Schisandra extract ameliorates arthritis pathogenesis by suppressing the NF‐κB and MAPK signalling pathways

Abstract Schisandra chinensis is a medicinal plant used to treat various diseases. Extracts from the leaves or fruits of S. chinensis and their components are used in osteoarthritis (OA). The OA inhibitory effect of schisandrol A, one of its components, has been previously confirmed. We aimed to confirm the OA inhibitory effect of Schisandra (including components like schisandrol A) to identify why the inhibitory effect of the Schisandra extract is better. First, we investigated the effects of the Schisandra extract on OA as a potential therapeutic. Experimental OA was induced in a mouse model via destabilized medial meniscus surgery. The animals were orally administered the Schisandra extract; the inhibition of cartilage destruction was confirmed using histological analysis. In vitro analysis showed that the Schisandra extract attenuated osteoarthritic cartilage destruction by regulating IL‐1β‐induced MMP3 and COX‐2 levels. The Schisandra extract inhibited IL‐1β‐induced degradation of IκB (NF‐κB pathway) and IL‐1β‐induced phosphorylation of p38 and JNK (mitogen‐activated protein kinase (MAPK) pathway). RNA‐sequencing analysis showed that the Schisandra extract decreased the expression of IL‐1β‐induced MAPK and NF‐κB signalling pathway‐related genes more than schisandrol A alone. Therefore, Schisandra extract may be more effective than schisandrol A in preventing OA progression by regulating MAPK and NF‐κB signalling.

is associated with the development of OA via the downstream activation of catabolic factors in articular chondrocytes, such as the increase in expressions of MMPs and COX-2. 9,10 In addition, MAPK subtypes (extracellular signal-regulated kinases1/2 [ERK1/2], p38, c-Jun N-terminal kinase [JNK]) are known to produce MMPs and COX-2 in chondrocytes and are involved in the progression of OA. 11,12 Thus, NF-κB and MAPK signalling pathways are activated by IL-1β, which increases the expression of catabolic factors such as MMP3 and COX-2, leading to the onset of OA. 13 Currently, natural plant-based treatments are being developed as alternatives for several diseases because they have fewer side effects than previously known drugs. 14,15 Previous studies have shown that extracts from some plants inhibit the development of OA. 16,17 Schisandra chinensis (Turcz.) Baill is cultivated in northeastern China, Korea, and Japan. As a traditional Chinese herb, it has been used as an antitussive and for the treatment of insomnia and hepatitis. [18][19][20] Recent studies have shown that the extracts of Schisandra possess biological activities, including anticancer, antioxidant, neuroprotective, hepatoprotective and anti-inflammatory activities. 21 In addition, studies have reported that the Schisandra leaf extract relieves pain and inflammation in a rat model of OA. 22,23 Schisandra is composed of several single compounds, one of which is schisandrol A, which is known to exert various pharmacological effects, including antioxidant, antiapoptotic and antiallergic effects. 24 Previous studies have shown that schisandrol A suppresses pro-inflammatory mediators (e.g. COX-2), which inhibit NF-κB activation. 24 In our previous study, we showed that schisandrol A inhibits OA progression by inhibiting NF-κB signalling. 25 Here, we aimed to investigate whether the use of the Schisandra extract containing various biologically active compounds affects OA progression more than that of a single substance, such as schisandrol A. To achieve this, we set up in vitro OA conditions using chondrocytes and compared the extent of changes in the expression of signalling genes involved in OA after treatment with schisandrol A (single compound) and Schisandra extracts containing various biologically active substances.

| Mouse model
The in vivo animal experiments were approved by the Animal Care and Use Committee of Ajou University, and the procedures adhered to the 8th edition of the Guide for the Care and Use of Laboratory Animals issued by the National Institutes of Health (protocol code 2016-0041). C57BL/6 mice and 5-day-old Institute of Cancer Research (ICR) mice were purchased from DBL Co. Ltd. C57BL/6J male mice weighing 18-20 g (10 weeks old) were housed at 23°C and exposed to a 12/12-h light-dark cycle; water and food were supplied regularly. Five-day-old ICR mice were used for articular primary chondrocyte cultures, and C57BL/6 mice were used for setting up the destabilized medial meniscus (DMM) mouse model.

| Reagents and treatment
Schisandrol A was purchased from Sigma-Aldrich. IL-1β was purchased from GenScript. Schisandrol A was dissolved in dimethyl sulfoxide for in vitro analysis, and the IL-1β recombinant protein was dissolved in sterile water. For oral administration to mice, various concentrations of schisandrol A (50, 100 and 500 mg/kg) were dissolved in phosphate-buffered saline (PBS). To induce OA in vitro, mouse articular chondrocytes were treated with IL-1β (1 ng/mL) or co-treated with IL-1β and Schisandra (50, 100 and 250 μg/mL) or schisandrol A (1000 μM, positive control) for 24 h prior to harvesting.

| Quantitative reverse transcriptionpolymerase chain reaction (qRT-PCR)
Total RNA was obtained from mouse articular chondrocytes using TRIzol reagent (Molecular Research Center Inc.). The sequences of primers used (for MMP, GAPDH and COX-2) are shown in Table S1.
The level of target gene amplification was determined using qRT-PCR with SYBR® Green fluorescence and Premix Ex Taq (TaKaRa Bio). The transcription level of each target gene was normalized to that of GAPDH and expressed as the fold change relative to the control group.

| Mouse model of experimental OA and oral administration of the extract
To create an OA model via medial meniscus tear, we performed DMM surgery to cut the meniscus of the mouse knee joint in 10-week-old male C57BL/6J mice. The mouse knee joint was processed for histological analysis 10 weeks after surgery. In the DMM-induced OA model, Schisandra (100, 200 or 500 mg/kg) was administered orally every other day for 6 weeks, and the mice were euthanized at the end of the 6-week regimen. Four treatment groups (DMM + PBS, DMM + 5 mg/kg extract, DMM + 10 mg/kg extract and DMM + 50 mg/kg extract) were used, with five animals in each treatment group.

| Evaluation of cartilage destruction
Cartilage destruction was evaluated via safranin O staining and scored using the Osteoarthritis Research Society International (OARSI) grading system. Mouse knee joints were fixed using 4% paraformaldehyde, dehydrated using 0.5 M EDTA (pH 8.0) for 2 weeks and embedded in paraffin. The paraffin blocks were cut into 5 μm sections and fixed on glass slides. The sections were hydrated using a graded ethanol series, and xylene was used to remove paraffin.
Immunohistochemistry was performed on mouse knee joint sections using anti-COX-2 (66351-I-Ig, Protein Tech).

| Culture of cartilage explants and Alcian blue staining
Schisandra extract (50, 100 and 250 μg/mL) or schisandrol A (400, 800 and 1000 μM) was administered to cartilage explants isolated from the knee joints of 5-day-old ICR mice (DBL) in combination with IL-1β for 48 h in DMEM. The cartilage explants were fixed using 4% paraformaldehyde, dehydrated using different concentrations of ethanol and embedded in paraffin blocks. The paraffin blocks were then cut into pieces of 5 μm thickness. The accumulation of sulphated proteoglycans was determined using Alcian blue staining. 14

| Statistical analysis
The data are presented as mean ± standard error of the mean. Two researchers independently prepared all the histological samples.
Each experiment was performed at least five times. One-way analysis of variance (anova) with Bonferroni's post hoc test was used for data analysis. The Prism 7 software (https://www.graph pad.com/ scien tific -softw are/prism/) was used to perform statistical analyses, and significance was defined at p ≤ 0.05.

| Schisandra extract was not cytotoxic for chondrocytes
First, we examined the cytotoxic effects of the Schisandra extract on chondrocytes. Chondrocytes were treated with 0, 50, 100, 250 and 500 μg/mL of the Schisandra extract for 24 h. As shown in Figure 1

| Schisandra extract suppressed the IL-1βinduced expression of catabolic factors in articular chondrocytes
We have already shown that the expression of catabolic factors such as MMP3 and COX-2 induces cartilage destruction. 9,10 Therefore, we examined whether the Schisandra extract suppressed the ex-  Figure 2C). Furthermore, the Schisandra extract inhibited IL-1βinduced PGE 2 production by chondrocytes ( Figure 2D). In the ex vivo experimental conditions of OA created using IL-1β, the degree of ECM degradation of the explant cartilage treated with IL-1β was reduced after treatment with the Schisandra extract and schisandrol A, as observed using Alcian blue staining ( Figure 2E,F). These results indicated that the Schisandra extract has the potential to block osteoarthritic progression by reducing MMP3 and COX-2 levels.

| Oral administration of the Schisandra extract suppressed cartilage destruction in the DMM-induced arthritis model
To examine whether oral administration of Schisandra suppressed arthritic cartilage degradation in vivo, we assessed the effect of the Schisandra extract in a mouse model of DMM-induced OA. The Schisandra extract in PBS (or the PBS-alone control) was orally administered to the mice thrice a week for 10 weeks after the DMM surgery ( Figure 3A). Oral administration of the Schisandra extract effectively reduced the degree of cartilage destruction when compared with that observed in the PBS control group ( Figure 3B). In addition, the OARSI grade and subchondral bone plate thickness in the group treated with the Schisandra extract were significantly lower than those in the PBS control group (Figure 3C,D). Moreover, we investigated the expression of COX-2 using immunohistochemistry.
Oral administration of the Schisandra extract reduced the expression of COX-2 in the cartilage of the mouse model of OA (Figure 3E,F). Therefore, the above results showed that the Schisandra extract inhibited cartilage destruction in a mouse model of OA.   We analysed how the expression pattern of these genes was altered by the Schisandra extract and schisandrol A. Lists of genes involved in each signalling pathway were derived from Ingenuity Pathway Analysis (IPA), and the RNA-seq data prepared using each gene list after treatment of IL-1β-stimulated chondrocytes with the Schisandra extract and schisandrol A were compared to obtain the pattern of gene expression ( Figure 5A). We observed that the expression of the NF-κB and MAPK signalling pathway (p38, JNK and ERK)-related genes, which was increased by IL-1β treatment, decreased considerably when the Schisandra extract was used compared to when schisandrol A alone was used ( Figure 5B,C). Thus, we suggest that the Schisandra extract may potentially inhibit IL-1β-activated MAPK and NF-κB signalling pathways more than schisandrol A ( Figure 6).

F I G U R E 1
Toxicity of Schisandra to chondrocytes. Cell viability was measured at various concentrations for 24 h and analysed using a lactate dehydrogenase (LDH) assay. Data were analysed using one-way analysis of variance with Bonferroni's test, and the plotted values are indicated as mean ± standard error of the mean.

F I G U R E 2
Schisandra inhibited the expression of MMPs and COX-2, and reduced IL-1β-induced production of PGE 2 and collagenase in mouse articular chondrocytes. Chondrocytes stimulated with IL-1β (1 ng/mL) were treated with different concentrations of Schisandra (0, 50, 100 or 250 μg/mL). The mRNA levels of MMP3 and COX-2 were measured using RT-PCR and qRT-PCR (A). COX-2 protein level was determined using western blotting and densitometry (B). PGE 2 (C) and collagenase (D) assays were performed with chondrocytes treated with various concentrations (0, 50, 100 or 250 μg/mL) of Schisandra after stimulation with IL-1β (1 ng/mL). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and extracellular signal-regulated kinase (ERK) were used as loading controls. Ex vivo cultured cartilage was cotreated with IL-1β and Schisandra extract (0, 50, 100 and 250 mg/mL) or schisandrol A (0, 400, 800 and 1000 μM) for 48 h and the cartilage area was analysed using Alcian blue staining (left); the stained area was quantified (right) (E, F). Data were analysed using one-way analysis of variance with Bonferroni's test, and the plotted values are indicated as mean ± standard error of the mean; *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 compared to the control group. Scale bar = 100 μm.

| DISCUSS ION
As damaged cartilage in OA cannot regenerate completely, painkillers with multiple negative effects are the mainstay of therapy. [26][27][28] Therefore, there is an urgent need for safe and efficient drugs for OA treatment. Recent reports have suggested the use of extracts from natural herbs or their components for treating OA. 29,30 Therefore, this study was conducted to confirm the possibility of using Schisandra extract as a potential treatment for OA. Several studies have found that herbal extracts alleviate chondrocyte inflammation, control pain and improve joint function. 30 35 Schisandrol B has been shown to inhibit the inflammatory response of microglia to lipopolysaccharide F I G U R E 4 Schisandra regulated IL-1β-induced the NF-κB and MAPK signalling pathways. Articular chondrocytes were pretreated with various concentrations of Schisandra for 24 h (n = 3) before being treated with IL-1β (1 ng/mL) for 12 min. Protein levels of phosphorylated JNK (pJNK), ERK (pERK) and p38 (pp38) were measured using western blotting. IκB level was detected using western blotting (A) and densitometry (B). Schisandrol A (1000 μM) was used as the positive control. Data were analysed using one-way analysis of variance with Bonferroni's test, and the plotted values are indicated as mean ± standard error of the mean; *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 compared to the control group.

