of action of 3′-SL as a treatment for OA. Oxidative stress is closely associated with cartilage destruction and OA progression (Sokolove and Lepus, 2013;Hu et al., 2017). The elevated production of ROS can make chondrocytes more vulnerable to oxidant-mediated cell death and lead to defective antioxidant mechanisms (Del Carlo and Loeser, 2003). Antioxidants, including SOD, catalase, and thioredoxin, are dysregulated and insufficient to detoxify ROS in OA patients (Altindag et al., 2007;Erturk et al., 2012;Gu et al., 2019;Zhong et al., 2019). Therefore, increasing the levels of these antioxidants can be used as a promising approach to prevent OA. Herein, our data revealed that 3′-SL significantly attenuated ROS production and changed intracellular redox status in the cell model of OA, as evidenced by decreased intracellular ROS production and increased oxidative stress defense system. These results demonstrated that 3′-SL can attenuate oxidative stress in IL-1β-treated chondrocytic cells.
Oxidative stress induces synovial inflammation, chondrocyte apoptosis, cartilage matrix synthesis, and intracellular signaling in OA progression (Lepetsos and Papavassiliou, 2016). Since a previous study showed that NLR family pyrin domain containing 3 (NLRP3) inflammasome activation in humans and mice is not involved in stressed-induced OA (Bougault et al., 2012), this study mainly focused on oxidative stress-related inflammation. It has been reported that nitric oxide catalyzed by iNOS and PGE2, produced from COX-2, could expedite the development of OA through ECM degradation (Sasaki et al., 1998). Moreover, IL-1β could induce an increase in iNOS and COX-2 (Daheshia and Yao, 2008;Sheu et al., 2015). Herein, the expression of iNOS, COX-2, NO, and PGE2 was usually enhanced in the cell model of OA. We found that the additional treatment with 3′-SL significantly downregulated the levels of iNOS, COX-2, NO, and PGE2. The synovial fluid and serum levels of IL-1β and IL-6 in OA patients are higher than in healthy normal subjects (Wojdasiewicz et al., 2014). In addition, it was observed that 3′-SL significantly suppressed the expression of IL-1 and IL-6 in the cell model of OA. These results demonstrated that 3′-SL can attenuate oxidative stressderived inflammatory responses in IL-1β-treated chondrocytic cells. Oxidative stress can cause mitochondrial apoptosis through the increased expression of the pro-apoptotic protein Bax and the decreased expression of the anti-apoptotic protein Bcl-2 (Pena-Blanco and Garcia-Saez, 2018). Our data revealed that 3′-SL significantly attenuated these changes in the expression of apoptotic proteins in IL-1β-treated chondrocytic cells. Likewise, the number of TUNEL-positive cells in the chondrocytic cells was significantly increased after IL-1β treatment. However, 3′-SL significantly decreased the number of TUNEL-positive cells. These results demonstrated that 3′-SL could reduce oxidative stress-derived apoptosis in IL-1β-treated chondrocytic cells.
Furthermore, the release of excess cartilage matrix degrading enzymes, such as MMP1, MMP3, and MMP13, is implicated in OA  progression (Ahmed et al., 2005;Wang et al., 2011;Maldonado and Nam, 2013). MMPs are a family of 23 enzymes with a specific function of inhibiting the synthesis of collagen II and aggrecan, which are critical for the synthesis of matrix-related proteins in cartilage (Dahlberg et al., 2000;Yamamoto et al., 2016). Proteolysis and pathological cartilage breakdown in OA are followed by abnormal expression of MMP members (Murphy et al., 2002). Among these MMP members, MMP1, MMP3, and MMP13 are responsible for the degradation of ECM in OA articular cartilage (Yoshihara et al., 2000). In this study, IL-1β increased the expression of MMP1, MMP3, and MMP13, while the expression of matrix related proteins, such as collagen II and aggrecan, was significantly downregulated in IL-1β-treated chondrocytic cells. However, all these changes could be restored by treatment with 3′-SL. These results demonstrate that 3′-SL could attenuate oxidative stressderived cartilage matrix degradation in IL-1β-treated chondrocytic cells. MAPK and PI3K/AKT/NF-κB signaling pathways are key mediators of cartilage degradation and OA progression (Sun et al., 2017;Zhang et al., 2019). MAPK signaling, which consists of ERK 1/2, P38, and JNK, can transduce extracellular stimuli into the nucleus (Keshet and Seger, 2010;Sugiura et al., 2011). In addition, MAPK activation is involved in the disruption of ECM (Sondergaard et al., 2010). Subsequently, the PI3K/AKT/NF-κB signaling cascade induces increased expression of catabolic factors and can contribute to cartilage degradation (Rigoglou and Papavassiliou, 2013;Jenei-Lanzl et al., 2019). Our data showed that the phosphorylation levels of P38, ERK, JNK, PI3K, and AKT, P65, and IκBα were significantly increased in the cell model of OA. However, these activated pathways were reversed by treatment with 3′-SL. The upstream regulators of NF-κB involve the MAPK and PI3K/AKT signaling pathways (Schulze-Osthoff et al., 1997;Yum et al., 2001). Thus, we evaluated the functional kinase activities using BAY 11-7082 in IL-1β-treated chondrocytic cells. In our study, the phosphorylation of IκBα and P65 was attenuated by pretreatment with Bay 11-7082 in IL-1β-induced chondrocytic cells. Furthermore, the inhibition of NF-kB activation by 3′- (E) Chondrocytic cells were pretreated with Bay 11-7,082 (10 μM) for 2 h, stimulated by IL-1β (10 ng/ml) for 24 h, and then treated with the different concentrations of 3′-SL (50 and 100 μM) for 24 h. The protein expression levels of p-IκBα, IκBα, p-P65, and P65 were determined by western blot and (F) quantification analysis. The values are mean ± standard error of the mean. *p < 0.05, **p < 0.01, and ***p < 0.001. PI3K, Phosphatidylinositol 3-kinase; AKT, protein kinase B; NF-κB, nuclear factor kappa light chain enhancer of activated B cells.
Frontiers in Pharmacology | www.frontiersin.org April 2021 | Volume 12 | Article 609817 8 SL and Bay 11-7082 was observed in IL-1β-induced chondrocytic cells. These observations highlight the importance and need to further investigate the detailed mechanisms for the activity of other kinases. Taken together, these results reveal that the regulation of MAPK and PI3K/ AKT/NF-κB signaling pathways plays a vital role in preserving the structural integrity of the matrix in the cell model of OA.
It should be note that although the chondrosarcoma cell line SW1353 is widely used as a substitute for primary adult articular chondrocytes, our results cannot be totally translated to primary OA chondrocytes considering the difference in gene expression between SW1353 and primary OA chondrocytes after treatment with IL-1β (Gebauer et al., 2005). This was the limitation of this study.
Sialyllactose is a representative human milk oligosaccharide in human breast milk. It can regulate immune homeostasis through receptor-mediated endocytosis and phagocytosis (Kim et al., 2019). Considering this and the multiple inhibitory effects of 3′-SL on IL-1β-mediated effects observed in this study, 3′-SL may mediate the receptor-mediated mechanism and could be used as a therapeutic agent for OA treatment. Further prospective studies are warranted to determine the accurate target of 3′-SL on IL-1βinduced oxidative stress and inflammation.
In conclusion, results from this study demonstrated that 3′-SL can counteract oxidative stress and inflammation via the suppression of activated MAPK and PI3K/AKT/NF-κB signaling pathways in IL-1β treated chondrocytic cells. Based on these findings, 3′-SL may be potentially used to protect against OA.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors.