Protective effects of Schisandrin C on chondrocyte damage by inhibiting MAPK and NF-κB signal pathways

Background: Osteoarthritis (OA) is a common joint disorder that affects the elderly population. The pathogenesis of OA is related to cartilage degradation and inammatory response. Schisandrin C (Sch C), a dibenzocyclooctadiene derivative of Schisandra chinensis, has been demonstrated to exert anti-inammatory effect in various inammation diseases. However, the effect of Sch C on OA remains unclear. Thus, we aimed to investigate its action on chondrocytes and explore the mechanism associated with the inammatory response. Methods: In this study, we chose rabbits and SW1353 cells as in vivo/in vitro models. Matrix metalloproteinase (MMP3), Nitric oxide (NO), IL-1β and TNF-α were detected by ELISA kits. The expression of MAPK/NF-κB related signaling molecules were determined by western blot. Results: In vitro, Sch C suppressed the IL-1β-induced production of NO and PGE2. Additionally, Sch C signicantly decreased IL-1β-induced p65 phosphorylation and mitogen-activated protein kinase (MAPK) activation, as evidenced by the reduced phosphorylation of p38, extracellular signal-regulated kinase (ERK), and c-Jun amino-terminal kinase (JNK). Moreover, Sch C prevented cartilage damage in rabbit OA model with lower Mankin’s score than the model group. Sch C also inhibited the level of inammatory cytokines in the articular cavity ushing uid in rabbit OA model. Conclusions: Our study suggested that Sch C inhibited the IL-1β-induced inammation and cartilage degradation through suppressing the MAPK and NF-κB signal pathways, indicating a potential in OA treatment. modied Eagle’s medium; CCK-8: cell counting kit-8; PVDF: polyvinylidene diuoride; HRP: horse-radish peroxidase; ECL: chemiluminescence; ACL-T: anterior cruciate ligament transection; HE: hematoxylin-eosin; NF-κB: nuclear factor-κB; IκB: inhibitory protein of κB; iNOS; inducible nitric oxide synthase; COX-2: cyclooxygenase-2.


Background
Osteoarthritis (OA) is the most frequently occurring joint disorder that affects diarthrodial joints such as the hand and knee joints. It is also a degenerative disease characterized by degradation of cartilage and in ammatory responses [1] . To date, the treatments of OA remain challenging, and its pathophysiology is still evolving. Present therapies such as Nonsteroidal anti-in ammatory drugs (NSAIDs) are at best moderately effective for the alleviation of the OA-related pain and swelling, which do not slow the progression of the disease [2] . Moreover, long-term use of NSAIDs can cause serious side effects. Therefore, the development of more effective and safe drugs for the treatment of OA is imperative.
During the progression of OA, in ammatory responses are considered to be risk factors for the loss of chondrocytes [3] . Previous Studies suggest that the pro-in ammatory cytokine IL-1β played an important role in the development of OA [4,5] . Patients who suffered from OA exhibited elevated levels of IL-1β in the synovial uid, synovial membrane, and cartilage [6] . It has been reported that IL-1β can induce reactive oxygen species production and increase caspase-3 and caspase-9 activities in chondrocytes, eventually leading to chondrocyte apoptosis [7] . Moreover, IL-1β has been demonstrated to induce the overproduction of nitric oxide (NO) and promote the expression of matrix-degrading genes such as matrix metalloproteinase (MMP), which are relevant to exacerbation of cartilage matrix degradation in OA [8] . It is well recognized that NF-κB and mitogen-activated protein kinase (MAPK) signal pathway are important in ammatory signaling in cartilage degradation and could be therapeutic targets in OA treatment [9,10] . Therefore, inhibition of in ammatory response may be a potential therapeutic target for future treatment of OA.
Schisandrin C (Sch C), which is extracted from Schisandra chinensis, was identi ed as the effective constituent, exhibiting anti-oxidative and anti-in ammatory effects [11,12] . Sch C is also found to reduce in ammation and oxidation through the MAPK pathway in lipopolysaccharide-stimulated human dental pulp cells [13] . However, the effect of Sch C on chondrocyte in ammation and OA progression has not been reported. The present study aimed to investigate the anti-in ammatory effects and mechanism of Sch C on IL-1β-induced chondrocyte in vitro. Furthermore, we investigated the effect of Sch C on rabbit OA model in vivo.

Cell culture and treatment
The human chondrosarcoma cell line SW-1353 was purchased from the Shanghai Institute of Cell Biology (Shanghai, China), and it was cultured in 5% DMEM supplemented with 10% FBS, L-glutamine, and antibiotics (50 U/ml penicillin and 50 µg/ml streptomycin). Cells grown to 70-80% con uency were washed twice with phosphate buffered saline prior to the treatment. Cells were incubated with various concentrations of Sch C in the absence or presence of IL-1β (10 ng/ml, Sigma-Aldrich, MO, USA). Control group were added with vehicle DMSO in the experiment. Cell viability was detected with a cell counting kit-8 (CCK-8, Wanlei Life Science, China) after 24 h of incubation. Matrix metalloproteinase (MMP3) in cell supernatants was detected by ELISA kits. The absorbance at 450 nm was measured using a microplate reader (Bio-Rad, CA, USA).

NO and PGE2 Measurement
To detect the level of NO and PGE2, SW-1353 cell supernatants were harvested after 24 h treatment with IL-1β (10 ng/ml) with or without different concentrations of Sch C. Griess reaction was conducted to measure the NO level and PGE2 concentration was detected with an ELISA kit (Elabscience, China) following the manufacturer's protocol.

Western blot
The total protein was extracted from SW-1353 cells using ice-cold RIPA buffer containing protease inhibitor (Boster Biological Technology, China) and stored at − 80 °C. The protein concentrations were quanti ed using the BCA assay (Beyotime Institute of Biotechnology, Shanghai, China). Aliquots (40 µg) were separated on 10% SDS-PAGE and transferred to the equilibrated polyvinylidene di uoride (PVDF) membrane (Millipore, MA, USA), which were blocked in 5% fat-free milk or 5% BSA for 2 h at room temperature and incubated at 4 °C overnight using the primary antibodies. Subsequently, blots were washed three times and incubated with horse-radish peroxidase (HRP)-labeled secondary antibody for 2 h at room temperature. Finally, the immunoreactive bands were detected with the chemiluminescence (ECL) kits (Millipore, MA, USA) and an imaging system (Bio-Rad, CA, USA). β-actin was used as the loading control and representative bands were shown.

Animals
Adult male New Zealand white rabbits (6 months old and weighing 3 ± 0.5 kg) were obtained from the Jinan Jinfeng Laboratory Animal Co. Ltd. The animals were housed singly and allowed freely with water and food. All animals were allowed to acclimate to their environment for 7 days before the experiment. All procedures were performed in agreement with the provision of Chinese Experimental Animals Administration Legislation and approved by the Animal Ethics Committee of Drug Nonclinical Evaluation and Research Center.
The animals were randomly divided into sham group and model group. Then rabbit OA models were established by open surgery including anterior cruciate ligament transection (ACL-T) and partial medial meniscectomy [14] . After 4 weeks, the animals that had undergone surgery were randomly divided into model group and Sch C group. After grouping, intra-articular injection with Sch C (50 µM) or vehicle (equal volume) was performed every 7 days. After 4 weeks, the animals were sacri ced, and the knee samples were xed with 4% paraformaldehyde solution.
Measurement of in ammatory cytokines in the articular cavity ushing uid 1 ml normal saline was injected into the articular cavity to ush, and the ushing solution was extracted, centrifuged at 2000 × g for 10 min, then the supernatant was absorbed and stored at -20 °C. The level of IL-1β and TNF-α in the articular cavity ushing uid were measured according to the instructions of ELISA kits.

Articular cartilage histopathology
Animals were euthanized and operated distal femur condyles were harvested. The specimens were xed with 10% formalin solution for 24 h and then decalci ed for 24 h. All specimens were cut into sections of 4 µm thickness and stained with hematoxylin-eosin (HE) for morphological analysis. Histological evaluation of OA was performed by eight individuals using modi ed Mankin's score [15] .

Statistical analysis
Data analysis was performed using Graph Pad Prism 5.0. Data are presented as mean ± SD and statistically analyzed using one-way analysis of variance, followed by the Tukey's test. P values of > 0.05 were considered statistically signi cant.

Results
Effects of Sch C on cell viability and production of MMP3 CCK-8 assay was used to determine the effects of Sch C on cell viability. As shown in Fig. 1A, Sch C exhibited no cytotoxicity on SW1353 cells with concentrations ≤ 100 µM at 24 hours. The effect of Sch C on IL-1β-induced MMP3 production was evaluated by ELISA kit. As shown in Fig. 1B, IL-1β (10 ng/ml) signi cantly induced upregulation of MMP3 level in cell supernatants. Cells pretreated with Sch C in the concentrations ranging from 25 to 75 µM signi cantly decreased the production of MMP3 induced by IL-1β in a concentration-dependent manner. Based on the results above, the concentration of 25 and 50 µM Sch C were used for in vitro studies and 50 µM was used for in vivo studies.
Effects of Sch C on IL-1β-induced production of NO and PGE2 The Griess reagent was used to measure the NO level and a ELISA kit was used to detect PGE2 level. IL-1β stimulation promoted production of NO and PGE2 in SW1353 cells, whereas Sch C at 25 and 50 µM decreased the production of NO and PGE2 ( Fig. 2A, B).

Effects of Sch C on activation IL-1β-induced MAPK signal activation in SW1353 cells
To explore the activation of MAPK in IL-1β-induced chondrocytes and its regulation by Sch C, we detected the phosphorylation of p38, ERK and JNK through western blot. IL-1β stimulation induced activation of MAPK pathway, as shown by increased expression of p-p38, p-ERK and p-JNK level, which were reduced by Sch C in a concentration-dependent manner (Fig. 3A). The results indicated that Sch C could suppress IL-1β-induced MAPK activation in SW1353 cells.

Effects of Sch C on IL-1β-induced NF-κB signal activation in SW1353 cells
The NF-κB signal activation was investigated by detecting the activation of p65 and degradation of IκBα. Western blot was used to detect activation of p65 and degradation of IκBα. The results demonstrated that IL-1β stimulation signi cantly induced phosphorylation of IκBα, and the upregulation of p-p65. However, pretreatment with 50 µM Sch C could signi cantly reduce the phosphorylation of IκBα and upregulation of p-p65 (Fig. 3B). The results indicated that Sch C could also suppress IL-1β-induced NF-κB signal activation in vitro.
Effects of Sch C on the level of IL-1β and TNF-α in the articular cavity ushing uid in rabbit OA model As shown in Fig. 4, the level of IL-1β and TNF-α in the ushing uid was signi cantly increased in the model group (p < 0.05). Treatment with Sch C signi cantly decreased the level of IL-1β and TNF-α in the ushing uid (p < 0.05).
Effects of Sch C on articular cartilage in rabbit OA model To evaluate the effect of Sch C on articular cartilage defects in vivo, rabbit animal model was constructed by removing ACL and partial medial meniscus. Compared with the model group, fewer ssuring was observed in cartilage from animals treated with 50 µM Sch C (Fig. 6A). The average Mankin's score of Sch C group was lower than the model group with signi cant difference (Fig. 6B). These data suggested that Sch C could ameliorate OA in the rabbit model.

Discussion
It is well recognized that OA is a disease characterized by whole joint with cartilage degeneration and synovial in ammation, which often leads to chronic pain and joint disability [16] . Evidence has shown the important role of in ammation in the pathophysiology of OA [17] . Inhibition of chondrocyte in ammation was a potential therapeutic strategy in OA [18] . In this study, we rst reported that the effects of Sch C on the IL-1β-induced in ammation and cartilage damage by inhibiting MAPK and NF-B signal pathway in SW1353 cells.
OA development is accompanied with local in ammatory responses. IL-1β, an important proin ammatory cytokine, induced in ammatory response in the chondrocytes, causing the damage to chondrocytes. Interference with IL-1β could ameliorate in ammation-induced damage to chondrocytes [19,20] . Previous study con rmed that IL-1β (10 ng/ml) could signi cantly increase the expression of in ammatory cytokines including NO and PGE2, causing damage to chondrocytes [21] . Our results revealed that IL-1β signi cantly upregulate the expression of NO and PGE2 and cause damage to SW1353 cells. However, Sch C could restore the changes induced by IL-1β stimulation. In addition, we construct rabbit OA model to observe the effects of Sch C on cartilage damage in vivo. The histological analysis further con rmed the protective effects of Sch C by ameliorating cartilage damage.
Due to the crucial role in chondrocyte in ammation and cartilage degradation in OA development [22,23] , MAPK and NF-κB were investigated in this study. In inactive state, NF-κB p65 presents with its inhibitor IκBα in cytoplasm. When treated by IL-1β, p65 is separated from IκBα with increased phosphorylation and translocate to nucleus, in which p65 promotes transcription of in ammation genes such as iNOS, COX-2 and cartilage degrading enzymes such as MMP3 [24,25] . In this study, Sch C decreased phosphorylation of p65 and its inhibitor IκBα in IL-1β-induced SW1353 cells. MAPK includes ERK1/2, p38 and JNK, which can be activated by phosphorylation. In accordance with the previous study [26] , our results revealed that Sch C decreased the phosphorylation of ERK1/2, p38 and JNK induced by IL-1β stimulation. These results indicated that the protective effect of Sch C on cartilage degradation is related to the suppression of MAPK and NF-κB pathways.

Conclusions
Our study is the rst paper illustrating that Sch C inhibited IL-1β-induced chondrocyte in ammation in vitro and ameliorated cartilage damage in vivo, which is related to the inhibition of NF-κB and MAPK signaling pathways. Our study indicated that Sch C may be a potential drug candidate in OA treatment.

Declarations
Availability of data and materials Author's contributions LRP conceived and designed the work, analyzed the data and wrote the original draft. All authors read and approved the nal manuscript. Figure 1 Effects of Sch C on cell viability and MMP3 production in cell supernatants. (A) SW-1353 cells were exposed to Sch C alone in the concentrations ranging from 12.5 to 100 μM for 24h (n = 5). (B) MMP3 production was detected by ELISA kit (n=3). Data was shown as means ± SD. #p < 0.05 when compared with the control group; *p < 0.05 when compared with the IL-1β treatment group.

Figure 2
Effects of Sch C on IL-1β-induced production of NO and PGE2 in SW-1353 cells. Cells were exposed with Sch C (25, 50μM) in the presence of IL-1β (10ng/ml) for 24h. Cell culture supernatants were harvested. (A) Griess reaction was used to detect NO concentration. (B) PGE2 level was measured by ELISA kit. Data was shown as means ± SD (n = 6). #p < 0.05 when compared with the control group; *p < 0.05 when compared with the IL-1β treatment group.

Figure 3
Effects of Sch C on MAPK pathway. Cells were exposed with Sch C (25, 50μM) with IL-1β. (A) phosphorylations of ERK, p38, JNK were detected by western blot. (B) Relative protein expression was quali ed by Image J software. ERK, p38 and JNK were used as the internal control, respectively (n=3). #p < 0.05 when compared with the control group; *p < 0.05 when compared with the IL-1β treatment group.

Figure 4
Effects of Sch C on NF-κB signaling pathway. Cells were exposed with Sch C (25, 50μM) with IL-1β. (A) Protein levels of p-IκBα, IκBα, p-p65, p65 were detected by western blot. (B) Relative protein expression was quali ed by Image J software. IκBα, p65 and β-actin were used as the loading control, respectively (n=3). #p < 0.05 when compared with the control group; *p < 0.05 when compared with the IL-1β treatment group.

Figure 5
Effects of Sch C on the level of IL-1β and TNF-α in the articular cavity ushing uid in rabbit OA model.
The concentrations of (A) IL-1β and (B) TNF-α were detected with ELISA kits (n = 8). #p < 0.05 when compared with the control group; *p < 0.05 when compared with the OA model group.