F I G U R E 5
Schisandra regulated IL-1β-induced NF-κB and MAPK signalling pathway-related genes more than schisandrol A in RNAsequencing (seq) analysis. RNA-seq analysis data was used to construct the heat map and it was visualized using Prism. (A) The related gene numbers were quantified and plotted using heat map data. (B, C) Data were analysed using one-way analysis of variance with Bonferroni's test, and the plotted values are indicated as mean ± standard error of the mean; *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 compared to the control group.
by inhibiting the NF-κB/MAPK pathway. 36 Schisandrol A inhibits OA progression via the NF-κB signalling pathway. 25 In addition, several studies have shown that Schisandrin A and B can inhibit cartilage degradation by inhibiting the NF-κB and MAPK signalling pathway. 37,38 Other studies have shown that the Schisandra leaf extract blocks inflammatory effects and could be useful for treating OA. 22,23 In the present study, oral administration of the Schisandra extract protected against cartilage breakdown in a DMM mouse model of OA.
Recent reports revealed that IL-1β controls the expression of catabolic factors in mouse chondrocytes. 39,40 Therefore, experimental OA conditions can be simulated in vitro using mouse chondrocytes.
IL-1β treatment of chondrocytes increases the expression of catabolic factors, including MMP3 and COX-2. 41,42 These proteases contribute to collagenase activity and the breakdown of type II collagen after cartilage destruction. 43 Prostaglandin synthesis mediated by COX-2 is a principal player of OA, and elevated levels of prostaglandins upregulate MMP synthesis. 44,45 Clinical evidence demonstrates that MMP and COX-2 are highly expressed in the cartilage of patients with arthritis, which causes OA. 46 In this study, the Schisandra extract decreased the expression of MMP3, COX-2 and PGE 2 induced by IL-1β stimulation in chondrocytes.
NF-κB and MAPK signalling play crucial roles in cellular responses to various factors, including stress, chemokines and pro-inflammatory cytokines, 47,48 and are critical for the progression of OA. Activation of NF-κB signalling is initiated by the degradation of the IκB protein inhibitor bound to NF-κB. 49 After IκB is degraded in response to numerous stimuli, p65, one of the subunits of the NF-κB complex, is phosphorylated, following which the NF-κB complex translocates to the nucleus, wherein it upregulates MMPs and COX-2, targets of NF-κB. 50,51 The downstream players of the MAPK signalling pathways include p38, JNK and ERK1/2. Studies have shown that IL-1β induces the phosphorylation of ERK, JNK and p38, which activate the transcription factors associated with MAPK signalling. 51,52 This is involved in the production of inflammatory factors, including COX-2 and MMPs. 53 Several studies have shown that the activation of the NF-κB and MAPK signalling pathways increases the expression of catabolic factors that disrupt joint cartilage, leading to OA progression. 41,53 In addition, the MAPK signalling pathway stimulates the activation of NF-κB by inducing the phosphorylation of IκB. 54 Previous studies have established that Schisandra has the ability to suppress the NF-κB signalling pathway and inhibit the phosphorylation of JNK and p38, while leaving the phosphorylation of ERK unaffected. This leads to a suppression of catabolic factors induced by IL-1β. 55 Consistent with these findings, our study demonstrated that treatment with Schisandra extract in IL-1β-stimulated chondrocytes resulted in the inhibition of JNK F I G U R E 6 A general summary of the findings: the Schisandra chinensis extract improved arthritis pathogenesis by inhibiting the IL-1βinduced NF-κB and MAPK signalling pathways, thereby significantly reducing the expression levels of MMP3 and COX-2. and p38 phosphorylation, alongside the blocking of the NF-κB signalling pathway. Additionally, it has been reported that the MAPK signalling pathway induces phosphorylation of IκB, resulting in its degradation and the subsequent activation of the NF-κB signalling pathway. 54 Hence, suppressing MAPK signalling can effectively inhibit NF-κB signalling. Overall, our study revealed that treatment with Schisandra extract blocked IκB degradation and repressed phosphorylation of both p38 and JNK, without affecting ERK phosphorylation. Consequently, both the MAPK and NF-κB signalling pathways were inhibited.
As described previously, IL-1β activates the MAPK and NF-κB signalling pathways. 51,52 Using IPA analysis on RNA-seq data, the pattern of changes in the expression of NF-κB and MAPK signallingrelated genes in chondrocytes under in vitro OA conditions, which were increased by IL-1β, was analysed. We found that the number of genes, whose expressions were elevated by IL-1β, decreased further when treated with the Schisandra extract than with schisandrol A.
RNA-seq is a widely used method for analysing mRNA expression ling. Schisandrol A, one of the various active compounds from the Schisandra extract, affected MAPK signalling and NF-κB-related genes more than schisandrol A alone. Therefore, the inhibitory effect of Schisandra extract on OA progression can be higher than that of single compounds. We suggest that the preparation of therapeutic agents using not only the Schisandra extract but also natural extracts containing various active compounds may be potentially used to treat OA. Furthermore, some studies have demonstrated the use of S. chinensis extract in clinical trials and applications for various diseases. 60,61 Therefore, further research is needed to confirm its efficacy in treating patients with OA. Hence, we anticipate that through the outcomes of this study and subsequent research endeavours, S. chinensis extract may effectively mitigate the underlying pathogenesis of human OA. In brief, our study suggests that the utilization of Schisandra extract holds promise as a potential drug worthy of investigation, capable of providing an effective treatment for various diseases associated with the NF-κB and MAPK signalling pathways, as well as OA treatment.

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors confirm that there are no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data used to support the findings of this study are included within the article.